A型口蹄疫病毒多表位纳米融合蛋白制备及全自动磁微粒CLIA抗体检测方法的建立

doi:10.11843/j.issn.0366-6964.2025.12.027

Abstract

Abstract: This experiment was conducted to prepare a multi-epitope nanofusion protein of foot-and-mouth disease virus serotype A (FMDV-A) and to establish a magnetic particle CLIA antibody detection method, which was to provide technical support for clinical immunization monitoring, prevention and control of FMDV. In this study, a magnetic particle chemiluminescent immunoassay (CLIA) for detecting antibodies against foot-and-mouth disease virus serotype A (FMDV-A) was established based on the expressed multi-epitope fusion protein of FMDV-A and its polyclonal antibody. Reaction conditions were optimized, and clinical samples were simultaneously tested using this method and commercial kits to compare the coincidence rate between the two methods. Finally, a highly soluble FMDV-A multi-epitope nanofusion protein and specific polyclonal antibody with a titer of 1∶5 120 were successfully prepared. A magnetic particle CLIA for FMDV-A antibodies detection was established based on the principle of competitive immunoassay. The optimal concentration of streptavidin magnetic beads (SA-Beads) was 0.5 mg·mL^-1, the optimal concentration of biotin-labeled recombinant FMDV-A protein antigen was 5 μg·L^-1, and the optimal concentration of acridinium ester (AE)-labeled polyclonal antibody was 2 μg·L^-1. The optimal sample volume was 10 μL, and the optimal reaction time was 15 minutes. The sensitivity test results show that the sensitivity of this method is 97.47%, and the sensitivity is good. No cross-reactivity was observed with other clinically similar pathogens, demonstrating strong specificity. The results of repeatability test demonstrated that coefficient of variation (CV) values were less than 5%, which had good stability and repeatability The results of the clinical trials indicated that the overall coincidence rate was 96.76% when compared with the LPB-ELISA method developed by Lanzhou Veterinary Research Institute. This research has established a technical foundation for the research and rapid diagnosis on type A foot-and-mouth disease in China. This study successfully prepared a VP1 multi epitope fusion nanoparticle protein of FMDV-A strain and obtained its high titer polyclonal antibody. The protein and polyclonal antibody were used to establish a detection method for FMDV-A magnetic particle CLIA antibody.

Key words: foot-and-mouth disease virus type A, ferritin, CLIA, magnetic particles chemiluminescence, nanoparticle

CLC Number:

S855.3

ZHENG Xueying, WANG Pei, SUN Weishan, ZHAO Haojun, YIN Yuqing, LI Jia, CHENG Rujia, LIU Haiying, WANG Dameng, MU Wei, LI Da, CHEN Xijun, ZHENG Bojun, MEI Li. Preparation of Multi-epitope Nanofusion Protein for Foot-and-Mouth Disease Virus Serotype A and Development for Antibodies Detection of a Full-Automated Magnetic Particle Chemiluminescent Immunoassay (CLIA)[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6257-6270.

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Preparation of Multi-epitope Nanofusion Protein for Foot-and-Mouth Disease Virus Serotype A and Development for Antibodies Detection of a Full-Automated Magnetic Particle Chemiluminescent Immunoassay (CLIA)

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