Quantification of Absolute IgG Concentration in Bat Sera

Quantification of Absolute IgG Concentration in Bat Sera | 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 Quantification of Absolute IgG Concentration in Bat Sera Frédéric Touzalin, Mads Frost Bertelsen, Stamatios Alan Tahas, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6811643/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bats are endowed with a remarkable capacity to withstand important pathogens through evolutionary adaptations in their immune systems. Antibodies are essential component of the adaptive immune response and serve as a crucial biomarker, indicating both present and past pathogen infections, as well as the overall physiological state of the organism. The main type of antibody found in the blood of mammals is IgG. It is produced as a result of specific T-cell-dependent antibody responses. Consequently, monitoring IgG in wild animals can yield valuable insights into pathogen dynamics and host responses. Currently, there is no simple technique for measuring absolute IgG concentration that can be generalized for different species of bats. The present study proposes a methodology to quantify total IgG levels in bats. The approach is based on an immunosorbent assay and employs only protein G as a detecting reagent for IgG. This method has the potential to be applied to diverse bat species, as well as other mammals. As a proof of concept, we present a detailed procedure to quantify serum IgG in Egyptian fruit bats ( Rousettus aegyptiacus ). The estimated concentration of IgG was found to be relatively high (5-6 mg/ml), highlighting the role of specific antibody responses in the immune defence of bats. To validate the method, we compare the results to an alternative approach based on SPR biosensor technology. Furthermore, data pertaining to the estimation of IgG levels in a different bat species, namely Myotis myotis , is presented. This simple and effective technique offers a valuable tool for advancing our understanding of immune function in bats and potentially other wild mammals, contributing to broader efforts in wildlife immunology and disease ecology. Biological sciences/Immunology Biological sciences/Immunology/Adaptive immunity Biological sciences/Zoology/Animal physiology Bats Antibodies IgG concentration ELISA immune assays Figures Figure 1 Figure 2 Figure 3 Introduction Bats harbour several virulent pathogens without appearing to manifest immunopathological symptoms, exhibit atypically prolonged lifespans, and suffer a low prevalence of metabolic diseases 1 , 2 , 3 , 4 . These characteristics of bats likely originate from a series of evolutionary adaptations that enabled active flight 5 , 6 , 7 . Understanding how bats tolerate pathogens may have important implications for human health 8 , 9 . Consequently, numerous studies have recently been conducted to elucidate the molecular mechanisms underlying the distinctive characteristics of the immune response in various bat species 10 . Much of this work has focused on the innate immune response, with a particular focus on the immune defence pathways triggered by viral infections. Collectively, these studies have revealed several evolutionary adaptations that result in dampened innate immune responses in bats 3 , 11 , 12 , 13 , 14 , 15 , 16 , 17 . Antibodies are the hallmark of the adaptive immune responses, and they play an essential role in pathogen neutralization 18 . Despite their central role in immune defence, there is a limited understanding of antibody responses in bats. Some studies have suggested that bats mount inefficient transient antibody responses 19 , 20 , 21 , 22 . Conversely, other studies have demonstrated highly specific neutralising antibody responses in experimental infection settings 23 . Antibodies from the IgG class are central components of T cell-dependent immune responses. The evaluation of total IgG levels in blood can provide important indications about the extent of the specific adaptive immune responses, as well as the influence of various physiological, ecological, and immunological factors on these responses. The estimation of absolute values of circulating IgG may allow for a comparison of the global extent of the IgG responses of bats with those of other species (e.g., humans). Furthermore, the universal approach for estimating of absolute IgG concentration may contribute to a comparative understanding of how life history or ecological differences among different species of bats lead to different immune profiles and tolerance capacity. However, to the best of our knowledge, there is no simple, quantitative method for determining absolute IgG concentrations in bat blood. This may be due to the lack of specific antibodies for detecting IgG in different species of bats. Previous studies have only presented arbitrary IgG levels in the blood of bats 24 , 25 , 26 , 27 . These levels were shown as variations in optical density generated by colorimetric assays, rather than as exact protein concentrations, as is usually done for humans 28 , 29 and experimental mice 30 . Here, a novel method was proposed for the straightforward quantification of the absolute IgG levels in serum samples of bats. The proposed method was based on an immune sorbent assay, a technique that has been successfully utilised in various research fields 31 . As a proof of concept, the assay was applied to serum samples from the Egyptian fruit bat ( Rousettus aegyptiacus, familly Pteropodidae ) and Bent-wing bat ( Miniopterus schreibersii , family Miniopteridae).. This bat species is the subject of extensive research and has contributed to significant advancements in our understanding of the bat immune system 11 , 12 , 32 , 33 , 34 . The proposed technique utilises protein G as an IgG detection component. Critical assay parameters are detailed, with attention given to adjustments required for species-specific implementation. Thus, as a proof of concept, we also estimated the concentration of a pool of sera from Greater mouse-eared bat ( Myotis myotis , family Vespertilionidae). The obtained data revealed that bats have relatively high levels of IgG from early stages of their life. This finding indicates that the bat species in question possesses the capacity to mount effective T cell-mediated antibody responses that are comparable to other mammals and humans. Results and Discussion The primary objective of this study is to develop a method for the quantification of the exact concentration of serum IgG that can be applied to different bat species. Given that Protein G has been demonstrated to bind to the constant region of IgG from a range of mammalian species with a high binding affinity, and that previously it has been shown to bind to IgG from certain bat species 35 , 36 , it was selected as the IgG detection reagent. The interaction of IgG with protein G was characterised by purifying total IgG from pooled human serum or serum pools of two bat species, captive R. aegyptiacus and wild Miniopterus schreibersii (family Miniopteridae), by ion exchange chromatography (Melon™ system). Subsequently, the binding affinity of IgG from different species to surface-immobilized protein G was evaluated using a surface plasmon resonance-based assay. The real-time interaction profiles indicated that human and R. aegyptiacus IgG bind to protein G. In contrast, M. schreibersii IgG did not show any binding to the protein, even when high concentration of IgG was injected (Fig. 1 ). The binding affinity of the human polyclonal IgG sample was further evaluated, and the equilibrium dissociation constant (K D ) was determined to be 5.5 (± 0.28 SD) nM. The affinity of IgG purified from R. aegyptiacus sera for protein G was lower, with an average value of 40.4 (± 0.83 SD) nM. This reduced affinity appears to result from decreased complex stability, as reflected by an increased dissociation rate (Fig. 1 ). These findings underscore the necessity for consideration of bat species-specific profiles when developing assays to assess total IgG concentration in bat sera. Next, we tested an experimental strategy based on an immunosorbent assay for assessment of total IgG concentration in bat sera (Fig. 2 a). This approach consists of direct immobilization of serially diluted (from 2000 to 2080000 folds) sera onto polystyrene ELISA plates and quantification of the IgG antibodies by using biotinylated protein G (Fig. 2 a). The advantage of direct immobilization of sera is that this approach does not necessitate the use of specific capture antibodies. The viability of the approach is supported by the fact that IgG is among the most abundant proteins in serum (the second most abundant in humans). A crucial element in conducting such an assay is the use of an IgG standard to enable the accurate quantification of the IgG concentration. If the binding affinity of protein G for human and bat IgG is identical, a pool of human sera from healthy donors could serve as an appropriate control; however, IgG from R. aegyptiacus exhibits approximately 8-fold lower affinity for protein G compared to human IgG (Fig. 1 ). To account for this discrepancy, we propose the implementation of a species-specific standard. Total IgG was purified from pooled R. aegyptiacus serum using a protein G-Sepharose matrix, and the concentration of purified IgG was determined by UV-Vis spectroscopy. The depleted bat serum was then reconstituted with known amounts of IgG to generate a calibration standard (Fig. 2 a). This bat-specific standard was subsequently used in all further experiments to estimate total IgG concentrations in bat sera. The reactivity of a fixed concentration (2 µg/ml) of biotinylated protein G to immobilized IgG present in serially diluted sera was measured separately for 32 R. aegyptiacus individuals (13 juveniles and 19 young adults) as well as in pooled sera prepared from the same samples (Fig. 2 b). The resulting data demonstrated that protein G binding was proportional to serum concentration across the dilution series. The binding curves for individual sera samples exhibited heterogeneity in terms of the level of protein G binding, as evidenced by the presence of more than one order difference in their inflex points. In certain cases, the binding did not reach saturation within the tested dilution range (Fig. 2 b). The interaction of protein G with IgG present in pools of human sera exhibited considerably higher reactivity, as would be expected based on the higher affinity of human IgG to protein G (Fig. 2 b). The overall reactivity of pools consisting of mixed sera from juvenile bats or young adults to protein G did not differ significantly (Fig. 2 b). To quantify the absolute IgG concentrations, present in bat samples, the standard bat serum was utilised, which had been reconstituted with a defined concentration of IgG from R. aegyptiacus (Fig. 2 c). Four points from the linear part of the binding curve were used for linear regression analyses and determination of the constants of the equation describing the fit. These values were then used to calculate the IgG concentration in bat sera. Two serum dilutions (16,000 and 32,000 ×) were selected as reference points on the basis that they fell within the linear part of the binding curves across all tested samples (see Fig. 2 b). The obtained results show individual heterogeneity in IgG in sera of R. aegyptiacus in both juvenile and young adult individuals (Fig. 2 d). The mean IgG concentrations in the sera of juvenile and young adult bats were 4.85 (± 3.6 SD) and 6.4 (± 3.2 SD) mg/ml, respectively. The concentration of IgG in these two age groups did not differ significantly and pooled sera had similar IgG concentrations (see Fig. 2 d). To further validate the utility of the proposed method for measuring IgG levels in other bat species, we assessed its application using serum samples from an unrelated bat species, Myotis myotis (family Vespertilionidae). The binding affinity of polyclonal IgG from M. myotis to immobilized protein G, K D value of 6.8 (± 0.6 SD) nM at 22°C (Fig. 2 e), was comparable to the one established for human IgG, K D of 5.5 (± 0.28 SD) nM. In contrast to the samples from R. aegyptiacus , the comparable binding behaviour of M. myotis and human IgG allows human serum pools to be used effectively as a standard for estimating absolute IgG concentrations in M. myotis sera. A comparison of the pooled sera from human (n = 20 individuals) and from M. myotis (n = 20 individuals) revealed nearly identical interaction profiles with protein G (Fig. 2 f). Utilising human sera as a standard, with a reference concentration of 10 mg/ml of IgG, it was determined that the total serum concentration of IgG in M. myotis was approximately 7.9 (± 0.14 SD) mg/ml. The higher absolute concentration of IgG in M. myotis as compared to the IgG in sera from R. aegyptiacus can be explained by the fact that the sera of the former species were collected from adult individuals whereas a large proportion of the latter animals were subadults. The findings of this study collectively imply that to estimate absolute IgG concentrations in bats, it is necessary to first define the appropriate species-specific standard. The standard in question can be either reconstituted sera with autologous IgG or human sera pools. Finally, to further validate the proposed approach for determining total IgG concentration in bat sera, an orthogonal technique was utilised. The changes in the resonance signal in the surface plasmon resonance-based technology are proportional to the amount of protein accumulated on the sensor's surface 37 . To obtain a standard curve, purified IgG from R. aegyptiacus was titrated on a sensor chip with immobilized protein G at high density. As anticipated, the IgG produced a clear concentration-dependent dose-response signal (Fig. 3 a). Subsequently, samples of the serum pool of R. aegyptiacus diluted to 2000-, 4000-, and 8000-fold were allowed to interact with immobilized protein G. The concentration-dependent signal obtained (Fig. 3 b) was then utilised to calculate the concentration of IgG in the serum pool using the standard curve. The estimated concentration of IgG in bat serum through this method was found to be well aligned with the one obtained by ELISA-based assay (Fig. 3 c). In summary, the present study details a straightforward procedure that enables the quantification of exact IgG concentrations in bat serum, with the potential applicability to other mammalian species. The assay was validated by using an orthogonal method to confirm its accuracy. The procedure consists of the following interconnected steps: 1) Direct coating of serially diluted serum samples and appropriate serum standards onto ELISA plates. 2) Incubation with a fixed concentration of biotinylated protein G. 3) Detection of bound protein G by streptavidin-conjugated reporter enzyme (horseradish peroxidase). 4) Data analysis. This assay applies to any species in which protein G can bind to IgG. A key advantage of the method is its adaptability, permitting preliminary steps to be completed in field settings where conventional laboratory equipment is not readily accessible. Step 1 can be carried out in the field where the plates can also be dried and sealed, and steps 2 to 4 can be carried out back in the laboratory. This approach eliminates the need for freezing or long-term storage of liquid serum samples, thereby addressing significant logistical challenges associated with research in remote or resource-limited locations. A critical aspect of the assay is the use of an appropriate IgG standard. In instances where the affinity of the IgG from the bat species under study is similar to that of human IgG, human serum can be used as a reference. However, if the affinity differs substantially, the bat serum should first be depleted of IgG. The IgG concentration should then be accurately measured, and the serum reconstituted with a defined amount of IgG. Therefore, to define the IgG standard for a given species, preliminary measurements of affinity are required. This may be regarded as a potential hindrance to the scalability of the assay across species, given that the IgG affinity for protein G remains unknown for the majority of bats. Nonetheless, the estimation of the affinity of intramolecular interactions is currently a trivial task. The advent of novel biosensor technologies has enabled the estimation of affinities of intermolecular interaction in high-throughput manner 38 . In this study, we demonstrated that in certain cases, such as with M. schreibersii IgG samples, the current methodological approach is not applicable due to the absence of detectable interaction between its IgG and protein G. Nevertheless, the underlying concept of this approach remains valid, involving the direct coating of serum dilution solutions on ELISA plates and the utilisation of species-specific control. This can be achieved by employing alternative IgG-specific reagents, such as commercially available bat-specific anti-IgG antibodies. The findings of this study indicate that two distinct bat species can produce relatively high levels of IgG antibodies. In the case of R. aegyptiacus , IgG levels were found to be comparatively high from early stages of life (1–2 years old). These findings underscore the significance of T cell-dependent adaptive immune responses in bats in their immune defence mechanisms. Those results corroborate with previous research demonstrating that bats have the capacity to produce proficient antibodies. Thus, studies have demonstrated that bats are indeed able to mount a neutralising antibody response against viruses such as influenza (H18N11), or various lyssaviruses 23 , 39 . Moreover, bats generated antibody repertoires with high antigen-binding diversity 40 , 41 , 42 . Alongside the relatively high concentrations of IgG observed in the bats examined here, these findings suggest that while the bat’s innate immune system is often highlighted as the primary mechanism balancing pathogen control and immune tolerance 3 , 4 , 10 , 17 , the adaptive immune response also plays a significant role in their overall strategy for combating infection. Research on bat IgG and more generally on bat humoral immunity, is critical for advancing our understanding of epidemiology, immunology, and zoonotic disease management 22 , 43 . Serum collection can be a minimally invasive method from which antibodies can be used for many purposes: screening for viral diversity 40 , 42 , conducting epidemiological surveillance through serological assay 44 , performing comparative functional assays to better understand IgG homeostasis 45 , or for the development of poly- or monoclonal antibodies for targeted studies 46 . In addition, individual variation of IgG in bats and other mammals is influenced by a complex interplay of endogenous as well as exogenous factors, which shape both the baseline and dynamic levels of IgG. For example, females generally exhibit higher basal IgG levels and more robust adaptive immune responses compared to males and have higher levels during pregnancy 47 , 48 . Age-related changes also affect IgG levels, reflecting the processes of immunosenescence and shifting immune competence over the lifespan 25 , 48 . Genetic variation plays a role as well, as demonstrated by heritability studies in livestock and free-ranking populations 49 , 50 . Pathogen exposure, and other external challenges can further modulate IgG concentrations 51 , 52 , making IgG a sensitive indicator of an individual’s immune status and overall health. Monitoring absolute IgG levels and comparing them can thus provide critical insights into the impacts of environmental change, disease prevalence, and population health dynamics in natural systems. Materials and methods Bat capture and sampling Collection of sera samples from the Greater mouse-eared bat Myotis myotis and Common bent-wing bat ( Miniopterus schreibersii ) was performed in compliance with ethical guidelines and under the permit issued by the Bulgarian Biodiversity Act (No 927/04.04.2022). The bats were captured at the overground bat roost Perla 2 ( M. myotis ) and Devetashka Cave ( M. schreibersii ), Bulgaria in September 2024. The animals were captured using mist nets and specific measures were taken to reduce stress and avoid injury as described in 53 . To collect blood samples, each bat was gently restrained and 30-50 µl of blood was collected from the uropatagium vein. The blood was collected using a sterile 27-gauge needles. The blood samples of Rousettus aegyptiacus bats were obtained from a captive colony in the Copenhagen Zoo, Denmark, following euthanasia by trained veterinarians. The euthanasia was performed in line with management decisions in the zoo to ensure the best welfare of the captive colony 54 . The bats were manually restrained and anaesthetic induction took place with sevoflurane (Sevotek 1000 mg/g, Alvira, Barcelona, Spain) in oxygen via facemask or by intramuscular administration of ketamine (10 mg/kg, Ketador Vet 100 mg/ml, Salfarm, Kolding, Denmark) and medetomidine (0.05 mg/kg, Domitor Vet 1 mg/ml, Orion Pharma, Copenhagen, denmark).. Once a plane of surgical anaesthesia was reached, euthanasia was carried out by lethal injection of pentobarbitone (200 mg/kg, Euthanimal 400 mg/ml, Scanvet, Fredensborg, denmark) into the brachial vein. Blood was then collected rapidly by cardiac puncture using a sterile 23-gauge needle. Serum was obtained by pipetting the supernatant after centrifuging the whole blood at 7500 rpm for 15min. Total IgG from M. myotis, M. schreibersii, and R. aegyptiacus was purified by the Melon TM system (Thermo Fisher Scientific, Waltham, MA, Ref # 1859850) as recommended by the manufacturer. As control, human IgG was purified from sera of healthy blood donors (n=20), obtained under the ethical convention between INSERM with Etablissement Français du Sang #18/EFS/033. Depletion of IgG from the serum of R. aegyptiacus Protein G agarose slurry (Thermo Fisher Scientific, Ref # 20397), 0.2 ml, was transferred to a 0.5 ml centrifuge column. The Protein G agarose was washed three times with PBS by centrifugation for 1 min at 100 ´ g. A serum sample from R. aegyptiacus (50 µL) was first diluted (10 ´) in PBS to a final volume of 500 µL and then incubated with Protein G agarose for 30 min at 20 °C with continuous rotation. After washing with PBS (5´), 0.5 ml of 0.2 M glycine pH 2.4 was added to the settled gel and incubated for 1 min. The eluate was collected by centrifugation and immediately neutralised by adding 1/10 volume of the eluate to Tris 1M, pH 9. The efficacy of IgG depletion from bat serum was confirmed by ELISA (as described later). The concentration of purified IgG was determined by measuring the absorbance at 280 nm using a UV-Vis spectrophotometer Nanodrop (Thermo Fisher Scientific). To prepare sera with standard IgG concentration, the protein was added to IgG-depleted serum, resulting in a reference sample of 1/20 diluted bat serum containing 0.13 mg/ml IgG. Real-time interaction analyses Evaluation of the binding affinity of IgG to Protein G The interaction of purified human and bat IgG with immobilised protein G was assessed using a surface plasmon resonance-based technology - Biacore 2000 (Cytiva, Biacore, Uppsala, Sweden). Recombinant protein G (Merck, Darmstadt, Germany, Ref# 19459) was conjugated to CM5 sensor chips (Cytiva, Biacore, 29149604) using an amine coupling kit (Cytiva, Biacore, #BR100050). Briefly, Protein G was diluted to a final concentration of 10 μg/ml in 5 mM maleic acid, pH 4, and injected over pre-activated sensor surfaces with a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. To inactivate the activated carboxyl groups after protein conjugation, the sensor surfaces were treated with a 1M solution of ethanolamine.HCl for 4 min. The immobilisation density of protein G was approximately 100 resonance units (RU). All interaction analyses were performed in HBS-EP buffer. The buffer was run at a constant flow rate of 30 µl/min. To evaluate the interaction of human and bat IgG with protein G, IgG purified from a pool of human, M. myotis and R. aegyptiacus sera was serially diluted in the running buffer in the range 125 - 3.91 nM and injected over the sensor surface. M. schreibersii IgG was diluted in the range of 1000 to 31.25 nM. The association and dissociation phases of IgG from different species to Protein G were monitored for 4 and 5 minutes respectively. After each injection, the sensor chip surface was regenerated by contact (30 s) with a solution of 1.5M NaSCN. All interaction analyses were performed at 22°C. Real-time binding profiles were obtained after subtraction of the response generated by IgG on a control surface. The binding kinetics and affinity of the interaction were calculated using BIAevaluation software v. 4.1.1 (Cytiva, Biacore), applying global kinetics analyses with the Langmuir binding model. Estimation of the IgG concentration in the serum of R. aegyptiacus For these analyses, a commercially available sensor chip with high density immobilised protein G was used (Sensor Chip Protein G, Biacore Cytiva, Ref #29149316). All interactions were performed in HBS-EP buffer at a flow rate of 30 µl/min. Initially, IgG purified from R. aegyptiacus serum pool was injected for 2 min contact time at increasing concentrations: 0.625, 1.25, 2.5, 5 and 10 µg/ml. The resonance response 30 s after the end of each injection was recorded and used to construct a standard curve with linear regression analysis. To estimate the total IgG concentration in the serum sample, the R. aegyptiacus serum pool was injected at dilutions of 2000, 4000 and 8000 ´ for 2 min contact time. The resonance response values obtained 30 s after the end of the injections were used to calculate the IgG concentration. All interactions were performed at 22°C. The sensor surface was regenerated by injection of 0.5% sodium dodecyl sulphate solution for a contact time of 30 seconds. The evaluation of the IgG concentration was performed by substituting the values of the resonance response after injection of diluted serum in the linear regression fit equation obtained by the standard IgG concentrations. Quantification of IgG in R. aegyptiacus and M. myotis serum Surface immobilisation of bat sera and controls Polystyrene 384-well ELISA plates (Nunc TM Maxisorb, Thermo Fisher Scientific) were coated with individual R. aegyptiacus sera or pooled R. aegyptiacus and M. myotis sera. Sera were initially diluted 2000 ´ followed by serial twofold dilutions to 2048000 ´. Pooled human sera diluted in the same range and reference serum from R. aegyptiacus with final IgG concentrations in the range 5 - 0.0045 µg/ml (2-fold dilution step) were coated as controls. The diluted sera were incubated on the plate at 20 °C for 90 minutes. Each wall of the ELISA plate was incubated with 20 µl of sample. Blocking All wells of the ELISA plates were incubated with PBS containing 0.25% Tween 20. Incubate for 1 hour at 20 °C. Incubation volume: 100 µl/well. Detection of IgG Biotinylated Protein G (Pierce™ Recombinant Protein G, Biotinylated, Thermo Fischer Scientific; Ref # 29988) was diluted to 2 µg/ml in PBS containing 0.05% Tween 20 (T-PBS) and the plates were incubated at 20 °C for 1 hour. Incubation volume: 20 µl/well. After incubation, the plates were washed 5 ´ with 100 µl/well T-PBS and Avidin-HRP (eBioscience, Ref # 00-4100-94) diluted 250 ´ in T-PBS was added at 20 µl/well and incubated for 1 hour at 20 °C. The plates were washed again 5 ´ with 100 µl/well of T-PBS and substrate solution (OPD SigmaFASTTM, Merck) was added to each well at a volume of 40 µl/well. Absorbance was measured at 492 nm after stopping the reaction by adding 40 µl/well of 2N HCl and using a TECAN Infinite 200 PRO plate reader (Tecan, Männedorf, Switzerland). Data analysis Standard R. aegyptiacus serum with a defined concentration of IgG was used to construct a standard dose-response curve. The linear part of the sigmoidal curve (IgG concentration range 0.039063 - 0.3125 µg/ml) was used for linear regression fitting. The absorbance values of the tested samples from R. aegyptiacus obtained after dilution of the sera at 16000 and 32000 ´ were used to calculate the IgG concentration by substituting the values in the linear regression equation. The concentration of IgG in M. myotis was calculated using the same approach using standard human serum as a control (n=20) with an approximate concentration of IgG of 10 mg/ml. All analyses were performed using Microsoft® Excel version 16.95.1 (Microsoft Corporation, Redmond, WA) and GraphPad Prism version 10.4.1 (GraphPad Software, Dotmatics, Boston, MA). Declarations All research activities were conducted in compliance with ethical guidelines and under the permit for work with wild animals issued by the Bulgarian Biodiversity Act (No 927/04.04.2022). Competing interests The authors declare no competing interests. Author Contribution F.T. and J.D.D. conceived the study and planned experiments; F.T., M.F.B., S.A.T., N.T. and S.D. collected and curated samples; F.T., R.V.L., M.L., N.T. and J.D.D performed experiments; F.T., M.W., J.D.D. analyzed data; E.T., M.W., D.S. provided infrastructural or intellectual support in preparation of study and contributed to interpretation of the results; F.T. and J.D.D. wrote the manuscript. All authors contributed to editing the manuscript. Acknowledgement The experimental work was by INSERM. N.T. was re supported by fellowships from the French Institute Bulgaria (Institut français de Bulgarie). The fieldwork for this study was supported by the Bulgarian National Science Fund, project КП-06-Н51/9 “Caves as a reservoir for novel and reoccurring zoonoses — ecological monitoring and metagenomic analysis in real-time.” F.T. has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska- Curie grant agreement No 101034345. D.G.S. was funded by a Wellcome Trust Senior Research Fellowship (217221/Z/19/Z). The zookeepers and veterinary staff of Copenhagen Zoo are thanked for their assistance in operations surrounding the restraint and anaesthesia of the captive bats. We are grateful to Angel Ivanov, Maxim Kolev and Stela-Teodora Trendafilova, who assisted in the collection of the blood samples in Bulgaria. 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Immune profile predicts survival and reflects senescence in a small, long-lived mammal, the greater sac-winged bat (Saccopteryx bilineata). PLoS One 9 ,e108268 (2014). Becker, D.J. et al. Predictors and immunological correlates of sublethal mercury exposure in vampire bats. R Soc Open Sci 4 ,170073 (2017). Becker, D.J. et al. Livestock abundance predicts vampire bat demography, immune profiles and bacterial infection risk. Philos Trans R Soc Lond B Biol Sci 373 (2018). Schauer, U. et al. IgG subclass concentrations in certified reference material 470 and reference values for children and adults determined with the binding site reagents. Clin Chem 49 ,1924-1929 (2003). Khan, S.R. et al. Determinants and Reference Ranges of Serum Immunoglobulins in Middle-Aged and Elderly Individuals: a Population-Based Study. J Clin Immunol 41 ,1902-1914 (2021). Klein-Schneegans, A.S., Kuntz, L., Fonteneau, P. & Loor, F. Serum concentrations of IgM, IgG1, IgG2b, IgG3 and IgA in C57BL/6 mice and their congenics at the lpr (lymphoproliferation) locus. J Autoimmun 2 ,869-875 (1989). Engvall, E. The ELISA, enzyme-linked immunosorbent assay. Clin Chem 56 ,319-320 (2010). Guito, J.C. et al. Asymptomatic Infection of Marburg Virus Reservoir Bats Is Explained by a Strategy of Immunoprotective Disease Tolerance. Curr Biol 31 ,257-270 e255 (2021). Larson, P.A. et al. Genomic features of humoral immunity support tolerance model in Egyptian rousette bats. Cell Rep 35 ,109140 (2021). Levinger, R. et al. Single-cell and Spatial Transcriptomics Illuminate Bat Immunity and Barrier Tissue Evolution. Mol Biol Evol 42 (2025). Akerstrom, B., Brodin, T., Reis, K. & Bjorck, L. Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies. J Immunol 135 ,2589-2592 (1985). Toshkova, N. et al. Temperature sensitivity of bat antibodies links metabolic state of bats with antigen-recognition diversity. Nat Commun 15 ,5878 (2024). Schuck, P. Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of interactions between biological macromolecules. Annu Rev Biophys Biomol Struct 26 ,541-566 (1997). McCann, B. et al. A Review on Perception of Binding Kinetics in Affinity Biosensors: Challenges and Opportunities. ACS Omega 10 ,4197-4216 (2025). Ameh, V.O. et al. Serum Neutralization Profiles of Straw-Colored Fruit Bats (Eidolon helvum) in Makurdi (Nigeria), against Four Lineages of Lagos Bat Lyssavirus. Viruses 13 (2021). Ruhs, E.C. et al. Applications of VirScan to broad serological profiling of bat reservoirs for emerging zoonoses. Front Public Health 11 ,1212018 (2023). Crowley, D.E. et al. Bats generate lower affinity but higher diversity antibody responses than those of mice, but pathogen-binding capacity increases if protein is restricted in their diet. PLoS Biol 22 ,e3002800 (2024). Toshkova, N. et al. An integrative approach for profiling antibody responses in bats to human pathogens. EMI: Animal & Environment 1 (2025). Gonzalez, V. et al. Studying bats using a One Health lens: bridging the gap between bat virology and disease ecology. J Virol ,e0145324 (2024). Barr, J. et al. Detection of filovirus-reactive antibodies in Australian bat species. J Gen Virol 103 (2022). Toshkova, N., Zhelyazkova, V., Justesen, S. & Dimitrov, J.D. Conservative pattern of interaction of bat and human IgG antibodies with FcRn. Dev Comp Immunol 139 ,104579 (2023). Wynne, J.W. et al. Purification and characterisation of immunoglobulins from the Australian black flying fox (Pteropus alecto) using anti-fab affinity chromatography reveals the low abundance of IgA. PLoS One 8 ,e52930 (2013). Ruoss, S., Becker, N.I., Otto, M.S., Czirjak, G.A. & Encarnacao, J.A. Effect of sex and reproductive status on the immunity of the temperate bat Myotis daubentonii. Mamm Biol 94 ,120-126 (2019). Hagg, S. & Jylhava, J. Sex differences in biological aging with a focus on human studies. Elife 10 (2021). Sparks, A.M. et al. The genetic architecture of helminth-specific immune responses in a wild population of Soay sheep (Ovis aries). PLoS Genet 15 ,e1008461 (2019). Altvater-Hughes, T.E. et al. Concentration and heritability of immunoglobulin G and natural antibody immunoglobulin M in dairy and beef colostrum along with serum total protein in their calves. J Anim Sci 100 (2022). Flies, A.S., Mansfield, L.S., Flies, E.J., Grant, C.K. & Holekamp, K.E. Socioecological predictors of immune defences in wild spotted hyenas. Funct Ecol 30 ,1549-1557 (2016). Abolins, S. et al. The ecology of immune state in a wild mammal, Mus musculus domesticus. PLoS Biol 16 ,e2003538 (2018). Smith, C.S., De Jong, C.E. & Field, H.E. Sampling Small Quantities of Blood from Microbats. Acta Chiropterologica 12 ,255-258, 254 (2010). Bertelsen, M.F. 23 - Issues Surrounding Surplus Animals in Zoos. . In: Miller, R.E., Lamberski, N. & Calle, P.P. (eds). Fowler’s Zoo and Wild Animal Medicine Current Therapy, , vol. 9. W.B. Saunders: Philadelphia, Pennsylvania, USA, 2019, pp 134–136. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6811643","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":472843072,"identity":"3cf11d27-1000-42cf-a2db-acffdbf57cfa","order_by":0,"name":"Frédéric Touzalin","email":"","orcid":"","institution":"University College Dublin","correspondingAuthor":false,"prefix":"","firstName":"Frédéric","middleName":"","lastName":"Touzalin","suffix":""},{"id":472843073,"identity":"550b07b8-a775-4bdc-9a85-bf8c01958334","order_by":1,"name":"Mads Frost Bertelsen","email":"","orcid":"","institution":"Center for Zoo and Wild Animal Health, Copenhagen Zoo","correspondingAuthor":false,"prefix":"","firstName":"Mads","middleName":"Frost","lastName":"Bertelsen","suffix":""},{"id":472843074,"identity":"39037af7-5c10-4863-a247-fa339c9fcad3","order_by":2,"name":"Stamatios Alan Tahas","email":"","orcid":"","institution":"Center for Zoo and Wild Animal Health, Copenhagen Zoo","correspondingAuthor":false,"prefix":"","firstName":"Stamatios","middleName":"Alan","lastName":"Tahas","suffix":""},{"id":472843075,"identity":"ead431c8-ff2f-473a-aba4-a2ad094823f7","order_by":3,"name":"Nia Toshkova","email":"","orcid":"","institution":"National Museum of Natural History, Bulgarian Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Nia","middleName":"","lastName":"Toshkova","suffix":""},{"id":472843076,"identity":"c8177f3a-6c1d-4a7c-8314-67e978108aa5","order_by":4,"name":"Stanimira Deleva","email":"","orcid":"","institution":"National Museum of Natural History, Bulgarian Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Stanimira","middleName":"","lastName":"Deleva","suffix":""},{"id":472843077,"identity":"bbec24eb-593e-49c8-bec1-f9add8fd572c","order_by":5,"name":"Maxime Lecerf","email":"","orcid":"","institution":"Centre de Recherche des Cordeliers, INSERM, CNRS, Sorbonne Université, Université Paris Cité","correspondingAuthor":false,"prefix":"","firstName":"Maxime","middleName":"","lastName":"Lecerf","suffix":""},{"id":472843078,"identity":"943f8546-31b6-44d1-a010-3177519b9f11","order_by":6,"name":"Robin V. Lacombe","email":"","orcid":"","institution":"Centre de Recherche des Cordeliers, INSERM, CNRS, Sorbonne Université, Université Paris Cité","correspondingAuthor":false,"prefix":"","firstName":"Robin","middleName":"V.","lastName":"Lacombe","suffix":""},{"id":472843081,"identity":"41b9e29a-f3fb-4cfd-bf8a-54245e3ad764","order_by":7,"name":"Maya Weinberg","email":"","orcid":"","institution":"University of Essex","correspondingAuthor":false,"prefix":"","firstName":"Maya","middleName":"","lastName":"Weinberg","suffix":""},{"id":472843083,"identity":"9e768662-bab9-4231-9aa0-6f04c5c24a9c","order_by":8,"name":"Emma Teeling","email":"","orcid":"","institution":"University College Dublin","correspondingAuthor":false,"prefix":"","firstName":"Emma","middleName":"","lastName":"Teeling","suffix":""},{"id":472843084,"identity":"98d2506a-d95e-4c8e-b96f-99ae1fa5f7a5","order_by":9,"name":"Daniel Streicker","email":"","orcid":"","institution":"University of Glasgow","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Streicker","suffix":""},{"id":472843088,"identity":"e7dd5e8c-c9b1-44e1-8968-cdb30702fadd","order_by":10,"name":"Jordan D. Dimitrov","email":"data:image/png;base64,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","orcid":"","institution":"Centre de Recherche des Cordeliers, INSERM, CNRS, Sorbonne Université, Université Paris Cité","correspondingAuthor":true,"prefix":"","firstName":"Jordan","middleName":"D.","lastName":"Dimitrov","suffix":""}],"badges":[],"createdAt":"2025-06-03 13:08:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6811643/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6811643/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84956374,"identity":"9dd2aa7a-556d-4c7d-bcea-02ef0a9a813d","added_by":"auto","created_at":"2025-06-19 08:19:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":295039,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction of IgG with Protein G. Real-time interaction profiles were obtained by SPR-based assay for the binding of IgG from human, \u003cem\u003eR. aegyptiacus\u003c/em\u003e, and \u003cem\u003eM. schreibersii\u003c/em\u003eto protein G immobilized on a sensor chip. Purified IgG was injected at two-fold dilutions ranging from 125 to 3.91 nM for human and \u003cem\u003eR. aegyptiacus\u003c/em\u003e, and from 1000 to 31.25 nM for \u003cem\u003eM. schreibersii\u003c/em\u003e. All measurements were performed at a temperature of 22 °C. The real-time binding profiles to the subtraction of reactivity to a control surface are depicted by black lines. The global kinetic fits using the Langmuir model are depicted by red lines.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6811643/v1/41219d3766d6e15c717d2dad.png"},{"id":84956178,"identity":"8c1858df-538f-460e-93b9-f7973297c652","added_by":"auto","created_at":"2025-06-19 08:11:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":418277,"visible":true,"origin":"","legend":"\u003cp\u003eProcedure for quantification of total IgG antibodies in bats. (A) The schematic representation illustrates the experimental approach for measurement of total IgG in the sera of bats. The scheme was created in https://BioRender.com (B) Binding curves obtained following immobilisation of individual sera samples or serum pools from \u003cem\u003eR. aegyptiacus\u003c/em\u003e diluted by a two-fold step in the range of 2000 – 2048000-folds and detected by biotinylated protein G (purple lines). The reactivity to human serum pool (two curves are obtained from two different ELISA plates) was also depicted (green lines). For each dilution, the mean of the optical density (OD) values from two technical replicates was utilised for curve construction. (C) The binding curve was obtained after immobilization of serum from \u003cem\u003eR. aegyptiacus\u003c/em\u003ereconstituted with autologous IgG. Each symbol represents the mean value of the mean OD with standard deviation (SD) from four technical replicates on two different ELISA plates. The final concentration of IgG presented in the sera was in the range of 5 - 0.0045 µg/ml (2-fold dilution step). The blue rectangle highlights the concentration range that was used for the linear regression fit (lower panel). The equation describing the linear regression fit is also shown. (D) The total serum concentration of IgG (mg/ml) was estimated for individual samples of juveniles and young adults of \u003cem\u003eR. aegyptiacus\u003c/em\u003e bats. Each circle in the diagram represents the total IgG concentration, calculated as the average of the concentrations obtained after dilution of sera at 16,000 and 32,000-fold. The calculations were performed by using the equation shown on panel C. The bar represents mean concentration of IgG with standard deviation (SD) obtained from all individuals in the groups. (E) The total serum concentration of IgG (mg/ml) was estimated for serum pools from samples of juveniles and young adults of \u003cem\u003eR. aegyptiacus\u003c/em\u003ebats. Each circle represents the IgG concentration obtained after performing the calculation after dilution of the serum pools to 16,000 and 32,000-fold. \u003cdel\u003e. \u003c/del\u003eThe bar represents the mean concentration of IgG. (F) Real-time interaction profiles for the binding of IgG from \u003cem\u003eM. myotis\u003c/em\u003e to protein G immobilized on a sensor chip. Purified bat IgG was injected at two-fold dilutions ranging from 125 to 3.91 nM. All measurements were performed at a temperature of 22 °C. The real-time binding profiles are depicted by black lines. The global kinetic fits using the Langmuir model are depicted by red lines. (G) The binding curves presented here were obtained following the immobilisation of serum pools from \u003cem\u003eM. myotis\u003c/em\u003e (orange line) and human (green line) diluted by a factor of two in the range of 2000 – 2048000-fold. For each dilution, the mean OD from two technical replicates of bat sera pool was used to construct the curves. The curve representing the binding of protein G to human serum pool was obtained after the two curves depicted on panel B were averaged.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6811643/v1/c63f5eb0dd3772f882f707ad.png"},{"id":84956180,"identity":"dd0f0f04-67e9-441d-8d34-088c176bb818","added_by":"auto","created_at":"2025-06-19 08:11:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":164242,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of total IgG in the serum of \u003cem\u003eR. aegyptiacus\u003c/em\u003e by surface plasmon resonance-based assay. (A) Titration of increasing concentrations (0.625, 1.25, 2.5, 5 and 10 µg/ml) of purified IgG from \u003cem\u003eR. aegyptiacus\u003c/em\u003e to immobilized high-density protein G. The red dashed line indicates the time point from the real-time binding curve at which the resonance response was used to construct the standard curve. (B) The linear regression fit of the sensor response versus the concentration of injected IgG. (C) The real-time binding curves that were generated following the injection of a serum pool of \u003cem\u003eR. aegyptiacus\u003c/em\u003e young adults, which had been diluted to 2000-, 4000-, and 8000-fold over the sensor with immobilised protein G. (D) A comparison of the total concentration of IgG in the serum pool of \u003cem\u003eR. aegyptiacus\u003c/em\u003e young adults, as determined by ELISA (as depicted in Figure 2) and surface plasmon resonance. The bars indicate the mean IgG concentration. Each circle represents the calculation of IgG levels following different serum dilutions in SPR-based and ELISA assays.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6811643/v1/1632c0f78026539005a0c29b.png"},{"id":96453327,"identity":"ac237ab9-7d0e-4c8b-bba6-4cbafc2c32b2","added_by":"auto","created_at":"2025-11-21 09:59:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1350947,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6811643/v1/07ab9549-c686-4db3-99e1-ccd4a4715f40.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Quantification of Absolute IgG Concentration in Bat Sera","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBats harbour several virulent pathogens without appearing to manifest immunopathological symptoms, exhibit atypically prolonged lifespans, and suffer a low prevalence of metabolic diseases \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. These characteristics of bats likely originate from a series of evolutionary adaptations that enabled active flight \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Understanding how bats tolerate pathogens may have important implications for human health \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Consequently, numerous studies have recently been conducted to elucidate the molecular mechanisms underlying the distinctive characteristics of the immune response in various bat species \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Much of this work has focused on the innate immune response, with a particular focus on the immune defence pathways triggered by viral infections. Collectively, these studies have revealed several evolutionary adaptations that result in dampened innate immune responses in bats \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAntibodies are the hallmark of the adaptive immune responses, and they play an essential role in pathogen neutralization \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Despite their central role in immune defence, there is a limited understanding of antibody responses in bats. Some studies have suggested that bats mount inefficient transient antibody responses \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Conversely, other studies have demonstrated highly specific neutralising antibody responses in experimental infection settings \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Antibodies from the IgG class are central components of T cell-dependent immune responses. The evaluation of total IgG levels in blood can provide important indications about the extent of the specific adaptive immune responses, as well as the influence of various physiological, ecological, and immunological factors on these responses. The estimation of absolute values of circulating IgG may allow for a comparison of the global extent of the IgG responses of bats with those of other species (e.g., humans). Furthermore, the universal approach for estimating of absolute IgG concentration may contribute to a comparative understanding of how life history or ecological differences among different species of bats lead to different immune profiles and tolerance capacity. However, to the best of our knowledge, there is no simple, quantitative method for determining absolute IgG concentrations in bat blood. This may be due to the lack of specific antibodies for detecting IgG in different species of bats. Previous studies have only presented arbitrary IgG levels in the blood of bats \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. These levels were shown as variations in optical density generated by colorimetric assays, rather than as exact protein concentrations, as is usually done for humans \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and experimental mice \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, a novel method was proposed for the straightforward quantification of the absolute IgG levels in serum samples of bats. The proposed method was based on an immune sorbent assay, a technique that has been successfully utilised in various research fields \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. As a proof of concept, the assay was applied to serum samples from the Egyptian fruit bat (\u003cem\u003eRousettus aegyptiacus, familly Pteropodidae\u003c/em\u003e) and Bent-wing bat (\u003cem\u003eMiniopterus schreibersii\u003c/em\u003e, family Miniopteridae).. This bat species is the subject of extensive research and has contributed to significant advancements in our understanding of the bat immune system \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The proposed technique utilises protein G as an IgG detection component. Critical assay parameters are detailed, with attention given to adjustments required for species-specific implementation. Thus, as a proof of concept, we also estimated the concentration of a pool of sera from Greater mouse-eared bat (\u003cem\u003eMyotis myotis\u003c/em\u003e, family Vespertilionidae). The obtained data revealed that bats have relatively high levels of IgG from early stages of their life. This finding indicates that the bat species in question possesses the capacity to mount effective T cell-mediated antibody responses that are comparable to other mammals and humans.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe primary objective of this study is to develop a method for the quantification of the exact concentration of serum IgG that can be applied to different bat species. Given that Protein G has been demonstrated to bind to the constant region of IgG from a range of mammalian species with a high binding affinity, and that previously it has been shown to bind to IgG from certain bat species \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, it was selected as the IgG detection reagent. The interaction of IgG with protein G was characterised by purifying total IgG from pooled human serum or serum pools of two bat species, captive \u003cem\u003eR. aegyptiacus\u003c/em\u003e and wild \u003cem\u003eMiniopterus schreibersii\u003c/em\u003e (family Miniopteridae), by ion exchange chromatography (Melon\u0026trade; system). Subsequently, the binding affinity of IgG from different species to surface-immobilized protein G was evaluated using a surface plasmon resonance-based assay. The real-time interaction profiles indicated that human and \u003cem\u003eR. aegyptiacus\u003c/em\u003e IgG bind to protein G. In contrast, \u003cem\u003eM. schreibersii\u003c/em\u003e IgG did not show any binding to the protein, even when high concentration of IgG was injected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The binding affinity of the human polyclonal IgG sample was further evaluated, and the equilibrium dissociation constant (K\u003csub\u003eD\u003c/sub\u003e) was determined to be 5.5 (\u0026plusmn; 0.28 SD) nM. The affinity of IgG purified from \u003cem\u003eR. aegyptiacus\u003c/em\u003e sera for protein G was lower, with an average value of 40.4 (\u0026plusmn; 0.83 SD) nM. This reduced affinity appears to result from decreased complex stability, as reflected by an increased dissociation rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These findings underscore the necessity for consideration of bat species-specific profiles when developing assays to assess total IgG concentration in bat sera.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we tested an experimental strategy based on an immunosorbent assay for assessment of total IgG concentration in bat sera (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). This approach consists of direct immobilization of serially diluted (from 2000 to 2080000 folds) sera onto polystyrene ELISA plates and quantification of the IgG antibodies by using biotinylated protein G (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The advantage of direct immobilization of sera is that this approach does not necessitate the use of specific capture antibodies. The viability of the approach is supported by the fact that IgG is among the most abundant proteins in serum (the second most abundant in humans).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA crucial element in conducting such an assay is the use of an IgG standard to enable the accurate quantification of the IgG concentration. If the binding affinity of protein G for human and bat IgG is identical, a pool of human sera from healthy donors could serve as an appropriate control; however, IgG from \u003cem\u003eR. aegyptiacus\u003c/em\u003e exhibits approximately 8-fold lower affinity for protein G compared to human IgG (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To account for this discrepancy, we propose the implementation of a species-specific standard. Total IgG was purified from pooled \u003cem\u003eR. aegyptiacus\u003c/em\u003e serum using a protein G-Sepharose matrix, and the concentration of purified IgG was determined by UV-Vis spectroscopy. The depleted bat serum was then reconstituted with known amounts of IgG to generate a calibration standard (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). This bat-specific standard was subsequently used in all further experiments to estimate total IgG concentrations in bat sera.\u003c/p\u003e \u003cp\u003eThe reactivity of a fixed concentration (2 \u0026micro;g/ml) of biotinylated protein G to immobilized IgG present in serially diluted sera was measured separately for 32 \u003cem\u003eR. aegyptiacus\u003c/em\u003e individuals (13 juveniles and 19 young adults) as well as in pooled sera prepared from the same samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The resulting data demonstrated that protein G binding was proportional to serum concentration across the dilution series. The binding curves for individual sera samples exhibited heterogeneity in terms of the level of protein G binding, as evidenced by the presence of more than one order difference in their inflex points. In certain cases, the binding did not reach saturation within the tested dilution range (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The interaction of protein G with IgG present in pools of human sera exhibited considerably higher reactivity, as would be expected based on the higher affinity of human IgG to protein G (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The overall reactivity of pools consisting of mixed sera from juvenile bats or young adults to protein G did not differ significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eTo quantify the absolute IgG concentrations, present in bat samples, the standard bat serum was utilised, which had been reconstituted with a defined concentration of IgG from \u003cem\u003eR. aegyptiacus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Four points from the linear part of the binding curve were used for linear regression analyses and determination of the constants of the equation describing the fit. These values were then used to calculate the IgG concentration in bat sera. Two serum dilutions (16,000 and 32,000 \u0026times;) were selected as reference points on the basis that they fell within the linear part of the binding curves across all tested samples (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The obtained results show individual heterogeneity in IgG in sera of \u003cem\u003eR. aegyptiacus\u003c/em\u003e in both juvenile and young adult individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The mean IgG concentrations in the sera of juvenile and young adult bats were 4.85 (\u0026plusmn;\u0026thinsp;3.6 SD) and 6.4 (\u0026plusmn;\u0026thinsp;3.2 SD) mg/ml, respectively. The concentration of IgG in these two age groups did not differ significantly and pooled sera had similar IgG concentrations (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eTo further validate the utility of the proposed method for measuring IgG levels in other bat species, we assessed its application using serum samples from an unrelated bat species, \u003cem\u003eMyotis myotis\u003c/em\u003e (family Vespertilionidae). The binding affinity of polyclonal IgG from \u003cem\u003eM. myotis\u003c/em\u003e to immobilized protein G, K\u003csub\u003eD\u003c/sub\u003e value of 6.8 (\u0026plusmn;\u0026thinsp;0.6 SD) nM at 22\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), was comparable to the one established for human IgG, K\u003csub\u003eD\u003c/sub\u003e of 5.5 (\u0026plusmn;\u0026thinsp;0.28 SD) nM. In contrast to the samples from \u003cem\u003eR. aegyptiacus\u003c/em\u003e, the comparable binding behaviour of \u003cem\u003eM. myotis\u003c/em\u003e and human IgG allows human serum pools to be used effectively as a standard for estimating absolute IgG concentrations in \u003cem\u003eM. myotis\u003c/em\u003e sera. A comparison of the pooled sera from human (n\u0026thinsp;=\u0026thinsp;20 individuals) and from \u003cem\u003eM. myotis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;20 individuals) revealed nearly identical interaction profiles with protein G (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Utilising human sera as a standard, with a reference concentration of 10 mg/ml of IgG, it was determined that the total serum concentration of IgG in \u003cem\u003eM. myotis\u003c/em\u003e was approximately 7.9 (\u0026plusmn;\u0026thinsp;0.14 SD) mg/ml. The higher absolute concentration of IgG in \u003cem\u003eM. myotis\u003c/em\u003e as compared to the IgG in sera from \u003cem\u003eR. aegyptiacus\u003c/em\u003e can be explained by the fact that the sera of the former species were collected from adult individuals whereas a large proportion of the latter animals were subadults. The findings of this study collectively imply that to estimate absolute IgG concentrations in bats, it is necessary to first define the appropriate species-specific standard. The standard in question can be either reconstituted sera with autologous IgG or human sera pools.\u003c/p\u003e \u003cp\u003eFinally, to further validate the proposed approach for determining total IgG concentration in bat sera, an orthogonal technique was utilised. The changes in the resonance signal in the surface plasmon resonance-based technology are proportional to the amount of protein accumulated on the sensor's surface \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. To obtain a standard curve, purified IgG from \u003cem\u003eR. aegyptiacus\u003c/em\u003e was titrated on a sensor chip with immobilized protein G at high density. As anticipated, the IgG produced a clear concentration-dependent dose-response signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Subsequently, samples of the serum pool of \u003cem\u003eR. aegyptiacus\u003c/em\u003e diluted to 2000-, 4000-, and 8000-fold were allowed to interact with immobilized protein G. The concentration-dependent signal obtained (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) was then utilised to calculate the concentration of IgG in the serum pool using the standard curve. The estimated concentration of IgG in bat serum through this method was found to be well aligned with the one obtained by ELISA-based assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, the present study details a straightforward procedure that enables the quantification of exact IgG concentrations in bat serum, with the potential applicability to other mammalian species. The assay was validated by using an orthogonal method to confirm its accuracy. The procedure consists of the following interconnected steps:\u003c/p\u003e \u003cp\u003e1) Direct coating of serially diluted serum samples and appropriate serum standards onto ELISA plates.\u003c/p\u003e \u003cp\u003e2) Incubation with a fixed concentration of biotinylated protein G.\u003c/p\u003e \u003cp\u003e3) Detection of bound protein G by streptavidin-conjugated reporter enzyme (horseradish peroxidase).\u003c/p\u003e \u003cp\u003e4) Data analysis.\u003c/p\u003e \u003cp\u003eThis assay applies to any species in which protein G can bind to IgG. A key advantage of the method is its adaptability, permitting preliminary steps to be completed in field settings where conventional laboratory equipment is not readily accessible. Step 1 can be carried out in the field where the plates can also be dried and sealed, and steps 2 to 4 can be carried out back in the laboratory. This approach eliminates the need for freezing or long-term storage of liquid serum samples, thereby addressing significant logistical challenges associated with research in remote or resource-limited locations.\u003c/p\u003e \u003cp\u003eA critical aspect of the assay is the use of an appropriate IgG standard. In instances where the affinity of the IgG from the bat species under study is similar to that of human IgG, human serum can be used as a reference. However, if the affinity differs substantially, the bat serum should first be depleted of IgG. The IgG concentration should then be accurately measured, and the serum reconstituted with a defined amount of IgG. Therefore, to define the IgG standard for a given species, preliminary measurements of affinity are required. This may be regarded as a potential hindrance to the scalability of the assay across species, given that the IgG affinity for protein G remains unknown for the majority of bats. Nonetheless, the estimation of the affinity of intramolecular interactions is currently a trivial task. The advent of novel biosensor technologies has enabled the estimation of affinities of intermolecular interaction in high-throughput manner \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrated that in certain cases, such as with \u003cem\u003eM. schreibersii\u003c/em\u003e IgG samples, the current methodological approach is not applicable due to the absence of detectable interaction between its IgG and protein G. Nevertheless, the underlying concept of this approach remains valid, involving the direct coating of serum dilution solutions on ELISA plates and the utilisation of species-specific control. This can be achieved by employing alternative IgG-specific reagents, such as commercially available bat-specific anti-IgG antibodies.\u003c/p\u003e \u003cp\u003eThe findings of this study indicate that two distinct bat species can produce relatively high levels of IgG antibodies. In the case of \u003cem\u003eR. aegyptiacus\u003c/em\u003e, IgG levels were found to be comparatively high from early stages of life (1\u0026ndash;2 years old). These findings underscore the significance of T cell-dependent adaptive immune responses in bats in their immune defence mechanisms. Those results corroborate with previous research demonstrating that bats have the capacity to produce proficient antibodies. Thus, studies have demonstrated that bats are indeed able to mount a neutralising antibody response against viruses such as influenza (H18N11), or various lyssaviruses \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Moreover, bats generated antibody repertoires with high antigen-binding diversity \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Alongside the relatively high concentrations of IgG observed in the bats examined here, these findings suggest that while the bat\u0026rsquo;s innate immune system is often highlighted as the primary mechanism balancing pathogen control and immune tolerance \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, the adaptive immune response also plays a significant role in their overall strategy for combating infection.\u003c/p\u003e \u003cp\u003eResearch on bat IgG and more generally on bat humoral immunity, is critical for advancing our understanding of epidemiology, immunology, and zoonotic disease management \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Serum collection can be a minimally invasive method from which antibodies can be used for many purposes: screening for viral diversity \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, conducting epidemiological surveillance through serological assay \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, performing comparative functional assays to better understand IgG homeostasis \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, or for the development of poly- or monoclonal antibodies for targeted studies \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition, individual variation of IgG in bats and other mammals is influenced by a complex interplay of endogenous as well as exogenous factors, which shape both the baseline and dynamic levels of IgG. For example, females generally exhibit higher basal IgG levels and more robust adaptive immune responses compared to males and have higher levels during pregnancy \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Age-related changes also affect IgG levels, reflecting the processes of immunosenescence and shifting immune competence over the lifespan \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Genetic variation plays a role as well, as demonstrated by heritability studies in livestock and free-ranking populations \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Pathogen exposure, and other external challenges can further modulate IgG concentrations \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, making IgG a sensitive indicator of an individual\u0026rsquo;s immune status and overall health. Monitoring absolute IgG levels and comparing them can thus provide critical insights into the impacts of environmental change, disease prevalence, and population health dynamics in natural systems.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cem\u003eBat capture and sampling\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCollection of sera samples from the Greater mouse-eared bat \u003cem\u003eMyotis myotis\u003c/em\u003e and Common bent-wing bat (\u003cem\u003eMiniopterus schreibersii\u003c/em\u003e) was performed in compliance with ethical guidelines and under the permit issued by the Bulgarian Biodiversity Act (No 927/04.04.2022). The bats were captured at the overground bat roost Perla 2 (\u003cem\u003eM. myotis\u003c/em\u003e) and Devetashka Cave (\u003cem\u003eM. schreibersii\u003c/em\u003e), Bulgaria in September 2024. The animals were captured using mist nets and specific measures were taken to reduce stress and avoid injury as described in\u003csup\u003e53\u003c/sup\u003e. To collect blood\u0026nbsp;samples, each bat was gently restrained and 30-50 \u0026micro;l of blood was collected from the uropatagium vein. The blood was collected using a sterile 27-gauge needles.\u003c/p\u003e\n\u003cp\u003eThe blood samples of \u003cem\u003eRousettus aegyptiacus\u0026nbsp;\u003c/em\u003ebats were obtained from a captive colony in the Copenhagen Zoo, Denmark, following euthanasia by trained veterinarians. The euthanasia was performed in line with management decisions in the zoo to ensure the best welfare of the captive colony\u003csup\u003e54\u003c/sup\u003e. The bats were manually restrained and anaesthetic induction took place with sevoflurane (Sevotek 1000 mg/g, Alvira, Barcelona, Spain) in oxygen via facemask or by intramuscular administration of ketamine (10 mg/kg, Ketador Vet 100 mg/ml, Salfarm, Kolding, Denmark) and medetomidine (0.05 mg/kg, Domitor Vet 1 mg/ml, Orion Pharma, Copenhagen, denmark).. Once a plane of surgical anaesthesia was reached, euthanasia was carried out by lethal injection of pentobarbitone (200 mg/kg, Euthanimal 400 mg/ml, Scanvet, Fredensborg, denmark) into the brachial vein. Blood was then collected rapidly by cardiac puncture using a sterile 23-gauge needle.\u003c/p\u003e\n\u003cp\u003eSerum was obtained by pipetting the supernatant after centrifuging the whole blood at 7500 rpm for 15min. Total IgG from \u003cem\u003eM. myotis, M. schreibersii,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eR. aegyptiacus\u0026nbsp;\u003c/em\u003ewas purified by the Melon\u003csup\u003eTM\u003c/sup\u003e system\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Thermo Fisher Scientific, Waltham, MA, Ref # 1859850) as recommended by the manufacturer. As control, human IgG was purified from sera of healthy blood donors (n=20), obtained under the ethical convention between INSERM with Etablissement Fran\u0026ccedil;ais du Sang #18/EFS/033.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDepletion of IgG from the serum of R. aegyptiacus\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eProtein G agarose slurry (Thermo Fisher Scientific, Ref # 20397), 0.2 ml, was transferred to a 0.5 ml centrifuge column. The Protein G agarose was washed three times with PBS by centrifugation for 1 min at 100 \u0026acute; g. A serum sample from \u003cem\u003eR. aegyptiacus\u003c/em\u003e (50 \u0026micro;L) was first diluted (10 \u0026acute;) in PBS to a final volume of 500 \u0026micro;L and then incubated with Protein G agarose for 30 min at 20 \u0026deg;C with continuous rotation. After washing with PBS (5\u0026acute;), 0.5 ml of 0.2 M glycine pH 2.4 was added to the settled gel and incubated for 1 min. The eluate was collected by centrifugation and immediately neutralised by adding 1/10 volume of the eluate to Tris 1M, pH 9. The efficacy of IgG depletion from bat serum was confirmed by ELISA (as described later).\u003c/p\u003e\n\u003cp\u003eThe concentration of purified IgG was determined by measuring the absorbance at 280 nm using a UV-Vis spectrophotometer Nanodrop (Thermo Fisher Scientific). To prepare sera with standard IgG concentration, the protein was added to IgG-depleted serum, resulting in a reference sample of 1/20 diluted bat serum containing 0.13 mg/ml IgG.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eReal-time interaction analyses\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003e\u003cu\u003eEvaluation of the binding affinity of IgG to Protein G\u003c/u\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe interaction of purified human and bat IgG with immobilised protein G was assessed using a surface plasmon resonance-based technology - Biacore 2000 (Cytiva, Biacore, Uppsala, Sweden). Recombinant protein G (Merck, Darmstadt, Germany, Ref# 19459) was conjugated to CM5 sensor chips (Cytiva, Biacore, 29149604) using an amine coupling kit (Cytiva, Biacore, #BR100050). Briefly, Protein G was diluted to a final concentration of 10 \u0026mu;g/ml in 5 mM maleic acid, pH 4, and injected over pre-activated sensor surfaces with a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. To inactivate the activated carboxyl groups after protein conjugation, the sensor surfaces were treated with a 1M solution of ethanolamine.HCl for 4 min. The immobilisation density of protein G was approximately 100 resonance units (RU).\u003c/p\u003e\n\u003cp\u003eAll interaction analyses were performed in HBS-EP buffer. The buffer was run at a constant flow rate of 30 \u0026micro;l/min. To evaluate the interaction of human and bat IgG with protein G, IgG purified from a pool of human, \u003cem\u003eM. myotis\u003c/em\u003e and \u003cem\u003eR. aegyptiacus\u003c/em\u003e sera was serially diluted in the running buffer in the range 125 - 3.91 nM and injected over the sensor surface. \u003cem\u003eM. schreibersii\u003c/em\u003e IgG was diluted in the range of 1000 to 31.25 nM. The association and dissociation phases of IgG from different species to Protein G were monitored for 4 and 5 minutes respectively. After each injection, the sensor chip surface was regenerated by contact (30 s) with a solution of 1.5M NaSCN. All interaction analyses were performed at 22\u0026deg;C. Real-time binding profiles were obtained after subtraction of the response generated by IgG on a control surface. The binding kinetics and affinity of the interaction were calculated using BIAevaluation software v. 4.1.1 (Cytiva, Biacore), applying global kinetics analyses with the Langmuir binding model.\u003c/p\u003e\n\u003col start=\"2\"\u003e\n \u003cli\u003e\u003cu\u003eEstimation of the IgG concentration in the serum of \u003cem\u003eR. aegyptiacus\u003c/em\u003e\u0026nbsp;\u003c/u\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eFor these analyses, a commercially available sensor chip with high density immobilised protein G was used (Sensor Chip Protein G, Biacore Cytiva, Ref #29149316). All interactions were performed in HBS-EP buffer at a flow rate of 30 \u0026micro;l/min. Initially, IgG purified from \u003cem\u003eR. aegyptiacus\u003c/em\u003e serum pool was injected for 2 min contact time at increasing concentrations: 0.625, 1.25, 2.5, 5 and 10 \u0026micro;g/ml. The resonance response 30 s after the end of each injection was recorded and used to construct a standard curve with linear regression analysis. To estimate the total IgG concentration in the serum sample, the \u003cem\u003eR. aegyptiacus\u003c/em\u003e serum pool was injected at dilutions of 2000, 4000 and 8000\u0026nbsp;\u0026acute;\u0026nbsp;for 2 min contact time. The resonance response values obtained 30 s after the end of the injections were used to calculate the IgG concentration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll interactions were performed at 22\u0026deg;C. The sensor surface was regenerated by injection of 0.5% sodium dodecyl sulphate solution for a contact time of 30 seconds.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe evaluation of the IgG concentration was performed by substituting the values of the resonance response after injection of diluted serum in the linear regression fit equation obtained by the standard IgG concentrations.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eQuantification of IgG in R. aegyptiacus and M. myotis\u003c/em\u003e \u003cem\u003eserum\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eSurface immobilisation of bat sera and controls\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003ePolystyrene 384-well ELISA plates (Nunc\u003csup\u003eTM\u003c/sup\u003e Maxisorb, Thermo Fisher Scientific) were coated with individual R. aegyptiacus sera or pooled \u003cem\u003eR. aegyptiacus\u003c/em\u003e and \u003cem\u003eM. myotis\u003c/em\u003e sera. Sera were initially diluted 2000\u0026nbsp;\u0026acute;\u0026nbsp;followed by serial twofold dilutions to 2048000\u0026nbsp;\u0026acute;. Pooled human sera diluted in the same range and reference serum from R. aegyptiacus with final IgG concentrations in the range 5 - 0.0045 \u0026micro;g/ml (2-fold dilution step) were coated as controls. The diluted sera were incubated on the plate at 20 \u0026deg;C for 90 minutes. Each wall of the ELISA plate was incubated with 20 \u0026micro;l of sample.\u0026nbsp;\u003c/p\u003e\n\u003col start=\"2\"\u003e\n \u003cli\u003eBlocking\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eAll wells of the ELISA plates were incubated with PBS containing 0.25% Tween 20. Incubate for 1 hour at 20 \u0026deg;C. Incubation volume: 100 \u0026micro;l/well.\u003c/p\u003e\n\u003col start=\"3\"\u003e\n \u003cli\u003eDetection of IgG\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eBiotinylated Protein G (Pierce\u0026trade; Recombinant Protein G, Biotinylated, Thermo Fischer Scientific; Ref # 29988) was diluted to 2 \u0026micro;g/ml in PBS containing 0.05% Tween 20 (T-PBS) and the plates were incubated at 20 \u0026deg;C for 1 hour. Incubation volume: 20 \u0026micro;l/well. After incubation, the plates were washed 5\u0026nbsp;\u0026acute;\u0026nbsp;with 100 \u0026micro;l/well T-PBS and Avidin-HRP (eBioscience, Ref # 00-4100-94) diluted 250\u0026nbsp;\u0026acute;\u0026nbsp;in T-PBS was added at 20 \u0026micro;l/well and incubated for 1 hour at 20 \u0026deg;C. The plates were washed again 5\u0026nbsp;\u0026acute;\u0026nbsp;with 100 \u0026micro;l/well of T-PBS and substrate solution (OPD SigmaFASTTM, Merck) was added to each well at a volume of 40 \u0026micro;l/well.\u003c/p\u003e\n\u003cp\u003eAbsorbance was measured at 492 nm after stopping the reaction by adding 40 \u0026micro;l/well of 2N HCl and using a TECAN Infinite 200 PRO plate reader (Tecan, M\u0026auml;nnedorf, Switzerland).\u003c/p\u003e\n\u003col start=\"4\"\u003e\n \u003cli\u003eData analysis\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eStandard \u003cem\u003eR. aegyptiacus\u003c/em\u003e serum with a defined concentration of IgG was used to construct a standard dose-response curve. The linear part of the sigmoidal curve (IgG concentration range 0.039063 - 0.3125 \u0026micro;g/ml) was used for linear regression fitting. The absorbance values of the tested samples from \u003cem\u003eR. aegyptiacus\u003c/em\u003e obtained after dilution of the sera at 16000 and 32000\u0026nbsp;\u0026acute;\u0026nbsp;were used to calculate the IgG concentration by substituting the values in the linear regression equation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe concentration of IgG in \u003cem\u003eM. myotis\u003c/em\u003e was calculated using the same approach using standard human serum as a control (n=20) with an approximate concentration of IgG of 10 mg/ml. All analyses were performed using Microsoft\u0026reg; Excel version 16.95.1 (Microsoft Corporation, Redmond, WA) and GraphPad Prism version 10.4.1 (GraphPad Software, Dotmatics, Boston, MA).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cspan\u003eAll research activities were conducted in compliance with ethical guidelines and under the permit for work with wild animals issued by the Bulgarian Biodiversity Act (No 927/04.04.2022).\u003c/span\u003e\u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eF.T. and J.D.D. conceived the study and planned experiments; F.T., M.F.B., S.A.T., N.T. and S.D. collected and curated samples; F.T., R.V.L., M.L., N.T. and J.D.D performed experiments; F.T., M.W., J.D.D. analyzed data; E.T., M.W., D.S. provided infrastructural or intellectual support in preparation of study and contributed to interpretation of the results; F.T. and J.D.D. wrote the manuscript. All authors contributed to editing the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe experimental work was by INSERM. N.T. was re supported by fellowships from the French Institute Bulgaria (Institut fran\u0026ccedil;ais de Bulgarie). The fieldwork for this study was supported by the Bulgarian National Science Fund, project КП-06-Н51/9 \u0026ldquo;Caves as a reservoir for novel and reoccurring zoonoses \u0026mdash; ecological monitoring and metagenomic analysis in real-time.\u0026rdquo; F.T. has received funding from the European Union\u0026rsquo;s Horizon 2020 research and innovation programme under the Marie Skłodowska- Curie grant agreement No 101034345. D.G.S. was funded by a Wellcome Trust Senior Research Fellowship (217221/Z/19/Z). The zookeepers and veterinary staff of Copenhagen Zoo are thanked for their assistance in operations surrounding the restraint and anaesthesia of the captive bats. We are grateful to Angel Ivanov, Maxim Kolev and Stela-Teodora Trendafilova, who assisted in the collection of the blood samples in Bulgaria.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003ehe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWilkinson, G.S. \u0026amp; Adams, D.M. Recurrent evolution of extreme longevity in bats. \u003cem\u003eBiol Lett\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e,20180860 (2019).\u003c/li\u003e\n\u003cli\u003eGorbunova, V., Seluanov, A. \u0026amp; Kennedy, B.K. The World Goes Bats: Living Longer and Tolerating Viruses. \u003cem\u003eCell Metab\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e,31-43 (2020).\u003c/li\u003e\n\u003cli\u003eIrving, A.T., Ahn, M., Goh, G., Anderson, D.E. \u0026amp; Wang, L.F. 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Saunders: Philadelphia, Pennsylvania, USA, 2019, pp 134\u0026ndash;136.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bats, Antibodies, IgG concentration, ELISA, immune assays","lastPublishedDoi":"10.21203/rs.3.rs-6811643/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6811643/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBats are endowed with a remarkable capacity to withstand important pathogens through evolutionary adaptations in their immune systems. Antibodies are essential component of the adaptive immune response and serve as a crucial biomarker, indicating both present and past pathogen infections, as well as the overall physiological state of the organism. The main type of antibody found in the blood of mammals is IgG. It is produced as a result of specific T-cell-dependent antibody responses. Consequently, monitoring IgG in wild animals can yield valuable insights into pathogen dynamics and host responses. Currently, there is no simple technique for measuring absolute IgG concentration that can be generalized for different species of bats. The present study proposes a methodology to quantify total IgG levels in bats. The approach is based on an immunosorbent assay and employs only protein G as a detecting reagent for IgG. This method has the potential to be applied to diverse bat species, as well as other mammals. As a proof of concept, we present a detailed procedure to quantify serum IgG in Egyptian fruit bats (\u003cem\u003eRousettus aegyptiacus\u003c/em\u003e). The estimated concentration of IgG was found to be relatively high (5-6 mg/ml), highlighting the role of specific antibody responses in the immune defence of bats. To validate the method, we compare the results to an alternative approach based on SPR biosensor technology. Furthermore, data pertaining to the estimation of IgG levels in a different bat species, namely \u003cem\u003eMyotis myotis\u003c/em\u003e, is presented. This simple and effective technique offers a valuable tool for advancing our understanding of immune function in bats and potentially other wild mammals, contributing to broader efforts in wildlife immunology and disease ecology.\u003c/p\u003e","manuscriptTitle":"Quantification of Absolute IgG Concentration in Bat Sera","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-19 08:11:06","doi":"10.21203/rs.3.rs-6811643/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"52ac56b2-4417-4bb3-8208-ddc198569617","owner":[],"postedDate":"June 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50214197,"name":"Biological sciences/Immunology"},{"id":50214198,"name":"Biological sciences/Immunology/Adaptive immunity"},{"id":50214199,"name":"Biological sciences/Zoology/Animal physiology"}],"tags":[],"updatedAt":"2025-12-15T14:23:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-19 08:11:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6811643","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6811643","identity":"rs-6811643","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}