中国兽医杂志2025，Vol.61Issue(6)：60-66,7.DOI:10.20157/j.cnki.zgsyzz.2025.06.008
O型口蹄疫病毒VLPs疫苗抗体竞争ELISA检测方法的建立
Establishment of a Competitive ELISA Method for Antibody Detection of O-Type Foot-and-Mouth Disease Virus(FMDV)Virus-like Particles(VLPs)Vaccine
摘要
Abstract
To effectively evaluate the immune efficacy of O-type foot-and-mouth disease virus(FMDV)virus-like particle(VLPs)vaccines,a competitive enzyme-linked immunosorbent assay(ELISA)was developed.In this study,O-type FMDV VLPs were used as the immunogen to produce monoclonal antibodies(mAbs)in mice.The optimal mAb was selected based on screening with both O-type FMDV VLPs and inactivated vaccine antigens,combined with ELISA four-parameter logistic curve fitting.The selected VLPs were used as coating antigens in a competitive ELISA,where HRP-conjugated mAb competed with test serum for binding.The assay was optimized for coating antigen concentration,HRP-mAb working dilution,and blocking buffer.A cutoff standard was established,and the sensitivity and specificity of the method were further analyzed.The practicality of the method was validated using 90 serum samples from pigs immunized with the O-type FMDV VLPs vaccine.The results identified mAb 1D3 as the specific antibody for O-type FMDV VLPs.The optimal assay conditions were as follows:coating concentration of VLPs at 0.2 µg/mL,HRP-conjugated 1D3 mAb at a dilution of 1∶1 000,and 1%gelatin as the optimal blocking solution.The interpretation criteria were:inhibition rate≥40%as positive;≤32%as negative;and between 32%-40%as borderline,requiring retesting after 14 days.Sensitivity testing showed a detection limit of serum titer at 1∶1 000.Only serum positive for O-type FMDV VLPs vaccination showed competitive binding,while sera from pigs immunized with inactivated O-type or A-type FMDV vaccines showed no competition.The inhibition rates of the 90 immunized pig serum samples followed the expected pattern of humoral immune response,demonstrating that the developed method is effective for evaluating antibody levels post-vaccination with the O-type FMDV VLPs vaccine.The competitive ELISA method established in this study for detecting antibodies against FMDV VLPs vaccine provides a technical foundation for the development of detection technologies for a broader range of VLP-based vaccines.关键词
O型口蹄疫/类病毒颗粒(VLPs)/竞争ELISAKey words
O-type foot-and-mouth disease/virus-like particles(VLPs)/competitive ELISA分类
农业科技引用本文复制引用
刘玲,李雪峰,戴伶俐,李晓艳,付玲芳,康斌,董鹏,武玉梅,尹忠良,吴蒙..O型口蹄疫病毒VLPs疫苗抗体竞争ELISA检测方法的建立[J].中国兽医杂志,2025,61(6):60-66,7.基金项目
呼和浩特市重大科技专项(2021-农-重-5) （2021-农-重-5）
