口蹄疫病毒O型中和抗体检测固相阻断ELISA方法的建立

Development of a solid-phase blocking ELISA for the detection of neutralizing antibodies against foot-and-mouth disease virus serotype O

摘要:

【目的】为评价O型口蹄疫病毒(foot-and-mouth disease virus，FMDV)灭活疫苗免疫后中和抗体(neutralizing antibodies，NA)水平，建立检测中和抗体的固相阻断ELISA (neutralizing antibodies solid-phase blocking ELISA，NA-SPBE)方法。【方法】本研究以本实验室前期制备的FMDV广谱反应性牛源单克隆抗体E32作为捕获抗体，以生物素标记的FMDV O型型内广谱中和牛源单克隆抗体C4作为检测抗体，经过条件优化建立了检测FMDV O型中和抗体的固相阻断ELISA方法，并对该方法进行了敏感性、特异性、重复性、交叉反应性与病毒中和试验(virus neutralization test，VNT)相关性等试验。【结果】抗体E32最佳包被浓度为0.5 μg/mL，O型FMDV灭活抗原最佳稀释浓度为0.25μg/mL，生物素标记抗体C4-Bio最佳工作浓度为0.06 μg/mL，链霉亲和素-HRP的最佳稀释度为1:40 000。以1.35 log₁₀作为效价判定临界值时，敏感性和特异性分别为97.14%和98.84%。利用该方法分别检测FMDV A型、FMDV Asia1型、BVDV、PRRSV、CSFV、PPRV抗体阳性血清时，均为阴性，未出现交叉反应。该方法批内和批间重复试验的变异系数均<10%，表明其重复性较好。利用该方法与VNT分别对160份血清样品进行检测，二者的相关系数r为0.807 5，P<0.000 1，相关性显著。【结论】该方法可以检测FMDV O型中和抗体，为FMDV O型灭活疫苗免疫效果评价提供有力技术支撑。

Abstract:

[Objective] To evaluate the level of neutralizing antibodies (NA) against foot-and-mouth disease virus (FMDV), a solid-phase blocking enzyme-linked immunosorbent assay (ELISA) based on bovine monoclonal antibodies was developed. [Methods] In this study, the bovine monoclonal antibody (mAb) E32 was used as the capture antibody and a biotinylated bovine mAb C4 as the detection antibody. Both mAbs had been produced in our laboratory. The mAb E32 is a cross-reactive antibody against FMDV and the mAb C4 is an intraserotype broadly neutralizing antibody against FMDV serotype O. With the two mAbs, a solid-phase blocking ELISA for detecting neutralizing antibody (NA-SPBE) against FMDV serotype O was developed. The sensitivity, specificity, repeatability, cross-reactivity, and correlation with virus neutralization test (VNT) of this assay were assessed. [Results] The optimum working concentrations of antibody E32, FMDV-inactivated antigen, and biotinylated C4 were 0.5 μg/mL, 0.25 μg/mL and 0.06 μg/mL, respectively, and the optimum dilution of streptavidin-HRP was 1:40 000. When 1.35 log₁₀ was used as the cut-off value, the sensitivity and specificity of the assay were determined as 97.14% and 98.84%, respectively. There was no cross reactivity with the antibodies specific to bovine viral diarrhea virus (BVDV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), peste des petits ruminant virus (PPRV), or FMDV serotypes A and Asia1. The intra-batch and inter-batch repeatability of the assay showed the coefficient of variation less than 10%. The detection of 160 serum samples demonstrated a correlation (r=0.807 5, P<0.000 1) between the titers obtained by NA-SPBE and VNT. [Conclusion] NA-SPBE can detect the neutralizing antibodies against FMDV serotype O and provides powerful technical support for evaluating the efficacy of FMDV serotype O-inactivated vaccine.