A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva

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By contrast, previous attempts to measure BDNF levels in readily accessible human body fluids such as saliva have been complicated by a lack of sensitivity and/or specificity of BDNF ELISAs (see Discussion). Recently, the suitability of a highly sensitive BDNF ELISA assay was validated using mouse plasma and serum where conventional BDNF ELISA fail to detect BDNF. In this report, we demonstrate that BDNF levels in human saliva are extremely low, in the low pg/mL range, yet detectable in all saliva samples tested. Methods Saliva samples were collected from healthy volunteers by a passive drool method. All samples were aliquoted and immediately frozen to keep at -80°C until use. At the time of use, the samples were thawed, centrifuged to remove any remaining particles and BDNF measurement conducted by using a previously validated BDNF ELISA assay (see below). Recombinant mature BDNF was used as a reference. Results The intra-assay variability was in the range of CV = 1.8 to 4.9%. Saliva samples could be kept frozen at -80°C for 2 months until use for measurements, but more than 4 freeze and thaw cycles caused BDNF losses presumably due to structural change of the antigen. The measurements were not affected by the method of collection provided the samples were diluted at least 2-fold. Conclusions The results indicate that human saliva samples collected in a non-invasive fashion can be used as a source of material to try and correlate BDNF levels with human conditions of interest. These results also confirm those of an independent study published recently using the same BDNF ELISA kit to measure BDNF levels in human saliva samples. 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F1000Research 2025, 14 :161 ( https://doi.org/10.12688/f1000research.160304.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva [version 1; peer review: 1 approved with reservations, 1 not approved] Fumie Akutsu https://orcid.org/0009-0007-0438-4602 1 , Shiro Sugino 1 , Mitsuo Watanabe https://orcid.org/0009-0006-0748-4863 1 , Yves-Alain Barde https://orcid.org/0000-0002-7627-461X 2 , Masaaki Kojima 1 Fumie Akutsu https://orcid.org/0009-0007-0438-4602 1 , Shiro Sugino 1 , [...] Mitsuo Watanabe https://orcid.org/0009-0006-0748-4863 1 , Yves-Alain Barde https://orcid.org/0000-0002-7627-461X 2 , Masaaki Kojima 1 PUBLISHED 04 Feb 2025 Author details Author details 1 FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan 2 School of Biosciences, Cardiff University, Cardiff, UK Fumie Akutsu Roles: Data Curation, Formal Analysis, Investigation, Writing – Original Draft Preparation, Writing – Review & Editing Shiro Sugino Roles: Conceptualization, Investigation, Writing – Review & Editing Mitsuo Watanabe Roles: Writing – Review & Editing Yves-Alain Barde Roles: Writing – Review & Editing Masaaki Kojima Roles: Conceptualization, Funding Acquisition, Investigation, Supervision, Validation, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS Abstract Background Hitherto, BDNF levels in humans have been primarily measured in serum and/or plasma where these levels are readily measurable, but primarily reflect the content of BDNF in blood platelets. By contrast, previous attempts to measure BDNF levels in readily accessible human body fluids such as saliva have been complicated by a lack of sensitivity and/or specificity of BDNF ELISAs (see Discussion). Recently, the suitability of a highly sensitive BDNF ELISA assay was validated using mouse plasma and serum where conventional BDNF ELISA fail to detect BDNF. In this report, we demonstrate that BDNF levels in human saliva are extremely low, in the low pg/mL range, yet detectable in all saliva samples tested. Methods Saliva samples were collected from healthy volunteers by a passive drool method. All samples were aliquoted and immediately frozen to keep at -80°C until use. At the time of use, the samples were thawed, centrifuged to remove any remaining particles and BDNF measurement conducted by using a previously validated BDNF ELISA assay (see below). Recombinant mature BDNF was used as a reference. Results The intra-assay variability was in the range of CV = 1.8 to 4.9%. Saliva samples could be kept frozen at -80°C for 2 months until use for measurements, but more than 4 freeze and thaw cycles caused BDNF losses presumably due to structural change of the antigen. The measurements were not affected by the method of collection provided the samples were diluted at least 2-fold. Conclusions The results indicate that human saliva samples collected in a non-invasive fashion can be used as a source of material to try and correlate BDNF levels with human conditions of interest. These results also confirm those of an independent study published recently using the same BDNF ELISA kit to measure BDNF levels in human saliva samples. READ ALL READ LESS Keywords ELISA, BDNF, Saliva, Immunoassay, Analytical Method Corresponding Author(s) Masaaki Kojima ( [email protected] ) Close Corresponding author: Masaaki Kojima Competing interests: FA, MW, SS, and MK are employees of FUJIFILM Wako Pure Chemical Corporation. YB is a consultant of FUJIFILM Wako Pure Chemical Corporation Grant information: This study was supported by FUJIFILM Wako Pure Chemical Corporation and no separate grants were involved in supporting this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 Akutsu F et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Akutsu F, Sugino S, Watanabe M et al. A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :161 ( https://doi.org/10.12688/f1000research.160304.1 ) First published: 04 Feb 2025, 14 :161 ( https://doi.org/10.12688/f1000research.160304.1 ) Latest published: 22 May 2025, 14 :161 ( https://doi.org/10.12688/f1000research.160304.2 )  There is a newer version of this article available. Suppress this message for one day. Introduction The role of Brain Derived Neurotrophic Factor (BDNF) in the function of the nervous system is well-established and a large body of work, primarily using mouse models, has long demonstrated this role to be essential for key aspects of brain function. 1 In humans, this essential role is supported by genetic studies including gene deletion and polymorphisms (for a recent review see Ateaque et al. 2 ). BDNF is also likely to be involved in a number of major neurological and psychiatric conditions including Major Depressive Disorder (MDD), 3 Alzheimer’s Disease, 4 Parkinson’s Disease, 5 and epilepsy. 6 What is less clear is the degree to which measurements of BDNF levels in accessible body fluids may be used to correlate these levels with brain function and dysfunction. While BDNF levels can be readily measured in human blood using traditional BDNF ELISAs, these levels primarily reflect the content of BDNF in blood platelets. 7 , 8 Thus, whether or not these levels are informative with regard to brain function and dysfunction in humans remains uncertain. This important question has been difficult to answer conclusively because of a major difference between mice and humans with regard to the presence of BDNF in blood. In mice, the most widely used animal model to explore the function and dysfunction of the nervous system, megakaryocytes, the progenitor cells of platelets, do not express the Bdnf gene at significant levels, unlike in the case in humans. 8 Very recently, minute levels of BDNF could be detected in mouse blood following the development of a much more sensitive BDNF ELISA. 9 The neurotrophins nerve growth factor (NGF) and neurotrophin-3 (NT3) share key structural features with BDNF but mouse serum incubation with anti-NGF and anti-NT3 antibodies did not reduce the BDNF ELISA signal. 9 By contrast, incubation with an anti-BDNF monoclonal antibody unrelated to the antibodies used in the BDNF ELISA did markedly reduce the signal. 9 The BDNF levels determined in mouse blood were found to be about 3 orders of magnitude lower than those determined in humans, with no difference between mouse plasma and serum, unlike is the case in humans. 9 This finding is consistent with the notion that BDNF in mouse blood does not originate from platelets, but from other sources, including the skeletal musculature as demonstrated by Fulgenzi et al. 10 Very recently, Ikenouchi et al. 11 conducted an independent study using the same BDNF ELISA described in the above and in an attempt to correlate BDNF levels in human saliva with psychological distress in healthcare workers 11 with the values in line with those reported here. Saliva samples can be readily collected and in a large number if needed, with minimum stress even for elderly patients. These straightforward points have attracted the attention of many in the biomedical community in the past as illustrated by a number of reports about measurement of BDNF in human saliva (see for example Nakagawa et al., 12 Kikuchi et al., 13 Zappella et al., 14 Biamonte et al., 15 Jasim et al., 16 and Zhang et al. 17 However, the BDNF values reported in these studies varied over a very wide range indicating that most BDNF measurement methods used thus far had either not the specificity and/or the sensitivity needed to reliably measure BDNF levels in human saliva, a conclusion already reached by Vrijen et al. 18 One recent exception was the use of a recently introduced and validated BDNF ELISA kit 9 that was independently used in a study conducted in parallel with ours (Ikenouchi et al., 11 see Discussion for details). In the present study, we report the supporting data indicating that the highly sensitive BDNF ELISA assay can be used to accurately measure levels of BDNF in human saliva samples, including after sample storage. Methods Samples Saliva samples were collected from 11 healthy volunteers at the company who did not have known significant health issues or drug usage. Written informed consents were obtained from all of the volunteers according to the company’s procedure which is set in accordance with Ethical Guidelines for Medical and Health Research Involving Human Subjects published by Ministry of Health, Labor, and Welfare, Japan. The participant were given a sterilized sample collection tube (2 mL cryovial (Salimetrics, Pennsylvania)) and instructed to rinse their mouth with a cup of water before saliva collection, saliva sample was collected into a sterilized sample collection tube by a passive drool method by using the Saliva Collection Aid (Salimetrics, Pennsylvania) according to the manufacturer’s instruction manual. All samples were collected in the same afternoon and then samples were aliquoted into 1.5 mL Eppendorf Safe-Lock Tube (Eppendorf, Tokyo) and then immediately frozen and kept at -80°C until use. At a time of sample use, the samples were thawed at room temperature and then centrifuged at 1500 × g for 15 min to remove remaining particles (pre-treated samples). Recombinant mature BDNF (PeproTech, Cranbury, New Jersey, USA) was purchased and used as a reference and standard material for the assays. Measurement of BDNF BDNF measurement was conducted by using Mature BDNF ELISA kit Wako, High Sensitive (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) according to the instructions provided with the kit. Briefly, samples were diluted 2-fold with the kit Buffer Solution. The BDNF standard stock solution was prepared by adding defined amount of purified water to the Mature BDNF Standard vial, and then a various concentration (0.000, 0.205, 0.512, 1.28, 3.20, 8.00, 20.0, 50.0, and 500 pg/mL) of standard solutions were prepared by diluting the 10 ng/mL Mature BDNF Standard stock solution with Buffer provided in the kit, according to the dilution scheme provided in the instruction. The solution initially filling the ELISA plate was discarded and the wells were washed 4 times with the Wash Solution (1×) included in the kit. The plate was inverted after each wash and gently tapped against clean paper towels to remove any excess liquid retained in the wells. 50 μL of diluted standard solution and of diluted samples were added to respective wells, with duplicate wells used for each standard and sample. After agitating the plate on a microplate mixer, the plate was then covered by Plate Seal (plastic film) and incubated for 2 hours at room temperature (20–25°C). After 2-hour incubation, the solution was discarded, and the wells were washed again 4 times with Wash Solution as above. 50 μL of Biotin-conjugated antibody solution was added to each well, the plate covered by Plate Seal and agitated and then incubated for one hour at room temperature. The solution was then discarded, and wells were washed 4 times with Wash Buffer as above. 50 μL of Peroxidase-conjugated Streptavidin Solution was added to each well, the plate was covered and incubated on for 30 min at room temperature. After a final series of 4 washes with the Wash Buffer, 50 μL of mixed luminescent reagents 1 and 2 (mixed at a ratio of 1:1 before use) were added to each well, the plate was placed on a shaker for 1 min and the luminescence was measured using a 96-well microplate reader Infinite200PRO MPlex from TECAN (Switzerland) at 10 min after the addition of the luminescent reagent. The standard solutions were used to generate a standard curve converting luminescence to BDNF concentrations used to determine the BDNF concentration in the experimental samples. All measurements were conducted twice (n=2) and average of 2 measurements was used for the evaluations. Results Spike test Saliva samples were spiked with 0, 20, or 100 pg/mL of known concentrations of recombinant mBDNF (reference material) at a 9:1 ratio, and then measurements conducted according to the instruction provided in the ELISA kit. The yields of the spiked mBDNF ranged from 85.1 to 102.0%, within the manufacturer’s specifications (within ±15%). The distribution of BDNF levels in undiluted samples were 0.296 to 4.09 pg/mL (average = 1.04 pg/mL, SD = 1.09 pg/mL) ( Table 1 ). Table 1. Recovery of spiked BDNF in saliva samples. Saliva sample ID Spiked BDNF (pg/mL) Measured BDNF (pg/mL) Recovered BDNF (pg/mL) Yield (%) #1 0 0.783 - - 20 18.4 17.7 88.5 100 90.3 89.6 89.6 #2 0 0.327 - - 20 18.2 17.9 89.5 100 91.6 91.3 91.3 #3 0 1.67 - - 20 22.1 20.4 102 100 98.8 97.1 97.1 #4 0 0.512 - - 20 18.1 17.6 88.0 100 86.9 86.4 86.4 #5 0 0.296 - - 20 18.7 18.4 92.0 100 95.7 95.4 95.4 #6 0 0.393 - - 20 17.5 17.1 85.5 100 85.5 85.1 85.1 #7 0 0.814 - - 20 18.4 17.6 88.0 100 86.4 85.6 85.6 #8 0 4.09 - - 20 23.9 19.8 99.0 100 91.1 87.0 87.0 #9 0 0.601 - - 20 18.6 18.0 90.0 100 101 100 100 #10 0 0.852 - - 20 18.3 17.4 87.0 100 86.3 85.4 85.4 #11 0 1.12 - - 20 18.5 17.4 87.0 100 100 98.9 98.9 Dilution linearity test Samples were serially diluted using the buffer included in the kit, and BDNF levels measured. As shown in Figure 1 , linearity was achieved when at least 2-fold sample dilution (one-to-one dilution) was conducted. Figure 1. Dilution linearity test. Samples were serially diluted by the buffer attached to the commercial kit. Precision Samples were spiked with 4 or 40 pg/mL of recombinant mBDNF at a ratio of 9:1. The resulted samples were measured 10 times (n=10) using the ELISA kit to calculate the within precision of the assay. The results showed that intra-assay repeatability was calculated to be within a range of CV =1.8 to 4.9% as shown in Table 2 . Table 2. Assay precision of the mBDNF ELISA. Sample ID #2 #8 #9 Spiked BDNF (pg/mL) 4.0 40.0 4.0 40.0 4.0 40.0 n Measured BDNF (pg/mL) 1 3.39 36.7 8.05 44.3 3.55 37.1 2 3.40 36.2 8.03 43.5 3.62 37.0 3 3.38 36.4 7.93 44.2 3.41 37.0 4 3.44 35.6 8.20 43.0 3.43 36.5 5 3.24 36.6 8.30 43.3 3.41 38.0 6 3.41 36.7 8.28 43.4 3.61 37.8 7 3.33 36.8 8.48 43.4 3.72 38.2 8 3.27 37.2 8.18 43.3 3.50 37.9 9 3.28 37.3 8.16 44.5 3.60 38.2 10 3.60 39.2 8.23 45.5 3.99 39.3 Mean 3.37 36.9 8.18 43.8 3.58 37.7 SD 0.10 0.95 0.16 0.77 0.18 0.82 CV (%) 3.1 2.6 1.9 1.8 4.9 2.2 Sample stability Long-term stability Long term BDNF stability was assessed at 2 different temperatures, -20 and -80°C by using 6 saliva samples spiked with the recombinant mBDNF. They were kept at the designated temperatures for 2 or 3 months, then remaining BDNF levels were measured by the ELISA kit of this study. As shown in Table 3 , degradation of BDNF at -20°C was significant and 30-40% reduction was observed. By contrast, BDNF reduction during 2-month storage was minimal at -80°C and the changes were within 15% although one outlier showed 20% reduction, which was still within the boundary of the acceptable range of 80 to 120% shown in ICH HARMONISED TRIPARTITE GUIDELINE, “Validation of Analytical Procedures: Methodology: Text and Methodology . Q2(R1)” (2005). These data indicates that measurement should be performed within 2 months after saliva sample collection. Table 3. Long term stability of BDNF in saliva. ID Day 0 2 months 3 months BDNF (pg/mL) (%) BDNF (pg/mL) (%) BDNF (pg/mL) (%) a) Stored at -20°C Spiked sample A 1.86 100% 1.17 62.9% 0.46 24.6% Spiked sample B 3.62 100% 2.23 61.6% 1.77 48.9% Spiked sample C 5.76 100% 5.71 99.1% 4.05 70.3% Spiked sample D 18.5 100% 14.2 76.8% 8.00 43.2% Spiked sample E 29.6 100% 16.9 57.1% 11.3 38.2% Spiked sample F 37.1 100% 24.0 64.7% 16.8 45.3% b) Stored at -80°C Spiked sample A 1.86 100% 1.70 91.4% 0.88 47.5% Spiked sample B 3.62 100% 3.43 94.8% 2.24 61.9% Spiked sample C 5.76 100% 5.21 90.5% 5.05 87.7% Spiked sample D 18.5 100% 16.4 88.6% 8.70 47.0% Spiked sample E 29.6 100% 23.6 79.7% 16.0 54.1% Spiked sample F 37.1 100% 34.7 93.5% 28.5 76.8% Repeated freeze and thawing test Impact of repeated freeze and thawing was assessed by repeating freeze and thawing cycle of the spiked saliva samples for 5 time and the remaining BDNF levels were measured. As shown in Figure 2 , more than 4 times freeze and thawing resulted in 15% or more BDNF reduction, therefore the maximum cycle number applicable to the frozen BDNF samples were determined to be 3 times or less. Figure 2. Effect of freeze and thaw cycles for the stability of BDNF in saliva samples. Evaluation of sample collection methods In order to standardize the sample collection procedure, 2 types of saliva collection devices, Salivette made of cotton (Sartstedt, Nümbrecht, Germany) and SalivaBio Infant Swab (Salimetrics, PA, USA) were tested for the mBDNF recovery. The tested devices were soaked with the saliva samples (ID #2, 4, and #11) spiked with known concentrations of recombinant mBDNF and then samples were recovered by centrifugation (1000 × g, 2 min for Salivette and 1500 × g, 15 min for SalivaBio Infant Swab), according to the manufacturers’ instructions. The resulted samples were diluted 2-fold and then mBDNF levels were measured. The average recovery of the mBDNF with these commercially available saliva collection devices were 97.8 and 102.3% with the cotton (Salivette) and inactivated polymer (SalivaBio Infant Swab) devices, respectively ( Table 4 ). Table 4. mBDNF recovery from swab samples. Saliva sample ID Spiked BDNF (pg/mL) Measured BDNF (pg/mL) Recovered BDNF (pg/mL) Yield (%) #2, cotton 0 0.224 20 18.6 18.4 92.0 100 102 102 102 #2, inactivated polymer 0 0.309 20 20.5 20.2 101 100 103 103 103 #4, cotton 0 0.432 20 18.2 17.8 89.0 100 109 109 109 #4, inactivated polymer 0 0.580 20 23.0 22.4 112 100 106 105 105 #11, cotton 0 0.804 20 18.0 17.2 86.0 100 110 109 109 #11, inactivated polymer 0 0.980 20 20.9 19.9 99.5 100 94.1 93.1 93.1 Discussion The main outcome of this study is that it confirms the notion that BDNF levels can be accurately quantified in human saliva samples with a commercially available, highly sensitive BDNF ELISA kit. The distribution of the BDNF levels in the saliva samples were 0.296 to 4.09 pg/mL, in line with the levels reported by Ikenouchi et al. 11 in a very recent study using the same BDNF ELISA. These levels are indeed extremely low and significantly below the detection limits of other commercially available ELISA kits, in agreement with conclusions reached by Vrijen et al. 18 These levels are also well below the limit of detection of notoriously insensitive methods such as Western blots. 19 , 20 Also, previous attempts to increase the sensitivity of BDNF ELISA measurements by the use of for example BDNF polyclonal antibodies raise questions about the specificity of such assays. 21 , 22 Not only do the levels of BDNF in human saliva reported in these previous studies vary greatly but also, they are 2 to 3 orders of magnitude higher than those reported here, i.e. in the ng/ml range (see for example Mandel et al. 21 or Bhat et al. 22 ). Obviously, saliva samples can be readily and repeatedly collected for BDNF level determinations and possible correlations explored with for example sex, age and physical exercise. Indeed, there is currently a great deal of interest reflected by multiple studies exploring possible relationships between various interventions such as physical exercise, cognitive training, dietary factors and the levels of “exerkines”, defined as secreted factors in response to exercise and carriers of information to various organs including brain (for a recent review, see Chow et al. 23 ). A possible role for exerkines is of great interest, especially in the context of the prevention of cognitive decline accompanying aging and neurodegenerative diseases including Alzheimer’s Disease. One such exerkine is platelet factor 4 (PF4), a factor derived from blood platelets that has recently been proposed to mediate the rejuvenating effects of young blood and to stimulate adult neurogenesis. 24 , 25 It has also been reported that platelets are a major source of extracellular vesicles (EVs) carrying PF4 26 as well as BDNF. 27 In theory then, it is possible that PF4 and BDNF may be transferred by platelet-derived EVs from platelet to brain whereby the reality of this proposition has not been demonstrated yet at the ultrastructural level using immuno-electron microscopy. Conclusion The results of this study using a highly sensitive BDNF ELISA demonstrate that saliva samples can be used to measure BDNF levels accurately in readily accessible human samples like saliva, thus confirming the very recent results of Ikenouchi et al. 11 Correlations can now be readily explored between BDNF levels in human saliva and a range of physiological and pathological conditions of interest, including exercise, ageing, depression and neurodegeneration. Ethical considerations Collection of the saliva samples from the volunteers was approved at the Expedited 27th Fujifilm Wako Pure Chemicals Life Science Ethics Review Committee with the approval number of #LS 011 on February 3, 2022. The company’s Ethics Review Committee was set in accordance with Ethical Guidelines for Medical and Health Research Involving Human Subjects published by Ministry of Health, Labor, and Welfare, Japan , and written i nformed consents were obtained from all of the volunteers. Author contributions Fumie Akutsu: Data curation; Formal analysis; Investigation; Validation; Writing—original draft; Writing—review & editing. Shiro Sugino: Conceptualization; Investigation; Writing—review & editing. Mitsuo Watanabe: Writing—review & editing. Yves-Alain Barde: Writing—review & editing. Masaaki Kojima: Conceptualization; Funding acquisition; Investigation; Supervision; Validation; Writing—review & editing. Roles Fumie Akutsu : Data curation; Formal analysis; Investigation; Validation; Writing—original draft; Writing—review & editing. Shiro Sugino : Conceptualization; Investigation; Writing—review & editing. Mitsuo Watanabe : Writing—review & editing. Yves-Alain Barde : Writing—review & editing. Masaaki Kojima : Conceptualization; Funding acquisition; Investigation; Supervision; Validation; Writing—review & editing. Data availability Figshare: BDNF measurement in saliva samples using highly sensitive ELISA, https://www.doi.org/10.6084/m9.figshare.27978699 28 This project contains the following underlying data: 1. Dataset for the tables and figures (Saliva validation data for manuscript_final.xlsx) Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). References 1. Huang EJ, Reichardt LF: Neurotrophins: roles in neuronal development and function. Annu. Rev. Neurosci. 2001; 24 : 677–736. PubMed Abstract | Publisher Full Text | Free Full Text 2. Ateaque S, Merkouris S, Barde Y-A: Neurotrophin signaling in the human nervous system. Front. Mol. Neurosci. 2023; 16 : 1225373. PubMed Abstract | Publisher Full Text | Free Full Text 3. Duman RS, Monteggia LM: A neurotrophic model for stress-related mood disorders. Biol. Psychiatry. 2006; 59 (12): 1116–1127. Publisher Full Text 4. Tapia-Arancibia L, Aliaga E, Silhol M, et al. : New insights into brain BDNF function in normal aging and Alzheimer disease. Brain Res. Rev. 2008; 59 : 201–220. PubMed Abstract | Publisher Full Text 5. Parain K, Murer MG, Yan Q, et al. : Reduced expression of brain-derived neurotrophic factor protein in Parkinson’s disease substantia nigra. Neuroreport. 1999; 10 (3): 557–561. Publisher Full Text 6. Takahashi M, Hayashi S, Kakita A, et al. : Patients with temporal lobe epilepsy show an increase in brain-derived neurotrophic factor protein and its correlation with neuropeptide Y. Brain Res. 1999; 818 (2): 579–582. PubMed Abstract | Publisher Full Text 7. Yamamoto H, Gurney ME: Human platelets contain brain-derived neurotrophic factor. J. Neurosci. 1990; 10 (11): 3469–3478. PubMed Abstract | Publisher Full Text | Free Full Text 8. Chacón-Fernández P, Säuberli K, Colzani M, et al. : Brain-derived Neurotrophic Factor in Megakaryocytes. J. Biol. Chem. 2016; 291 (19): 9872–9881. PubMed Abstract | Publisher Full Text | Free Full Text 9. Want A, Morgan JE, Barde Y-A: Brain-derived neurotrophic factor measurements in mouse serum and plasma using a sensitive and specific enzyme-linked immunosorbent assay. Sci. Rep. 2023; 13 : 7740. PubMed Abstract | Publisher Full Text | Free Full Text 10. Fulgenzi G, Hong Z, Tomassoni-Ardori F, et al. : Novel metabolic role for BDNF in pancreatic β-cell insulin secretion. Nat. Commun. 2020; 11 : 1950. PubMed Abstract | Publisher Full Text | Free Full Text 11. Ikenouchi A, Okamoto N, Hamada S, et al. : Association between salivary mature brain-derived neurotrophic factor and psychological distress in healthcare workers. Brain Behav. 2023; 13 : e3278. PubMed Abstract | Publisher Full Text | Free Full Text 12. Nakagawa Y, To M, Saruta J, et al. : Effect of social isolation stress on saliva BDNF in rat. J. Oral Sci. 2019; 61 (4): 516–520. PubMed Abstract | Publisher Full Text 13. Kikuchi T, Sakaguchi W, Saruta J, et al. : Hypertriglyceridemia-induced brain-derived neurotrophic factor in rat submandibular glands. J. Oral Biosci. 2020; 62 : 327–335. PubMed Abstract | Publisher Full Text 14. Zappella M, Biamonte F, Balzamino BO, et al. : Relaxation Response in Stressed Volunteers: Psychometric Tests and Neurotrophin Changes in Biological Fluids. Front. Psych. 2021; 12 : 655453. PubMed Abstract | Publisher Full Text | Free Full Text 15. Biamonte F, Re A, Balzamino BO, et al. : Circulating and Salivary NGF and BDNF Levels in SARS-CoV-2 Infection: Potential Predictor Biomarkers of COVID-19 Disease—Preliminary Data. J. Pers.Med. 2022; 12 : 1877. PubMed Abstract | Publisher Full Text | Free Full Text 16. Jasim H, Ghafouri B, Gerdle B, et al. : Altered levels of salivary and plasma pain related markers in temporomandibular disorders. J. Headache Pain. 2020; 21 : 105. PubMed Abstract | Publisher Full Text | Free Full Text 17. Zhang Y, Ye S, Zhang Y, et al. : Potential salivary and serum biomarkers for burning mouth syndrome and their relationship with anxiety/depression. J. Dent. Sci. 2024; 19 : 1052–1060. PubMed Abstract | Publisher Full Text | Free Full Text 18. Vrijen C, Schenk HM, Hartman CA, et al. : Measuring BDNF in saliva using commercial ELISA: Results from a small pilot study. Psychiatry Res. 2017; 254 : 340–346. PubMed Abstract | Publisher Full Text 19. Mandel AL, Ozdener H, Utermohlen V: Identification of pro- and mature brain-derived neurotrophic factor in human saliva. Arch. Oral Biol. 2009; 54 (7): 689–695. PubMed Abstract | Publisher Full Text | Free Full Text 20. Jasim H, Anders Carlsson A, Hedenberg-Magnusson B, et al. : Saliva as a medium to detect and measure biomarkers related to pain. Sci. Rep. 2018; 8 : 3220. PubMed Abstract | Publisher Full Text | Free Full Text 21. Mandel AL, Ozdener H, Utermohlen V: Brain-derived neurotrophic factor in human saliva: ELISA optimization and biological correlates. J. Immunoassay Immunochem. 2011; 32 (1): 18–30. PubMed Abstract | Publisher Full Text | Free Full Text 22. Bhat SS, Revankar AV, Naik RD: Human salivary concentrations of brain derived neurotrophic factor correlates with subjective pain intensity associated with initial orthodontic therapy. Sci. Rep. 2023; 13 (1): 1752. PubMed Abstract | Publisher Full Text | Free Full Text 23. Chow LS, Gerszten RE, Taylor JM, et al. : Exerkines in health, resilience and disease. Nat. Rev. Endocrinol. 2022; 18 (5): 273–289. PubMed Abstract | Publisher Full Text | Free Full Text 24. Leiter O, Brici D, Fletcher SJ, et al. : Platelet-derived exerkine CXCL4/platelet factor 4 rejuvenates hippocampal neurogenesis and restores cognitive function in aged mice. Nat. Commun. 2023; 14 : 4375. PubMed Abstract | Publisher Full Text | Free Full Text 25. Schroer AB, Ventura PB, Sucharov J, et al. : Platelet factors attenuate inflammation and rescue cognition in ageing. Nature. 2023; 620 (7976): 1071–1079. PubMed Abstract | Publisher Full Text | Free Full Text 26. Antwi-Baffour S, Adjei J, Aryeh C, et al. : Understanding the biosynthesis of platelets-derived extracellular vesicles. Immun. Inflamm. Dis. 2015; 3 (3): 133–140. Publisher Full Text 27. Aatonen MT, Ohman T, Nyman TA, et al. : Isolation and characterization of platelet-derived extracellular vesicles. J. Extracell. Vesicles. 2014; 3 : 3. PubMed Abstract | Publisher Full Text | Free Full Text 28. Watanabe M, Akutsu F, Sugino S, et al. : BDNF measurement in saliva samples using highly sensitive ELISA. Dataset. figshare. 2025. Publisher Full Text Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 04 Feb 2025 ADD YOUR COMMENT Comment Author details Author details 1 FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan 2 School of Biosciences, Cardiff University, Cardiff, UK Fumie Akutsu Roles: Data Curation, Formal Analysis, Investigation, Writing – Original Draft Preparation, Writing – Review & Editing Shiro Sugino Roles: Conceptualization, Investigation, Writing – Review & Editing Mitsuo Watanabe Roles: Writing – Review & Editing Yves-Alain Barde Roles: Writing – Review & Editing Masaaki Kojima Roles: Conceptualization, Funding Acquisition, Investigation, Supervision, Validation, Writing – Review & Editing Competing interests FA, MW, SS, and MK are employees of FUJIFILM Wako Pure Chemical Corporation. YB is a consultant of FUJIFILM Wako Pure Chemical Corporation Grant information This study was supported by FUJIFILM Wako Pure Chemical Corporation and no separate grants were involved in supporting this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (2) version 2 Revised Published: 22 May 2025, 14:161 https://doi.org/10.12688/f1000research.160304.2 version 1 Published: 04 Feb 2025, 14:161 https://doi.org/10.12688/f1000research.160304.1 Copyright © 2025 Akutsu F et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Akutsu F, Sugino S, Watanabe M et al. A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :161 ( https://doi.org/10.12688/f1000research.160304.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 04 Feb 2025 Views 0 Cite How to cite this report: Thomas EA. Reviewer Report For: A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :161 ( https://doi.org/10.5256/f1000research.176178.r371741 ) The direct URL for this report is: https://f1000research.com/articles/14-161/v1#referee-response-371741 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 04 Apr 2025 Elizabeth A Thomas , University of California Irvine, Irvine, CA, USA Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.176178.r371741 The paper “A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva” presents important assay details for quantifying BDNF levels in saliva samples. These include spike and recovery data for the saliva matrix, ... Continue reading READ ALL The paper “A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva” presents important assay details for quantifying BDNF levels in saliva samples. These include spike and recovery data for the saliva matrix, the effects of different saliva collection protocols, linearity of dilution in the saliva matrix, precision and reproducibility of the BDNF measures and information regarding sample stability under long-term storage and the effects of multiple freeze thaws on BDNF detection. These are all essential considerations to show that saliva can be used for BDNF measurements. Saliva is a non-invasive biofluid that has gained recent attention for use in a wide range of human studies; hence knowing assay specifics for quantification of a very popular physiological peptide by ELISA has important consequences for others wanting to measure BDNF in saliva. The authors list previous studies that have quantified BDNF in human saliva samples and state that “BDNF values reported in these studies varied over a very wide range, indicating that most BDNF measurement methods used thus far had either not the specificity and/or the sensitivity needed to reliably measure BDNF levels in human saliva”. Aside from using different ELISA assays, it is also very likely that these past studies used different saliva collection methods and storage conditions, which the authors show can affect BDNF levels in saliva. Hence, the current findings could possibly explain some of the variability observed in past studies. This is another relevant aspect of the work. A few other corrections/suggestions: -In the Introduction, they include Nakagawa et al. (ref 12) and Kikuchi et al., (ref 13) as human saliva studies, when these were carried out in rats. These should be omitted, but there are several other studies that have measured BDNF in saliva samples in humans that could be mentioned. -In particular, one past study (Mandel A et a., 2011) also had worked out optimized conditions for another BDNF ELISA (although not as in depth as the current paper) and this study should be mentioned somewhere. -In the Abstract, it is not necessary to mention the mouse study: “Recently, the suitability of a highly sensitive BDNF ELISA assay was validated using mouse plasma and serum where conventional BDNF ELISA fail to detect BDNF”. The fact that it was validated in mouse is not as important as the fact that the assay works well in human saliva. -The introductory text regarding the origin of plasma/serum-derived BDNF levels is interesting, as the author make the suggestion that BDNF levels measures in blood might not inform about brain dysfunction in humans. Two previous studies (Ikenouchi A et al., 2023 and Gutierrez A et al., 2020). shown that saliva and plasma levels of BDNF are not correlated, possibly suggesting that saliva measures could be more relevant. This is worth mentioning in the Discussion. -The time frame (i.e. 2 pm to 5 pm) of saliva sample collection should be stated. Also, the amount of saliva (i.e. 1 ml) collected should be stated. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: biomarkers, peripheral biofluids (plasma, saliva), neurodegenerative diseases I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Thomas EA. Reviewer Report For: A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :161 ( https://doi.org/10.5256/f1000research.176178.r371741 ) The direct URL for this report is: https://f1000research.com/articles/14-161/v1#referee-response-371741 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 22 May 2025 Fumie Akutsu , FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan 22 May 2025 Author Response Dear Dr. Thomas, Please find our responses written below. Reviewer’s comments: The paper “A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in ... Continue reading Dear Dr. Thomas, Please find our responses written below. Reviewer’s comments: The paper “A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva” presents important assay details for quantifying BDNF levels in saliva samples. These include spike and recovery data for the saliva matrix, the effects of different saliva collection protocols, linearity of dilution in the saliva matrix, precision and reproducibility of the BDNF measures and information regarding sample stability under long-term storage and the effects of multiple freeze thaws on BDNF detection. These are all essential considerations to show that saliva can be used for BDNF measurements. Saliva is a non-invasive biofluid that has gained recent attention for use in a wide range of human studies; hence knowing assay specifics for quantification of a very popular physiological peptide by ELISA has important consequences for others wanting to measure BDNF in saliva. The authors list previous studies that have quantified BDNF in human saliva samples and state that “BDNF values reported in these studies varied over a very wide range, indicating that most BDNF measurement methods used thus far had either not the specificity and/or the sensitivity needed to reliably measure BDNF levels in human saliva”. Aside from using different ELISA assays, it is also very likely that these past studies used different saliva collection methods and storage conditions, which the authors show can affect BDNF levels in saliva. Hence, the current findings could possibly explain some of the variability observed in past studies. This is another relevant aspect of the work. A few other corrections/suggestions: -In the Introduction, they include Nakagawa et al. (ref 12) and Kikuchi et al., (ref 13) as human saliva studies, when these were carried out in rats. These should be omitted, but there are several other studies that have measured BDNF in saliva samples in humans that could be mentioned. 1. We agree to your comment to remove Nakagawa et al (ref 12) and Kikuchi et al (ref 13), since their studies are not for human but for rat saliva BDNF levels. We included the Tahiroglu et al. in the Discussion section (new ref 22) as the most recent report discussing measurement of salivary BDNF. -In particular, one past study (Mandel A et al., 2011) also had worked out optimized conditions for another BDNF ELISA (although not as in depth as the current paper) and this study should be mentioned somewhere. 2. We add some description about Mandel A et al in Discussion: “and significant number (up to 35%) of saliva samples needed to be excluded from the analysis due to the lack of appropriate detection sensitivity in the case of Mandel et al 20 .” -In the Abstract, it is not necessary to mention the mouse study: “Recently, the suitability of a highly sensitive BDNF ELISA assay was validated using mouse plasma and serum where conventional BDNF ELISA fail to detect BDNF”. The fact that it was validated in mouse is not as important as the fact that the assay works well in human saliva. 3. Although the reviewer suggests removing the sentence about the mouse study in the Abstract, we believe this is an important improvement in measurement of BDNF in various samples which was not done by commercially available BDNF assays in the past. So, we would like to keep the sentence in the Abstract as it specifically refers to validation, an all-important aspect. Sensitivity is of course key but so is validation. Readers may be interested to go back to the study we cite in our manuscript and check for themselves what is meant by validation. Briefly, beyond the reduction of the BDNF signal using a BDNF monoclonal antibody not used in the BDNF ELISA. the finding that BDNF levels in mouse plasma and serum are indistinguishable is key. Indeed, the process of platelet degranulation leads to the release of a number of cytokines and growth factors from platelets and the finding that the BDNF values remain unchanged compared with the plasma values is an important aspect of the validation. -The introductory text regarding the origin of plasma/serum-derived BDNF levels is interesting, as the author make the suggestion that BDNF levels measures in blood might not inform about brain dysfunction in humans. Two previous studies (Ikenouchi A et al., 2023 and Gutierrez A et al., 2020). shown that saliva and plasma levels of BDNF are not correlated, possibly suggesting that saliva measures could be more relevant. This is worth mentioning in the Discussion. 4. Per reviewer’s suggestion, we modify the discussion section: “Obviously, saliva samples can be readily and repeatedly collected for BDNF level determinations and possible correlations explored with various neurological and psychiatric conditions. While the origin of BDNF in saliva is unclear, the study by Ikenouchi et al. 12 demonstrated that there is no correlation between the levels of mBDNF in saliva and plasma. One possible source of BDNF in saliva samples are the sensory nerves innervating the buccal cavity including the tongue and gingiva as the levels of BDNF in sensory afferents are comparatively high (for review, see 2). Given that it is now possible to measure BDNF levels in human saliva, it would be interesting to know whether BDNF levels in saliva correlate with a range of physiological and pathological conditions of interest using appropriate study designs.” -The time frame (i.e. 2 pm to 5 pm) of saliva sample collection should be stated. Also, the amount of saliva (i.e. 1 ml) collected should be stated. Is the work clearly and accurately presented and does it cite the current literature? 5. We modify the part about the sample collection by adding the time frame and amount of the saliva sample collection to the original manuscript: All samples were collected in the same afternoon (noon to 5 pm) and then 6 to 10 mL of the samples were aliquoted into 1.5 mL Dear Dr. Thomas, Please find our responses written below. Reviewer’s comments: The paper “A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva” presents important assay details for quantifying BDNF levels in saliva samples. These include spike and recovery data for the saliva matrix, the effects of different saliva collection protocols, linearity of dilution in the saliva matrix, precision and reproducibility of the BDNF measures and information regarding sample stability under long-term storage and the effects of multiple freeze thaws on BDNF detection. These are all essential considerations to show that saliva can be used for BDNF measurements. Saliva is a non-invasive biofluid that has gained recent attention for use in a wide range of human studies; hence knowing assay specifics for quantification of a very popular physiological peptide by ELISA has important consequences for others wanting to measure BDNF in saliva. The authors list previous studies that have quantified BDNF in human saliva samples and state that “BDNF values reported in these studies varied over a very wide range, indicating that most BDNF measurement methods used thus far had either not the specificity and/or the sensitivity needed to reliably measure BDNF levels in human saliva”. Aside from using different ELISA assays, it is also very likely that these past studies used different saliva collection methods and storage conditions, which the authors show can affect BDNF levels in saliva. Hence, the current findings could possibly explain some of the variability observed in past studies. This is another relevant aspect of the work. A few other corrections/suggestions: -In the Introduction, they include Nakagawa et al. (ref 12) and Kikuchi et al., (ref 13) as human saliva studies, when these were carried out in rats. These should be omitted, but there are several other studies that have measured BDNF in saliva samples in humans that could be mentioned. 1. We agree to your comment to remove Nakagawa et al (ref 12) and Kikuchi et al (ref 13), since their studies are not for human but for rat saliva BDNF levels. We included the Tahiroglu et al. in the Discussion section (new ref 22) as the most recent report discussing measurement of salivary BDNF. -In particular, one past study (Mandel A et al., 2011) also had worked out optimized conditions for another BDNF ELISA (although not as in depth as the current paper) and this study should be mentioned somewhere. 2. We add some description about Mandel A et al in Discussion: “and significant number (up to 35%) of saliva samples needed to be excluded from the analysis due to the lack of appropriate detection sensitivity in the case of Mandel et al 20 .” -In the Abstract, it is not necessary to mention the mouse study: “Recently, the suitability of a highly sensitive BDNF ELISA assay was validated using mouse plasma and serum where conventional BDNF ELISA fail to detect BDNF”. The fact that it was validated in mouse is not as important as the fact that the assay works well in human saliva. 3. Although the reviewer suggests removing the sentence about the mouse study in the Abstract, we believe this is an important improvement in measurement of BDNF in various samples which was not done by commercially available BDNF assays in the past. So, we would like to keep the sentence in the Abstract as it specifically refers to validation, an all-important aspect. Sensitivity is of course key but so is validation. Readers may be interested to go back to the study we cite in our manuscript and check for themselves what is meant by validation. Briefly, beyond the reduction of the BDNF signal using a BDNF monoclonal antibody not used in the BDNF ELISA. the finding that BDNF levels in mouse plasma and serum are indistinguishable is key. Indeed, the process of platelet degranulation leads to the release of a number of cytokines and growth factors from platelets and the finding that the BDNF values remain unchanged compared with the plasma values is an important aspect of the validation. -The introductory text regarding the origin of plasma/serum-derived BDNF levels is interesting, as the author make the suggestion that BDNF levels measures in blood might not inform about brain dysfunction in humans. Two previous studies (Ikenouchi A et al., 2023 and Gutierrez A et al., 2020). shown that saliva and plasma levels of BDNF are not correlated, possibly suggesting that saliva measures could be more relevant. This is worth mentioning in the Discussion. 4. Per reviewer’s suggestion, we modify the discussion section: “Obviously, saliva samples can be readily and repeatedly collected for BDNF level determinations and possible correlations explored with various neurological and psychiatric conditions. While the origin of BDNF in saliva is unclear, the study by Ikenouchi et al. 12 demonstrated that there is no correlation between the levels of mBDNF in saliva and plasma. One possible source of BDNF in saliva samples are the sensory nerves innervating the buccal cavity including the tongue and gingiva as the levels of BDNF in sensory afferents are comparatively high (for review, see 2). Given that it is now possible to measure BDNF levels in human saliva, it would be interesting to know whether BDNF levels in saliva correlate with a range of physiological and pathological conditions of interest using appropriate study designs.” -The time frame (i.e. 2 pm to 5 pm) of saliva sample collection should be stated. Also, the amount of saliva (i.e. 1 ml) collected should be stated. Is the work clearly and accurately presented and does it cite the current literature? 5. We modify the part about the sample collection by adding the time frame and amount of the saliva sample collection to the original manuscript: All samples were collected in the same afternoon (noon to 5 pm) and then 6 to 10 mL of the samples were aliquoted into 1.5 mL Competing Interests: FA, MW, SS, and MK are employees of FUJIFILM Wako Pure Chemical Corporation. YB is a consultant of FUJIFILM Wako Pure Chemical Corporation Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 22 May 2025 Fumie Akutsu , FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan 22 May 2025 Author Response Dear Dr. Thomas, Please find our responses written below. Reviewer’s comments: The paper “A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in ... Continue reading Dear Dr. Thomas, Please find our responses written below. Reviewer’s comments: The paper “A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva” presents important assay details for quantifying BDNF levels in saliva samples. These include spike and recovery data for the saliva matrix, the effects of different saliva collection protocols, linearity of dilution in the saliva matrix, precision and reproducibility of the BDNF measures and information regarding sample stability under long-term storage and the effects of multiple freeze thaws on BDNF detection. These are all essential considerations to show that saliva can be used for BDNF measurements. Saliva is a non-invasive biofluid that has gained recent attention for use in a wide range of human studies; hence knowing assay specifics for quantification of a very popular physiological peptide by ELISA has important consequences for others wanting to measure BDNF in saliva. The authors list previous studies that have quantified BDNF in human saliva samples and state that “BDNF values reported in these studies varied over a very wide range, indicating that most BDNF measurement methods used thus far had either not the specificity and/or the sensitivity needed to reliably measure BDNF levels in human saliva”. Aside from using different ELISA assays, it is also very likely that these past studies used different saliva collection methods and storage conditions, which the authors show can affect BDNF levels in saliva. Hence, the current findings could possibly explain some of the variability observed in past studies. This is another relevant aspect of the work. A few other corrections/suggestions: -In the Introduction, they include Nakagawa et al. (ref 12) and Kikuchi et al., (ref 13) as human saliva studies, when these were carried out in rats. These should be omitted, but there are several other studies that have measured BDNF in saliva samples in humans that could be mentioned. 1. We agree to your comment to remove Nakagawa et al (ref 12) and Kikuchi et al (ref 13), since their studies are not for human but for rat saliva BDNF levels. We included the Tahiroglu et al. in the Discussion section (new ref 22) as the most recent report discussing measurement of salivary BDNF. -In particular, one past study (Mandel A et al., 2011) also had worked out optimized conditions for another BDNF ELISA (although not as in depth as the current paper) and this study should be mentioned somewhere. 2. We add some description about Mandel A et al in Discussion: “and significant number (up to 35%) of saliva samples needed to be excluded from the analysis due to the lack of appropriate detection sensitivity in the case of Mandel et al 20 .” -In the Abstract, it is not necessary to mention the mouse study: “Recently, the suitability of a highly sensitive BDNF ELISA assay was validated using mouse plasma and serum where conventional BDNF ELISA fail to detect BDNF”. The fact that it was validated in mouse is not as important as the fact that the assay works well in human saliva. 3. Although the reviewer suggests removing the sentence about the mouse study in the Abstract, we believe this is an important improvement in measurement of BDNF in various samples which was not done by commercially available BDNF assays in the past. So, we would like to keep the sentence in the Abstract as it specifically refers to validation, an all-important aspect. Sensitivity is of course key but so is validation. Readers may be interested to go back to the study we cite in our manuscript and check for themselves what is meant by validation. Briefly, beyond the reduction of the BDNF signal using a BDNF monoclonal antibody not used in the BDNF ELISA. the finding that BDNF levels in mouse plasma and serum are indistinguishable is key. Indeed, the process of platelet degranulation leads to the release of a number of cytokines and growth factors from platelets and the finding that the BDNF values remain unchanged compared with the plasma values is an important aspect of the validation. -The introductory text regarding the origin of plasma/serum-derived BDNF levels is interesting, as the author make the suggestion that BDNF levels measures in blood might not inform about brain dysfunction in humans. Two previous studies (Ikenouchi A et al., 2023 and Gutierrez A et al., 2020). shown that saliva and plasma levels of BDNF are not correlated, possibly suggesting that saliva measures could be more relevant. This is worth mentioning in the Discussion. 4. Per reviewer’s suggestion, we modify the discussion section: “Obviously, saliva samples can be readily and repeatedly collected for BDNF level determinations and possible correlations explored with various neurological and psychiatric conditions. While the origin of BDNF in saliva is unclear, the study by Ikenouchi et al. 12 demonstrated that there is no correlation between the levels of mBDNF in saliva and plasma. One possible source of BDNF in saliva samples are the sensory nerves innervating the buccal cavity including the tongue and gingiva as the levels of BDNF in sensory afferents are comparatively high (for review, see 2). Given that it is now possible to measure BDNF levels in human saliva, it would be interesting to know whether BDNF levels in saliva correlate with a range of physiological and pathological conditions of interest using appropriate study designs.” -The time frame (i.e. 2 pm to 5 pm) of saliva sample collection should be stated. Also, the amount of saliva (i.e. 1 ml) collected should be stated. Is the work clearly and accurately presented and does it cite the current literature? 5. We modify the part about the sample collection by adding the time frame and amount of the saliva sample collection to the original manuscript: All samples were collected in the same afternoon (noon to 5 pm) and then 6 to 10 mL of the samples were aliquoted into 1.5 mL Dear Dr. Thomas, Please find our responses written below. Reviewer’s comments: The paper “A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva” presents important assay details for quantifying BDNF levels in saliva samples. These include spike and recovery data for the saliva matrix, the effects of different saliva collection protocols, linearity of dilution in the saliva matrix, precision and reproducibility of the BDNF measures and information regarding sample stability under long-term storage and the effects of multiple freeze thaws on BDNF detection. These are all essential considerations to show that saliva can be used for BDNF measurements. Saliva is a non-invasive biofluid that has gained recent attention for use in a wide range of human studies; hence knowing assay specifics for quantification of a very popular physiological peptide by ELISA has important consequences for others wanting to measure BDNF in saliva. The authors list previous studies that have quantified BDNF in human saliva samples and state that “BDNF values reported in these studies varied over a very wide range, indicating that most BDNF measurement methods used thus far had either not the specificity and/or the sensitivity needed to reliably measure BDNF levels in human saliva”. Aside from using different ELISA assays, it is also very likely that these past studies used different saliva collection methods and storage conditions, which the authors show can affect BDNF levels in saliva. Hence, the current findings could possibly explain some of the variability observed in past studies. This is another relevant aspect of the work. A few other corrections/suggestions: -In the Introduction, they include Nakagawa et al. (ref 12) and Kikuchi et al., (ref 13) as human saliva studies, when these were carried out in rats. These should be omitted, but there are several other studies that have measured BDNF in saliva samples in humans that could be mentioned. 1. We agree to your comment to remove Nakagawa et al (ref 12) and Kikuchi et al (ref 13), since their studies are not for human but for rat saliva BDNF levels. We included the Tahiroglu et al. in the Discussion section (new ref 22) as the most recent report discussing measurement of salivary BDNF. -In particular, one past study (Mandel A et al., 2011) also had worked out optimized conditions for another BDNF ELISA (although not as in depth as the current paper) and this study should be mentioned somewhere. 2. We add some description about Mandel A et al in Discussion: “and significant number (up to 35%) of saliva samples needed to be excluded from the analysis due to the lack of appropriate detection sensitivity in the case of Mandel et al 20 .” -In the Abstract, it is not necessary to mention the mouse study: “Recently, the suitability of a highly sensitive BDNF ELISA assay was validated using mouse plasma and serum where conventional BDNF ELISA fail to detect BDNF”. The fact that it was validated in mouse is not as important as the fact that the assay works well in human saliva. 3. Although the reviewer suggests removing the sentence about the mouse study in the Abstract, we believe this is an important improvement in measurement of BDNF in various samples which was not done by commercially available BDNF assays in the past. So, we would like to keep the sentence in the Abstract as it specifically refers to validation, an all-important aspect. Sensitivity is of course key but so is validation. Readers may be interested to go back to the study we cite in our manuscript and check for themselves what is meant by validation. Briefly, beyond the reduction of the BDNF signal using a BDNF monoclonal antibody not used in the BDNF ELISA. the finding that BDNF levels in mouse plasma and serum are indistinguishable is key. Indeed, the process of platelet degranulation leads to the release of a number of cytokines and growth factors from platelets and the finding that the BDNF values remain unchanged compared with the plasma values is an important aspect of the validation. -The introductory text regarding the origin of plasma/serum-derived BDNF levels is interesting, as the author make the suggestion that BDNF levels measures in blood might not inform about brain dysfunction in humans. Two previous studies (Ikenouchi A et al., 2023 and Gutierrez A et al., 2020). shown that saliva and plasma levels of BDNF are not correlated, possibly suggesting that saliva measures could be more relevant. This is worth mentioning in the Discussion. 4. Per reviewer’s suggestion, we modify the discussion section: “Obviously, saliva samples can be readily and repeatedly collected for BDNF level determinations and possible correlations explored with various neurological and psychiatric conditions. While the origin of BDNF in saliva is unclear, the study by Ikenouchi et al. 12 demonstrated that there is no correlation between the levels of mBDNF in saliva and plasma. One possible source of BDNF in saliva samples are the sensory nerves innervating the buccal cavity including the tongue and gingiva as the levels of BDNF in sensory afferents are comparatively high (for review, see 2). Given that it is now possible to measure BDNF levels in human saliva, it would be interesting to know whether BDNF levels in saliva correlate with a range of physiological and pathological conditions of interest using appropriate study designs.” -The time frame (i.e. 2 pm to 5 pm) of saliva sample collection should be stated. Also, the amount of saliva (i.e. 1 ml) collected should be stated. Is the work clearly and accurately presented and does it cite the current literature? 5. We modify the part about the sample collection by adding the time frame and amount of the saliva sample collection to the original manuscript: All samples were collected in the same afternoon (noon to 5 pm) and then 6 to 10 mL of the samples were aliquoted into 1.5 mL Competing Interests: FA, MW, SS, and MK are employees of FUJIFILM Wako Pure Chemical Corporation. YB is a consultant of FUJIFILM Wako Pure Chemical Corporation Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Balzamino BO. Reviewer Report For: A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :161 ( https://doi.org/10.5256/f1000research.176178.r367703 ) The direct URL for this report is: https://f1000research.com/articles/14-161/v1#referee-response-367703 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 24 Feb 2025 Bijorn Omar Balzamino , IRCCS Fondazione Bietti, Rome, Italy Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.176178.r367703 Report 1 . Importance of the idea . In this short paper the authors analyze the effectiveness of a new ELISA kit for BDNF, using saliva as sample. Unfortunately, the idea is not new and ... Continue reading READ ALL Report 1 . Importance of the idea . In this short paper the authors analyze the effectiveness of a new ELISA kit for BDNF, using saliva as sample. Unfortunately, the idea is not new and there are several kits on the market for the analysis of BDNF from, which have also been used for the study of BDNF in saliva (see Nakagawa et al., 2019; Ye et al., 2020, Jasim et al., 2020; Kikuchi et al., 2020; Zappella et al., 2021; Biamonte et al., 2022 and Zhang et al., 2024) for example. The method section is well written so for statistical results. 2. Recommending a Revision and Resubmission: I think that the entire work should be set up differently, specifying better how other kits already exist on the market and how the BDNF has already been evaluated with these kits, therefore it’s more likely to describe a comparison between them. The results and discussion are well written and justified, the only thing that is not well illustrated is the abstract part where it is said that BDNF is not detectable in saliva, and this, as previously stated, is not true. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly References 1. Nakagawa Y, To M, Saruta J, Yamamoto Y, et al.: Effect of social isolation stress on saliva BDNF in rat. J Oral Sci . 2019; 61 (4): 516-520 PubMed Abstract | Publisher Full Text 2. Ye D, Gajendra S, Lawyer G, Jadeja N, et al.: Inflammatory biomarkers and growth factors in saliva and gingival crevicular fluid of e-cigarette users, cigarette smokers, and dual smokers: A pilot study. J Periodontol . 2020; 91 (10): 1274-1283 PubMed Abstract | Publisher Full Text 3. Jasim H, Ghafouri B, Carlsson A, Hedenberg-Magnusson B, et al.: Daytime changes of salivary biomarkers involved in pain. J Oral Rehabil . 2020; 47 (7): 843-850 PubMed Abstract | Publisher Full Text 4. Kikuchi T, Sakaguchi W, Saruta J, Yamamoto Y, et al.: Hypertriglyceridemia-induced brain-derived neurotrophic factor in rat submandibular glands. J Oral Biosci . 2020; 62 (4): 327-335 PubMed Abstract | Publisher Full Text 5. Zappella M, Biamonte F, Balzamino BO, Manieri R, et al.: Relaxation Response in Stressed Volunteers: Psychometric Tests and Neurotrophin Changes in Biological Fluids. Front Psychiatry . 2021; 12 : 655453 PubMed Abstract | Publisher Full Text 6. Biamonte F, Re A, Balzamino BO, Ciasca G, et al.: Circulating and Salivary NGF and BDNF Levels in SARS-CoV-2 Infection: Potential Predictor Biomarkers of COVID-19 Disease-Preliminary Data. J Pers Med . 2022; 12 (11). PubMed Abstract | Publisher Full Text Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Balzamino BO. Reviewer Report For: A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :161 ( https://doi.org/10.5256/f1000research.176178.r367703 ) The direct URL for this report is: https://f1000research.com/articles/14-161/v1#referee-response-367703 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 28 Feb 2025 Fumie Akutsu , FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan 28 Feb 2025 Author Response Dear reviewer, “We never claimed that measuring BDNF levels in human saliva is a new idea. Our intention was instead to demonstrate that this is a feasible objective using ... Continue reading Dear reviewer, “We never claimed that measuring BDNF levels in human saliva is a new idea. Our intention was instead to demonstrate that this is a feasible objective using a BDNF ELISA kit that has the necessary sensitivity and specificity . Our study confirmed the results of just one previous, recent study using the same BDNF ELISA kit by Ikenouchi and colleagues. This kit is the one BDNF ELISA kit that has been fully validated in a previous study (PMID: 37173369) in which Want and colleagues thoroughly documents both the specificity and sensitivity of this ELISA kit. The other BDNF ELISA kits mentioned by the reviewer report values that are implausibly high for BDNF levels in human saliva, with no demonstration of specificity. We hope that these additional comments will help clarifying what appears to be a misunderstanding about the objective of our study.” In order to clarify our intention, we will modify the Conclusion to: Our results indicate that the BDNF ELISA kit previously validated both with regard to specificity and sensitivity (Want et al.) 9 can be used to accurately determine the minute amounts of BDNF present in human saliva. These results also confirm those recently published by Ikenouchi and colleagues 11 using the very same BDNF ELISA kit. Correlations can now be readily explored between BDNF levels in human saliva and a range of physiological and pathological conditions of interest, including exercise, ageing, depression and neurodegeneration. We would appreciate your kind review. Dear reviewer, “We never claimed that measuring BDNF levels in human saliva is a new idea. Our intention was instead to demonstrate that this is a feasible objective using a BDNF ELISA kit that has the necessary sensitivity and specificity . Our study confirmed the results of just one previous, recent study using the same BDNF ELISA kit by Ikenouchi and colleagues. This kit is the one BDNF ELISA kit that has been fully validated in a previous study (PMID: 37173369) in which Want and colleagues thoroughly documents both the specificity and sensitivity of this ELISA kit. The other BDNF ELISA kits mentioned by the reviewer report values that are implausibly high for BDNF levels in human saliva, with no demonstration of specificity. We hope that these additional comments will help clarifying what appears to be a misunderstanding about the objective of our study.” In order to clarify our intention, we will modify the Conclusion to: Our results indicate that the BDNF ELISA kit previously validated both with regard to specificity and sensitivity (Want et al.) 9 can be used to accurately determine the minute amounts of BDNF present in human saliva. These results also confirm those recently published by Ikenouchi and colleagues 11 using the very same BDNF ELISA kit. Correlations can now be readily explored between BDNF levels in human saliva and a range of physiological and pathological conditions of interest, including exercise, ageing, depression and neurodegeneration. We would appreciate your kind review. Competing Interests: No competing interests were disclosed. Close Report a concern Author Response 22 May 2025 Fumie Akutsu , FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan 22 May 2025 Author Response Dear Dr. Thomas, Please find our responses written below. Reviewer’s comments: The paper “A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in ... Continue reading Dear Dr. Thomas, Please find our responses written below. Reviewer’s comments: The paper “A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva” presents important assay details for quantifying BDNF levels in saliva samples. These include spike and recovery data for the saliva matrix, the effects of different saliva collection protocols, linearity of dilution in the saliva matrix, precision and reproducibility of the BDNF measures and information regarding sample stability under long-term storage and the effects of multiple freeze thaws on BDNF detection. These are all essential considerations to show that saliva can be used for BDNF measurements. Saliva is a non-invasive biofluid that has gained recent attention for use in a wide range of human studies; hence knowing assay specifics for quantification of a very popular physiological peptide by ELISA has important consequences for others wanting to measure BDNF in saliva. The authors list previous studies that have quantified BDNF in human saliva samples and state that “BDNF values reported in these studies varied over a very wide range, indicating that most BDNF measurement methods used thus far had either not the specificity and/or the sensitivity needed to reliably measure BDNF levels in human saliva”. Aside from using different ELISA assays, it is also very likely that these past studies used different saliva collection methods and storage conditions, which the authors show can affect BDNF levels in saliva. Hence, the current findings could possibly explain some of the variability observed in past studies. This is another relevant aspect of the work. A few other corrections/suggestions: -In the Introduction, they include Nakagawa et al. (ref 12) and Kikuchi et al., (ref 13) as human saliva studies, when these were carried out in rats. These should be omitted, but there are several other studies that have measured BDNF in saliva samples in humans that could be mentioned. à We agree to your comment to remove Nakagawa et al (ref 12) and Kikuchi et al (ref 13), since their studies are not for human but for rat saliva BDNF levels. We included the Tahiroglu et al. in the Discussion section (new ref 22) as the most recent report discussing measurement of salivary BDNF. -In particular, one past study (Mandel A et al., 2011) also had worked out optimized conditions for another BDNF ELISA (although not as in depth as the current paper) and this study should be mentioned somewhere. à We add some description about Mandel A et al in Discussion: “and significant number (up to 35%) of saliva samples needed to be excluded from the analysis due to the lack of appropriate detection sensitivity in the case of Mandel et al 20 .” -In the Abstract, it is not necessary to mention the mouse study: “Recently, the suitability of a highly sensitive BDNF ELISA assay was validated using mouse plasma and serum where conventional BDNF ELISA fail to detect BDNF”. The fact that it was validated in mouse is not as important as the fact that the assay works well in human saliva. à Although the reviewer suggests removing the sentence about the mouse study in the Abstract, we believe this is an important improvement in measurement of BDNF in various samples which was not done by commercially available BDNF assays in the past. So, we would like to keep the sentence in the Abstract as it specifically refers to validation, an all-important aspect. Sensitivity is of course key but so is validation. Readers may be interested to go back to the study we cite in our manuscript and check for themselves what is meant by validation. Briefly, beyond the reduction of the BDNF signal using a BDNF monoclonal antibody not used in the BDNF ELISA. the finding that BDNF levels in mouse plasma and serum are indistinguishable is key. Indeed, the process of platelet degranulation leads to the release of a number of cytokines and growth factors from platelets and the finding that the BDNF values remain unchanged compared with the plasma values is an important aspect of the validation. -The introductory text regarding the origin of plasma/serum-derived BDNF levels is interesting, as the author make the suggestion that BDNF levels measures in blood might not inform about brain dysfunction in humans. Two previous studies (Ikenouchi A et al., 2023 and Gutierrez A et al., 2020). shown that saliva and plasma levels of BDNF are not correlated, possibly suggesting that saliva measures could be more relevant. This is worth mentioning in the Discussion. à Per reviewer’s suggestion, we modify the discussion section: “Obviously, saliva samples can be readily and repeatedly collected for BDNF level determinations and possible correlations explored with various neurological and psychiatric conditions. While the origin of BDNF in saliva is unclear, the study by Ikenouchi et al. 12 demonstrated that there is no correlation between the levels of mBDNF in saliva and plasma. One possible source of BDNF in saliva samples are the sensory nerves innervating the buccal cavity including the tongue and gingiva as the levels of BDNF in sensory afferents are comparatively high (for review, see 2). Given that it is now possible to measure BDNF levels in human saliva, it would be interesting to know whether BDNF levels in saliva correlate with a range of physiological and pathological conditions of interest using appropriate study designs.” -The time frame (i.e. 2 pm to 5 pm) of saliva sample collection should be stated. Also, the amount of saliva (i.e. 1 ml) collected should be stated. Is the work clearly and accurately presented and does it cite the current literature? à We modify the part about the sample collection by adding the time frame and amount of the saliva sample collection to the original manuscript: All samples were collected in the same afternoon (noon to 5 pm) and then 6 to 10 mL of the samples were aliquoted into 1.5 mL Dear Dr. Thomas, Please find our responses written below. Reviewer’s comments: The paper “A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva” presents important assay details for quantifying BDNF levels in saliva samples. These include spike and recovery data for the saliva matrix, the effects of different saliva collection protocols, linearity of dilution in the saliva matrix, precision and reproducibility of the BDNF measures and information regarding sample stability under long-term storage and the effects of multiple freeze thaws on BDNF detection. These are all essential considerations to show that saliva can be used for BDNF measurements. Saliva is a non-invasive biofluid that has gained recent attention for use in a wide range of human studies; hence knowing assay specifics for quantification of a very popular physiological peptide by ELISA has important consequences for others wanting to measure BDNF in saliva. The authors list previous studies that have quantified BDNF in human saliva samples and state that “BDNF values reported in these studies varied over a very wide range, indicating that most BDNF measurement methods used thus far had either not the specificity and/or the sensitivity needed to reliably measure BDNF levels in human saliva”. Aside from using different ELISA assays, it is also very likely that these past studies used different saliva collection methods and storage conditions, which the authors show can affect BDNF levels in saliva. Hence, the current findings could possibly explain some of the variability observed in past studies. This is another relevant aspect of the work. A few other corrections/suggestions: -In the Introduction, they include Nakagawa et al. (ref 12) and Kikuchi et al., (ref 13) as human saliva studies, when these were carried out in rats. These should be omitted, but there are several other studies that have measured BDNF in saliva samples in humans that could be mentioned. à We agree to your comment to remove Nakagawa et al (ref 12) and Kikuchi et al (ref 13), since their studies are not for human but for rat saliva BDNF levels. We included the Tahiroglu et al. in the Discussion section (new ref 22) as the most recent report discussing measurement of salivary BDNF. -In particular, one past study (Mandel A et al., 2011) also had worked out optimized conditions for another BDNF ELISA (although not as in depth as the current paper) and this study should be mentioned somewhere. à We add some description about Mandel A et al in Discussion: “and significant number (up to 35%) of saliva samples needed to be excluded from the analysis due to the lack of appropriate detection sensitivity in the case of Mandel et al 20 .” -In the Abstract, it is not necessary to mention the mouse study: “Recently, the suitability of a highly sensitive BDNF ELISA assay was validated using mouse plasma and serum where conventional BDNF ELISA fail to detect BDNF”. The fact that it was validated in mouse is not as important as the fact that the assay works well in human saliva. à Although the reviewer suggests removing the sentence about the mouse study in the Abstract, we believe this is an important improvement in measurement of BDNF in various samples which was not done by commercially available BDNF assays in the past. So, we would like to keep the sentence in the Abstract as it specifically refers to validation, an all-important aspect. Sensitivity is of course key but so is validation. Readers may be interested to go back to the study we cite in our manuscript and check for themselves what is meant by validation. Briefly, beyond the reduction of the BDNF signal using a BDNF monoclonal antibody not used in the BDNF ELISA. the finding that BDNF levels in mouse plasma and serum are indistinguishable is key. Indeed, the process of platelet degranulation leads to the release of a number of cytokines and growth factors from platelets and the finding that the BDNF values remain unchanged compared with the plasma values is an important aspect of the validation. -The introductory text regarding the origin of plasma/serum-derived BDNF levels is interesting, as the author make the suggestion that BDNF levels measures in blood might not inform about brain dysfunction in humans. Two previous studies (Ikenouchi A et al., 2023 and Gutierrez A et al., 2020). shown that saliva and plasma levels of BDNF are not correlated, possibly suggesting that saliva measures could be more relevant. This is worth mentioning in the Discussion. à Per reviewer’s suggestion, we modify the discussion section: “Obviously, saliva samples can be readily and repeatedly collected for BDNF level determinations and possible correlations explored with various neurological and psychiatric conditions. While the origin of BDNF in saliva is unclear, the study by Ikenouchi et al. 12 demonstrated that there is no correlation between the levels of mBDNF in saliva and plasma. One possible source of BDNF in saliva samples are the sensory nerves innervating the buccal cavity including the tongue and gingiva as the levels of BDNF in sensory afferents are comparatively high (for review, see 2). Given that it is now possible to measure BDNF levels in human saliva, it would be interesting to know whether BDNF levels in saliva correlate with a range of physiological and pathological conditions of interest using appropriate study designs.” -The time frame (i.e. 2 pm to 5 pm) of saliva sample collection should be stated. Also, the amount of saliva (i.e. 1 ml) collected should be stated. Is the work clearly and accurately presented and does it cite the current literature? à We modify the part about the sample collection by adding the time frame and amount of the saliva sample collection to the original manuscript: All samples were collected in the same afternoon (noon to 5 pm) and then 6 to 10 mL of the samples were aliquoted into 1.5 mL Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 28 Feb 2025 Fumie Akutsu , FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan 28 Feb 2025 Author Response Dear reviewer, “We never claimed that measuring BDNF levels in human saliva is a new idea. Our intention was instead to demonstrate that this is a feasible objective using ... Continue reading Dear reviewer, “We never claimed that measuring BDNF levels in human saliva is a new idea. Our intention was instead to demonstrate that this is a feasible objective using a BDNF ELISA kit that has the necessary sensitivity and specificity . Our study confirmed the results of just one previous, recent study using the same BDNF ELISA kit by Ikenouchi and colleagues. This kit is the one BDNF ELISA kit that has been fully validated in a previous study (PMID: 37173369) in which Want and colleagues thoroughly documents both the specificity and sensitivity of this ELISA kit. The other BDNF ELISA kits mentioned by the reviewer report values that are implausibly high for BDNF levels in human saliva, with no demonstration of specificity. We hope that these additional comments will help clarifying what appears to be a misunderstanding about the objective of our study.” In order to clarify our intention, we will modify the Conclusion to: Our results indicate that the BDNF ELISA kit previously validated both with regard to specificity and sensitivity (Want et al.) 9 can be used to accurately determine the minute amounts of BDNF present in human saliva. These results also confirm those recently published by Ikenouchi and colleagues 11 using the very same BDNF ELISA kit. Correlations can now be readily explored between BDNF levels in human saliva and a range of physiological and pathological conditions of interest, including exercise, ageing, depression and neurodegeneration. We would appreciate your kind review. Dear reviewer, “We never claimed that measuring BDNF levels in human saliva is a new idea. Our intention was instead to demonstrate that this is a feasible objective using a BDNF ELISA kit that has the necessary sensitivity and specificity . Our study confirmed the results of just one previous, recent study using the same BDNF ELISA kit by Ikenouchi and colleagues. This kit is the one BDNF ELISA kit that has been fully validated in a previous study (PMID: 37173369) in which Want and colleagues thoroughly documents both the specificity and sensitivity of this ELISA kit. The other BDNF ELISA kits mentioned by the reviewer report values that are implausibly high for BDNF levels in human saliva, with no demonstration of specificity. We hope that these additional comments will help clarifying what appears to be a misunderstanding about the objective of our study.” In order to clarify our intention, we will modify the Conclusion to: Our results indicate that the BDNF ELISA kit previously validated both with regard to specificity and sensitivity (Want et al.) 9 can be used to accurately determine the minute amounts of BDNF present in human saliva. These results also confirm those recently published by Ikenouchi and colleagues 11 using the very same BDNF ELISA kit. Correlations can now be readily explored between BDNF levels in human saliva and a range of physiological and pathological conditions of interest, including exercise, ageing, depression and neurodegeneration. We would appreciate your kind review. Competing Interests: No competing interests were disclosed. Close Report a concern Author Response 22 May 2025 Fumie Akutsu , FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan 22 May 2025 Author Response Dear Dr. Thomas, Please find our responses written below. Reviewer’s comments: The paper “A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in ... Continue reading Dear Dr. Thomas, Please find our responses written below. Reviewer’s comments: The paper “A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva” presents important assay details for quantifying BDNF levels in saliva samples. These include spike and recovery data for the saliva matrix, the effects of different saliva collection protocols, linearity of dilution in the saliva matrix, precision and reproducibility of the BDNF measures and information regarding sample stability under long-term storage and the effects of multiple freeze thaws on BDNF detection. These are all essential considerations to show that saliva can be used for BDNF measurements. Saliva is a non-invasive biofluid that has gained recent attention for use in a wide range of human studies; hence knowing assay specifics for quantification of a very popular physiological peptide by ELISA has important consequences for others wanting to measure BDNF in saliva. The authors list previous studies that have quantified BDNF in human saliva samples and state that “BDNF values reported in these studies varied over a very wide range, indicating that most BDNF measurement methods used thus far had either not the specificity and/or the sensitivity needed to reliably measure BDNF levels in human saliva”. Aside from using different ELISA assays, it is also very likely that these past studies used different saliva collection methods and storage conditions, which the authors show can affect BDNF levels in saliva. Hence, the current findings could possibly explain some of the variability observed in past studies. This is another relevant aspect of the work. A few other corrections/suggestions: -In the Introduction, they include Nakagawa et al. (ref 12) and Kikuchi et al., (ref 13) as human saliva studies, when these were carried out in rats. These should be omitted, but there are several other studies that have measured BDNF in saliva samples in humans that could be mentioned. à We agree to your comment to remove Nakagawa et al (ref 12) and Kikuchi et al (ref 13), since their studies are not for human but for rat saliva BDNF levels. We included the Tahiroglu et al. in the Discussion section (new ref 22) as the most recent report discussing measurement of salivary BDNF. -In particular, one past study (Mandel A et al., 2011) also had worked out optimized conditions for another BDNF ELISA (although not as in depth as the current paper) and this study should be mentioned somewhere. à We add some description about Mandel A et al in Discussion: “and significant number (up to 35%) of saliva samples needed to be excluded from the analysis due to the lack of appropriate detection sensitivity in the case of Mandel et al 20 .” -In the Abstract, it is not necessary to mention the mouse study: “Recently, the suitability of a highly sensitive BDNF ELISA assay was validated using mouse plasma and serum where conventional BDNF ELISA fail to detect BDNF”. The fact that it was validated in mouse is not as important as the fact that the assay works well in human saliva. à Although the reviewer suggests removing the sentence about the mouse study in the Abstract, we believe this is an important improvement in measurement of BDNF in various samples which was not done by commercially available BDNF assays in the past. So, we would like to keep the sentence in the Abstract as it specifically refers to validation, an all-important aspect. Sensitivity is of course key but so is validation. Readers may be interested to go back to the study we cite in our manuscript and check for themselves what is meant by validation. Briefly, beyond the reduction of the BDNF signal using a BDNF monoclonal antibody not used in the BDNF ELISA. the finding that BDNF levels in mouse plasma and serum are indistinguishable is key. Indeed, the process of platelet degranulation leads to the release of a number of cytokines and growth factors from platelets and the finding that the BDNF values remain unchanged compared with the plasma values is an important aspect of the validation. -The introductory text regarding the origin of plasma/serum-derived BDNF levels is interesting, as the author make the suggestion that BDNF levels measures in blood might not inform about brain dysfunction in humans. Two previous studies (Ikenouchi A et al., 2023 and Gutierrez A et al., 2020). shown that saliva and plasma levels of BDNF are not correlated, possibly suggesting that saliva measures could be more relevant. This is worth mentioning in the Discussion. à Per reviewer’s suggestion, we modify the discussion section: “Obviously, saliva samples can be readily and repeatedly collected for BDNF level determinations and possible correlations explored with various neurological and psychiatric conditions. While the origin of BDNF in saliva is unclear, the study by Ikenouchi et al. 12 demonstrated that there is no correlation between the levels of mBDNF in saliva and plasma. One possible source of BDNF in saliva samples are the sensory nerves innervating the buccal cavity including the tongue and gingiva as the levels of BDNF in sensory afferents are comparatively high (for review, see 2). Given that it is now possible to measure BDNF levels in human saliva, it would be interesting to know whether BDNF levels in saliva correlate with a range of physiological and pathological conditions of interest using appropriate study designs.” -The time frame (i.e. 2 pm to 5 pm) of saliva sample collection should be stated. Also, the amount of saliva (i.e. 1 ml) collected should be stated. Is the work clearly and accurately presented and does it cite the current literature? à We modify the part about the sample collection by adding the time frame and amount of the saliva sample collection to the original manuscript: All samples were collected in the same afternoon (noon to 5 pm) and then 6 to 10 mL of the samples were aliquoted into 1.5 mL Dear Dr. Thomas, Please find our responses written below. Reviewer’s comments: The paper “A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva” presents important assay details for quantifying BDNF levels in saliva samples. These include spike and recovery data for the saliva matrix, the effects of different saliva collection protocols, linearity of dilution in the saliva matrix, precision and reproducibility of the BDNF measures and information regarding sample stability under long-term storage and the effects of multiple freeze thaws on BDNF detection. These are all essential considerations to show that saliva can be used for BDNF measurements. Saliva is a non-invasive biofluid that has gained recent attention for use in a wide range of human studies; hence knowing assay specifics for quantification of a very popular physiological peptide by ELISA has important consequences for others wanting to measure BDNF in saliva. The authors list previous studies that have quantified BDNF in human saliva samples and state that “BDNF values reported in these studies varied over a very wide range, indicating that most BDNF measurement methods used thus far had either not the specificity and/or the sensitivity needed to reliably measure BDNF levels in human saliva”. Aside from using different ELISA assays, it is also very likely that these past studies used different saliva collection methods and storage conditions, which the authors show can affect BDNF levels in saliva. Hence, the current findings could possibly explain some of the variability observed in past studies. This is another relevant aspect of the work. A few other corrections/suggestions: -In the Introduction, they include Nakagawa et al. (ref 12) and Kikuchi et al., (ref 13) as human saliva studies, when these were carried out in rats. These should be omitted, but there are several other studies that have measured BDNF in saliva samples in humans that could be mentioned. à We agree to your comment to remove Nakagawa et al (ref 12) and Kikuchi et al (ref 13), since their studies are not for human but for rat saliva BDNF levels. We included the Tahiroglu et al. in the Discussion section (new ref 22) as the most recent report discussing measurement of salivary BDNF. -In particular, one past study (Mandel A et al., 2011) also had worked out optimized conditions for another BDNF ELISA (although not as in depth as the current paper) and this study should be mentioned somewhere. à We add some description about Mandel A et al in Discussion: “and significant number (up to 35%) of saliva samples needed to be excluded from the analysis due to the lack of appropriate detection sensitivity in the case of Mandel et al 20 .” -In the Abstract, it is not necessary to mention the mouse study: “Recently, the suitability of a highly sensitive BDNF ELISA assay was validated using mouse plasma and serum where conventional BDNF ELISA fail to detect BDNF”. The fact that it was validated in mouse is not as important as the fact that the assay works well in human saliva. à Although the reviewer suggests removing the sentence about the mouse study in the Abstract, we believe this is an important improvement in measurement of BDNF in various samples which was not done by commercially available BDNF assays in the past. So, we would like to keep the sentence in the Abstract as it specifically refers to validation, an all-important aspect. Sensitivity is of course key but so is validation. Readers may be interested to go back to the study we cite in our manuscript and check for themselves what is meant by validation. Briefly, beyond the reduction of the BDNF signal using a BDNF monoclonal antibody not used in the BDNF ELISA. the finding that BDNF levels in mouse plasma and serum are indistinguishable is key. Indeed, the process of platelet degranulation leads to the release of a number of cytokines and growth factors from platelets and the finding that the BDNF values remain unchanged compared with the plasma values is an important aspect of the validation. -The introductory text regarding the origin of plasma/serum-derived BDNF levels is interesting, as the author make the suggestion that BDNF levels measures in blood might not inform about brain dysfunction in humans. Two previous studies (Ikenouchi A et al., 2023 and Gutierrez A et al., 2020). shown that saliva and plasma levels of BDNF are not correlated, possibly suggesting that saliva measures could be more relevant. This is worth mentioning in the Discussion. à Per reviewer’s suggestion, we modify the discussion section: “Obviously, saliva samples can be readily and repeatedly collected for BDNF level determinations and possible correlations explored with various neurological and psychiatric conditions. While the origin of BDNF in saliva is unclear, the study by Ikenouchi et al. 12 demonstrated that there is no correlation between the levels of mBDNF in saliva and plasma. One possible source of BDNF in saliva samples are the sensory nerves innervating the buccal cavity including the tongue and gingiva as the levels of BDNF in sensory afferents are comparatively high (for review, see 2). Given that it is now possible to measure BDNF levels in human saliva, it would be interesting to know whether BDNF levels in saliva correlate with a range of physiological and pathological conditions of interest using appropriate study designs.” -The time frame (i.e. 2 pm to 5 pm) of saliva sample collection should be stated. Also, the amount of saliva (i.e. 1 ml) collected should be stated. Is the work clearly and accurately presented and does it cite the current literature? à We modify the part about the sample collection by adding the time frame and amount of the saliva sample collection to the original manuscript: All samples were collected in the same afternoon (noon to 5 pm) and then 6 to 10 mL of the samples were aliquoted into 1.5 mL Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 04 Feb 2025 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 4 Version 2 (revision) 22 May 25 read read read read Version 1 04 Feb 25 read read Bijorn Omar Balzamino , IRCCS Fondazione Bietti, Rome, Italy Elizabeth A Thomas , University of California Irvine, Irvine, USA Sedat Yildiz , University of Inonu, Malatya, Turkey Emad Al-Dujaili , University of Edinburgh, Edinburgh, UK Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Al-Dujaili E. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 22 Jul 2025 | for Version 2 Emad Al-Dujaili , Queens Research Medical Institute, University of Edinburgh, Edinburgh, Honorary Professor, UK 0 Views copyright © 2025 Al-Dujaili E. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The assay seems to be very well conducted and followed the guidelines to develop and evaluate an immunoassay method. I have the following the comments The researcher does not have to freeze and thaw 4 times. He must prepare aliquots and use one at a time! Add some important results to the abstract such as the specificity and sensitivity. Collection and handling of saliva samples were properly conducted; however, the number of participants was small. What is the lower detection limit and sensitivity of this ELISA? Is there some specificity data performed? I did not see any. In the abstract, the authors said that saliva samples were collected by a passive drool method. However, in the results, they use the Salivette made of cotton (Sartstedt, Nümbrecht, Germany) and SalivaBio Infant Swab (Salimetrics, PA, USA). These methods are different! Can the authors explain? The discussion is short and require further clarifications. The nature of the volunteers, and samples from males verses females etc. What are the applications of this assay? Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise BiochemistryNutrition and DieteticsNutritional Biochemistry, EndocrinologyPharmacy I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Al-Dujaili E. Peer Review Report For: A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :161 ( https://doi.org/10.5256/f1000research.181316.r388831) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-161/v2#referee-response-388831 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Yildiz S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 28 Jun 2025 | for Version 2 Sedat Yildiz , University of Inonu, Malatya, Turkey 0 Views copyright © 2025 Yildiz S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions I thank to the authors for this simple yet important presentation. As BDNF is associated with many physiological/pathological functions in the body, its non-invasive determination in saliva samples provides a valuable tool for reasearch and developments in associated areas. The authors have used FUJIFILM Wako's chemiluminescence assay. Although features of this assay is given in the website of the producer, some details need to be given in this manuscript. These are: 1. Please provide reference curve that you have obtained and used for calculation of the results. The readership would like to see the shape of that curve. My imagination, after reading the data in the commercial website, is that the curve is very linear and suitable for determination of low levels of BDNF (such as in saliva). 2. Please provide cross-reactivity of the antibody/test towards the similar molecules such as NGF-beta and NT, as presented in the commercial website. This will help readership to understand specificity of the antibody/test. 3. Please provide the data about the specific recognition of BDNF by the test as revealed by the BDNF knockout mice. This further provides data about reliability of the test developed. 4. Please state (in materials methods section, under mmeasurement of BDNF) thar this test is a chemiluminescence test. So that, the readership can understand it is a sensitive test kit. 5. If it is feasable to you, please provide data that increased saliva level does not result in an interference in the assay. Although dilution study is quite parallel, we need to know matrix effect as the standards are prepared in buffer instead of saliva or artificial saliva. In order to solve this issue, please add increasing amounts of saliva to reference curve and check whether the linearity and parallelism with original standard curve continues. If it is not feasible for you, you can omit this comment. There are some othe minor corrections that needed to be carried out: 1. In the abstract section, please remove "see discussion" part as it is not necessary in the abstarct 2. In the abstract section, beginf the results with the mean levels of the BDNF, rather than the validation data. Please provide the levels first, then the validation. Because, as a reader, I would like to see the real/quantitative results in the saliva, as it is said to be very low compared to the blood samples. 3.In the results section, please add the reference curve first, as explained above. 4. In the discussion section, please use a a careful language when referring to the products of the other companies. In that respect, in terms of research oriented kits, no one should expect similar levels accross the products of the different companies. Because, this generally is due to the purity and structure of the test types. But this does not mean that the other test kits are not able to measure the analyte. The level might be different but the measurement might be more accurate. As far as I understand from the producer's web site, this kit is not a "diagnostically approved kit". Therefore, "different levels accross the companies" should not be the backbone of the discussion section. Rather, the usability of these test kits might be discussed or the areas where all these test kits might be used should be explained more. 4.In the first sentence of the discussion, rather than using "confirm", please use "provides evidence" as there should be much more comprehensive studies that the data presented in the current study. 5. In the discussion section, please remove the term "notoriously" as it is not an academic explanation. Please use more direct language like "... detection by the methods like Western blots". 6. Please use a careful language for the test kits that use polyclonal antibodies. Because, in ELISA, sometimes the best results are obtained by the use of polyclonal antibodies or cocktail of the monoclonal antibodies. 7. In conclusions section, please remove "...thus confirming ...". There is no need for the last part of this sentence. There should not be a "confirming" situation taking into account the variables in the test systems and biology itself. 8. Please remove the words "readily" from the conclusion section. Instead, the terms "easily" or "non-invasively" should be preferred. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Immunoassay development, stress physiology, antibody production, assay validation, antibody or antigen tagging with enzymes, immunoaffinity purification, I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Yildiz S. Peer Review Report For: A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :161 ( https://doi.org/10.5256/f1000research.181316.r390268) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-161/v2#referee-response-390268 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Balzamino B. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 09 Jun 2025 | for Version 2 Bijorn Omar Balzamino , IRCCS Fondazione Bietti, Rome, Italy 0 Views copyright © 2025 Balzamino B. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The author claims that other kits show very variable values ​​of bdnf in saliva, according to him, very high and therefore influenced by various factors, including the non-specificity of the kit used. I appreciate the author's response, but I still can't understand how he can justify that this product is more specific than an R&D mab248 duo-set for example. Any kit used for research has a certificate of specificity of the marker, specifying no cross-reactivity with other similar markers. if the author then wants to make this kit usable in diagnostics, the results must be demonstrated differently. Competing Interests No competing interests were disclosed. Reviewer Expertise Research and development laboratory for biochemical, molecular and cellular applications in ophthalmological sciences I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (0) Balzamino BO. Peer Review Report For: A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :161 ( https://doi.org/10.5256/f1000research.181316.r386656) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-161/v2#referee-response-386656 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Thomas E. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 06 Jun 2025 | for Version 2 Elizabeth A Thomas , University of California Irvine, Irvine, CA, USA 0 Views copyright © 2025 Thomas E. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions I have no further comments. Competing Interests No competing interests were disclosed. Reviewer Expertise biomarkers, peripheral biofluids (plasma, saliva), neurodegenerative diseases I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Thomas EA. Peer Review Report For: A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :161 ( https://doi.org/10.5256/f1000research.181316.r386655) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-161/v2#referee-response-386655 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Thomas E. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 04 Apr 2025 | for Version 1 Elizabeth A Thomas , University of California Irvine, Irvine, CA, USA 0 Views copyright © 2025 Thomas E. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The paper “A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva” presents important assay details for quantifying BDNF levels in saliva samples. These include spike and recovery data for the saliva matrix, the effects of different saliva collection protocols, linearity of dilution in the saliva matrix, precision and reproducibility of the BDNF measures and information regarding sample stability under long-term storage and the effects of multiple freeze thaws on BDNF detection. These are all essential considerations to show that saliva can be used for BDNF measurements. Saliva is a non-invasive biofluid that has gained recent attention for use in a wide range of human studies; hence knowing assay specifics for quantification of a very popular physiological peptide by ELISA has important consequences for others wanting to measure BDNF in saliva. The authors list previous studies that have quantified BDNF in human saliva samples and state that “BDNF values reported in these studies varied over a very wide range, indicating that most BDNF measurement methods used thus far had either not the specificity and/or the sensitivity needed to reliably measure BDNF levels in human saliva”. Aside from using different ELISA assays, it is also very likely that these past studies used different saliva collection methods and storage conditions, which the authors show can affect BDNF levels in saliva. Hence, the current findings could possibly explain some of the variability observed in past studies. This is another relevant aspect of the work. A few other corrections/suggestions: -In the Introduction, they include Nakagawa et al. (ref 12) and Kikuchi et al., (ref 13) as human saliva studies, when these were carried out in rats. These should be omitted, but there are several other studies that have measured BDNF in saliva samples in humans that could be mentioned. -In particular, one past study (Mandel A et a., 2011) also had worked out optimized conditions for another BDNF ELISA (although not as in depth as the current paper) and this study should be mentioned somewhere. -In the Abstract, it is not necessary to mention the mouse study: “Recently, the suitability of a highly sensitive BDNF ELISA assay was validated using mouse plasma and serum where conventional BDNF ELISA fail to detect BDNF”. The fact that it was validated in mouse is not as important as the fact that the assay works well in human saliva. -The introductory text regarding the origin of plasma/serum-derived BDNF levels is interesting, as the author make the suggestion that BDNF levels measures in blood might not inform about brain dysfunction in humans. Two previous studies (Ikenouchi A et al., 2023 and Gutierrez A et al., 2020). shown that saliva and plasma levels of BDNF are not correlated, possibly suggesting that saliva measures could be more relevant. This is worth mentioning in the Discussion. -The time frame (i.e. 2 pm to 5 pm) of saliva sample collection should be stated. Also, the amount of saliva (i.e. 1 ml) collected should be stated. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise biomarkers, peripheral biofluids (plasma, saliva), neurodegenerative diseases I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 22 May 2025 Fumie Akutsu, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan Dear Dr. Thomas, Please find our responses written below. Reviewer’s comments: The paper “A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva” presents important assay details for quantifying BDNF levels in saliva samples. These include spike and recovery data for the saliva matrix, the effects of different saliva collection protocols, linearity of dilution in the saliva matrix, precision and reproducibility of the BDNF measures and information regarding sample stability under long-term storage and the effects of multiple freeze thaws on BDNF detection. These are all essential considerations to show that saliva can be used for BDNF measurements. Saliva is a non-invasive biofluid that has gained recent attention for use in a wide range of human studies; hence knowing assay specifics for quantification of a very popular physiological peptide by ELISA has important consequences for others wanting to measure BDNF in saliva. The authors list previous studies that have quantified BDNF in human saliva samples and state that “BDNF values reported in these studies varied over a very wide range, indicating that most BDNF measurement methods used thus far had either not the specificity and/or the sensitivity needed to reliably measure BDNF levels in human saliva”. Aside from using different ELISA assays, it is also very likely that these past studies used different saliva collection methods and storage conditions, which the authors show can affect BDNF levels in saliva. Hence, the current findings could possibly explain some of the variability observed in past studies. This is another relevant aspect of the work. A few other corrections/suggestions: -In the Introduction, they include Nakagawa et al. (ref 12) and Kikuchi et al., (ref 13) as human saliva studies, when these were carried out in rats. These should be omitted, but there are several other studies that have measured BDNF in saliva samples in humans that could be mentioned. 1. We agree to your comment to remove Nakagawa et al (ref 12) and Kikuchi et al (ref 13), since their studies are not for human but for rat saliva BDNF levels. We included the Tahiroglu et al. in the Discussion section (new ref 22) as the most recent report discussing measurement of salivary BDNF. -In particular, one past study (Mandel A et al., 2011) also had worked out optimized conditions for another BDNF ELISA (although not as in depth as the current paper) and this study should be mentioned somewhere. 2. We add some description about Mandel A et al in Discussion: “and significant number (up to 35%) of saliva samples needed to be excluded from the analysis due to the lack of appropriate detection sensitivity in the case of Mandel et al 20 .” -In the Abstract, it is not necessary to mention the mouse study: “Recently, the suitability of a highly sensitive BDNF ELISA assay was validated using mouse plasma and serum where conventional BDNF ELISA fail to detect BDNF”. The fact that it was validated in mouse is not as important as the fact that the assay works well in human saliva. 3. Although the reviewer suggests removing the sentence about the mouse study in the Abstract, we believe this is an important improvement in measurement of BDNF in various samples which was not done by commercially available BDNF assays in the past. So, we would like to keep the sentence in the Abstract as it specifically refers to validation, an all-important aspect. Sensitivity is of course key but so is validation. Readers may be interested to go back to the study we cite in our manuscript and check for themselves what is meant by validation. Briefly, beyond the reduction of the BDNF signal using a BDNF monoclonal antibody not used in the BDNF ELISA. the finding that BDNF levels in mouse plasma and serum are indistinguishable is key. Indeed, the process of platelet degranulation leads to the release of a number of cytokines and growth factors from platelets and the finding that the BDNF values remain unchanged compared with the plasma values is an important aspect of the validation. -The introductory text regarding the origin of plasma/serum-derived BDNF levels is interesting, as the author make the suggestion that BDNF levels measures in blood might not inform about brain dysfunction in humans. Two previous studies (Ikenouchi A et al., 2023 and Gutierrez A et al., 2020). shown that saliva and plasma levels of BDNF are not correlated, possibly suggesting that saliva measures could be more relevant. This is worth mentioning in the Discussion. 4. Per reviewer’s suggestion, we modify the discussion section: “Obviously, saliva samples can be readily and repeatedly collected for BDNF level determinations and possible correlations explored with various neurological and psychiatric conditions. While the origin of BDNF in saliva is unclear, the study by Ikenouchi et al. 12 demonstrated that there is no correlation between the levels of mBDNF in saliva and plasma. One possible source of BDNF in saliva samples are the sensory nerves innervating the buccal cavity including the tongue and gingiva as the levels of BDNF in sensory afferents are comparatively high (for review, see 2). Given that it is now possible to measure BDNF levels in human saliva, it would be interesting to know whether BDNF levels in saliva correlate with a range of physiological and pathological conditions of interest using appropriate study designs.” -The time frame (i.e. 2 pm to 5 pm) of saliva sample collection should be stated. Also, the amount of saliva (i.e. 1 ml) collected should be stated. Is the work clearly and accurately presented and does it cite the current literature? 5. We modify the part about the sample collection by adding the time frame and amount of the saliva sample collection to the original manuscript: All samples were collected in the same afternoon (noon to 5 pm) and then 6 to 10 mL of the samples were aliquoted into 1.5 mL View more View less Competing Interests FA, MW, SS, and MK are employees of FUJIFILM Wako Pure Chemical Corporation. YB is a consultant of FUJIFILM Wako Pure Chemical Corporation reply Respond Report a concern Thomas EA. Peer Review Report For: A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :161 ( https://doi.org/10.5256/f1000research.176178.r371741) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-161/v1#referee-response-371741 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Balzamino B. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 24 Feb 2025 | for Version 1 Bijorn Omar Balzamino , IRCCS Fondazione Bietti, Rome, Italy 0 Views copyright © 2025 Balzamino B. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (2) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Report 1 . Importance of the idea . In this short paper the authors analyze the effectiveness of a new ELISA kit for BDNF, using saliva as sample. Unfortunately, the idea is not new and there are several kits on the market for the analysis of BDNF from, which have also been used for the study of BDNF in saliva (see Nakagawa et al., 2019; Ye et al., 2020, Jasim et al., 2020; Kikuchi et al., 2020; Zappella et al., 2021; Biamonte et al., 2022 and Zhang et al., 2024) for example. The method section is well written so for statistical results. 2. Recommending a Revision and Resubmission: I think that the entire work should be set up differently, specifying better how other kits already exist on the market and how the BDNF has already been evaluated with these kits, therefore it’s more likely to describe a comparison between them. The results and discussion are well written and justified, the only thing that is not well illustrated is the abstract part where it is said that BDNF is not detectable in saliva, and this, as previously stated, is not true. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly References 1. Nakagawa Y, To M, Saruta J, Yamamoto Y, et al.: Effect of social isolation stress on saliva BDNF in rat. J Oral Sci . 2019; 61 (4): 516-520 PubMed Abstract | Publisher Full Text 2. Ye D, Gajendra S, Lawyer G, Jadeja N, et al.: Inflammatory biomarkers and growth factors in saliva and gingival crevicular fluid of e-cigarette users, cigarette smokers, and dual smokers: A pilot study. J Periodontol . 2020; 91 (10): 1274-1283 PubMed Abstract | Publisher Full Text 3. Jasim H, Ghafouri B, Carlsson A, Hedenberg-Magnusson B, et al.: Daytime changes of salivary biomarkers involved in pain. J Oral Rehabil . 2020; 47 (7): 843-850 PubMed Abstract | Publisher Full Text 4. Kikuchi T, Sakaguchi W, Saruta J, Yamamoto Y, et al.: Hypertriglyceridemia-induced brain-derived neurotrophic factor in rat submandibular glands. J Oral Biosci . 2020; 62 (4): 327-335 PubMed Abstract | Publisher Full Text 5. Zappella M, Biamonte F, Balzamino BO, Manieri R, et al.: Relaxation Response in Stressed Volunteers: Psychometric Tests and Neurotrophin Changes in Biological Fluids. Front Psychiatry . 2021; 12 : 655453 PubMed Abstract | Publisher Full Text 6. Biamonte F, Re A, Balzamino BO, Ciasca G, et al.: Circulating and Salivary NGF and BDNF Levels in SARS-CoV-2 Infection: Potential Predictor Biomarkers of COVID-19 Disease-Preliminary Data. J Pers Med . 2022; 12 (11). PubMed Abstract | Publisher Full Text Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (2) Author Response 28 Feb 2025 Fumie Akutsu, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan Dear reviewer, “We never claimed that measuring BDNF levels in human saliva is a new idea. Our intention was instead to demonstrate that this is a feasible objective using a BDNF ELISA kit that has the necessary sensitivity and specificity . Our study confirmed the results of just one previous, recent study using the same BDNF ELISA kit by Ikenouchi and colleagues. This kit is the one BDNF ELISA kit that has been fully validated in a previous study (PMID: 37173369) in which Want and colleagues thoroughly documents both the specificity and sensitivity of this ELISA kit. The other BDNF ELISA kits mentioned by the reviewer report values that are implausibly high for BDNF levels in human saliva, with no demonstration of specificity. We hope that these additional comments will help clarifying what appears to be a misunderstanding about the objective of our study.” In order to clarify our intention, we will modify the Conclusion to: Our results indicate that the BDNF ELISA kit previously validated both with regard to specificity and sensitivity (Want et al.) 9 can be used to accurately determine the minute amounts of BDNF present in human saliva. These results also confirm those recently published by Ikenouchi and colleagues 11 using the very same BDNF ELISA kit. Correlations can now be readily explored between BDNF levels in human saliva and a range of physiological and pathological conditions of interest, including exercise, ageing, depression and neurodegeneration. We would appreciate your kind review. View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Author Response 22 May 2025 Fumie Akutsu, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan Dear Dr. Thomas, Please find our responses written below. Reviewer’s comments: The paper “A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva” presents important assay details for quantifying BDNF levels in saliva samples. These include spike and recovery data for the saliva matrix, the effects of different saliva collection protocols, linearity of dilution in the saliva matrix, precision and reproducibility of the BDNF measures and information regarding sample stability under long-term storage and the effects of multiple freeze thaws on BDNF detection. These are all essential considerations to show that saliva can be used for BDNF measurements. Saliva is a non-invasive biofluid that has gained recent attention for use in a wide range of human studies; hence knowing assay specifics for quantification of a very popular physiological peptide by ELISA has important consequences for others wanting to measure BDNF in saliva. The authors list previous studies that have quantified BDNF in human saliva samples and state that “BDNF values reported in these studies varied over a very wide range, indicating that most BDNF measurement methods used thus far had either not the specificity and/or the sensitivity needed to reliably measure BDNF levels in human saliva”. Aside from using different ELISA assays, it is also very likely that these past studies used different saliva collection methods and storage conditions, which the authors show can affect BDNF levels in saliva. Hence, the current findings could possibly explain some of the variability observed in past studies. This is another relevant aspect of the work. A few other corrections/suggestions: -In the Introduction, they include Nakagawa et al. (ref 12) and Kikuchi et al., (ref 13) as human saliva studies, when these were carried out in rats. These should be omitted, but there are several other studies that have measured BDNF in saliva samples in humans that could be mentioned. à We agree to your comment to remove Nakagawa et al (ref 12) and Kikuchi et al (ref 13), since their studies are not for human but for rat saliva BDNF levels. We included the Tahiroglu et al. in the Discussion section (new ref 22) as the most recent report discussing measurement of salivary BDNF. -In particular, one past study (Mandel A et al., 2011) also had worked out optimized conditions for another BDNF ELISA (although not as in depth as the current paper) and this study should be mentioned somewhere. à We add some description about Mandel A et al in Discussion: “and significant number (up to 35%) of saliva samples needed to be excluded from the analysis due to the lack of appropriate detection sensitivity in the case of Mandel et al 20 .” -In the Abstract, it is not necessary to mention the mouse study: “Recently, the suitability of a highly sensitive BDNF ELISA assay was validated using mouse plasma and serum where conventional BDNF ELISA fail to detect BDNF”. The fact that it was validated in mouse is not as important as the fact that the assay works well in human saliva. à Although the reviewer suggests removing the sentence about the mouse study in the Abstract, we believe this is an important improvement in measurement of BDNF in various samples which was not done by commercially available BDNF assays in the past. So, we would like to keep the sentence in the Abstract as it specifically refers to validation, an all-important aspect. Sensitivity is of course key but so is validation. Readers may be interested to go back to the study we cite in our manuscript and check for themselves what is meant by validation. Briefly, beyond the reduction of the BDNF signal using a BDNF monoclonal antibody not used in the BDNF ELISA. the finding that BDNF levels in mouse plasma and serum are indistinguishable is key. Indeed, the process of platelet degranulation leads to the release of a number of cytokines and growth factors from platelets and the finding that the BDNF values remain unchanged compared with the plasma values is an important aspect of the validation. -The introductory text regarding the origin of plasma/serum-derived BDNF levels is interesting, as the author make the suggestion that BDNF levels measures in blood might not inform about brain dysfunction in humans. Two previous studies (Ikenouchi A et al., 2023 and Gutierrez A et al., 2020). shown that saliva and plasma levels of BDNF are not correlated, possibly suggesting that saliva measures could be more relevant. This is worth mentioning in the Discussion. à Per reviewer’s suggestion, we modify the discussion section: “Obviously, saliva samples can be readily and repeatedly collected for BDNF level determinations and possible correlations explored with various neurological and psychiatric conditions. While the origin of BDNF in saliva is unclear, the study by Ikenouchi et al. 12 demonstrated that there is no correlation between the levels of mBDNF in saliva and plasma. One possible source of BDNF in saliva samples are the sensory nerves innervating the buccal cavity including the tongue and gingiva as the levels of BDNF in sensory afferents are comparatively high (for review, see 2). Given that it is now possible to measure BDNF levels in human saliva, it would be interesting to know whether BDNF levels in saliva correlate with a range of physiological and pathological conditions of interest using appropriate study designs.” -The time frame (i.e. 2 pm to 5 pm) of saliva sample collection should be stated. Also, the amount of saliva (i.e. 1 ml) collected should be stated. Is the work clearly and accurately presented and does it cite the current literature? à We modify the part about the sample collection by adding the time frame and amount of the saliva sample collection to the original manuscript: All samples were collected in the same afternoon (noon to 5 pm) and then 6 to 10 mL of the samples were aliquoted into 1.5 mL View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Balzamino BO. Peer Review Report For: A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :161 ( https://doi.org/10.5256/f1000research.176178.r367703) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. 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