Development of Lateral Flow Immunochromatographic Assay with Anti-Pythium insidiosum Antibodies for Point-of-Care Testing of Vascular Pythiosis

Development of Lateral Flow Immunochromatographic Assay with Anti-Pythium insidiosum Antibodies for Point-of-Care Testing of Vascular Pythiosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Development of Lateral Flow Immunochromatographic Assay with Anti-Pythium insidiosum Antibodies for Point-of-Care Testing of Vascular Pythiosis Panwad Tongchai, Ati Burassakarn, Nattapong Langsiri, Navaporn Worasilchai, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4596892/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Jan, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract The pathogenic oomycete Pythium insidiosum causes a fatal infectious illness known as pythiosis, impacting humans and certain animals in numerous countries in the tropics and subtropics. Delayed diagnosis is a primary factor contributing to the heightened morbidity and mortality associated with the disease. Several new serodiagnostic methods have been developed to improve the identification of pythiosis. However, these assays provide only indirect evidence of pythiosis and are not readily available in the commercial market. Here, we have developed an affordable point-of-care test (POCT) kit based on an immunochromatographic assay for the direct detection of P. insidiosum antigens. Our recent findings reveal that the lateral flow sandwich immunological testing cassette can accurately identify vascular pythiosis antigens using a small volume of patient’s plasma, accomplishing 100% accuracy and a limit of detection (LOD) of 8 ng/mL. This prototype cartridge represents a significant stride toward the advancement of enriched POCT for pythiosis serodiagnosis. Biological sciences/Microbiology/Infectious disease diagnostics Health sciences/Diseases/Infectious diseases/Fungal infection Figures Figure 1 Figure 2 Figure 3 Figure 4 Research Highlights Point-of-care detection (POCT) of vascular pythiosis in less than 30 minutes. An anti-PIA mAbs is leveraged to specifically differentiate between P. insidiosum and other antigens. Equipment-free readout using a lateral-flow immunochromatographic strip. The assay diagnoses pythiosis in clinical samples with 100% specificity. Introduction Pythiosis is a severe infectious illness affecting humans and animals, particularly horses, pets, and livestock. The causative agent is the pathogenic aquatic fungus-like oomycete Pythium insidiosum 1 . The incidence of this infection is mainly detailed in tropical, subtropical, and temperate area 1 – 3 . While the infected animals commonly exhibit clinical manifestations, involving cutaneous/subcutaneous lesions and disorders of the gastrointestinal tract, the symptoms in humans, especially those with hematological disorders like thalassemia hemoglobinopathy, typically present with arterial occlusions in the lower extremities (vascular pythiosis) and ocular infections 1 – 3 . Treatment with common antifungal agent demonstrates ineffective against P. insidiosum infections. The desired management for pythiosis occupies the surgical removal of the infected organ, for instance, the eye or leg. Unfortunately, numerous pythiosis patients accede to advanced infections or inadequate treatment 4 . The prognosis for pythiosis patients is notably unfavorable when there is a delay in diagnosis, leading to late treatment 4 , 5 . The gold-standard diagnostic approach for pythiosis occupies a fungal culture aimed at isolating P. insidiosum from infected samples. This method, while labor-intensive and time-consuming, requires expertise for accomplishment and result interpretation 1 , 6 . Additionally, it frequently evidences unsuccessful in separating the organism, particularly when sporadically present in such samples, and may yield to low temperatures during transportation and storage 1 , 7 , 8 . Hence, a more sensitive method is very important for early P. insidiosum detection, supporting timely patient management. Various alternative detection approaches, utilizing sensitive molecular and immunological technologies, have been proposed to address the limitations of the microbial culture method in identifying P. insidiosum 6 , 9 . In addition, the identification of a target pathogen's DNA or RNA sequence through nucleic acid-based tests (NATs) emerges as a highly effective method for ensuring robust microbial identification 10 , 11 , enabling the rapid capture of minute quantities of the pathogen's nucleic acid in a clinical specimen, enhancing diagnostic sensitivity. Despite the appropriateness and efficiency of existing immunological methods, distinguishing between antibodies indicative of a past or recent P. insidiosum infection can be challenging. Furthermore, these methods cannot detect anti- P. insidiosum antibodies in patients with localized infections, such as in the cornea, an immunologically privileged site lacking specific types of host immune responses, such as antibody production 12 , 13 . Notably, there is currently no commercially available immunological test for pythiosis, and its in-house development is hindered by the absence of a P. insidiosum -specific antigen. This unavailability and inaccessibility limit the incorporation of immunological tests in clinical laboratories. Unlike in-house immunological assays, all essential reagents (such as primers, probes, nucleotides, and DNA polymerases) and necessary equipment for developing NATs are readily accessible in molecular diagnostic laboratories or can be procured from commercial sources. The lateral flow immunochromatographic assay (LFA) has grown extensively in popularity for the serodiagnosis of numerous infectious diseases due to its user-friendly format, rapid result turnaround, and high levels of detection sensitivity and specificity. Notably, this test is particularly valuable in faint or endemic areas where diagnostic facilities for pythiosis may be lacking, thus addressing critical healthcare needs in underserved regions 14 . Over the decades, immunotherapeutic P. insidiosum -antigen (PIA) has exhibited efficacy in activating immune responses and managing pythiosis in both human and animal cases 15 – 18 . The success of PIA immunotherapy in pythiosis has been postulated to stalk from its ability to induce a shift from a T-helper lymphocyte type 2 (Th2) to a T-helper lymphocyte type 1 (Th1) response 16 . In vascular pythiosis treatment, for instance, the administration of PIA has demonstrated that an enzyme-linked immunosorbent assay (ELISA) value of P. insidiosum -specific antibody exceeding 8 correlates with improved patient survival, signifying a robust host immune response to PIA in this contex 19 . Utilizing immunotherapy for vascular pythiosis represents a unique strategy leveraging the innate potentials of the patient's immune system. Due to the distributed evidence of published information on LFA in fungal detection strategy, we aimed to develop a prototype of the point-of-care testing (POCT) with mouse monoclonal anti-PIA immunoglobulin G (IgG) for P. insidiosum antigens detection in serum samples of a patient with vascular pythiosis. It is noteworthy that recent research has identified specific monoclonal antibodies, such as anti-PIA IgG, as a potential tool for detecting P. insidiosum . However, our study exclusively focuses on the prototype test, signaling a forthcoming shift in the technology utilized for diagnosing vascular pythiosis. Results Principle and Designing of PyT-LFA Detection System . The proof-of-concept approach outlined in our recent work utilizes lateral-flow immunochromatography assay for pythiosis diagnosis, referred to as PyT-LFA, for Pythium insidiosu m detection. The system, designed as a broadly-detected Pythium insidiosum antigen (PIA) platform, successfully handles notable sample types, particularly, patient serum. Figure 1 depicts the configuration and compartment of PyT-LFA device (i.e., the sample pad, conjugate pad, testing line, and control line). Designed as a versatile antigen-antibody reaction dogma, the colloidal gold-conjugated mouse anti- P. insidiosum monoclonal IgG scheme demonstrates applicability across PIA in serum from the whole blood of each patient is separated with the standard centrifugation method 20 . The encountered serum samples, specifically from vascular pythiosis was specifically targeted and effectively applied with the developed platform, highlighting the system's utility for the vascular pythiosis, as illustrated in. The whole testing process takes approximately 10 minutes for sample preparation and 15 minutes for detection. The outcomes include semi-quantitative results – positive and negative yields with their definite intensity. The serum samples, when added to the sample pads of the PyT-LFA strips, combine with mouse anti-PIA mAb-conjugated colloidal gold (colloidal AG-anti-PIA) on the conjugate pad due to the specific binding of a PIA (no.1). As the products migrate along the strip, the complexes are captured by PIA-specific mAb at the test line (T line), resulting in a red band at the T line (no.2). The remaining colloidal gold-anti-PIA continue to move and are captured by goat anti-mouse IgG at the control line (C line), forming another red band that confirms the LFA system's efficacy (no.3). In the absence of target PIA, no red band is observed at the T line as shown in Figs. 1 A. Results can be visually assessed based on the presence of a red band at the T line. The LFA procedure can be completed within 25 minutes from serum preparation to result in readout as indicated in Fig. 1 B. Therefore, in Fig. 1 C, LFA employs a cassette strip containing five components: a sample pad, a conjugate pad, a nitrocellulose membrane, a Wick (absorbent) pad, and a plastic cushion. Serum samples are applied to the sample pad and can be visually detected with the PIA-specific mAb, which is colloidal gold-conjugated. Establishment of PyT-LFA Platform Technical challenges in the LFA context from various factors such as antibody clones, running buffers, pretreated sample pad reagents, and surfactants being enclosed during the development step and commercialization process. These factors can impact the specificity and sensitivity of the tested results. Failure to assemble optimized components or suitable materials in the device can lead to demanding accuracy requirements and detection limit thresholds. Various substances, including those utilizing valuable materials or complex solutions, have been utilized to enhance LFA performance. While these materials have proven effective, their practicality remains suboptimal. Therefore, the search for improved alternatives continues. Here, we propose simple yet powerful materials and reagents, devoid of costly materials and complex solutions, for LFA improvement. The optimization of P. insidiosum -specific mAb clones was initially explored. Table 1 shown the presence of a diverse set of P. insidiosum -specific mAb clones, including 1.0 mg/mL of each PyT 1F5, PyT 3–24, and PyT 3–29, at the test (T) line. Variations were observed in the outcomes of different antibody clones when different running buffers were employed. Interestingly, all strips, except Strips No. 7 (which depicted a true negative result, TN), showed the false positive (FP) results when tested with 100 µL of 1xPBS + 0.1% TX405 buffer only (data not shown). However, Strip No. 9 exhibited a positive band (+ 1), Strip No. 3 and No. 6 resulted in false negative (FN) when 95 µL CMF1 (pH 8.0) buffer with 5 µL of PIA-spiked serum (10 µg/mL; Positive test) was used. Conversely, Strip No. 1 (+ 3.5) and No. 4 (+ 1) displayed positive outcomes alongside FN results in the other strips when using the 95 µL of FL (pH 8.0) buffer with 5 µL of PIA-spiked serum (10 µg/mL; Positive test) as shown in Fig. 2 A. Therefore, the PyT 1F5 clone and FL (pH 8.0) buffer were selected for the downstream process. We then enhanced the efficiency of the sample pad (Cytosep® 1662) using several pretreated reagents. When the pooled negative serum (20 µL) with FL buffer (pH 8.0, 80 µL) was applied to the V228 (pH 9.6)-pretreated sample pad, no band was observed (true negative, TN), whereas an inverse result (false positive; FP) was noted in the sample pad pretreated with CMF-1 or F1 supplemented with 0.5% casein solution at 15 minutes testing as demonstrated in Fig. 2 B). Moreover, the maximum volume of the serum sample, resulting in the true negative (TN) outcome was defined as 40 µL as indicated in Fig. 2 C. Thus, the pretreatment of the sample pad with V228 (pH 9.6) reagent and the maximum serum sample volume were optimized in the current study. To improve the detection sensitivity of the apparatus, we subsequently evaluated the potential of several surfactants added to the running buffer. Similar TN results were observed when testing pooled negative serum with FL (pH 8.0) without surfactants and FL (pH 8.0) supplemented with Tween 20 or 0.1% Biot constituents. However, a band of undemanding intensity was observed when experimenting with the FL (pH 8.0) buffer supplemented with TX100 as shown in Fig. 2 D; upper. Testing with PIA-spiked serum (final conc. 1,000 ng/mL) revealed that both the no surfactant buffer and all supplementation exhibited a positive band (True positive, TP) as demonstrated in Fig. 2 D; lower. Additionally, 0.1% Biot (pH 8.0) enhanced the TN result when testing with pooled negative serum samples, compared to other concentrations. In conclusion, we utilized the PyT 1F5 clone as the captured antibody system, FL with 0.1% Biot (pH 8.0) solution, and V228 (pH 9.6) reagent as the running buffer surfactant and pretreated sample pad reagents, respectively, for the developed device. Evaluation and Validation of PyT-LFA tool To assess the limit of detection (LOD) of the PyT-LFA system, we conducted assays on serial dilutions of PIA-spiked serum using a signal reader to ensure precision. For a direct and semi-quantitative determination of the LOD, we prepared serial dilutions of the samples in physiological serum and analyzed them with the PyT-LFA platform. Figure 3 illustrates the results, with serum samples containing different concentrations of PIA. The LFA efficiency (observed as band intensity) notably improved with increasing sensitivity of the running buffer surfactant. Even at a low concentration of 8 ng/mL, the test exhibited high specificity, with no FN results. Typically, the concentration of PIA in serum falls within the range of 4–8 ng/mL, indicating that the LOD of the PyT-LFA system is suitable for clinical testing. Furthermore, Fig. 3 demonstrates an imaged band for detection, exhibited as the measurable signal at the T line relative to the respective concentrations of PIA. We also evaluated the stability (Reproducibility) of the PyT-LFA platform by analyzing the intensity band of the samples with a long-term storage period (30 days, 90 days, and 120 days) of the device. The triplicate of the same sample was prepared, followed by visual detection using conditioned lateral flow strips. The three spiked- and healthy serum pairs were prepared using three batches of strips under the same protocol. The results presented in Fig. 4 indicate no differences among the three pairs of strips. This study thus demonstrates the high reproducibility, sensitivity, and reliability of our PyT-LFA platform, offering a valuable method for diagnosing pythiosis in clinical samples. Discussion Human and animal pythiosis is endemic in several tropical and subtropical countries 3 . In human pythiosis, four distinct pathological forms have been documented. While cutaneous pythiosis is characterized by granulomatous and ulcerating lesions typically affecting the face or limbs, vascular pythiosis involves arteries and can lead to arterial occlusion or aneurysm formation. The major clinical manifestation of ocular pythiosis is presented as keratitis. However, disseminated pythiosis is marked by the infection of internal organs 2 , 21 . In the fashionable microbial infection, both immunological and nucleic acid-based methods have been utilized for pathogen Identification. Several serodiagnostic tests have been developed to aid in the early diagnosis of pythiosis 6 . In-house enzyme-linked immunosorbent assay (ELISA) and Immunoblotting assays have demonstrated high sensitivity and specificity for diagnosing pythiosis 22 – 25 . However, these tests require skilled personnel, stable and reproducible reagents, expensive equipment, and lengthy turnaround times. Immunodiffusion (ID) is a straightforward serological test commonly utilized in laboratories for pythiosis diagnosis and is considered a standard serodiagnostic method 26 – 28 . While the ID test is easy to conduct and exhibits high specificity, its sensitivity is often limited, and the prolonged turnaround time may result in false-negative outcomes and delayed treatment. Consequently, there is a pressing need to enhance diagnostic procedures to improve patient care in managing pythiosis. The lateral flow immunochromatography has gained significant acceptance for the serodiagnosis of various infectious diseases owing to its user-friendly format, rapid result generation, and high levels of detection sensitivity and specificity 14 . Krajaejun et.al. 13 introduced an immunochromatographic test (ICT) for human pythiosis diagnosis (target to antibodies), with evaluation revealing a rapid turnaround time of less than 30 minutes for obtaining results. In contrast to the ID assay, the ICT revealed 88% sensitivity and 100% specificity, whereas the ID assay demonstrated 61% sensitivity and 100% specificity. While existing immunological methods are deemed appropriate and efficient, there are still limits in their applications. The distinguishing between antibodies indicative of past or recent P. insidiosum infections can pose challenges. Moreover, these methods are difficult to detect anti-P. insidiosum antibodies in patients with localized infections, particularly in an immunologically privileged site such as the eyes, notably antibody production (actually it can detect by ELISA with titier < 1:800). Moreover, there is currently no commercially available immunological test for pythiosis, and the development of in-house assays is impeded by the absence of a P. insidiosum -specific antigen. Consequently, the unavailability and inaccessibility of such tests hinder their integration into clinical laboratories 12 , 13 . Due to the distributed evidence of published information on P. insidiosum -antigen (PIA) in the context of pythiosis therapy, we, therefore, aimed to develop a prototype of the point-of-care testing with mouse monoclonal anti-PIA immunoglobulin G (IgG) for P. insidiosum antigens detection in serum samples collected from the patients with vascular pythiosis (PyT-LFA). The limit of detection (LOD) of the PyT-LFA prototype for detecting P. insidiosum antigens in all pythiosis sera was 8 ng/mL. Sera from all vascular pythiosis patients tested positive by our device. However, the failure to detect P. insidiosum antigens in these patients likely occurred due to long-term sera storage and inadequate presentation of antigens in localized bloodstream infections 30 – 32 . Therefore, the serodiagnosis of vascular infection should be approached cautiously, considering the expected rate of false-negative results. The prototype could serve as a point-of-care instrument for vascular pythiosis, while other serodiagnosis approaches require specific compartment preparation before testing. The turnaround time of the device was remarkably shorter compared to others (15–20 minutes. For the PyT-LFA; 1–24 hours for others). Therefore, the PyT-LFA demonstrates better performance and greater convenience than previous tools, making it suitable for the serodiagnosis of vascular pythiosis. However, further evaluation is necessary to assess its performance in diagnosing ocular and cutaneous pythiosis. Our findings show that the devices, which had been stored for 6 months, were suitable for future investigation with the vascular pythiosis sera assays. The assay’s repeatability was shown to be stable, with only 100% of collected positive samples yielding positive results when reanalyzed. Additionally, our tests on the same specimens produced positive results. Other investigations revealed a significant association between the LFA and 1,3-Beta-D glucan (BG) assay results when tests were performed on the same day. However, this correlation decreased after a week of sample storage. Interestingly, the existing studies have also found a decline in BG test accuracy following both short- and long-term freezing. The BG test identifies Beta-D glucan, which can be hydrolyzed or degraded over time due to acidic conditions, even when samples are maintained at low temperatures to prevent these processe 19 , 33 – 37 . Furthermore, subsequent PyT-LFA test findings differed from earlier tests when samples were held at -80°C, implying that the test’s diagnostic performance may be stabilized. It has been postulated that the P. insidiosum antigen identified by the PyT-LFA test becomes stable over time due to autolytic breakdown. As a result, it is assumed that the PyT-LFA test be performed as soon as feasible to ensure a good correlation with the BG assay results. The LOD and specificity of the prototype developed in this study might be in line with previous reports. However, enhancements to the PyT-LFA test could be achieved through its combination with other tests, such as PCR or the CRISPR/Cas system. The rationed LOD observed in the LFD test in our study may be linked to patients being exposed to multiple antibiotics and/or antifungal agents before sample collection. However, nucleic acid-based methods, particularly PCR, offer distinct advantages over immunological techniques due to their superior sensitivity, specificity, and rapidity 38 , 39 Moreover, advancements in biosensor technology have further enhanced the sensitivity, specificity, and simplicity of nucleic acid-based assays 40 , paving the way for the development of convenient, cost-effective, and specific diagnostic tools for Pythium spp. pathogen monitoring applications. This can greatly aid in implementing management practices aimed at reducing the risk of epidemics. Collectively, our prototype offers several notable advantages. This can greatly aid in implementing management practices aimed at reducing the risk of epidemics. Limitations of the study Here, we have successfully developed a sensitive, cost-effective, and user-friendly PIA detection platform by lateral flow immunochromatography. This platform allows for detection results to be obtained within 25 minutes using clinically relevant samples (such as serum) directly without the complicated steps. We anticipate that the PyT-LFA system could be readily adapted for detecting various types of human pythiosis, particularly, vascular and systemic pythiosis. However, detecting variations in PIA number remains a challenge. We plan to further refine the LOD value and read-out system to enable the LFA system to detect the lowest PIA number variations effectively. Declarations ACKNOWLEDGMENTS This work is funded by Thailand Science Research and Innovation (TSRI) Fund Chulalongkorn University (Grant no. HEA663000027). AUTHOR CONTRIBUTIONS N.W. and P.T. carried out assays and analyzed the results; P.T., A.B., NL, and A.C. supported the optimization of assays and prepared data; A.C. developed the strip; A.B., and A.C. performed analyses of assay results; N.W., A.B., and A.C. conceived and designed the experiments; P.T., A.B., and A.C. wrote the manuscript. DECLARATION OF INTERESTS The authors declare no competing interests. References Gaastra, W., Lipman, L.J., De Cock, A.W., Exel, T.K., Pegge, R.B., Scheurwater, J., Vilela, R., and Mendoza, L. (2010). Pythium insidiosum: an overview. Vet Microbiol 146 , 1–16. 10.1016/j.vetmic.2010.07.019 . Krajaejun, T., Sathapatayavongs, B., Pracharktam, R., Nitiyanant, P., Leelachaikul, P., Wanachiwanawin, W., Chaiprasert, A., Assanasen, P., Saipetch, M., Mootsikapun, P., et al. (2006). Clinical and epidemiological analyses of human pythiosis in Thailand. Clin Infect Dis 43 , 569–576. 10.1086/506353 . Yolanda, H., and Krajaejun, T. (2022). 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Subcutaneous pythiosis in human treated successfully with antimicrobial treatment, debridement and immunotherapy. BMJ Case Rep 17 . 10.1136/bcr-2023-258587 . Keeratijarut, A., Lohnoo, T., Rujirawat, T., Yingyong, W., Kalambaheti, T., Miller, S., Phuntumart, V., and Krajaejun, T. (2015). The Immunoreactive Exo-1,3-beta-Glucanase from the Pathogenic Oomycete Pythium insidiosum Is Temperature Regulated and Exhibits Glycoside Hydrolase Activity. PLoS One 10 , e0135239. 10.1371/journal.pone.0135239 . Tondolo, J.S.M., Ledur, P.C., Loreto, E.S., Verdi, C.M., Bitencourt, P.E.R., de Jesus, F.P.K., Rocha, J.P., Alves, S.H., Sassaki, G.L., and Santurio, J.M. (2017). Extraction, characterization and biological activity of a (1,3)(1,6)-beta-d-glucan from the pathogenic oomycete Pythium insidiosum. Carbohydr Polym 157 , 719–727. 10.1016/j.carbpol.2016.10.053 . Pickering, J.W., Sant, H.W., Bowles, C.A., Roberts, W.L., and Woods, G.L. (2005). Evaluation of a (1->3)-beta-D-glucan assay for diagnosis of invasive fungal infections. J Clin Microbiol 43 , 5957–5962. 10.1128/JCM.43.12.5957-5962.2005 . Singh, S., Kaur, H., Choudhary, H., Sethi, S., Malhotra, P., Gupta, K.L., Rudramurthy, S.M., and Chakrabarti, A. (2018). Evaluation of biomarkers: Galactomannan and 1,3-beta-D-glucan assay for the diagnosis of invasive fungal infections in immunocompromised patients from a tertiary care centre. Indian J Med Microbiol 36 , 557–563. 10.4103/ijmm.IJMM_18_366 . Tran, T., and Beal, S.G. (2016). Application of the 1,3-beta-D-Glucan (Fungitell) Assay in the Diagnosis of Invasive Fungal Infections. Arch Pathol Lab Med 140 , 181–185. 10.5858/arpa.2014-0230-RS . Schurko, A.M., Mendoza, L., de Cock, A.W., Bedard, J.E., and Klassen, G.R. (2004). Development of a species-specific probe for Pythium insidiosum and the diagnosis of pythiosis. J Clin Microbiol 42 , 2411–2418. 10.1128/JCM.42.6.2411-2418.2004 . Zhang, C., Liu, X., Yao, Y., Liu, K., Hui, W., Zhu, J., Dou, Y., Hua, K., Peng, M., Wang, Z., et al. (2018). Genotyping of Multiple Clinical Samples with a Combined Direct PCR and Magnetic Lateral Flow Assay. iScience 7 , 170–179. 10.1016/j.isci.2018.09.005 . Sridapan, T., and Krajaejun, T. (2022). Nucleic Acid-Based Detection of Pythium insidiosum: A Systematic Review. J Fungi (Basel) 9 . 10.3390/jof9010027 . Cha, S.H., Kim, S.H., Bischoff, K., Kim, H.J., Son, S.W., and Kang, H.G. (2012). Production of a highly group-specific monoclonal antibody against zearalenone and its application in an enzyme-linked immunosorbent assay. J Vet Sci 13 , 119–125. 10.4142/jvs.2012.13.2.119 . Li, P., Wu, J., Zhang, L., Fan, Z., Yu, T., Jiang, F., Tang, X., Zhang, Z., Zhang, W., and Zhang, Q. (2017). Doses of Immunogen Contribute to Specificity Spectrums of Antibodies against Aflatoxin. Toxins (Basel) 9 . 10.3390/toxins9050172 . Gao, Y., Huang, X., Zhu, Y., and Lv, Z. (2018). A brief review of monoclonal antibody technology and its representative applications in immunoassays. J Immunoassay Immunochem 39 , 351–364. 10.1080/15321819.2018.1515775 . Johnston, S.C., Bowles, M., Winzor, D.J., and Pond, S.M. (1988). Comparison of paraquat-specific murine monoclonal antibodies produced by in vitro and in vivo immunization. Fundam Appl Toxicol 11 , 261–267. 10.1016/0272–0590(88)90150-9 . Mitra, S., and Tomar, P.C. (2021). Hybridoma technology; advancements, clinical significance, and future aspects. J Genet Eng Biotechnol 19 , 159. 10.1186/s43141-021-00264-6 . Mazurkevych, A., Malyuk, M., Bezdieniezhnykh, N., Starodub, L., Kharkevych, Y., Jakubczak, A., and Gryzinska, M. (2017). Immunophenotypic characteristics and karyotype analysis of bone marrow-derived mesenchymal stem cells of rabbits during in vitro cultivation. Pol J Vet Sci 20 , 687–695. 10.1515/pjvs-2017-0086 . Beatty, J.D., Beatty, B.G., and Vlahos, W.G. (1987). Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Methods 100 , 173–179. 10.1016/0022-1759(87)90187-6 . STAR METHODS Lead contact Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Ariya Chindamporn ( [email protected] .). Materials availability This study did not generate unique reagents. Data availability The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS METHOD DETAILS Healthy blood collection Fresh human whole blood samples were collected from 5 healthy volunteers using clotted-blood tubes. Serum was separated from each sample using standard centrifugation method 20 . Each individual provided informed consent, and the study received approval from the Institutional Review Board of the faculty of Medicine, Chulalongkorn University, a WHO-certified ethics committee (IRB No. 0427/67). All methods adhered to these approved guidelines. Preparation of mouse monoclonal anti- P. insidiosum antibodies Pythium insidiosum antigens (PIA) was prepared from clade 1 P. insidiosum strain as previously described 15 and used as an inoculum in our study. A 20 mg/mL PIA stock solution was prepared with PCR-grade water and stored at −80°C. Construction of a PyT-LFA cassette (i) Conjugation of antibody to colloidal gold. The 40-nanometer-particle colloidal gold suspension (Arista, Allentown, PA, USA) was adjusted to pH 9.65 using 0.2 M Na 2 CO 3 . Subsequently, 3 µg of mouse anti- P.insidiodum (PyT) mAb was added to each 500 µL of colloidal gold, followed by a 30-minute incubation at room temperature. The residual surfaces of the colloidal gold particles were then blocked by incubating with 5% (w/v) bovine serum albumin (Sigma, St. Louis, MO, USA) for 10 minutes. After centrifugation at 6,000 × g for 15 minutes, the supernatant was discarded, and the conjugate pellet was washed in 0.5% (w/v) casein. This was followed by another centrifugation at 6,000 × g for 15 minutes, after which the supernatant was removed. The conjugate was resuspended in a solution containing 0.5% (w/v) casein and 20% (w/v) sucrose in 0.02 M Tris-HCl (pH 8.0), with a volume 40 times smaller than the original suspension. A 2.5 by 2.5-millimeter piece of glass fiber (GF33; Whatman Schleicher & Schuell, Dassel, Germany) was impregnated with 2.5 µL of this IgG-colloidal gold conjugate and dried in a dehumidifier cabinet for an hour. (ii) Immobilization of antigen and antibody onto a nitrocellulose membrane. A nitrocellulose membrane measuring 1.5 centimeters wide (CN140; Whatman Schleicher & Schuell, Dassel, Germany) was coated with PyT (1 mg/mL) to create the test line and goat anti-mouse IgG (1 mg/mL) in PBS to create the control line, each at a volume of 1 μL per centimeter using a dispenser (ZX 1000; BioDot, Irvine, CA). Subsequently, the membrane was dried, blocked with 1% (w/v) bovine serum albumin, and dried again in a dehumidifier cabinet. (iii) Assembly of PyT-LFA strips. The nitrocellulose membrane, glass fiber with colloidal gold conjugate, sample pad (Cytosep 1622 / V228, pH 9.6), and wicking pad (18 mm.; 470 chromatography paper; Whatman, Maidstone, England) were carefully arranged and mounted onto a plastic backing (60 mm.). This assembly was then precisely cut into strips measuring 25 mm. in width using a strip-cutting machine (CM 4000 R; BioDot, Irvine, CA). (iv) Detection of human P. insidiosum antigens by the PyT-LFA. Each serum sample (40 µL) underwent an optimized dilution in 60 µL running buffer (FL with 0.1% Biot pH 8.0). Subsequently, the lateral flow immunochromatographic test (LFA) was conducted in duplicate using 100 μL of the diluted serum in the indicated buffer. Following a 15-minute incubation period, the test signal of each LFA strip was evaluated by three independent laboratory personnel using visual inspection. To quantify the LFA signal, each strip was scanned using a scanner (Epson Perfection 1670 photo scanner; Seiko Epson Corp., Japan) to generate a tagged image file format picture. Test and background signal intensities were analyzed utilizing the Image J software (https://imagej.net/ij/docs/intro.html). The resulting intensity value, obtained by subtracting the background signal from the test signal. Quantification and statistical analysis All statistical analyses were conducted using GraphPad Prism version 10.2.1 for Windows (GraphPad Software, www.graphpad.com). Differences between the two groups were assessed using an independent sample t-test (unpaired, two-tailed). Error bars represent ±SD. p -values < .05 were considered statistically significant. The signal of each LFA strip was evaluated by three independent experiments. Tables Table 1. Detail of each component in the PyT-LFA cassette. Strip No. Capture (Test line) Detector (Conjugate) Strip 1 Clone PyT 1F5 Clone PyT 1F5 Strip 2 Clone PyT 3-24 Strip 3 Clone PyT 3-29 Strip 4 Clone PyT 3-24 Clone PyT 1F5 Strip 5 Clone PyT 3-24 Strip 6 Clone PyT 3-29 Strip 7 Clone PyT 3-29 Clone PyT 1F5 Strip 8 Clone PyT 3-24 Strip 9 Clone PyT 3-29 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 04 Jan, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 09 Oct, 2024 Reviews received at journal 07 Oct, 2024 Reviews received at journal 01 Oct, 2024 Reviewers agreed at journal 28 Sep, 2024 Reviewers agreed at journal 19 Sep, 2024 Reviewers invited by journal 30 Jun, 2024 Editor assigned by journal 24 Jun, 2024 Editor invited by journal 24 Jun, 2024 Submission checks completed at journal 21 Jun, 2024 First submitted to journal 17 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4596892","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":323257969,"identity":"487f082e-bbf8-4a09-a625-ebb2944d3af8","order_by":0,"name":"Panwad Tongchai","email":"","orcid":"","institution":"Chulalongkorn University","correspondingAuthor":false,"prefix":"","firstName":"Panwad","middleName":"","lastName":"Tongchai","suffix":""},{"id":323257971,"identity":"f7658a70-9ce0-407d-8fa4-a5585de66d38","order_by":1,"name":"Ati Burassakarn","email":"","orcid":"","institution":"Chulalongkorn University","correspondingAuthor":false,"prefix":"","firstName":"Ati","middleName":"","lastName":"Burassakarn","suffix":""},{"id":323257974,"identity":"c7c0144f-74b7-4e5f-821d-94ec4c0c90e0","order_by":2,"name":"Nattapong Langsiri","email":"","orcid":"","institution":"Chulalongkorn University","correspondingAuthor":false,"prefix":"","firstName":"Nattapong","middleName":"","lastName":"Langsiri","suffix":""},{"id":323257979,"identity":"3c07e0c1-d671-4f95-8ef3-95743b7fbfd4","order_by":3,"name":"Navaporn Worasilchai","email":"","orcid":"","institution":"Chulalongkorn University","correspondingAuthor":false,"prefix":"","firstName":"Navaporn","middleName":"","lastName":"Worasilchai","suffix":""},{"id":323257980,"identity":"e55ef74f-7048-4462-a77c-cc0214779157","order_by":4,"name":"Ariya Chindamporn","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIie3OMQuCQBTA8SeGLZLrSeJ9hZPGoM+iCLUoNAoNCYGT0Sr4JeobXDi0HM0FLe0OjQ0OPREaT8eg+w/H43g/7gBUqp9MS9vTBQIa727MYWSGBDpi9JKuIB1MrLLKyBsWq0OZM44DBbrnUkKuQWbnEMbFQ7AzDl5qTHz5M0LLiAk8PpKIcRN0Hz/GpIIisRvgK4bk3MC2nzAkU3zFb0llQtVPPKHt5g4LveK2XFcOu3iZEcmJK8bPe50sqFWEp2edbKilCznBRgS+OzgYffuY/hqwpFKpVP/cB36xPOkLbSJsAAAAAElFTkSuQmCC","orcid":"","institution":"Chulalongkorn University","correspondingAuthor":true,"prefix":"","firstName":"Ariya","middleName":"","lastName":"Chindamporn","suffix":""}],"badges":[],"createdAt":"2024-06-18 03:01:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4596892/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4596892/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-84833-y","type":"published","date":"2025-01-04T15:57:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60619727,"identity":"da9c00a7-83c8-4b38-a27e-3f110b5b3bbc","added_by":"auto","created_at":"2024-07-18 20:46:21","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1907222,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic Illustration of the PyT-LFA prototype. \u003c/strong\u003e(A) PIA detection using a specifically designed sandwich immunoassay (corresponding to T and C lines). (B) Result interpretation: when the serum sample is added to the LFA cassette, they are driven forward by buffer action, and colloidal gold signals and color appear at the control line (C line) or both the control and test lines (T line). The results can be read visually. (C) Configuration and compartment of PyT-LFA\u003cstrong\u003e \u003c/strong\u003edevice.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4596892/v1/79df64740297418d4ca0b53f.jpg"},{"id":60619729,"identity":"c6134c8f-7b45-4611-8809-9ec803628b22","added_by":"auto","created_at":"2024-07-18 20:46:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2580166,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptimization of major compartments in PyT-LFA development. \u003c/strong\u003eThe variation of antibody clones (A), pretreated sample pad (Cytosep\u003csup\u003e®\u003c/sup\u003e 1662) reagents (B), running buffer usage volume (C), and surfactant reagents (D) was indicated. The intensity of the band at the T and C lines was observed when testing with serum samples (pooled negative and spiked serum), Consequently, the results were analyzed for each ratio, with \"+\" representing the band intensity at the T line.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4596892/v1/cf7c9223703456570aaf9d96.jpg"},{"id":60619728,"identity":"5b783f85-23a0-4a27-9d20-bee44ca65db9","added_by":"auto","created_at":"2024-07-18 20:46:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1976909,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of PyT-LFA tool. \u003c/strong\u003eThe band image and its intensity at the T and C lines were employed to determine the detection limit (LOD) range of the tool in the testing with PIA spiked serum. The PIA concentration was indicated and results were analyzed for each ratio, with \"+\" representing the band intensity at the T line.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4596892/v1/f93a2a320837ec5ccdab0b65.jpg"},{"id":60619730,"identity":"c69692c6-e5ab-49a6-8fcf-38a67889f1f9","added_by":"auto","created_at":"2024-07-18 20:46:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1371071,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStability of PyT-LFA device. \u003c/strong\u003eThe detected results of the cassette stored for 30 days (A), 90 days (B), and 120 days (C) are presented. Triplicate tests were conducted using the same sample types (spiked serum; PS and Negative serum; NS).\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4596892/v1/306cae25171638b1a10d131e.jpg"},{"id":73093187,"identity":"0e51b407-5c6c-4ad7-bcff-e8f96ff27eb2","added_by":"auto","created_at":"2025-01-06 16:09:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8466780,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4596892/v1/7b381053-a50b-4887-a5af-04f024af51ae.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of Lateral Flow Immunochromatographic Assay with Anti-Pythium insidiosum Antibodies for Point-of-Care Testing of Vascular Pythiosis","fulltext":[{"header":"Research Highlights","content":"\u003cul\u003e\n \u003cli\u003ePoint-of-care detection (POCT) of vascular pythiosis in less than 30 minutes.\u003c/li\u003e\n \u003cli\u003eAn anti-PIA mAbs is leveraged to specifically differentiate between \u003cem\u003eP. insidiosum\u003c/em\u003e and other antigens.\u003c/li\u003e\n \u003cli\u003eEquipment-free readout using a lateral-flow immunochromatographic strip.\u003c/li\u003e\n \u003cli\u003eThe assay diagnoses pythiosis in clinical samples with 100% specificity.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003ePythiosis is a severe infectious illness affecting humans and animals, particularly horses, pets, and livestock. The causative agent is the pathogenic aquatic fungus-like oomycete \u003cem\u003ePythium insidiosum\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The incidence of this infection is mainly detailed in tropical, subtropical, and temperate area\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. While the infected animals commonly exhibit clinical manifestations, involving cutaneous/subcutaneous lesions and disorders of the gastrointestinal tract, the symptoms in humans, especially those with hematological disorders like thalassemia hemoglobinopathy, typically present with arterial occlusions in the lower extremities (vascular pythiosis) and ocular infections\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Treatment with common antifungal agent demonstrates ineffective against \u003cem\u003eP. insidiosum\u003c/em\u003e infections. The desired management for pythiosis occupies the surgical removal of the infected organ, for instance, the eye or leg. Unfortunately, numerous pythiosis patients accede to advanced infections or inadequate treatment\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The prognosis for pythiosis patients is notably unfavorable when there is a delay in diagnosis, leading to late treatment\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe gold-standard diagnostic approach for pythiosis occupies a fungal culture aimed at isolating \u003cem\u003eP. insidiosum\u003c/em\u003e from infected samples. This method, while labor-intensive and time-consuming, requires expertise for accomplishment and result interpretation\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Additionally, it frequently evidences unsuccessful in separating the organism, particularly when sporadically present in such samples, and may yield to low temperatures during transportation and storage\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Hence, a more sensitive method is very important for early \u003cem\u003eP. insidiosum\u003c/em\u003e detection, supporting timely patient management. Various alternative detection approaches, utilizing sensitive molecular and immunological technologies, have been proposed to address the limitations of the microbial culture method in identifying \u003cem\u003eP. insidiosum\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In addition, the identification of a target pathogen's DNA or RNA sequence through nucleic acid-based tests (NATs) emerges as a highly effective method for ensuring robust microbial identification\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, enabling the rapid capture of minute quantities of the pathogen's nucleic acid in a clinical specimen, enhancing diagnostic sensitivity.\u003c/p\u003e \u003cp\u003eDespite the appropriateness and efficiency of existing immunological methods, distinguishing between antibodies indicative of a past or recent \u003cem\u003eP. insidiosum\u003c/em\u003e infection can be challenging. Furthermore, these methods cannot detect anti-\u003cem\u003eP. insidiosum\u003c/em\u003e antibodies in patients with localized infections, such as in the cornea, an immunologically privileged site lacking specific types of host immune responses, such as antibody production\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Notably, there is currently no commercially available immunological test for pythiosis, and its in-house development is hindered by the absence of a \u003cem\u003eP. insidiosum\u003c/em\u003e-specific antigen. This unavailability and inaccessibility limit the incorporation of immunological tests in clinical laboratories. Unlike in-house immunological assays, all essential reagents (such as primers, probes, nucleotides, and DNA polymerases) and necessary equipment for developing NATs are readily accessible in molecular diagnostic laboratories or can be procured from commercial sources.\u003c/p\u003e \u003cp\u003eThe lateral flow immunochromatographic assay (LFA) has grown extensively in popularity for the serodiagnosis of numerous infectious diseases due to its user-friendly format, rapid result turnaround, and high levels of detection sensitivity and specificity. Notably, this test is particularly valuable in faint or endemic areas where diagnostic facilities for pythiosis may be lacking, thus addressing critical healthcare needs in underserved regions\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOver the decades, immunotherapeutic \u003cem\u003eP. insidiosum\u003c/em\u003e-antigen (PIA) has exhibited efficacy in activating immune responses and managing pythiosis in both human and animal cases\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The success of PIA immunotherapy in pythiosis has been postulated to stalk from its ability to induce a shift from a T-helper lymphocyte type 2 (Th2) to a T-helper lymphocyte type 1 (Th1) response\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In vascular pythiosis treatment, for instance, the administration of PIA has demonstrated that an enzyme-linked immunosorbent assay (ELISA) value of \u003cem\u003eP. insidiosum\u003c/em\u003e-specific antibody exceeding 8 correlates with improved patient survival, signifying a robust host immune response to PIA in this contex\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Utilizing immunotherapy for vascular pythiosis represents a unique strategy leveraging the innate potentials of the patient's immune system.\u003c/p\u003e \u003cp\u003eDue to the distributed evidence of published information on LFA in fungal detection strategy, we aimed to develop a prototype of the point-of-care testing (POCT) with mouse monoclonal anti-PIA immunoglobulin G (IgG) for \u003cem\u003eP. insidiosum\u003c/em\u003e antigens detection in serum samples of a patient with vascular pythiosis. It is noteworthy that recent research has identified specific monoclonal antibodies, such as anti-PIA IgG, as a potential tool for detecting \u003cem\u003eP. insidiosum\u003c/em\u003e. However, our study exclusively focuses on the prototype test, signaling a forthcoming shift in the technology utilized for diagnosing vascular pythiosis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePrinciple and Designing of PyT-LFA Detection System\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe proof-of-concept approach outlined in our recent work utilizes lateral-flow immunochromatography assay for pythiosis diagnosis, referred to as PyT-LFA, for \u003cem\u003ePythium insidiosu\u003c/em\u003em detection. The system, designed as a broadly-detected \u003cem\u003ePythium insidiosum\u003c/em\u003e antigen (PIA) platform, successfully handles notable sample types, particularly, patient serum. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e depicts the configuration and compartment of PyT-LFA device (i.e., the sample pad, conjugate pad, testing line, and control line). Designed as a versatile antigen-antibody reaction dogma, the colloidal gold-conjugated mouse anti-\u003cem\u003eP. insidiosum\u003c/em\u003e monoclonal IgG scheme demonstrates applicability across PIA in serum from the whole blood of each patient is separated with the standard centrifugation method\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The encountered serum samples, specifically from vascular pythiosis was specifically targeted and effectively applied with the developed platform, highlighting the system's utility for the vascular pythiosis, as illustrated in. The whole testing process takes approximately 10 minutes for sample preparation and 15 minutes for detection. The outcomes include semi-quantitative results \u0026ndash; positive and negative yields with their definite intensity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe serum samples, when added to the sample pads of the PyT-LFA strips, combine with mouse anti-PIA mAb-conjugated colloidal gold (colloidal AG-anti-PIA) on the conjugate pad due to the specific binding of a PIA (no.1). As the products migrate along the strip, the complexes are captured by PIA-specific mAb at the test line (T line), resulting in a red band at the T line (no.2). The remaining colloidal gold-anti-PIA continue to move and are captured by goat anti-mouse IgG at the control line (C line), forming another red band that confirms the LFA system's efficacy (no.3). In the absence of target PIA, no red band is observed at the T line as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. Results can be visually assessed based on the presence of a red band at the T line. The LFA procedure can be completed within 25 minutes from serum preparation to result in readout as indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. Therefore, in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, LFA employs a cassette strip containing five components: a sample pad, a conjugate pad, a nitrocellulose membrane, a Wick (absorbent) pad, and a plastic cushion. Serum samples are applied to the sample pad and can be visually detected with the PIA-specific mAb, which is colloidal gold-conjugated.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of PyT-LFA Platform\u003c/h2\u003e \u003cp\u003eTechnical challenges in the LFA context from various factors such as antibody clones, running buffers, pretreated sample pad reagents, and surfactants being enclosed during the development step and commercialization process. These factors can impact the specificity and sensitivity of the tested results. Failure to assemble optimized components or suitable materials in the device can lead to demanding accuracy requirements and detection limit thresholds. Various substances, including those utilizing valuable materials or complex solutions, have been utilized to enhance LFA performance. While these materials have proven effective, their practicality remains suboptimal. Therefore, the search for improved alternatives continues. Here, we propose simple yet powerful materials and reagents, devoid of costly materials and complex solutions, for LFA improvement.\u003c/p\u003e \u003cp\u003eThe optimization of \u003cem\u003eP. insidiosum\u003c/em\u003e-specific mAb clones was initially explored. Table\u0026nbsp;1 shown the presence of a diverse set of \u003cem\u003eP. insidiosum\u003c/em\u003e-specific mAb clones, including 1.0 mg/mL of each PyT 1F5, PyT 3\u0026ndash;24, and PyT 3\u0026ndash;29, at the test (T) line. Variations were observed in the outcomes of different antibody clones when different running buffers were employed. Interestingly, all strips, except Strips No. 7 (which depicted a true negative result, TN), showed the false positive (FP) results when tested with 100 \u0026micro;L of 1xPBS\u0026thinsp;+\u0026thinsp;0.1% TX405 buffer only (data not shown). However, Strip No. 9 exhibited a positive band (+\u0026thinsp;1), Strip No. 3 and No. 6 resulted in false negative (FN) when 95 \u0026micro;L CMF1 (pH 8.0) buffer with 5 \u0026micro;L of PIA-spiked serum (10 \u0026micro;g/mL; Positive test) was used. Conversely, Strip No. 1 (+\u0026thinsp;3.5) and No. 4 (+\u0026thinsp;1) displayed positive outcomes alongside FN results in the other strips when using the 95 \u0026micro;L of FL (pH 8.0) buffer with 5 \u0026micro;L of PIA-spiked serum (10 \u0026micro;g/mL; Positive test) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. Therefore, the PyT 1F5 clone and FL (pH 8.0) buffer were selected for the downstream process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then enhanced the efficiency of the sample pad (Cytosep\u0026reg; 1662) using several pretreated reagents. When the pooled negative serum (20 \u0026micro;L) with FL buffer (pH 8.0, 80 \u0026micro;L) was applied to the V228 (pH 9.6)-pretreated sample pad, no band was observed (true negative, TN), whereas an inverse result (false positive; FP) was noted in the sample pad pretreated with CMF-1 or F1 supplemented with 0.5% casein solution at 15 minutes testing as demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Moreover, the maximum volume of the serum sample, resulting in the true negative (TN) outcome was defined as 40 \u0026micro;L as indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC. Thus, the pretreatment of the sample pad with V228 (pH 9.6) reagent and the maximum serum sample volume were optimized in the current study.\u003c/p\u003e \u003cp\u003eTo improve the detection sensitivity of the apparatus, we subsequently evaluated the potential of several surfactants added to the running buffer. Similar TN results were observed when testing pooled negative serum with FL (pH 8.0) without surfactants and FL (pH 8.0) supplemented with Tween 20 or 0.1% Biot constituents. However, a band of undemanding intensity was observed when experimenting with the FL (pH 8.0) buffer supplemented with TX100 as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD; upper. Testing with PIA-spiked serum (final conc. 1,000 ng/mL) revealed that both the no surfactant buffer and all supplementation exhibited a positive band (True positive, TP) as demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD; lower. Additionally, 0.1% Biot (pH 8.0) enhanced the TN result when testing with pooled negative serum samples, compared to other concentrations. In conclusion, we utilized the PyT 1F5 clone as the captured antibody system, FL with 0.1% Biot (pH 8.0) solution, and V228 (pH 9.6) reagent as the running buffer surfactant and pretreated sample pad reagents, respectively, for the developed device.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation and Validation of PyT-LFA tool\u003c/h2\u003e \u003cp\u003eTo assess the limit of detection (LOD) of the PyT-LFA system, we conducted assays on serial dilutions of PIA-spiked serum using a signal reader to ensure precision. For a direct and semi-quantitative determination of the LOD, we prepared serial dilutions of the samples in physiological serum and analyzed them with the PyT-LFA platform. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrates the results, with serum samples containing different concentrations of PIA. The LFA efficiency (observed as band intensity) notably improved with increasing sensitivity of the running buffer surfactant. Even at a low concentration of 8 ng/mL, the test exhibited high specificity, with no FN results. Typically, the concentration of PIA in serum falls within the range of 4\u0026ndash;8 ng/mL, indicating that the LOD of the PyT-LFA system is suitable for clinical testing. Furthermore, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e demonstrates an imaged band for detection, exhibited as the measurable signal at the T line relative to the respective concentrations of PIA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also evaluated the stability (Reproducibility) of the PyT-LFA platform by analyzing the intensity band of the samples with a long-term storage period (30 days, 90 days, and 120 days) of the device. The triplicate of the same sample was prepared, followed by visual detection using conditioned lateral flow strips. The three spiked- and healthy serum pairs were prepared using three batches of strips under the same protocol. The results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e indicate no differences among the three pairs of strips. This study thus demonstrates the high reproducibility, sensitivity, and reliability of our PyT-LFA platform, offering a valuable method for diagnosing pythiosis in clinical samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHuman and animal pythiosis is endemic in several tropical and subtropical countries\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In human pythiosis, four distinct pathological forms have been documented. While cutaneous pythiosis is characterized by granulomatous and ulcerating lesions typically affecting the face or limbs, vascular pythiosis involves arteries and can lead to arterial occlusion or aneurysm formation. The major clinical manifestation of ocular pythiosis is presented as keratitis. However, disseminated pythiosis is marked by the infection of internal organs\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In the fashionable microbial infection, both immunological and nucleic acid-based methods have been utilized for pathogen Identification.\u003c/p\u003e \u003cp\u003eSeveral serodiagnostic tests have been developed to aid in the early diagnosis of pythiosis\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In-house enzyme-linked immunosorbent assay (ELISA) and Immunoblotting assays have demonstrated high sensitivity and specificity for diagnosing pythiosis\u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, these tests require skilled personnel, stable and reproducible reagents, expensive equipment, and lengthy turnaround times. Immunodiffusion (ID) is a straightforward serological test commonly utilized in laboratories for pythiosis diagnosis and is considered a standard serodiagnostic method\u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. While the ID test is easy to conduct and exhibits high specificity, its sensitivity is often limited, and the prolonged turnaround time may result in false-negative outcomes and delayed treatment. Consequently, there is a pressing need to enhance diagnostic procedures to improve patient care in managing pythiosis.\u003c/p\u003e \u003cp\u003eThe lateral flow immunochromatography has gained significant acceptance for the serodiagnosis of various infectious diseases owing to its user-friendly format, rapid result generation, and high levels of detection sensitivity and specificity\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Krajaejun et.al.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e introduced an immunochromatographic test (ICT) for human pythiosis diagnosis (target to antibodies), with evaluation revealing a rapid turnaround time of less than 30 minutes for obtaining results. In contrast to the ID assay, the ICT revealed 88% sensitivity and 100% specificity, whereas the ID assay demonstrated 61% sensitivity and 100% specificity.\u003c/p\u003e \u003cp\u003eWhile existing immunological methods are deemed appropriate and efficient, there are still limits in their applications. The distinguishing between antibodies indicative of past or recent \u003cem\u003eP. insidiosum\u003c/em\u003e infections can pose challenges. Moreover, these methods are difficult to detect anti-P. \u003cem\u003einsidiosum\u003c/em\u003e antibodies in patients with localized infections, particularly in an immunologically privileged site such as the eyes, notably antibody production (actually it can detect by ELISA with titier\u0026thinsp;\u0026lt;\u0026thinsp;1:800). Moreover, there is currently no commercially available immunological test for pythiosis, and the development of in-house assays is impeded by the absence of a \u003cem\u003eP. insidiosum\u003c/em\u003e-specific antigen. Consequently, the unavailability and inaccessibility of such tests hinder their integration into clinical laboratories\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDue to the distributed evidence of published information on \u003cem\u003eP. insidiosum\u003c/em\u003e-antigen (PIA) in the context of pythiosis therapy, we, therefore, aimed to develop a prototype of the point-of-care testing with mouse monoclonal anti-PIA immunoglobulin G (IgG) for \u003cem\u003eP. insidiosum\u003c/em\u003e antigens detection in serum samples collected from the patients with vascular pythiosis (PyT-LFA).\u003c/p\u003e \u003cp\u003eThe limit of detection (LOD) of the PyT-LFA prototype for detecting \u003cem\u003eP. insidiosum\u003c/em\u003e antigens in all pythiosis sera was 8 ng/mL. Sera from all vascular pythiosis patients tested positive by our device. However, the failure to detect \u003cem\u003eP. insidiosum\u003c/em\u003e antigens in these patients likely occurred due to long-term sera storage and inadequate presentation of antigens in localized bloodstream infections \u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Therefore, the serodiagnosis of vascular infection should be approached cautiously, considering the expected rate of false-negative results. The prototype could serve as a point-of-care instrument for vascular pythiosis, while other serodiagnosis approaches require specific compartment preparation before testing. The turnaround time of the device was remarkably shorter compared to others (15\u0026ndash;20 minutes. For the PyT-LFA; 1\u0026ndash;24 hours for others). Therefore, the PyT-LFA demonstrates better performance and greater convenience than previous tools, making it suitable for the serodiagnosis of vascular pythiosis. However, further evaluation is necessary to assess its performance in diagnosing ocular and cutaneous pythiosis.\u003c/p\u003e \u003cp\u003eOur findings show that the devices, which had been stored for 6 months, were suitable for future investigation with the vascular pythiosis sera assays. The assay\u0026rsquo;s repeatability was shown to be stable, with only 100% of collected positive samples yielding positive results when reanalyzed. Additionally, our tests on the same specimens produced positive results. Other investigations revealed a significant association between the LFA and 1,3-Beta-D glucan (BG) assay results when tests were performed on the same day. However, this correlation decreased after a week of sample storage. Interestingly, the existing studies have also found a decline in BG test accuracy following both short- and long-term freezing. The BG test identifies Beta-D glucan, which can be hydrolyzed or degraded over time due to acidic conditions, even when samples are maintained at low temperatures to prevent these processe\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan additionalcitationids=\"CR34 CR35 CR36\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Furthermore, subsequent PyT-LFA test findings differed from earlier tests when samples were held at -80\u0026deg;C, implying that the test\u0026rsquo;s diagnostic performance may be stabilized. It has been postulated that the \u003cem\u003eP. insidiosum\u003c/em\u003e antigen identified by the PyT-LFA test becomes stable over time due to autolytic breakdown. As a result, it is assumed that the PyT-LFA test be performed as soon as feasible to ensure a good correlation with the BG assay results. The LOD and specificity of the prototype developed in this study might be in line with previous reports. However, enhancements to the PyT-LFA test could be achieved through its combination with other tests, such as PCR or the CRISPR/Cas system. The rationed LOD observed in the LFD test in our study may be linked to patients being exposed to multiple antibiotics and/or antifungal agents before sample collection.\u003c/p\u003e \u003cp\u003eHowever, nucleic acid-based methods, particularly PCR, offer distinct advantages over immunological techniques due to their superior sensitivity, specificity, and rapidity\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e Moreover, advancements in biosensor technology have further enhanced the sensitivity, specificity, and simplicity of nucleic acid-based assays\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, paving the way for the development of convenient, cost-effective, and specific diagnostic tools for \u003cem\u003ePythium\u003c/em\u003e spp. pathogen monitoring applications. This can greatly aid in implementing management practices aimed at reducing the risk of epidemics.\u003c/p\u003e \u003cp\u003eCollectively, our prototype offers several notable advantages. This can greatly aid in implementing management practices aimed at reducing the risk of epidemics.\u003c/p\u003e"},{"header":"Limitations of the study","content":"\u003cp\u003eHere, we have successfully developed a sensitive, cost-effective, and user-friendly PIA detection platform by lateral flow immunochromatography. This platform allows for detection results to be obtained within 25 minutes using clinically relevant samples (such as serum) directly without the complicated steps. We anticipate that the PyT-LFA system could be readily adapted for detecting various types of human pythiosis, particularly, vascular and systemic pythiosis. However, detecting variations in PIA number remains a challenge. We plan to further refine the LOD value and read-out system to enable the LFA system to detect the lowest PIA number variations effectively.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is funded by Thailand Science Research and Innovation (TSRI) Fund Chulalongkorn University (Grant no. HEA663000027).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN.W. and P.T. carried out assays and analyzed the results; P.T., A.B., NL, and A.C. supported the optimization of assays and prepared data; A.C. developed the strip; A.B., and A.C. performed analyses of assay results; N.W., A.B., and A.C. conceived and designed the experiments; P.T., A.B., and A.C. wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDECLARATION OF INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGaastra, W., Lipman, L.J., De Cock, A.W., Exel, T.K., Pegge, R.B., Scheurwater, J., Vilela, R., and Mendoza, L. (2010). Pythium insidiosum: an overview. 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(2012). Production of a highly group-specific monoclonal antibody against zearalenone and its application in an enzyme-linked immunosorbent assay. J Vet Sci \u003cem\u003e13\u003c/em\u003e, 119\u0026ndash;125. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4142/jvs.2012.13.2.119\u003c/span\u003e\u003cspan address=\"10.4142/jvs.2012.13.2.119\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, P., Wu, J., Zhang, L., Fan, Z., Yu, T., Jiang, F., Tang, X., Zhang, Z., Zhang, W., and Zhang, Q. (2017). Doses of Immunogen Contribute to Specificity Spectrums of Antibodies against Aflatoxin. 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J Genet Eng Biotechnol \u003cem\u003e19\u003c/em\u003e, 159. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s43141-021-00264-6\u003c/span\u003e\u003cspan address=\"10.1186/s43141-021-00264-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMazurkevych, A., Malyuk, M., Bezdieniezhnykh, N., Starodub, L., Kharkevych, Y., Jakubczak, A., and Gryzinska, M. (2017). Immunophenotypic characteristics and karyotype analysis of bone marrow-derived mesenchymal stem cells of rabbits during in vitro cultivation. Pol J Vet Sci \u003cem\u003e20\u003c/em\u003e, 687\u0026ndash;695. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1515/pjvs-2017-0086\u003c/span\u003e\u003cspan address=\"10.1515/pjvs-2017-0086\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeatty, J.D., Beatty, B.G., and Vlahos, W.G. (1987). Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Methods \u003cem\u003e100\u003c/em\u003e, 173\u0026ndash;179. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/0022-1759(87)90187-6\u003c/span\u003e\u003cspan address=\"10.1016/0022-1759(87)90187-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"STAR METHODS","content":"\u003cp\u003e\u003cstrong\u003eLead contact\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther information and requests for resources should be directed\u003c/p\u003e\n\u003cp\u003eto and will be fulfilled by the lead contact, Ariya Chindamporn ([email protected].).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not generate unique reagents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMETHOD DETAILS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHealthy blood collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh human whole blood samples were collected from 5 healthy volunteers using clotted-blood tubes. Serum was separated from each sample using standard centrifugation method\u003csup\u003e20\u003c/sup\u003e. Each individual provided informed consent, and the study received approval from the Institutional Review Board of the faculty of Medicine, Chulalongkorn University, a WHO-certified ethics committee (IRB No. 0427/67). All methods adhered to these approved guidelines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of mouse monoclonal anti-\u003cem\u003eP. insidiosum\u003c/em\u003e antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePythium insidiosum\u003c/em\u003e antigens (PIA) was prepared from clade 1 \u003cem\u003eP. insidiosum\u003c/em\u003e strain as previously described\u003csup\u003e15\u003c/sup\u003e and used as an inoculum in our study. A 20 mg/mL PIA stock solution was prepared with PCR-grade water and stored at \u0026minus;80\u0026deg;C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of a PyT-LFA cassette \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(i) Conjugation of antibody to colloidal gold.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 40-nanometer-particle colloidal gold suspension (Arista, Allentown, PA, USA) was adjusted to pH 9.65 using 0.2 M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e. Subsequently, 3 \u0026micro;g of mouse anti-\u003cem\u003eP.insidiodum\u003c/em\u003e (PyT) mAb was added to each 500 \u0026micro;L of colloidal gold, followed by a 30-minute incubation at room temperature. The residual surfaces of the colloidal gold particles were then blocked by incubating with 5% (w/v) bovine serum albumin (Sigma, St. Louis, MO, USA) for 10 minutes. After centrifugation at 6,000 \u0026times; g for 15 minutes, the supernatant was discarded, and the conjugate pellet was washed in 0.5% (w/v) casein. This was followed by another centrifugation at 6,000 \u0026times; g for 15 minutes, after which the supernatant was removed. The conjugate was resuspended in a solution containing 0.5% (w/v) casein and 20% (w/v) sucrose in 0.02 M Tris-HCl (pH 8.0), with a volume 40 times smaller than the original suspension. A 2.5 by 2.5-millimeter piece of glass fiber (GF33; Whatman Schleicher \u0026amp; Schuell, Dassel, Germany) was impregnated with 2.5 \u0026micro;L of this IgG-colloidal gold conjugate and dried in a dehumidifier cabinet for an hour.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(ii) Immobilization of antigen and antibody onto a nitrocellulose membrane.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA nitrocellulose membrane measuring 1.5 centimeters wide (CN140; Whatman Schleicher \u0026amp; Schuell, Dassel, Germany) was coated with PyT (1 mg/mL) to create the test line and goat anti-mouse IgG (1 mg/mL) in PBS to create the control line, each at a volume of 1 \u0026mu;L per centimeter using a dispenser (ZX 1000; BioDot, Irvine, CA). Subsequently, the membrane was dried, blocked with 1% (w/v) bovine serum albumin, and dried again in a dehumidifier cabinet.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(iii) Assembly of PyT-LFA strips.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe nitrocellulose membrane, glass fiber with colloidal gold conjugate, sample pad (Cytosep 1622 / V228, pH 9.6), and wicking pad (18 mm.; 470 chromatography paper; Whatman, Maidstone, England) were carefully arranged and mounted onto a plastic backing (60 mm.). This assembly was then precisely cut into strips measuring 25 mm. in width using a strip-cutting machine (CM 4000 R; BioDot, Irvine, CA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(iv) Detection of human \u003cem\u003eP. insidiosum\u003c/em\u003e antigens by the PyT-LFA.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach serum sample (40 \u0026micro;L) underwent an optimized dilution in 60 \u0026micro;L running buffer (FL with 0.1% Biot pH 8.0). Subsequently, the lateral flow immunochromatographic test (LFA) was conducted in duplicate using 100 \u0026mu;L of the diluted serum in the indicated buffer. Following a 15-minute incubation period, the test signal of each LFA strip was evaluated by three independent laboratory personnel using visual inspection. To quantify the LFA signal, each strip was scanned using a scanner (Epson Perfection 1670 photo scanner; Seiko Epson Corp., Japan) to generate a tagged image file format picture. Test and background signal intensities were analyzed utilizing the Image J software (https://imagej.net/ij/docs/intro.html). The resulting intensity value, obtained by subtracting the background signal from the test signal.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification and statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were conducted using GraphPad Prism version 10.2.1 for Windows (GraphPad Software, www.graphpad.com). Differences between the two groups were assessed using an independent sample t-test (unpaired, two-tailed). Error bars represent \u0026plusmn;SD. \u003cem\u003ep\u003c/em\u003e-values \u0026lt; .05 were considered statistically significant. The signal of each LFA strip was evaluated by three independent experiments.\u0026nbsp;\u003c/p\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Detail of each component in the PyT-LFA cassette.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.428571428571427%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrip No.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"44.89795918367347%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCapture (Test line)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.673469387755105%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDetector (Conjugate)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.428571428571427%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrip 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"44.89795918367347%\" rowspan=\"3\"\u003e\n \u003cp\u003eClone PyT 1F5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.673469387755105%\" valign=\"top\"\u003e\n \u003cp\u003eClone PyT 1F5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.888888888888886%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrip 2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.111111111111114%\" valign=\"top\"\u003e\n \u003cp\u003eClone PyT 3-24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.888888888888886%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrip 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.111111111111114%\" valign=\"top\"\u003e\n \u003cp\u003eClone PyT 3-29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.428571428571427%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrip 4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"44.89795918367347%\" rowspan=\"3\"\u003e\n \u003cp\u003eClone PyT 3-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.673469387755105%\" valign=\"top\"\u003e\n \u003cp\u003eClone PyT 1F5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.888888888888886%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrip 5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.111111111111114%\" valign=\"top\"\u003e\n \u003cp\u003eClone PyT 3-24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.888888888888886%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrip 6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.111111111111114%\" valign=\"top\"\u003e\n \u003cp\u003eClone PyT 3-29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.428571428571427%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrip 7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"44.89795918367347%\" rowspan=\"3\"\u003e\n \u003cp\u003eClone PyT 3-29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.673469387755105%\" valign=\"top\"\u003e\n \u003cp\u003eClone PyT 1F5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.888888888888886%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrip 8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.111111111111114%\" valign=\"top\"\u003e\n \u003cp\u003eClone PyT 3-24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.888888888888886%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrip 9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.111111111111114%\" valign=\"top\"\u003e\n \u003cp\u003eClone PyT 3-29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4596892/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4596892/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe pathogenic oomycete \u003cem\u003ePythium insidiosum\u003c/em\u003e causes a fatal infectious illness known as pythiosis, impacting humans and certain animals in numerous countries in the tropics and subtropics. Delayed diagnosis is a primary factor contributing to the heightened morbidity and mortality associated with the disease. Several new serodiagnostic methods have been developed to improve the identification of pythiosis. However, these assays provide only indirect evidence of pythiosis and are not readily available in the commercial market. Here, we have developed an affordable point-of-care test (POCT) kit based on an immunochromatographic assay for the direct detection of \u003cem\u003eP. insidiosum\u003c/em\u003e antigens. Our recent findings reveal that the lateral flow sandwich immunological testing cassette can accurately identify vascular pythiosis antigens using a small volume of patient\u0026rsquo;s plasma, accomplishing 100% accuracy and a limit of detection (LOD) of 8 ng/mL. This prototype cartridge represents a significant stride toward the advancement of enriched POCT for pythiosis serodiagnosis.\u003c/p\u003e","manuscriptTitle":"Development of Lateral Flow Immunochromatographic Assay with Anti-Pythium insidiosum Antibodies for Point-of-Care Testing of Vascular Pythiosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-18 20:46:16","doi":"10.21203/rs.3.rs-4596892/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-09T06:46:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-07T09:34:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-01T08:46:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"232560685468193200963946118008358310284","date":"2024-09-28T23:22:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16897472226689349225580457507514442793","date":"2024-09-19T15:11:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-30T16:05:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-24T11:43:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-06-24T10:40:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-21T13:48:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-06-18T03:00:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"81d837d6-4258-4c16-a679-327cba0dd033","owner":[],"postedDate":"July 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34190374,"name":"Biological sciences/Microbiology/Infectious disease diagnostics"},{"id":34190375,"name":"Health sciences/Diseases/Infectious diseases/Fungal infection"}],"tags":[],"updatedAt":"2025-01-06T15:59:43+00:00","versionOfRecord":{"articleIdentity":"rs-4596892","link":"https://doi.org/10.1038/s41598-024-84833-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-01-04 15:57:09","publishedOnDateReadable":"January 4th, 2025"},"versionCreatedAt":"2024-07-18 20:46:16","video":"","vorDoi":"10.1038/s41598-024-84833-y","vorDoiUrl":"https://doi.org/10.1038/s41598-024-84833-y","workflowStages":[]},"version":"v1","identity":"rs-4596892","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4596892","identity":"rs-4596892","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}