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. 2025 Oct 18;109(1):232. doi: 10.1007/s00253-025-13616-w

A chemiluminescence immunoassay for detecting neutralizing antibodies of foot-and-mouth disease virus serotype A

Mengyang Sun 1, Yanfang Bao 2, Kun Li 1, Yili Zuo 2, Huiyan Zhang 1, Yuanfang Fu 1, Pinghua Li 1, Pu Sun 1, Zhixun Zhao 1, Tao Jiang 2, Xingwen Bai 1, Mi Lin 2,✉, Zengjun Lu 1,✉, Yimei Cao 1,✉
PMCID: PMC12535544  PMID: 41108374

Abstract

Abstract

Foot-and-mouth disease (FMD) is a highly contagious viral disease affecting cloven-hoofed animals, posing significant threats to global livestock industries. Post-vaccination neutralizing antibody levels reflect vaccine efficacy, but traditional virus neutralization tests (VNT) for detecting neutralizing antibodies requires restrictive biocontainment facilities to handle live virus, is time-consuming and laborious. In this study, we successfully established a high-efficiency magnetic particle-based chemiluminescence immunoassay (MP-CLIA) using two previously characterized monoclonal antibodies (W125 and W145) generated through single B cell antibody technology. The MP-CLIA method exhibited a sensitivity of 95.93% and specificity of 100%, with a cut-off value of 41.395 activity units (U) by detecting the known 221 positive and 122 negative sera. The positive/negative coincidence rate between the MP-CLIA and VNT was 92.2% and the kappa coefficient was 78.19%, indicating a relatively high level of consistency. When integrated with a fully automated chemiluminescence immunoassay analyzer, this method only takes 20 min for a single test. These results show that MP-CLIA is a promising tool for the safe, rapid, and fully automatic detection of neutralizing antibodies against FMD virus (FMDV) serotype A.

Key points

  • A MP-CLIA using two bovine single B cell antibodies was established.

  • The MP-CLIA method exhibited a sensitivity of 95.93% and specificity of 100%.

  • The MP-CLIA specifically detects neutralizing antibodies against FMDV serotype A.

Keywords: Foot-and-mouth disease virus, Serotype A, Neutralizing antibodies, Magnetic particle, Chemiluminescence immunoassay

Introduction

Foot-and-mouth disease (FMD), caused by the foot-and-mouth disease virus (FMDV), is an extremely infectious disease that affects animals possessing cloven hooves. The disease spreads rapidly over long distances, with over 70 susceptible species, posing severe threats to global livestock industries (Kitching et al. 2007; Arzt et al. 2024). FMDV, a small RNA virus with high genetic variability, is classified into seven serotypes: O, A, C, Asia 1, SAT 1, SAT 2, and SAT 3 (Bachrach 1968). There is a lack of cross-protection between different serotypes of the virus. Moreover, even within the same serotype, antigenic disparities among various lineages restrict the cross-protection against heterologous strains (Belsham 2005). In China, FMDV serotype A and serotype O are predominant. FMDV serotype A demonstrates the most significant antigenic structural variability, resulting in limited cross-protection among its different lineages (Fry et al. 1999; Kitching et al. 2007; Islam et al. 2021). In regions where FMD is endemic, inactivated vaccines continue to be the principal preventive approach (Lu et al. 2022). As the principal protective immune components mediating host defense against FMDV infection, neutralizing antibodies serve as critical correlates for post-vaccination immune monitoring. Quantitative assessment of these antibodies provides an evidence-based foundation for evaluating vaccine effectiveness and optimizing immunization protocols (Amadori et al. 1991; Pay and Hingley 1992). The World Organization for Animal Health (WOAH) recommended three serological assays: virus neutralization test (VNT), liquid-phase blocking enzyme-linked immunosorbent assay (LPB-ELISA) and solid-phase competitive enzyme-linked immunosorbent assay (SPC-ELISA) (WOAH 2024). VNT is the “gold standard” for detecting neutralizing antibodies; however, this method requires using live viruses, posing a risk of virus dissemination. Moreover, it has a long experimental cycle and poor repeatability and is not suitable for batch testing of serum samples (Jamal and Belsham 2013). LPB-ELISA is well suited for large-scale detection, but it has a propensity for non-specific reactions, which frequently leads to false positive results (Hamblin et al. 1986). SPC-ELISA exhibits operational simplicity and robust reproducibility; however, its low-throughput design significantly hinders efficient processing of large sample volumes (Mackay et al. 1998; Brocchi et al. 2006). Therefore, there is a need to develop a safe, precise, and rapid novel method for the detection of neutralizing antibodies.

Chemiluminescence immunoassay (CLIA) is a detection method that integrates high-sensitivity chemiluminescence technology with high-specificity immune reactions. Through leveraging immunolabeling technology, substrates or catalysts (enzymes or inorganic catalysts) of chemiluminescent reactions are labeled onto pre-prepared specific antigens or antibodies. Through the immunoreaction bridge, a correlation is established between the concentration of analytes and chemiluminescent intensity, enabling the quantitative detection of target substances (Azim et al. 2018). In recent years, nanomaterials have demonstrated promising application outcomes in chemiluminescent detection methods (Rosi and Mirkin 2005; Wang et al. 2012; Liu et al. 2016), with magnetic particles (MPs) being particularly widely utilized. MPs are primarily functionalized with antigens/antibodies to capture target molecules, forming immune complexes. Under an external magnetic field, these complexes are efficiently separated from the reaction mixture. Compared to traditional microplates, MPs offer larger surface areas, easier washing and separation, and covalent bonding (replacing physical adsorption) for stable antigen/antibody immobilization, these features accelerate immune reactions and improve sensitivity (Xie et al. 2017). CLIA boasts advantages such as rapid processing, high throughput, user-friendly operation, and compatibility with automation. And it has been extensively applied in clinical diagnostics (Liu et al. 2013; Satyaputra et al. 2021; Li et al. 2023b), food safety monitoring (Kovács and Rásky 2001; Wang et al. 2013; Zhu et al. 2021), and animal disease detection (Vidziunaité et al. 1995; Zhou et al. 2014; Shi et al. 2023), demonstrating its versatility and reliability.

The single B cell technology enables the isolation of FMDV-specific monoclonal antibodies (mAbs) directly from natural hosts such as cattle and pig (Li et al. 2020, 2021), this method avoids the complexity of hybridoma fusion and screening, offering a simpler, faster, and more efficient method for antibody production. Notably, antibodies generated through this technology retain unique antibody gene sequence information, allowing permanent digital archiving and eliminating the risk of data loss (Li et al. 2020), this ensures a stable and abundant supply of antibodies for experimental applications. Using this technology, two bovine-derived monoclonal antibodies (mAbs) W125 and W145 were isolated previously in our laboratory, both of which were broadly neutralizing antibodies against FMDV serotype A and could neutralize all representative strains circulating in China, including sublineages G1 (represented by strain FMDV A/WH/CHA/09) and G2 (represented by strain FMDV A/GDMM/CHA/2013) within the SEA97 lineage, as well as the AF72 vaccine strain (subtype A22) (Li et al. 2023a).

In this study, we developed a safe, efficient, and fully automated magnetic particle-based chemiluminescence immunoassay (MP-CLIA) using mAbs W125 and W145 for detecting neutralizing antibodies against FMDV serotype A in animal sera. The cutoff, sensitivity, and specificity of the MP-CLIA were determined using sera with a known status. In addition, the positive/negative coincidence rate between the MP-CLIA and VNT was also evaluated.

Materials and methods

Virus, antibodies, and chemical materials

The FMDV A/WH/CHA/09 146S antigen were prepared and stored by the National Foot-and-Mouth Disease Reference Laboratory of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The bovine-derived broadly neutralizing antibodies W125 and W145 against FMDV serotype A were prepared using single B-cell antibody technology and preserved in our laboratory (Li et al. 2023a).

The magnetic particles (MPs) were purchased from JSR Corporation, Tokyo, Japan; alkaline phosphatase (AP) and adamantyl-1,2-dioxetane phenyl phosphate (AMPPD) were obtained from Sigma-Aldrich, St. Louis, MO, USA; 1-ethyl-3-(3-dimethylaminopropyl)-carbohydrate (EDC) and N-hydroxysuccinimide (NHS) were purchased from Thermo Fisher Scientific, Waltham, MA, USA.

Serum samples

  • (i)

    Serum samples from unvaccinated animals: A total of 122 serum samples were collected from healthy pigs and cattle unvaccinated against FMDV, including 27 pig sera and 95 cattle sera, to determine cut-off values and specificity. All these sera tested negative by O type liquid-phase blocking ELISA (O-LPBE) and A type liquid-phase blocking ELISA (A-LPBE) (titers < 0.6 log₁₀).

  • (ii)

    Positive serum samples with known immune backgrounds: A total of 221 serum samples were collected from pigs and cattle vaccinated against FMDV vaccines, including 80 samples from cattle vaccinated with inactivated FMDV serotype A vaccine, 39 samples from cattle vaccinated with bivalent inactivated vaccine against FMDV serotypes O and A, 2 samples from pigs vaccinated with inactivated FMDV serotype A vaccine, and 100 samples from pigs vaccinated with bivalent inactivated FMDV serotype O and A vaccine. These sera had VNT titers ≥ 1.65 log₁₀.

  • (iii)

    Collection of field serum samples: A total of 296 serum samples were collected from animals vaccinated with FMDV vaccines for comparison with VNT results. These include 170 samples from pigs vaccinated with inactivated FMDV vaccines (serotype A or bivalent O/A) and 126 from cattle vaccinated with inactivated FMDV vaccines (serotype A or bivalent O/A).

  • (iv)

    Positive serum samples for other FMDV serotypes and other viruses: Thirty serum samples were from pigs vaccinated with inactivated FMDV serotype O vaccine; 18 samples were from animals infected with FMDV serotype Asia 1; additionally, samples included 8 sera from pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV), 4 sera from sheep infected with peste des petits ruminants virus (PPRV), 3 sera from pigs infected with Seneca Valley virus (SVV), 3 sera from pigs infected with classical swine fever virus (CSFV), 3 sera from cattle infected with Mycoplasma, and 3 sera from cattle infected with bovine viral diarrhea virus (BVDV).

Magnetic particles (MPs) coupled with antibody W125

2 mg of MPs were adsorbed using a biological magnetic separator for 1 min. After the solution was discarded, the MPs were resuspended in an appropriate volume of 0.1 mol·L⁻1 2-(N-morpholino) ethanesulfonic acid (MES) (pH 5.0). This washing step was repeated four times. After the final wash, the MPs were resuspended in 200 μL of 0.1 mol·L⁻1 MES (pH 5.0). 20 μL of NHS (10 mg/mL) and 20 μL of EDC (10 mg/mL) were each added, and the mixture was incubated in a shaker at room temperature for 30 min to activate the MPs. Following activation, the MPs were washed four times with 0.1 mol·L⁻1 MES (pH 7.0) and resuspended in 200 μL of 0.1 mol·L⁻1 MES (pH 7.0). Then 10 μg of FMDV serotype A antibody W125 was added, and the coupling reaction was carried out by shaking at room temperature for 3 h. Subsequently, 10% bovine serum albumin (BSA) was added to block the MPs by incubation with shaking for 3 h at room temperature. After washing three times, the MPs were resuspended in 4 mL phosphate-buffered saline with Tween® 20 (PBST) containing 1% BSA and 0.05% Proclin-300 (Sigma-Aldrich, St. Louis, MO, USA) and stored at 4 ℃ until use.

Alkaline phosphatase (AP) labeling of antibody W145

NaIO₄ and alkaline phosphatase (AP) were mixed at a mass ratio of 1.5:1 and reacted at 4 °C for 1 h. An equal volume of ethylene glycol (1%) was then added to the mixture to reduce excess NaIO₄, followed by incubation at 4 °C for 2 h, completing the activation of AP. Subsequently, antibody W145 and activated AP were mixed at a mass ratio of 1:2 and dialyzed overnight at 4 °C in 0.5 M carbonate buffer (pH 9.6). After dialysis, NaBH₄ was added, and the reaction was allowed to proceed at 4 °C for 1 h. The mixture was then dialyzed overnight at 4 °C in 0.1 M phosphate-buffered saline (PBS) (pH 7.4) to prepare the alkaline phosphatase-labeled antibody (AP-W145). Finally, the dialyzed product was collected, mixed with PBS containing 50% glycerol, and stored at − 20 °C or below for future use.

Preparation of standards

A serum sample with a VNT titer of 1:128 against FMDV serotype A was defined as containing 100 active units (100 U), which means the antibody content of this serum is 100 U. The standard serum used in this study was prepared by immunizing a healthy pig with an inactivated FMDV serotype A vaccine (containing 10 μg/mL of 146S antigen, 2 mL per immunization). A booster immunization was administered 28 days after the primary immunization. Blood was collected 28 days after the booster dose, and the serum was separated and tested using both VNT and a commercial competitive ELISA kit (Lanzhou Shouyan Biotechnology Co., Ltd, Lanzhou, China) for FMDV serotype A neutralizing antibodies. The serum showed a neutralizing antibody titer of 1:4096 in both assays, confirming its accuracy and qualifying it as a reference standard for the evaluation of serum neutralizing antibody titers. Therefore, the standard was assigned 3200 U. The standard was serially diluted to generate antibody contents of 3200 U (1:4096), 1600 U (1:2048), 800 U (1:1024), 400 U (1:512), 200 U (1:256), 100 U (1:128), 50 U (1:64), 25 U (1:32), 12.5 U (1:16), and 6.25 U (1:8), stored at − 20 °C or below.

Using antibody titer (1:X) as the abscissa and antibody contents (U) as the ordinate, linear fitting had been performed to obtain the line and equation representing the correspondence between the antibody contents (U) and the antibody titer (1:X).

Assay optimization

The optimal reaction conditions were selected using a single-factor screening method. Standard samples and negative/positive control sera were tested with different MPs-W125 concentrations (0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL), A/WH/CHA/09 antigen concentrations (0.1 µg/mL, 0.25 µg/mL, 0.5 µg/mL, 1 µg/mL, and 2 µg/mL), AP-W145 dilution ratios (1:500, 1:1000, 1:2000, 1:5000, and 1:10,000), reaction times (5 min, 10 min, 15 min, and 20 min), and sample volumes (10 µL, 20 µL, 30 µL, 40 µL, and 50 µL). The optimal conditions were determined based on the R2 value of the standard curve (prepared by detecting standard samples with antibody contents (U) as the X-axis and the relative light unit (RLU) as the Y-axis to plot a four-parameter standard curve) and the luminescence intensity ratio (N/P ratio) of negative and positive control sera.

Using optimized conditions, the standard substance was detected, and a four-parameter standard curve was fitted, finally obtaining a curve and an equation reflecting the corresponding relationship between antibody contents (U) and RLU.

Development of the MP-CLIA

As illustrated in Fig. 1, 40 μL of the sample to be tested, 25 μL of MPs-W125 (0.5 mg/mL), 100 μL of AP-W145 (1:1000), and 100 μL of A/WH/CHA/09 146S antigen (0.25 μg/mL) were simultaneously added to the reaction cup, thoroughly mixed, and incubated at 37 °C for 15 min. The MPs were adsorbed using a magnetic plate for 1 min, and the reaction solution was discarded. Washing buffer was added to resuspend the MPs, and the adsorption-washing step was repeated four times, after which the final washing buffer was discarded, 100 μL of the chemiluminescent substrate AMPPD was added, mixed, and incubated at 37 °C for 3 min. RLU was measured, and antibody content was calculated. A MP-CLIA method for detecting the neutralizing antibody of FMDV serotype A was preliminarily established.

Fig. 1.

Fig. 1

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Schematic illustration of the magnetic particle-based chemiluminescence immunoassay (MP-CLIA) for quantitative detection of FMDV serotype A

Cut-off value, diagnostic sensitivity, and diagnostic specificity

Using the optimized MP-CLIA, we detected standards to fit a standard curve, tested 343 sera with clear backgrounds and analyzed the data via receiver operating characteristic curve (ROC curve) and interactive dot plot. The optimal cut-off value for the MP-CLIA was determined through ROC curve analysis and interactive dot plot visualization using the Youden index as the evaluation metric, which was subsequently established as the diagnostic criterion for distinguishing positive and negative results in detecting neutralizing antibodies against FMDV serotype A.

The diagnostic sensitivity of the MP-CLIA was calculated by analyzing the detection rate of 221 positive samples among 343 total sera. The diagnostic specificity was determined through evaluation of test results from 122 negative serum samples.

Serotype specificity and cross-reactivity

The serotype specificity of the MP-CLIA was validated by testing 30 FMDV serotype O antibody-positive serum samples and 18 FMDV serotype Asia 1 antibody-positive serum samples. Cross-reactivity evaluation included 8 PRRSV antibody-positive sera, 4 PPRV antibody-positive sera, and 3 serum samples each positive for SVV, Mycoplasma, BVDV, and CSFV antibodies, to assess potential cross-reactivity with antibodies against heterologous pathogens.

Repeatability

To calculate intra-batch and inter-batch repeatability, four serum samples were selected: two negative, one weakly positive, and one strongly positive, arranged in ascending order of antibody concentration. These samples were tested using the same batch of magnetic particles for intra-batch assessment and three different batches of magnetic particles for inter-batch assessment. The coefficients of variation were subsequently calculated for both intra-batch and inter-batch measurements.

Virus neutralization test (VNT)

Baby Hamster Kidney-21 (BHK-21) cell is a fibroblast cell line derived from the kidneys of Syrian hamsters. It is an adherent continuous cell line with high proliferative capacity and viral susceptibility. Its high sensitivity makes it the preferred cell line for the isolation and diagnosis of FMDV (de la Torre et al. 1988). The virus neutralizing activity of the test serum samples was determined using a BHK-21 cell micro-neutralization test with FMDV A/WH/CHA/09. The procedure was briefly summarized as follows: 100 tissue culture infectious doses (TCID₅₀) of virus were incubated with serially twofold diluted serum in 96-well plates at 37 °C for 1 h. Subsequently, 5 × 104 cells/well were added to each well and cultured at 37 °C for 3 days. Observe and record the cytopathic effect (CPE). The endpoint titer was calculated as the reciprocal of the last serum dilution that neutralized 100 TCID₅₀ FMDV in 50% of the wells. The VNT adopted a cutoff value of 1.65 log₁₀ (1:45), which was established based on the standard operating procedures from the World Reference Laboratory for Foot-and-Mouth Disease (WRLFMD) at The Pirbright Institute, United Kingdom.

Consistency analysis between the MP-CLIA and VNT

Antibody contents in 296 serum samples were detected using the established MP-CLIA, while titers were measured via VNT. The results were systematically compared and analyzed to assess the concordance rate of positivity/negativity determinations between the two methods. The consistency between the two detection methods was compared by calculating the kappa coefficient.

Statistical analysis

The linear fitting line/equation (Taylor 2023) was used for the conversion between the antibody contents (U) and the antibody titer (1:X).

Receiver operating characteristic (ROC) curve analysis (Fawcett 2006) was used for determining the cutoff value, sensitivity, and specificity of the assay.

The four-parameter standard curve (Findlay and Dillard 2007) was used for the conversion between RLU and antibody contents (U).

Linear regression and ROC curve analysis was performed using GraphPad Prism 10.1.2 software (San Diego, CA, USA). The four-parameter standard curve was fitted using Origin 2024 (OriginLab Corporation, Northampton, MA, USA).

Results

Assay optimization

Under different conditions, a series of twofold diluted standard sera, along with one positive and one negative control serum, were detected. The R2 value of the standard curve and the luminescence intensity ratio (N/P ratio) of negative and positive control sera were calculated. Optimization of the concentration of MPs-W125 showed that MPs-W125 at a concentration of 0.5 mg/mL had the highest N/P ratio and R2 value. Therefore, the optimal concentration of MPs-W125 was determined to be 0.5 mg/mL (Fig. 2A). Through optimization of the A/WH/CHA/09 antigen concentration, the results indicated that the optimal antigen concentration was 0.25 µg/mL (Fig. 2B). Similarly, the optimal dilution of AP-W145 was found to be 1:1000 (Fig. 2C), the optimal sample volume was 40 µL (Fig. 2D). Regarding the reaction time, since extending the duration from 15 to 20 min showed minimal improvement in the N/P ratio while incurring additional time costs, we ultimately selected 15 min as the optimal reaction time (Fig. 2E).

Fig. 2.

Fig. 2

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Optimization of reaction conditions of MP-CLIA for detecting neutralizing antibodies against FMDV Serotype A. The concentration of MPs-W125 (A), the concentration of A/WH/CHA/09 antigen (B), the concentration of alkaline phosphatase (AP)-W145 (C), sample volume (D), and reaction time (E) were optimized based on R2 and N/P

The corresponding relationship between antibody contents (U) and VNT titer

The standard antibody contents (U) and corresponding neutralization titer (1:X) were.

as follows: 3200 U (1:4096), 1600 U (1:2048), 800 U (1:1024), 400 U (1:512), 200 U (1:256), 100 U (1:128), 50 U (1:64), 25 U (1:32), 12.5 U (1:16), and 6.25 U (1:8). The corresponding relationship between the standard antibody contents (U) and neutralization titer (1:X) was determined as U = 0.78125X (Fig. 3). After the U value of the serum to be tested was detected by the automatic chemiluminescence immunoassay analyzer, if you want to obtain the corresponding neutralizing titer, you could substitute the U value into the above equation to get the corresponding neutralizing titer.

Fig. 3.

Fig. 3

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Scatter plot and regression line of the MP-CLIA antibody contents (U) and VNT titers. The neutralizing antibody titer (1:X) of the standard positive serum at different dilutions was taken as the X-axis, and the MP-CLIA-measured antibody contents (U) were taken as the Y-axis, with R2 = 1 and Y = 0.78125X

Determination of the cut-off value, diagnostic sensitivity, and diagnostic specificity

A four-parameter logistic standard curve was established using the optimized MP-CLIA method by detecting the standards (Fig. 4), afterwards, it was entered into the automatic chemiluminescence immunoassay analyzer. When testing samples, the instrument would convert the RLU into the antibody contents (U) according to this curve/equation. The U value could then be converted into the antibody titer using the equation in Fig. 3, thereby completing the final conversion from the RLU to the antibody contents (U), and then to the antibody titer. The cut-off value, diagnostic sensitivity, and diagnostic specificity were determined through ROC curve (Fig. 5A) and interactive dot plot (Fig. 5B) analyses of 343 serum samples (221 positive and 122 negative) with clear background. The maximum Youden index was 0.959, corresponding to a cut-off value of 41.395 U (corresponding to a titer of 1:52.9856), with a sensitivity of 95.93%, specificity of 100%, an area under the ROC curve (AUC) of 0.994 (95% CI: 0.988–0.999), and p < 0.0001. The diagnostic criteria were defined as follows: samples with values ≥ 41.395 U are classified as FMDV serotype A neutralizing antibody positive, while those < 41.395 U are considered negative.

Fig. 4.

Fig. 4

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The four-parameter standard curve representing the conversion relationship between antibody contents (U) and the relative light unit (RLU). The X-axis represents antibody contents (U), and the Y-axis represents the relative light unit (RLU). Perform fitting using Origin 2024 (OriginLab Corporation, Northampton, MA, USA)

Fig. 5.

Fig. 5

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Receiver operating characteristic (ROC) analysis for the determination of the cut-off value, sensitivity, and specificity of the MP-CLIA. A ROC curve of the MP-CLIA for the detection of FMDV serotype A neutralizing antibodies. The X-axis represents 100% – specificity % (false positive rate), and the Y-axis represents sensitivity % (true positive rate). B Interactive dot plot of MP-CLIA that represents the dispersion of individual antibody contents (U) in positive sera (n = 221) and negative sera (n = 122). The dashed line represents the selected cut-off value

Serotype specificity and cross-reactivity

To test the serotype specificity of the established MP-CLIA method, 30 serum samples positive for FMDV serotype O antibodies and 18 serum samples positive for FMDV serotype Asia 1 antibodies were detected, among which one serotype O serum sample tested positive (Fig. 6A). To evaluate cross-reactivity with other pathogens, using the MP-CLIA to detect 8 PRRSV-positive sera, 4 PPRV-positive sera, and 3 serum samples each positive for SVV, Mycoplasma, BVDV, and CSFV antibodies (Fig. 6B). All test results were negative, indicating no cross-reactivity of the MP-CLIA with other pathogens.

Fig. 6.

Fig. 6

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Serotype specificity of MP-CLIA with FMDV serotypes Asia 1 and O, and cross-reactivity with other pathogens. A Dispersion of individual antibody contents (U) for FMDV serotype Asia 1-positive sera (n = 18) and serotype O-positive sera (n = 30). B Dispersion of individual antibody contents (U) for PRRSV-positive sera (n = 8), PPRV-positive sera (n = 4), Mycoplasma-positive sera (n = 3), SVV-positive sera (n = 3), CSFV-positive sera (n = 3), and BVDV-positive sera (n = 3). The dashed line represents the cut-off value

Repeatability assay

Using magnetic particles from the same batch and three different batches, four serum samples (two negative, one weakly positive, and one strongly positive) were tested in ascending order of antibody concentration. Coefficient of variation (% CV) values between intra-batch and inter-batch were evaluated. The results showed that all CV values were less than 10% (Table 1), indicating that the MP-CLIA method has good repeatability.

Table 1.

Reproducibility of the MP-CLIA determined by intra- and inter-batch CV values

Serum number Intra-batch Inter-batch
Meana1 SDb1 CVc1 Meana2 SDb2 CVc2
1 6.68 0.071 1.07% 6.79 0.094 1.39%
2 23.60 0.445 1.89% 23.59 0.764 3.24%
3 50.84 0.938 1.84% 51.03 0.250 0.49%
4 703.31 0.481 0.07% 707.66 3.440 0.49%
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c1CV represents the intra-batch coefficients of variation, and the calculation method is as follows: c1=b1a1 × 100%

c2CV represents the inter-batch coefficients of variation, and the calculation method is as follows: c2=b2a2 × 100%

Concordance between the MP-CLIA and VNT

A total of 296 serum samples were tested using both VNT and the established MP-CLIA (Fig. 7). As shown in Table 2, the MP-CLIA detected 216 positive results and 57 negative results, with an overall agreement rate of 92.2% compared to VNT. The Kappa coefficient between MP-CLIA antibody contents and VNT titers was calculated to be 78.19%, indicating high consistency between the two methods’ detection results.

Fig. 7.

Fig. 7

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Dispersion of individual values obtained using MP-CLIA and VNT (n = 296). The antibody contents (U) detected by MP-CLIA were converted into neutralizing titers using the equation U = 0.78125X. VNT titers below the assay sensitivity (0.9 log10) are considered to be 0. The —— line represents the cut-off value of MP-CLIA, the …… line represents the cut-off value of VNT

Table 2.

Positive/negative relationship between MP-CLIA and VNT

MP-CLIA
Positive Negative Total
VNT Positive 216 16 232
Negative 7 57 64
Total 223 73 296
The consistence rate 92.2%
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Discussion

As a new-generation technology following radioimmunoassays (RIA), CLIA has experienced rapid development. With recent advancements in nanomaterials and automatic detection systems, CLIA has been widely adopted across various testing fields and demonstrated significant advantages (Zhong et al. 2019; Xiao and Xu 2020). Particularly, the clinical testing field has become the core application area of CLIA (Xiao and Lin 2015). Moreover, its high sensitivity, efficient processing speed, and operational efficiency demonstrated in various testing fields have also enabled it to exhibit great potential in large-scale serological monitoring and vaccine efficacy evaluation. At present, China still adopts immunization with inactivated vaccines as the main prevention and control strategy for FMD. The neutralizing antibody titer in animals after immunization is an important indicator for evaluating the immunoefficacy of vaccines. Traditional methods like VNT and ELISA are time-consuming and labor-intensive. There is an unmet clinical need for innovative diagnostic approaches to enable rapid and efficient detection of neutralizing antibody titers. In view of this, we developed a high-efficiency MP-CLIA method for detecting neutralizing antibodies of FMDV serotype A, thereby enabling more efficient evaluation of the immunoefficacy of vaccines.

The sensitivity and specificity of antibody-based diagnostic assays are significantly influenced by the selection of detection antibodies. The capacity of antibodies recognizing a single epitope to reflect the comprehensive antibody response against diverse epitopes on the FMDV capsid remains a critical consideration. Therefore, the epitopes recognized by detection antibodies must be immunodominant and overlap with those recognized by the majority of FMDV antibodies generated in the host animal (Jeong et al. 2021). The receptor-binding site represents the most critical neutralizing epitope and the most immunodominant neutralizing region (Li et al. 2024). Precise detection of antibody responses targeting this site can effectively reflect the overall neutralizing antibody response levels. Previously, our laboratory successfully generated bovine monoclonal antibodies W145 and W125 against FMDV serotype A using single B-cell antibody technology. W145 recognizes the RGD + 2 critical amino acid in VP1 of serotype A viruses and binds to the viral receptor-binding site to block viral entry into host cells (Li et al. 2023a). Additionally, binding of antibodies targeting adjacent neutralizing epitopes in VP3 and VP2 may exert steric hindrance on W145 binding, enabling accurate reflection of neutralizing antibody response levels (Aggarwal and Barnett 2002). W125 recognizes antigenic sites spanning the B-C, E–F, and H-I loops of VP2, as well as the B-B knob and H-I loop of VP3 (Li et al. 2023a). When used in combination, W145 and W125 cover nearly all immunodominant neutralizing epitopes on FMDV structural proteins (Li et al. 2023a), minimizing the likelihood of missed detection and enhancing the sensitivity of the assay.

By combining monoclonal antibodies W145 and W125 with the CLIA method, we successfully developed a FMDV serotype A neutralizing antibody detection method with 95.93% sensitivity, 100% specificity, and 92.2% concordance with VNT. The kappa coefficient between the MP-CLIA and VNT reached 78.19%, demonstrating substantial agreement according to Landis and Koch’s classification criteria (Landis and Koch 1977). Cross-reactivity testing showed no cross-reactivity with FMDV serotype Asia 1, PRRSV, PPRV, SVV, Mycoplasma, BVDV, and CSFV, but exhibited slight cross-reactivity with serum samples from pigs vaccinated with the FMDV serotype O vaccine, potentially due to nonspecific reactions or heterotypic humoral immune responses.

Traditional VNT and ELISA methods require labor-intensive serial serum dilutions and are constrained by limited sample throughput per microplate, making quantitative detection cumbersome and time-consuming. In contrast, the tube-based MP-CLIA developed in this study eliminates the need for manual gradient dilution, sample loading, washing, and result calculation. When integrated with a fully automated chemiluminescence analyzer, it not only enables batch processing but also significantly reduces detection time, achieving rapid, efficient, and high-throughput testing. These advancements provide robust technical support for large-scale FMD surveillance and prevention.

It was important to acknowledge that this study had certain limitations. Regarding the selection of antigens, although FMDV A/WH/CHA/09 did not represent all prevalent strains in China, this particular strain was used exclusively to establish the detection method. This decision was based on the fact that most inactivated vaccines used in China at the time employed the A/WH/CHA/09 strain. To maintain consistency with the vaccine strain, both the MP-CLIA and VNT assays utilized A/WH/CHA/09. This approach ensured that the antibodies detected by both methods were homologous, leading to a high degree of agreement. However, when the A/WH/CHA/09 antigen was used to detect heterologous antibodies, such as antibodies against A/GDMM/CHA/2013 or AF72, the resulting antibody titers were lower than those obtained with homologous strains. Therefore, in order to accurately detect antibodies against other lineages, it was necessary to replace the 146S antigen with the corresponding strain.

In conclusion, by using two strains of bovine-derived broadly neutralizing antibodies, an MP-CLIA method for detecting neutralizing antibodies against FMDV serotype A was successfully established. This method enables accurate quantification of neutralizing antibody levels in serum and allows conversion of antibody contents (U) to titers. This highly efficient and convenient method achieves rapid detection within 20 min per test, and demonstrating both high sensitivity and specificity. It exhibits strong concordance with VNT results, establishing its utility as a robust alternative to VNT for quantifying FMDV serotype A-specific protective antibodies and assessing vaccine-induced immune efficacy.

Author contribution

MYS performed experimental operations, data analysis, and wrote the original manuscript. YFB, KL, HYZ, and YFF provided experimental materials. PHL, PS, ZXZ, TJ, and XWB were responsible for methodology. ML, ZJL, and YMC contributed to conceptualization and funding acquisition. YLZ, ZJL and YMC undertook manuscript review and editing. All authors read and approved the manuscript.

Funding

This work was supported by grants from the National Key R&D Program of China (2021YFD1800300), the Major Science and Technology Project of Gansu Province (23ZDNA007), and the talent Innovation and Entrepreneurship Project of lanzhou (2024-HL-7).

Data availability

Data and materials are available from the corresponding authors upon reasonable request.

Declarations

Ethics approval

All procedures involving animals have been approved by the Animal Ethics Committee of the Lanzhou Veterinary Research Institute (LVRI), Chinese Academy of Agricultural Sciences.

Conflict of interest

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Mi Lin, Email: linmi@caas.cn.

Zengjun Lu, Email: luzengjun@caas.cn.

Yimei Cao, Email: caoyimei@caas.cn.

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Data Availability Statement

Data and materials are available from the corresponding authors upon reasonable request.

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