Establishment of a blocking ELISA for assessing neutralizing antibody levels against porcine epidemic diarrhea virus

Establishment of a blocking ELISA for assessing neutralizing antibody levels against porcine epidemic diarrhea virus | 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 Research Article Establishment of a blocking ELISA for assessing neutralizing antibody levels against porcine epidemic diarrhea virus Peiyang Ding, Shuoqi Dong, Linyi Bai, Shulei Li, Xiao Liu, Jingming Zhou, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9143664/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract A highly specific blocking ELISA was developed for serological diagnosis of porcine epidemic diarrhea (PED) using monoclonal antibodies (mAbs) against the S1 protein of PEDV. The S1 protein was expressed in HEK293F cells and used to immunize BALB/c mice. Through hybridoma screening, a high-affinity monoclonal antibody designated 3E3 was obtained. Indirect ELISA, Western blot, and immunofluorescence assays confirmed its significant neutralizing activity, with a neutralization titer of 1:2¹⁰. A blocking ELISA was subsequently established using HRP-labeled 3E3. The assay showed no cross-reactivity with other porcine viruses (African swine fever virus (ASFV), porcine circovirus 2 (PCV2), porcine deltacoronavirus (PDCoV), porcine reproductive and respiratory syndrome virus (PRRSV), Porcine rotavirus (PoRV) and porcine transmissible gastroenteritis virus (TGEV) ) and exhibited good reproducibility, with intra- and inter-batch coefficients of variation below 10%. Compared with the virus neutralization test, the blocking ELISA demonstrated higher sensitivity. Results from the two methods showed a strong positive correlation, with a positive agreement of 88.63%, a negative agreement of 88.89%, and an overall agreement of 88.8%. In conclusion, the established blocking ELISA is specific, sensitive, and reliable, suitable for serological monitoring of PEDV and evaluation of vaccine-induced immunity. Monoclonal antibody Porcine epidemic diarrhea virus Neutralizing antibody Blocking ELISA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Porcine epidemic diarrhea virus (PEDV), a member of the Alphacoronavirus genus within the Coronaviridae family, is a single-stranded positive-sense RNA virus and the causative agent of porcine epidemic diarrhea (PED) (Chen et al. 2013 ; Lee and Lee 2014 ). Infection is characterized by diarrhea, vomiting, dehydration, and high mortality, particularly in suckling piglets, where fatalities can reach 100% (Debouck and Pensaert 1980 ). Since its first recognition in the 1970s, PEDV has caused repeated outbreaks worldwide, leading to substantial economic losses in the swine industry (Pensaert and de Bouck 1978 ; Song and Park 2012 ). The PEDV genome is approximately 28 kb in length and encodes four major structural proteins: spike (S), envelope, membrane, and nucleocapsid (Yang et al., 2014 ; Kocherhans et al., 2001 ). The S protein mediates viral entry into host cells and is a key target for neutralizing antibodies (Liu et al. 2015 ). It is cleaved into S1 (1–735 aa) and S2 (736–1383 aa) subunits (Deng et al. 2016 ). The S1 subunit contains major antigenic determinants and neutralizing epitopes and is crucial for inducing protective immunity by blocking virus–receptor interactions (Chang et al. 2019 ). Monoclonal antibodies against S1 are therefore valuable tools for developing specific diagnostic assays and studying viral entry (Makadiya et al. 2016 ; Oh et al. 2014 ). The continuous evolution of PEDV, co-circulation of multiple genotypes, and potential serological cross-reactivity with other porcine coronaviruses such as TGEV and PDCoV challenge the performance of current diagnostic methods (Ma et al. 2024 ). In PED control, assessing vaccine immunogenicity is essential. Protective efficacy correlates with levels of virus-neutralizing antibodies, making neutralizing antibody titer a key indicator of vaccine performance (Lee 2015 ). However, the virus neutralization test (VNT), considered the gold standard, is labor-intensive, time-consuming, and low-throughput, unsuitable for large-scale serosurveillance ( Mariën et al. 2021 ). Although ELISAs based on whole virus or structural proteins (nucleocapsid or membrane) are widely used, they often fail to differentiate neutralizing from non-neutralizing antibodies and may cross-react with antibodies against related coronaviruses, compromising specificity (Li et al. 2016 ). Blocking ELISA based on monoclonal antibodies offers a promising alternative. This format uses competition between serum antibodies and a defined neutralizing mAb for binding to a specific epitope ( Sayee et al. 2024 ). The degree of signal inhibition correlates with the level of functional antibodies in serum. Studies have shown good agreement between blocking ELISA and VNT results, enabling efficient evaluation of neutralizing antibody levels (Zhao et al. 2025 ). Therefore, developing a blocking ELISA targeting the PEDV S1 protein with a well-characterized neutralizing mAb would support accurate vaccine evaluation, swine herd immunity monitoring, and epidemiological studies (Rasmussen et al. 2018 ). In this study, we expressed the S1 protein in HEK293F cells, generated murine monoclonal antibodies, and selected a high-potency neutralizing mAb (3E3). Using HRP-labeled 3E3, we established a blocking ELISA that is sensitive, specific, reproducible, and suitable for high-throughput detection of PEDV antibodies in clinical samples. This assay provides a practical tool for vaccine immunogenicity assessment and lays groundwork for further studies on PEDV neutralization mechanisms and therapeutic antibody development. 2. Materials and Methods 2.1 Materials and Reagents The PEDV strain China/Hubei/2016 (GenBank KY928065.1), HEK293F cells, Vero cells, and SP2/0 myeloma cells were maintained in the Molecular Immunology Laboratory of Zhengzhou University. A recombinant plasmid carrying the S1 gene was stored at − 80°C. 293T-II medium was purchased from SinoBiological, and linear polyethylenimine (PEI) transfection reagent (MW 40000) was from Yisheng Biotechnology. Female BALB/c mice (6–8 weeks old) were obtained from Huaxing Animal Experimental Center. 2.2 Expression and Purification of PEDV-S1 Protein The S1 protein was expressed in HEK293F cells. Cells in logarithmic growth phase with viability > 90% were seeded at 2×10⁶ cells/mL in fresh medium and cultured at 37°C, 5% CO₂ with shaking (150–175 rpm). At the time of transfection, cell density was adjusted to 3×10⁶ cells/mL. Transfection was performed using SMM293-TII according to the manufacturer’s protocol. Culture supernatant was harvested 5–7 days post-transfection by centrifugation at 8,000 ×g for 30 min. S1 expression was confirmed by SDS-PAGE and Western blot. The protein was purified by Ni-affinity chromatography using equilibration buffer (20 mM Tris-HCl, 150 mM NaCl, pH 8.0) and eluted with a linear imidazole gradient (20–500 mM) in the same buffer. Eluted fractions containing S1 were pooled, dialyzed, and concentrated using a 30-kDa ultrafiltration device. Purity and identity were verified by SDS-PAGE and Western blot. 2.3 Production of monoclonal antibodies against PEDV-S1 protein Four female BALB/c mice (6–8 weeks old) were immunized subcutaneously with 20 µg purified S1 protein emulsified in Freund’s complete adjuvant. Boosters with the same antigen dose emulsified in Freund’s incomplete adjuvant were given on days 14 and 28. Serum antibody titers were measured by indirect ELISA one week after the third immunization. The mouse with the highest titer received an intraperitoneal injection of 50 µg antigen without adjuvant. Three to five days later, splenocytes were fused with SP2/0 myeloma cells using PEG 1500. Hybridomas secreting anti-S1 antibodies were selected by indirect ELISA and subcloned three times by limiting dilution. For ascites production, Freund's incomplete adjuvant-primed BALB/c mice were injected intraperitoneally with 1×10⁷ hybridoma cells. Ascitic fluid was collected 7–10 days later, clarified by centrifugation, and purified using a Protein A affinity column. 2.4 Immunofluorescence Assay (IFA) The reactivity of mAbs with PEDV was tested by IFA. Vero cells grown in 96-well plates to 80% confluence were infected with PEDV (10⁴·⁶ TCID₅₀/mL) in DMEM containing 6 µg/mL trypsin. After 24 h incubation at 37°C, 5% CO₂, cells showing syncytia were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100, and blocked with 5% skim milk in PBST overnight at 4°C. Cells were incubated with anti-S1 mAb or mouse pre-immune serum (control) at 37°C for 1 h, followed by FITC-conjugated goat anti-mouse IgG (1:200) for 1 h. Nuclei were stained with DAPI. Images were captured using a fluorescence microscope. 2.5 Virus Neutralization Test (VNT) Vero cells were seeded in 96-well plates one day before the assay. Mouse ascitic fluid was serially diluted twofold in serum-free DMEM starting at 1:5. An equal volume of PEDV containing 200 TCID₅₀ was added to each well. After 1 h incubation at 37°C, the virus–antibody mixture was transferred to Vero cell monolayers and adsorbed for 1 h. Cells were washed and maintained in DMEM with 2% FBS for 48 h. Infection was assessed by IFA as above. The neutralization titer was defined as the highest dilution that inhibited > 90% of infection. 2.6 Development of the Blocking ELISA Purified S1 protein was used as the coating antigen and HRP-conjugated mAb 3E3 as the competing antibody. Checkerboard titration was performed to determine optimal antigen coating concentration and HRP-3E3 dilution. S1 protein was diluted in carbonate buffer (2, 1, 0.5, 0.25 µg/mL), added to 96-well plates (100 µL/well), and incubated overnight at 4°C. Plates were blocked with 5% skim milk in PBST for 2 h at 37°C. Test sera (diluted 1:20 in PBST) were added (100 µL/well) and incubated for 1 h at 37°C. After washing, HRP-3E3 (optimal dilution in PBST) was added and incubated for 1 h. TMB substrate was added, and after 15 min the reaction was stopped with 2 M H₂SO₄. Absorbance was measured at 450 nm. The percent inhibition (PI) was calculated as: PI = [(OD₍negative serum₎ − OD₍test serum₎) / OD₍negative serum₎] × 100%. Parameters optimized by univariate analysis included: coating conditions (4°C/12 h, 4°C/16 h, 37°C/2 h, 37°C/4 h), blocking solution (5% skim milk, 5% BSA, 10% FBS) and blocking time (4°C/12 h, 4°C/16 h, 37°C/1 h, 37°C/2 h), serum dilution (1:2 to 1:80), serum incubation time (60, 90, 120 min), HRP-3E3 incubation time (30, 60, 90 min), and TMB development time (5, 10, 15, 20 min). 2.7 Cut-off Value Determination Based on the optimization described above, the final reaction conditions and procedural steps for the blocking ELISA were established. To define the assay cut-off, 80 sera confirmed as negative for PEDV by both virus neutralization test and a commercial antibody detection kit were analyzed using the optimized protocol. The mean percent inhibition (PI) and standard deviation (SD) were calculated from the OD₄₅₀ values. The cut-off values were determined as follows: Cut-off₁ = mean PI + 2×SD; Cut-off₂ = mean PI + 3×SD. The interpretation criteria were set as follows: samples with a PI < Cut-off₁ were considered negative; samples with a PI ≥ Cut-off₂ were considered positive. Samples yielding a PI between Cut-off₁ and Cut-off₂ were classified as suspect and were retested. If the repeat result remained within the suspect range, the sample was judged as negative. 2.8 Evaluation of specificity, repeatability, and sensitivity of blocking ELISA To assess the specificity of the assay, the optimized protocol was used to test serum samples positive for ASFV, PCV2, PDCoV, PRRSV, PoRV, and TGEV. PEDV-positive serum served as the control, and the blocking rate was calculated for each sample. For repeatability evaluation, eight serum samples (three negative and five positive) were analyzed in triplicate within the same run and across different runs. The intra- and inter-batch coefficients of variation were calculated from the blocking rates to determine assay reproducibility. Sensitivity was evaluated by testing fourteen PEDV-neutralizing antibody-positive sera, which were first identified by virus neutralization test and then serially diluted two-fold for measurement of blocking activity using the established ELISA. 2.9 Compliance Test To assess agreement between the blocking ELISA and the virus neutralization test, 107 pig serum samples were tested in parallel using both methods, and their concordance was calculated. 3. Results 3.1 Identification of PEDV S1 protein The PEDV S1 recombinant protein was expressed in HEK293F cells and purified by nickel-affinity chromatography. As shown in Figure 1A, SDS-PAGE analysis revealed a characteristic diffuse band at approximately 100 kDa, corresponding to the expected molecular weight of the S1 protein. Specificity was further confirmed by Western blot. Using either a mouse anti-His tag monoclonal antibody (Fig. 1B) or PEDV-positive swine serum (Fig. 1C) as the primary antibody, followed by an HRP-conjugated goat anti-mouse secondary antibody, a single immunoreactive band was observed at the same position. The banding pattern was consistent with the SDS-PAGE result, confirming the identity and integrity of the His-tagged S1 recombinant protein. These data demonstrate that the nickel-column purification yielded a high-purity S1 protein with correct antigenicity, suitable for monoclonal antibody production and subsequent development of the blocking ELISA. 3.2 Characterization of Monoclonal Antibodies Against PEDV S1 Mice were immunized with purified S1 protein. Following cell fusion and three rounds of subcloning, five hybridoma lines secreting monoclonal antibodies (mAbs) specific for PEDV S1 were established (Fig. 2A). Immunofluorescence assay (IFA) confirmed that all five mAbs bound to native PEDV in infected Vero cells (Fig. 2B). Western blot analysis further demonstrated specific recognition of the denatured S1 protein, indicating that the mAbs target linear epitopes (Fig. 2C). Virus neutralization testing identified mAb 3E3 as possessing the highest neutralizing activity, with a titer of 1:2¹⁰, and it effectively inhibited PEDV-induced cytopathic effect (Fig. 2D). Consequently, 3E3 was selected for subsequent assay development. The antibody was purified by Protein A affinity chromatography. SDS-PAGE under reducing conditions showed the expected heavy- and light-chain bands at approximately 50 kDa and 25 kDa, respectively, confirming successful purification (Fig. 2E). The purified 3E3 was then conjugated to horseradish peroxidase (HRP) using the sodium periodate method. The conjugate retained high antigen-binding activity, yielding an indirect ELISA titer of 1:256,000 or greater (Fig. 2F), confirming that the labeling process did not compromise immunoreactivity. 3.3 Development and Optimization of the Blocking ELISA Checkerboard titration identified an optimal antigen coating concentration of 1 µg/mL for the S1 protein and a working dilution of 1:2000 for the HRP‑conjugated 3E3 antibody, which yielded the highest blocking rate for positive serum (Table 1). Although coating at 4 °C for 16 h gave the highest absolute blocking rate, the corresponding negative control OD values were too low, reducing the assay’s ability to discriminate weakly positive samples (Fig. 3A). Therefore, following the principle of maximizing the signal‑to‑noise ratio and resolution, coating at 37 °C for 4 h was selected as the standard condition, as it provided a high blocking rate while maintaining robust differentiation. The use of 5% BSA as the blocking solution with a 2‑h incubation at 37 °C resulted in the highest blocking efficiency (Fig. 3B and 3C). For sample and conjugate incubation, a combination of 60 min for swine serum and 90 min for HRP‑3E3 was found to be optimal (Fig. 3D). A serum dilution of 1:20 produced the maximal blocking rate (Fig. 3E), and a TMB substrate development time of 15 min was chosen based on the plateau of the signal response (Fig. 3F). 3.4 Determination of the Assay Cut‑off The optimized blocking ELISA was tested on 80 PEDV‑negative swine sera. The mean percent inhibition (PI) was 10.36% with a standard deviation (SD) of 8.06% (Fig. 4). Based on these results, the following cut‑off values were established: Cut‑off₁ = 26.48% (mean + 2×SD) Cut‑off₂ = 34.54% (mean + 3×SD) Samples with PI < Cut‑off₁ were considered negative, and those with PI ≥ Cut‑off₂ were classified as positive. Samples yielding PI values between Cut‑off₁ and Cut‑off₂ were regarded as suspect and were retested. If the repeat result remained within the suspect range, the sample was ultimately judged as negative. 3.5 Specificity, Sensitivity, and Repeatability of the Blocking ELISA The specificity of the assay was evaluated using sera positive for ASFV, PCV2, PDCoV, PRRSV, PoRV and TGEV. All heterologous sera yielded blocking rates below the positive cut‑off, confirming no cross‑reactivity (Fig. 5). Sensitivity was assessed using 14 PEDV‑positive sera previously titrated by virus neutralization test. The blocking ELISA titers correlated strongly with the neutralization titers, and the ELISA displayed a higher analytical sensitivity, detecting antibodies at greater dilutions (Table 2). Repeatability was determined by testing eight sera (three negative, five positive) in triplicate within the same run and across three independent runs. The maximum intra‑batch and inter‑batch coefficients of variation were 5.46% and 7.67%, respectively, both well below 10% (Table 3), demonstrating excellent assay reproducibility. 3.6 Consistency Between Blocking ELISA and Virus Neutralization Test The consistency between the blocking ELISA and the virus neutralization test (VNT) was evaluated using 107 clinical pig serum samples tested in parallel by both methods. The blocking ELISA identified 46 positive and 61 negative samples, while the VNT identified 44 positive and 63 negative samples. Using the VNT as the reference standard, the positive agreement rate of the blocking ELISA was 88.63% (39/44), the negative agreement rate was 88.89% (56/63), and the overall agreement rate was 88.8% (95/107) (Table 4). These results demonstrate a high level of concordance between the two assays, indicating that the blocking ELISA reliably reflects the serum neutralizing antibody level. 4. Discussion Neutralizing antibodies are a well-established correlate of protection for many viral diseases, serving as a primary indicator of vaccine efficacy(Gilbert et al., 2021). These antibodies function by binding specifically to viral surface proteins, thereby blocking attachment and entry into host cells(Adams et al., 2023). Consequently, serum neutralizing antibody titers provide a direct measure of an individual’s ability to resist infection. In the context of PEDV, the level of circulating neutralizing antibodies in sows is closely linked to the passive immunity transferred to piglets via colostrum(Gerber et al., 2014; Li et al., 2017). This relationship underscores the importance of accurate serological monitoring for predicting neonatal protection and managing herd health. Studies have shown that high post-vaccination neutralizing antibody titers correlate with reduced clinical signs, milder intestinal pathology, and lower viral shedding following challenge(Lin et al., 2018). These findings reinforce the central role of neutralizing antibodies in evaluating vaccine performance(Lang et al., 2024). Traditional virus neutralization tests, while considered the gold standard, are labor-intensive, time-consuming, and require biosafety containment, limiting their use in large-scale surveillance(Tao et al., 2022). To address this, we developed a blocking ELISA based on a recombinant S1 protein and a neutralizing monoclonal antibody, 3E3. The assay is designed to detect antibodies that compete with 3E3 for binding to a key epitope on the S1 subunit, thereby offering a functional measure of virus-neutralizing activity. Our results demonstrate that the blocking ELISA exhibits high sensitivity, specificity, and reproducibility. It showed no cross-reactivity with antibodies against other common porcine pathogens, including ASFV, PCV2, PDCoV, PRRSV, PoRV，and TGEV. Furthermore, the assay displayed strong agreement with the virus neutralization test, supporting its reliability as a surrogate method for detecting PEDV-specific neutralizing antibodies. The established ELISA provides a practical tool for rapid, high-throughput screening of swine sera. It can be applied in various settings, such as monitoring vaccine-induced immunity in breeding herds, evaluating sow colostrum quality, and conducting epidemiological surveys. Regular testing could help identify gaps in herd immunity and guide timely interventions(Tesfagaber et al., 2024). A limitation of this study is the use of a single monoclonal antibody directed against a specific epitope. Given the ongoing evolution of PEDV and the emergence of variant strains, future work should assess the assay’s performance against diverse viral genotypes(Yang et al., 2025). Incorporating additional monoclonal antibodies targeting conserved neutralizing epitopes could further enhance the breadth and robustness of the assay(Li et al., 2023). In conclusion, the blocking ELISA described here offers a reliable and efficient alternative to traditional neutralization tests for measuring PEDV-specific antibodies. With further validation against contemporary field strains, this method could become a valuable asset in PED control and prevention programs. 5. Conclusion This study established a blocking ELISA for the detection of PEDV-specific neutralizing antibodies. The assay employs recombinant S1 protein as the solid-phase antigen and HRP-conjugated neutralizing monoclonal antibody 3E3 as the competitor. Validation results confirmed the high specificity of the assay, with no observed cross-reactivity against antibodies to other major swine viruses. The method also demonstrated good sensitivity, reproducibility, and a strong correlation with the conventional virus neutralization test. This blocking ELISA provides a reliable, rapid, and high-throughput tool for PEDV serology. It is suited for applications such as monitoring vaccine-induced immunity in swine herds and assessing maternal antibody levels in sows, which is critical for evaluating passive immune protection in suckling piglets. While the developed ELISA shows excellent performance for current diagnostic needs, its reactivity against emerging PEDV variants should be continually evaluated to maintain its utility. In summary, this assay represents a practical and valuable tool to support PED control programs and vaccine efficacy evaluation. Declarations Funding : The study was supported by the Regional Innovation and Development Joint Fund of the National Natural Science Foundation of China (Grant No. U24A20448), the Major Science and Technology Projects in Henan Province (Grant No. 241110310200), the Major Research Program of Longhu Laboratory of Advanced Immunology (Grant No. LHLab_ZD20230012), and the Key R&D and Promotion Projects in Henan Province of China (Grant No. 252102111011). Declaration of competing interest: The authors declare no conflicts of interest. CRediT authorship contribution statement Peiyang Ding : Conceptualization, Writing-review and editing, Investigation, Supervision, Methodology, Resources. Shuoqi Dong : Writing-original draft, Formal analysis, Visualization, Validation, Data curation. Linyi Bai: Investigation, Supervision. Shulei Li : Investigation, Supervision. Xiao Liu : Investigation, Supervision. Jingming Zhou : Investigation, Supervision. Aiping Wang : Project administration, Investigation, Supervision, Funding acquisition. Data availability The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Ethical approval for mouse experiments All animal experiments were approved by the Ethics Committee of Henan Longhu Laboratory of Advanced Immunology(Approval No. LHLab-HNZD-2023003) and conducted in accordance with the guidelines for the care and use of laboratory animals. 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A competitive ELISA based on nanobodies for the detection of serum neutralizing antibodies against porcine epidemic diarrhea virus. Animal Diseases, 5, 7. https://doi.org/10.1186/s44149-025-00161-2 Tables Table 1 Determination of optimal antigen coating concentration and antibody dilution Antigen coating concentration （ ug/ml ） Dilutionof HRP-3E3 monoclonal antibody 1 ： 2000 1 ： 4000 1 ： 8000 1 ： 16000 1 ： 32000 1 ： 64000 0.25 Blocking rate（PI） 69.82% 56.83% 10.67% 17.92% 53.80% 40.67% 0.5 Blocking rate（PI） 83.19% 76.30% 48.10% 10.63% 48.31% 46.06% 1 Blocking rate（PI） 90.37% 86.25% 86.06% 67.53% 51.44% 62.58% 2 Blocking rate（PI） 88.99% 87.64% 83.54% 83.38% 63.94% 42.23% Table 2 Sensitivity of Blocking ELISA Serum Neutralizing antibody titer Blocking ELISA 1 4 10 2 8 20 3 8 20 4 32 80 5 32 80 6 64 160 7 128 320 8 128 320 9 256 640 10 256 640 11 512 640 12 512 640 13 1024 1280 14 1024 1280 Table 3 Repeatability of Blocking ELISA Serum Intra batch repetition (PI/%) Inter batch repetition (PI/%) X SD CV/% X SD CV/% a 92.38 0.004 0.45 92.72% 0.004 0.40 b 82.20 0.009 1.06 82.58% 0.005 0.58 c 96.41 0.004 0.42 96.75% 0.004 0.37 d 90.44 0.005 0.51 90.08% 0.003 0.35 e 53.87 0.011 2.04 53.69% 0.028 5.26 f 30.26 0.009 3.06 28.82% 0.015 5.26 g 32.05 0.010 3.12 34.79% 0.027 7.67 h 12.71 0.007 5.46 13.11% 0.005 3.57 Table 4 Compliance rate between the blocking ELISA and the neutralization test Detection method Serum neutralization test Positive Negative Total Blocking ELISA Positive 39 7 46 Negative 5 56 61 Total 44 63 107 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 31 Mar, 2026 Editor assigned by journal 31 Mar, 2026 Submission checks completed at journal 31 Mar, 2026 First submitted to journal 16 Mar, 2026 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-9143664","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":615232041,"identity":"850635a2-8ae1-45b8-a9ec-fafa4bf5e760","order_by":0,"name":"Peiyang Ding","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Peiyang","middleName":"","lastName":"Ding","suffix":""},{"id":615232044,"identity":"202fc66b-7ada-452b-8c0e-0c2d010d474a","order_by":1,"name":"Shuoqi Dong","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Shuoqi","middleName":"","lastName":"Dong","suffix":""},{"id":615232045,"identity":"12e195bb-46b3-433b-a9bf-816fdaf1fb40","order_by":2,"name":"Linyi Bai","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Linyi","middleName":"","lastName":"Bai","suffix":""},{"id":615232047,"identity":"47e14c94-6610-45cd-a6b3-999c7d3b75b1","order_by":3,"name":"Shulei Li","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Shulei","middleName":"","lastName":"Li","suffix":""},{"id":615232048,"identity":"fada01ad-b2bb-45a2-843d-b4ac4eb4f52f","order_by":4,"name":"Xiao Liu","email":"","orcid":"","institution":"Longhu Laboratory of Advanced Immunology, Zhengzhou, 450046, China.","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Liu","suffix":""},{"id":615232049,"identity":"73f82e08-0399-4050-bb5b-99589a79f7a3","order_by":5,"name":"Jingming Zhou","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Jingming","middleName":"","lastName":"Zhou","suffix":""},{"id":615232050,"identity":"d8510547-fab9-4d91-88dc-bebc383aff79","order_by":6,"name":"Aiping Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYDACZjBpw8PP3kCaljQZyZ4DpNl12MbghgORas3ZeQ+/+Nl2nofhBgPjh485RGixbOZLs+xtu83DOLuBWXLmNiK0GBzmMTPg3Xabh1nmABszL7FaDP9uO8fDJpFAvBbjx7zbDvDwEK3FspnHjFn2XzKPBM/BZuL8Ys5/xvjjmzN29vbHmw9++EiUwxgY2CQgTMYGItRDtDB/IE7pKBgFo2AUjFgAAM9eMffAi8rcAAAAAElFTkSuQmCC","orcid":"","institution":"Zhengzhou University","correspondingAuthor":true,"prefix":"","firstName":"Aiping","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-03-17 03:54:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9143664/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9143664/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106915832,"identity":"1a7c5695-5411-485d-946a-0315f8864a85","added_by":"auto","created_at":"2026-04-14 18:02:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":162629,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of PEDV-S1 recombinant protein SDS-PAGE validation of S1 protein, where M is the protein marker, 1 is the 293f supernatant before purification, and 2 is the purified and concentrated S1 protein. B. Western blot validation of S1 protein (His monoclonal antibody detection), M is the protein marker, 1: S1 protein; 2: PCGS3 empty load C. Western blot validation (PEDV positive blood test), M: marker; 1: S1 protein; 2: PCGS3 unloaded methods.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9143664/v1/5b1f16b409634a3dfa2840f9.png"},{"id":106960786,"identity":"d8de8d8a-9b6c-4c92-8df5-57a40df5ef2e","added_by":"auto","created_at":"2026-04-15 09:23:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":866998,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of anti-PEDV-S1 monoclonal antibody A. B. Western blot was used to detect the reactivity of monoclonal antibodies with S1 protein, where M is the marker and 1 is the monoclonal antibody. C. Immunofluorescence identification of monoclonal antibodies and PEDV, with PC positive control being mouse multi-antibody serum and NC negative control being Vero cell control. D. The virus neutralization experiment showed that the neutralization potency of 11E4 was 1:2\u003csup\u003e3\u003c/sup\u003e, 8H4 was 1:2\u003csup\u003e4\u003c/sup\u003e, and 3E3 was 1:2\u003csup\u003e10\u003c/sup\u003e. PC was used as the virus control, and NC was used as the cell control. E. Purification of monoclonal antibody 3E3 M: Marker is 1, pre-purified ascites is 2, and purified monoclonal antibody F is 3. The titer of HRP-3E3 was determined, and the ELISA titer could reach 1:256000.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9143664/v1/7e05987dbda4e2a14c6cbb69.png"},{"id":106915836,"identity":"2e87bf49-ef7f-4e2c-bc55-f7d2b19b9203","added_by":"auto","created_at":"2026-04-14 18:02:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":400562,"visible":true,"origin":"","legend":"\u003cp\u003eEstablishment and optimization of blocking ELISA. A. Optimization of antigen coating time B. Optimization of blocking time C. Selection of blocking solution D. Optimization of serum incubation time and HRP-3E3 incubation time E. Selection of serum dilution F. Optimization of color development time.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9143664/v1/7ef2ee5d36215f17594380ec.png"},{"id":106915834,"identity":"58a01d1a-97ea-44cc-a2cf-8bbcee4b301e","added_by":"auto","created_at":"2026-04-14 18:02:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135904,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of the critical value for blocking ELISA. 80 PEDV-negative samples were tested using an optimized detection method. Calculate the mean and standard deviation (SD) of the blocking rate, and The cut-off values were determined as follows: Cut-off₁ = mean PI + 2×SD; Cut-off₂ = mean PI + 3×SD.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9143664/v1/dd990169341c5e2645f4c19e.png"},{"id":106961828,"identity":"dc5dac3b-a381-4c04-b65b-fc3f90bef473","added_by":"auto","created_at":"2026-04-15 09:27:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":78674,"visible":true,"origin":"","legend":"\u003cp\u003eBlocking the specificity of ELISA. Use this blocking ELISA method to detect the blocking rate of ASFV, PCV2, PDCoV, PRRSV, PoRV and TGEV positive pig serum.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9143664/v1/43ba1096c185019d94960f08.png"},{"id":106963482,"identity":"d824ec3d-88ed-4feb-8429-759203959f84","added_by":"auto","created_at":"2026-04-15 09:44:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2735179,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9143664/v1/cf9fd941-2b14-43cb-ad50-0cbb3428f850.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Establishment of a blocking ELISA for assessing neutralizing antibody levels against porcine epidemic diarrhea virus","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePorcine epidemic diarrhea virus (PEDV), a member of the Alphacoronavirus genus within the Coronaviridae family, is a single-stranded positive-sense RNA virus and the causative agent of porcine epidemic diarrhea (PED) (Chen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lee and Lee \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Infection is characterized by diarrhea, vomiting, dehydration, and high mortality, particularly in suckling piglets, where fatalities can reach 100% (Debouck and Pensaert \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). Since its first recognition in the 1970s, PEDV has caused repeated outbreaks worldwide, leading to substantial economic losses in the swine industry (Pensaert and de Bouck \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Song and Park \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe PEDV genome is approximately 28 kb in length and encodes four major structural proteins: spike (S), envelope, membrane, and nucleocapsid (Yang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kocherhans et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The S protein mediates viral entry into host cells and is a key target for neutralizing antibodies (Liu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). It is cleaved into S1 (1\u0026ndash;735 aa) and S2 (736\u0026ndash;1383 aa) subunits (Deng et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The S1 subunit contains major antigenic determinants and neutralizing epitopes and is crucial for inducing protective immunity by blocking virus\u0026ndash;receptor interactions (Chang et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Monoclonal antibodies against S1 are therefore valuable tools for developing specific diagnostic assays and studying viral entry (Makadiya et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Oh et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe continuous evolution of PEDV, co-circulation of multiple genotypes, and potential serological cross-reactivity with other porcine coronaviruses such as TGEV and PDCoV challenge the performance of current diagnostic methods (Ma et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In PED control, assessing vaccine immunogenicity is essential. Protective efficacy correlates with levels of virus-neutralizing antibodies, making neutralizing antibody titer a key indicator of vaccine performance (Lee \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, the virus neutralization test (VNT), considered the gold standard, is labor-intensive, time-consuming, and low-throughput, unsuitable for large-scale serosurveillance \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eMari\u0026euml;n et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although ELISAs based on whole virus or structural proteins (nucleocapsid or membrane) are widely used, they often fail to differentiate neutralizing from non-neutralizing antibodies and may cross-react with antibodies against related coronaviruses, compromising specificity (Li et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBlocking ELISA based on monoclonal antibodies offers a promising alternative. This format uses competition between serum antibodies and a defined neutralizing mAb for binding to a specific epitope \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eSayee et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The degree of signal inhibition correlates with the level of functional antibodies in serum. Studies have shown good agreement between blocking ELISA and VNT results, enabling efficient evaluation of neutralizing antibody levels (Zhao et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Therefore, developing a blocking ELISA targeting the PEDV S1 protein with a well-characterized neutralizing mAb would support accurate vaccine evaluation, swine herd immunity monitoring, and epidemiological studies (Rasmussen et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we expressed the S1 protein in HEK293F cells, generated murine monoclonal antibodies, and selected a high-potency neutralizing mAb (3E3). Using HRP-labeled 3E3, we established a blocking ELISA that is sensitive, specific, reproducible, and suitable for high-throughput detection of PEDV antibodies in clinical samples. This assay provides a practical tool for vaccine immunogenicity assessment and lays groundwork for further studies on PEDV neutralization mechanisms and therapeutic antibody development.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials and Reagents\u003c/h2\u003e \u003cp\u003eThe PEDV strain China/Hubei/2016 (GenBank KY928065.1), HEK293F cells, Vero cells, and SP2/0 myeloma cells were maintained in the Molecular Immunology Laboratory of Zhengzhou University. A recombinant plasmid carrying the S1 gene was stored at \u0026minus;\u0026thinsp;80\u0026deg;C. 293T-II medium was purchased from SinoBiological, and linear polyethylenimine (PEI) transfection reagent (MW 40000) was from Yisheng Biotechnology. Female BALB/c mice (6\u0026ndash;8 weeks old) were obtained from Huaxing Animal Experimental Center.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Expression and Purification of PEDV-S1 Protein\u003c/h2\u003e \u003cp\u003eThe S1 protein was expressed in HEK293F cells. Cells in logarithmic growth phase with viability\u0026thinsp;\u0026gt;\u0026thinsp;90% were seeded at 2\u0026times;10⁶ cells/mL in fresh medium and cultured at 37\u0026deg;C, 5% CO₂ with shaking (150\u0026ndash;175 rpm). At the time of transfection, cell density was adjusted to 3\u0026times;10⁶ cells/mL. Transfection was performed using SMM293-TII according to the manufacturer\u0026rsquo;s protocol. Culture supernatant was harvested 5\u0026ndash;7 days post-transfection by centrifugation at 8,000 \u0026times;g for 30 min. S1 expression was confirmed by SDS-PAGE and Western blot. The protein was purified by Ni-affinity chromatography using equilibration buffer (20 mM Tris-HCl, 150 mM NaCl, pH 8.0) and eluted with a linear imidazole gradient (20\u0026ndash;500 mM) in the same buffer. Eluted fractions containing S1 were pooled, dialyzed, and concentrated using a 30-kDa ultrafiltration device. Purity and identity were verified by SDS-PAGE and Western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Production of monoclonal antibodies against PEDV-S1 protein\u003c/h2\u003e \u003cp\u003eFour female BALB/c mice (6\u0026ndash;8 weeks old) were immunized subcutaneously with 20 \u0026micro;g purified S1 protein emulsified in Freund\u0026rsquo;s complete adjuvant. Boosters with the same antigen dose emulsified in Freund\u0026rsquo;s incomplete adjuvant were given on days 14 and 28. Serum antibody titers were measured by indirect ELISA one week after the third immunization. The mouse with the highest titer received an intraperitoneal injection of 50 \u0026micro;g antigen without adjuvant. Three to five days later, splenocytes were fused with SP2/0 myeloma cells using PEG 1500. Hybridomas secreting anti-S1 antibodies were selected by indirect ELISA and subcloned three times by limiting dilution. For ascites production, Freund's incomplete adjuvant-primed BALB/c mice were injected intraperitoneally with 1\u0026times;10⁷ hybridoma cells. Ascitic fluid was collected 7\u0026ndash;10 days later, clarified by centrifugation, and purified using a Protein A affinity column.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Immunofluorescence Assay (IFA)\u003c/h2\u003e \u003cp\u003eThe reactivity of mAbs with PEDV was tested by IFA. Vero cells grown in 96-well plates to 80% confluence were infected with PEDV (10⁴\u0026middot;⁶ TCID₅₀/mL) in DMEM containing 6 \u0026micro;g/mL trypsin. After 24 h incubation at 37\u0026deg;C, 5% CO₂, cells showing syncytia were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100, and blocked with 5% skim milk in PBST overnight at 4\u0026deg;C. Cells were incubated with anti-S1 mAb or mouse pre-immune serum (control) at 37\u0026deg;C for 1 h, followed by FITC-conjugated goat anti-mouse IgG (1:200) for 1 h. Nuclei were stained with DAPI. Images were captured using a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Virus Neutralization Test (VNT)\u003c/h2\u003e \u003cp\u003eVero cells were seeded in 96-well plates one day before the assay. Mouse ascitic fluid was serially diluted twofold in serum-free DMEM starting at 1:5. An equal volume of PEDV containing 200 TCID₅₀ was added to each well. After 1 h incubation at 37\u0026deg;C, the virus\u0026ndash;antibody mixture was transferred to Vero cell monolayers and adsorbed for 1 h. Cells were washed and maintained in DMEM with 2% FBS for 48 h. Infection was assessed by IFA as above. The neutralization titer was defined as the highest dilution that inhibited\u0026thinsp;\u0026gt;\u0026thinsp;90% of infection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Development of the Blocking ELISA\u003c/h2\u003e \u003cp\u003ePurified S1 protein was used as the coating antigen and HRP-conjugated mAb 3E3 as the competing antibody. Checkerboard titration was performed to determine optimal antigen coating concentration and HRP-3E3 dilution. S1 protein was diluted in carbonate buffer (2, 1, 0.5, 0.25 \u0026micro;g/mL), added to 96-well plates (100 \u0026micro;L/well), and incubated overnight at 4\u0026deg;C. Plates were blocked with 5% skim milk in PBST for 2 h at 37\u0026deg;C. Test sera (diluted 1:20 in PBST) were added (100 \u0026micro;L/well) and incubated for 1 h at 37\u0026deg;C. After washing, HRP-3E3 (optimal dilution in PBST) was added and incubated for 1 h. TMB substrate was added, and after 15 min the reaction was stopped with 2 M H₂SO₄. Absorbance was measured at 450 nm. The percent inhibition (PI) was calculated as:\u003c/p\u003e \u003cp\u003ePI = [(OD₍negative serum₎ \u0026minus; OD₍test serum₎) / OD₍negative serum₎] \u0026times; 100%.\u003c/p\u003e \u003cp\u003eParameters optimized by univariate analysis included: coating conditions (4\u0026deg;C/12 h, 4\u0026deg;C/16 h, 37\u0026deg;C/2 h, 37\u0026deg;C/4 h), blocking solution (5% skim milk, 5% BSA, 10% FBS) and blocking time (4\u0026deg;C/12 h, 4\u0026deg;C/16 h, 37\u0026deg;C/1 h, 37\u0026deg;C/2 h), serum dilution (1:2 to 1:80), serum incubation time (60, 90, 120 min), HRP-3E3 incubation time (30, 60, 90 min), and TMB development time (5, 10, 15, 20 min).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cut-off Value Determination\u003c/h2\u003e \u003cp\u003eBased on the optimization described above, the final reaction conditions and procedural steps for the blocking ELISA were established. To define the assay cut-off, 80 sera confirmed as negative for PEDV by both virus neutralization test and a commercial antibody detection kit were analyzed using the optimized protocol. The mean percent inhibition (PI) and standard deviation (SD) were calculated from the OD₄₅₀ values. The cut-off values were determined as follows: Cut-off₁ = mean PI\u0026thinsp;+\u0026thinsp;2\u0026times;SD; Cut-off₂ = mean PI\u0026thinsp;+\u0026thinsp;3\u0026times;SD. The interpretation criteria were set as follows: samples with a PI\u0026thinsp;\u0026lt;\u0026thinsp;Cut-off₁ were considered negative; samples with a PI\u0026thinsp;\u0026ge;\u0026thinsp;Cut-off₂ were considered positive. Samples yielding a PI between Cut-off₁ and Cut-off₂ were classified as suspect and were retested. If the repeat result remained within the suspect range, the sample was judged as negative.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Evaluation of specificity, repeatability, and sensitivity of blocking ELISA\u003c/h2\u003e \u003cp\u003eTo assess the specificity of the assay, the optimized protocol was used to test serum samples positive for ASFV, PCV2, PDCoV, PRRSV, PoRV, and TGEV. PEDV-positive serum served as the control, and the blocking rate was calculated for each sample. For repeatability evaluation, eight serum samples (three negative and five positive) were analyzed in triplicate within the same run and across different runs. The intra- and inter-batch coefficients of variation were calculated from the blocking rates to determine assay reproducibility. Sensitivity was evaluated by testing fourteen PEDV-neutralizing antibody-positive sera, which were first identified by virus neutralization test and then serially diluted two-fold for measurement of blocking activity using the established ELISA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Compliance Test\u003c/h2\u003e \u003cp\u003eTo assess agreement between the blocking ELISA and the virus neutralization test, 107 pig serum samples were tested in parallel using both methods, and their concordance was calculated.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Identification of PEDV S1 protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PEDV S1 recombinant protein was expressed in HEK293F cells and purified by nickel-affinity chromatography. As shown in Figure 1A, SDS-PAGE analysis revealed a characteristic diffuse band at approximately 100 kDa, corresponding to the expected molecular weight of the S1 protein. Specificity was further confirmed by Western blot. Using either a mouse anti-His tag monoclonal antibody (Fig. 1B) or PEDV-positive swine serum (Fig. 1C) as the primary antibody, followed by an HRP-conjugated goat anti-mouse secondary antibody, a single immunoreactive band was observed at the same position. The banding pattern was consistent with the SDS-PAGE result, confirming the identity and integrity of the His-tagged S1 recombinant protein. These data demonstrate that the nickel-column purification yielded a high-purity S1 protein with correct antigenicity, suitable for monoclonal antibody production and subsequent development of the blocking ELISA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Characterization of Monoclonal Antibodies Against PEDV S1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were immunized with purified S1 protein. Following cell fusion and three rounds of subcloning, five hybridoma lines secreting monoclonal antibodies (mAbs) specific for PEDV S1 were established (Fig. 2A). Immunofluorescence assay (IFA) confirmed that all five mAbs bound to native PEDV in infected Vero cells (Fig. 2B). Western blot analysis further demonstrated specific recognition of the denatured S1 protein, indicating that the mAbs target linear epitopes (Fig. 2C). Virus neutralization testing identified mAb 3E3 as possessing the highest neutralizing activity, with a titer of 1:2\u0026sup1;⁰, and it effectively inhibited PEDV-induced cytopathic effect (Fig. 2D). Consequently, 3E3 was selected for subsequent assay development. The antibody was purified by Protein A affinity chromatography. SDS-PAGE under reducing conditions showed the expected heavy- and light-chain bands at approximately 50 kDa and 25 kDa, respectively, confirming successful purification (Fig. 2E). The purified 3E3 was then conjugated to horseradish peroxidase (HRP) using the sodium periodate method. The conjugate retained high antigen-binding activity, yielding an indirect ELISA titer of 1:256,000 or greater (Fig. 2F), confirming that the labeling process did not compromise immunoreactivity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Development and Optimization of the Blocking ELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCheckerboard titration identified an optimal antigen coating concentration of 1 \u0026micro;g/mL for the S1 protein and a working dilution of 1:2000 for the HRP‑conjugated 3E3 antibody, which yielded the highest blocking rate for positive serum (Table 1). Although coating at 4 \u0026deg;C for 16 h gave the highest absolute blocking rate, the corresponding negative control OD values were too low, reducing the assay\u0026rsquo;s ability to discriminate weakly positive samples (Fig. 3A). Therefore, following the principle of maximizing the signal‑to‑noise ratio and resolution, coating at 37 \u0026deg;C for 4 h was selected as the standard condition, as it provided a high blocking rate while maintaining robust differentiation. The use of 5% BSA as the blocking solution with a 2‑h incubation at 37 \u0026deg;C resulted in the highest blocking efficiency (Fig. 3B and 3C). For sample and conjugate incubation, a combination of 60 min for swine serum and 90 min for HRP‑3E3 was found to be optimal (Fig. 3D). A serum dilution of 1:20 produced the maximal blocking rate (Fig. 3E), and a TMB substrate development time of 15 min was chosen based on the plateau of the signal response (Fig. 3F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Determination of the Assay Cut‑off\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe optimized blocking ELISA was tested on 80 PEDV‑negative swine sera. The mean percent inhibition (PI) was 10.36% with a standard deviation (SD) of 8.06% (Fig. 4). Based on these results, the following cut‑off values were established:\u003c/p\u003e\n\u003cp\u003eCut‑off₁ = 26.48% (mean + 2\u0026times;SD)\u003c/p\u003e\n\u003cp\u003eCut‑off₂ = 34.54% (mean + 3\u0026times;SD)\u003c/p\u003e\n\u003cp\u003eSamples with PI \u0026lt; Cut‑off₁ were considered negative, and those with PI \u0026ge; Cut‑off₂ were classified as positive. Samples yielding PI values between Cut‑off₁ and Cut‑off₂ were regarded as suspect and were retested. If the repeat result remained within the suspect range, the sample was ultimately judged as negative.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Specificity, Sensitivity, and Repeatability of the Blocking ELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe specificity of the assay was evaluated using sera positive for ASFV, PCV2, PDCoV, PRRSV, PoRV and TGEV. All heterologous sera yielded blocking rates below the positive cut‑off, confirming no cross‑reactivity (Fig. 5).\u003c/p\u003e\n\u003cp\u003eSensitivity was assessed using 14 PEDV‑positive sera previously titrated by virus neutralization test. The blocking ELISA titers correlated strongly with the neutralization titers, and the ELISA displayed a higher analytical sensitivity, detecting antibodies at greater dilutions (Table 2).\u003c/p\u003e\n\u003cp\u003eRepeatability was determined by testing eight sera (three negative, five positive) in triplicate within the same run and across three independent runs. The maximum intra‑batch and inter‑batch coefficients of variation were 5.46% and 7.67%, respectively, both well below 10% (Table 3), demonstrating excellent assay reproducibility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Consistency Between Blocking ELISA and Virus Neutralization Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe consistency between the blocking ELISA and the virus neutralization test (VNT) was evaluated using 107 clinical pig serum samples tested in parallel by both methods. The blocking ELISA identified 46 positive and 61 negative samples, while the VNT identified 44 positive and 63 negative samples. Using the VNT as the reference standard, the positive agreement rate of the blocking ELISA was 88.63% (39/44), the negative agreement rate was 88.89% (56/63), and the overall agreement rate was 88.8% (95/107) (Table 4). These results demonstrate a high level of concordance between the two assays, indicating that the blocking ELISA reliably reflects the serum neutralizing antibody level.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eNeutralizing antibodies are a well-established correlate of protection for many viral diseases, serving as a primary indicator of vaccine efficacy(Gilbert et al., 2021). These antibodies function by binding specifically to viral surface proteins, thereby blocking attachment and entry into host cells(Adams et al., 2023). Consequently, serum neutralizing antibody titers provide a direct measure of an individual\u0026rsquo;s ability to resist infection.\u003c/p\u003e\n\u003cp\u003eIn the context of PEDV, the level of circulating neutralizing antibodies in sows is closely linked to the passive immunity transferred to piglets via colostrum(Gerber et al., 2014; Li et al., 2017). This relationship underscores the importance of accurate serological monitoring for predicting neonatal protection and managing herd health. Studies have shown that high post-vaccination neutralizing antibody titers correlate with reduced clinical signs, milder intestinal pathology, and lower viral shedding following challenge(Lin et al., 2018). These findings reinforce the central role of neutralizing antibodies in evaluating vaccine performance(Lang et al., 2024).\u003c/p\u003e\n\u003cp\u003eTraditional virus neutralization tests, while considered the gold standard, are labor-intensive, time-consuming, and require biosafety containment, limiting their use in large-scale surveillance(Tao et al., 2022). To address this, we developed a blocking ELISA based on a recombinant S1 protein and a neutralizing monoclonal antibody, 3E3. The assay is designed to detect antibodies that compete with 3E3 for binding to a key epitope on the S1 subunit, thereby offering a functional measure of virus-neutralizing activity.\u003c/p\u003e\n\u003cp\u003eOur results demonstrate that the blocking ELISA exhibits high sensitivity, specificity, and reproducibility. It showed no cross-reactivity with antibodies against other common porcine pathogens, including ASFV, PCV2, PDCoV, PRRSV, PoRV，and TGEV. Furthermore, the assay displayed strong agreement with the virus neutralization test, supporting its reliability as a surrogate method for detecting PEDV-specific neutralizing antibodies.\u003c/p\u003e\n\u003cp\u003eThe established ELISA provides a practical tool for rapid, high-throughput screening of swine sera. It can be applied in various settings, such as monitoring vaccine-induced immunity in breeding herds, evaluating sow colostrum quality, and conducting epidemiological surveys. Regular testing could help identify gaps in herd immunity and guide timely interventions(Tesfagaber et al., 2024).\u003c/p\u003e\n\u003cp\u003eA limitation of this study is the use of a single monoclonal antibody directed against a specific epitope. Given the ongoing evolution of PEDV and the emergence of variant strains, future work should assess the assay\u0026rsquo;s performance against diverse viral genotypes(Yang et al., 2025). Incorporating additional monoclonal antibodies targeting conserved neutralizing epitopes could further enhance the breadth and robustness of the assay(Li et al., 2023).\u003c/p\u003e\n\u003cp\u003eIn conclusion, the blocking ELISA described here offers a reliable and efficient alternative to traditional neutralization tests for measuring PEDV-specific antibodies. With further validation against contemporary field strains, this method could become a valuable asset in PED control and prevention programs.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study established a blocking ELISA for the detection of PEDV-specific neutralizing antibodies. The assay employs recombinant S1 protein as the solid-phase antigen and HRP-conjugated neutralizing monoclonal antibody 3E3 as the competitor. Validation results confirmed the high specificity of the assay, with no observed cross-reactivity against antibodies to other major swine viruses. The method also demonstrated good sensitivity, reproducibility, and a strong correlation with the conventional virus neutralization test. This blocking ELISA provides a reliable, rapid, and high-throughput tool for PEDV serology. It is suited for applications such as monitoring vaccine-induced immunity in swine herds and assessing maternal antibody levels in sows, which is critical for evaluating passive immune protection in suckling piglets. While the developed ELISA shows excellent performance for current diagnostic needs, its reactivity against emerging PEDV variants should be continually evaluated to maintain its utility. In summary, this assay represents a practical and valuable tool to support PED control programs and vaccine efficacy evaluation.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003eThe study was supported by the Regional Innovation and Development Joint Fund of the National Natural Science Foundation of China (Grant No. U24A20448), the Major Science and Technology Projects in Henan Province (Grant No. 241110310200), the Major Research Program of Longhu Laboratory of Advanced Immunology (Grant No. LHLab_ZD20230012), and the Key R\u0026amp;D and Promotion Projects in Henan Province of China (Grant No. 252102111011).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest:\u003c/strong\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeiyang Ding\u003c/strong\u003e: Conceptualization, Writing-review and editing, Investigation, Supervision, Methodology, Resources. \u003cstrong\u003eShuoqi Dong\u003c/strong\u003e: Writing-original draft, \u0026nbsp;Formal analysis, Visualization, Validation, Data curation.\u003cstrong\u003eLinyi Bai:\u0026nbsp;\u003c/strong\u003eInvestigation, Supervision. \u003cstrong\u003eShulei Li\u003c/strong\u003e: Investigation, Supervision. \u003cstrong\u003eXiao Liu\u003c/strong\u003e: Investigation, Supervision. \u003cstrong\u003eJingming Zhou\u003c/strong\u003e : Investigation, Supervision.\u003cstrong\u003eAiping Wang\u003c/strong\u003e: Project administration, Investigation, Supervision, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval for mouse experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Ethics Committee of Henan Longhu Laboratory of Advanced Immunology(Approval No. LHLab-HNZD-2023003) and conducted in accordance with the guidelines for the care and use of laboratory animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work, the author used DeepSeek AI (https://www.deepseek.com/) to refine the English expressions to enhance the fluency. After using this tool, the author will review and edit the content as needed and will be fully responsible for the content of the published articles.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdams, C., Wen, J., Yan, Y., et al. (2023). Structure and neutralization mechanism of a human antibody targeting a complex Epitope on Zika virus. PLoS Pathogens, *19*(1), e1010814. https://doi.org/10.1371/journal.ppat.1010814\u003c/li\u003e\n\u003cli\u003eChang, C. Y., Cheng, I. C., Chang, Y. C., et al. (2019). Identification of neutralizing monoclonal antibodies targeting novel conformational epitopes of the porcine epidemic diarrhoea virus spike protein. Scientific Reports, 9(1), 2529. \u003cstrong\u003ehttps://doi.org/10.1038/s41598-019-39844-5\u003c/strong\u003e\u003c/li\u003e\n\u003cli\u003eChen, J., Liu, X., Shi, D., et al. (2013). Detection and molecular diversity of spike gene of porcine epidemic diarrhea virus in China. 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Proteomics, 22, e2200155.https://doi.org/10.1002/pmic.202200155\u003c/li\u003e\n\u003cli\u003eTesfagaber, W., Wang, W., Wang, L., et al. (2024). A highly efficient blocking ELISA based on p72 monoclonal antibody for the detection of African swine fever virus antibodies and identification of its linear B cell epitope. International Journal of Biological Macromolecules, 268(Pt 1), 131695.https://doi.org/10.1016/j.ijbiomac.2024.131695\u003c/li\u003e\n\u003cli\u003eYang, D. Q., Ge, F. F., Ju, H. B., et al. (2014). Whole-genome analysis of porcine epidemic diarrhea virus (PEDV) from eastern China. Archives of Virology, 159(10), 2777\u0026ndash;2785. \u003cu\u003e \u003c/u\u003ehttps://doi.org/10.1007/s00705-014-2102-7\u003c/li\u003e\n\u003cli\u003eYang, X., Yang, J., Su, X., et al. (2025). Nanobody-based epitope mapping and establishment of a blocking ELISA for S protein of porcine epidemic diarrhea virus. International Journal of Biological Macromolecules, 257(Pt 2), 131652.https://doi.org/10.1016/j.ijbiomac.2025.148110\u003c/li\u003e\n\u003cli\u003eZhao, J., Zhang, L., Kong, Y., Dan, M., Xiri, Y., Ji, P., Jiang, S., Sun, Y., \u0026amp; Zhao, Q. (2025). A competitive ELISA based on nanobodies for the detection of serum neutralizing antibodies against porcine epidemic diarrhea virus. Animal Diseases, 5, 7. https://doi.org/10.1186/s44149-025-00161-2\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eDetermination of optimal antigen coating concentration and antibody dilution\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"548\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"2\" rowspan=\"2\" style=\"width: 164px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntigen coating concentration\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eug/ml\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"6\" style=\"width: 384px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDilutionof HRP-3E3 monoclonal antibody\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"66\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003cstrong\u003e2000\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003cstrong\u003e4000\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003cstrong\u003e8000\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003cstrong\u003e16000\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003cstrong\u003e32000\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003cstrong\u003e64000\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"59\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 41px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 122px;\"\u003e\n \u003cp\u003eBlocking rate（PI）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e69.82%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e56.83%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e10.67%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 65px;\"\u003e\n \u003cp\u003e17.92%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 65px;\"\u003e\n \u003cp\u003e53.80%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 65px;\"\u003e\n \u003cp\u003e40.67%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"48\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"48\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 41px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 122px;\"\u003e\n \u003cp\u003eBlocking rate（PI）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e83.19%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e76.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e48.10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 65px;\"\u003e\n \u003cp\u003e10.63%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 65px;\"\u003e\n \u003cp\u003e48.31%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 65px;\"\u003e\n \u003cp\u003e46.06%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"48\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"48\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 41px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 122px;\"\u003e\n \u003cp\u003eBlocking rate（PI）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e90.37%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e86.25%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e86.06%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 65px;\"\u003e\n \u003cp\u003e67.53%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 65px;\"\u003e\n \u003cp\u003e51.44%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 65px;\"\u003e\n \u003cp\u003e62.58%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"48\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"48\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 41px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 122px;\"\u003e\n \u003cp\u003eBlocking rate（PI）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e88.99%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e87.64%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e83.54%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 65px;\"\u003e\n \u003cp\u003e83.38%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 65px;\"\u003e\n \u003cp\u003e63.94%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 65px;\"\u003e\n \u003cp\u003e42.23%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"48\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"51\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003eSensitivity of Blocking ELISA\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"566\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSerum\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 285px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNeutralizing antibody titer\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 191px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlocking ELISA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 285px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 191px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 285px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 191px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 285px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 191px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 285px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 191px;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 285px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 191px;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 285px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 191px;\"\u003e\n \u003cp\u003e160\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 285px;\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 191px;\"\u003e\n \u003cp\u003e320\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 285px;\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 191px;\"\u003e\n \u003cp\u003e320\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 285px;\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 191px;\"\u003e\n \u003cp\u003e640\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 285px;\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 191px;\"\u003e\n \u003cp\u003e640\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 285px;\"\u003e\n \u003cp\u003e512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 191px;\"\u003e\n \u003cp\u003e640\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 285px;\"\u003e\n \u003cp\u003e512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 191px;\"\u003e\n \u003cp\u003e640\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 285px;\"\u003e\n \u003cp\u003e1024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 191px;\"\u003e\n \u003cp\u003e1280\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 285px;\"\u003e\n \u003cp\u003e1024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 191px;\"\u003e\n \u003cp\u003e1280\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e Repeatability of Blocking ELISA\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"566\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSerum\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIntra batch repetition (PI/%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInter batch repetition (PI/%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eX\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCV/%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eX\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCV/%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 69px;\"\u003e\n \u003cp\u003ea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e92.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.004\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e92.72%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.004\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e0.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 69px;\"\u003e\n \u003cp\u003eb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e82.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.009\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e82.58%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.005\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 69px;\"\u003e\n \u003cp\u003ec\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e96.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.004\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e96.75%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.004\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 69px;\"\u003e\n \u003cp\u003ed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e90.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.005\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e90.08%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.003\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 69px;\"\u003e\n \u003cp\u003ee\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e53.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.011\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e2.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e53.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.028\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e5.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 69px;\"\u003e\n \u003cp\u003ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e30.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.009\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e3.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e28.82%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.015\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e5.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 69px;\"\u003e\n \u003cp\u003eg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e32.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.010\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e3.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e34.79%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.027\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e7.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 69px;\"\u003e\n \u003cp\u003eh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e12.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0.007\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 90px;\"\u003e\n \u003cp\u003e5.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e13.11%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.005\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 86px;\"\u003e\n \u003cp\u003e3.57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u0026nbsp;\u003c/strong\u003eCompliance rate between the blocking ELISA and the neutralization test\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"566\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"2\" rowspan=\"2\" style=\"width: 280px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDetection method\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 287px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSerum neutralization test\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 95px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 96px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 95px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" rowspan=\"3\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlocking ELISA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 78px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 95px;\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 96px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 95px;\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 78px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 95px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 96px;\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 95px;\"\u003e\n \u003cp\u003e61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 78px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 95px;\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 96px;\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 95px;\"\u003e\n \u003cp\u003e107\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Monoclonal antibody, Porcine epidemic diarrhea virus, Neutralizing antibody, Blocking ELISA","lastPublishedDoi":"10.21203/rs.3.rs-9143664/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9143664/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA highly specific blocking ELISA was developed for serological diagnosis of porcine epidemic diarrhea (PED) using monoclonal antibodies (mAbs) against the S1 protein of PEDV. The S1 protein was expressed in HEK293F cells and used to immunize BALB/c mice. Through hybridoma screening, a high-affinity monoclonal antibody designated 3E3 was obtained. Indirect ELISA, Western blot, and immunofluorescence assays confirmed its significant neutralizing activity, with a neutralization titer of 1:2\u0026sup1;⁰. A blocking ELISA was subsequently established using HRP-labeled 3E3. The assay showed no cross-reactivity with other porcine viruses (African swine fever virus (ASFV), porcine circovirus 2 (PCV2), porcine deltacoronavirus (PDCoV), porcine reproductive and respiratory syndrome virus (PRRSV), Porcine rotavirus (PoRV) and porcine transmissible gastroenteritis virus (TGEV) ) and exhibited good reproducibility, with intra- and inter-batch coefficients of variation below 10%. Compared with the virus neutralization test, the blocking ELISA demonstrated higher sensitivity. Results from the two methods showed a strong positive correlation, with a positive agreement of 88.63%, a negative agreement of 88.89%, and an overall agreement of 88.8%. In conclusion, the established blocking ELISA is specific, sensitive, and reliable, suitable for serological monitoring of PEDV and evaluation of vaccine-induced immunity.\u003c/p\u003e","manuscriptTitle":"Establishment of a blocking ELISA for assessing neutralizing antibody levels against porcine epidemic diarrhea virus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-14 18:02:39","doi":"10.21203/rs.3.rs-9143664/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-31T11:16:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-31T10:27:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-31T10:26:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Veterinary Research Communications","date":"2026-03-17T03:37:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"be644053-1310-43aa-9a42-8d4b426e81be","owner":[],"postedDate":"April 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-14T18:02:39+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-14 18:02:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9143664","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9143664","identity":"rs-9143664","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}