非洲猪瘟病毒p54蛋白单克隆抗体的制备及阻断ELISA方法的建立

doi:10.11843/j.issn.0366-6964.2022.03.014

Abstract

Abstract: To develop a blocking ELISA for the detection of antibodies against African swine fever virus (ASFV), ASFV P54 protein expressed in E. coli was used as the coating antigen, and monoclonal antibody against P54 protein was prepared. The optimum reaction conditions of blocking ELISA were determined by square matrix titration, and the sensitivity, specificity, repeatability and conformity were evaluated. The results showed that the optimum conditions of the assay were as follows:the antigen coating concentration was 2.0 μg·mL^-1, antigen coating temperature and time were 4℃ overnight, the dilution of tested serum was 1:40, the dilution of enzyme-labelling mAb was 1:1 000, the action time of tested serum was 1 h, the action time of enzyme-labelling mAb was 30 min, and the action time of substrate was 10 min. Through the detection of negative and positive samples, as well as specificity and sensitivity tests, it was determined that the criteria for determining the negativity and positivity of the assay were:Results determined to be positive at cut-off blocking rate ≥ 55.2%, negative at blocking rate <43.4%, and suspicious between the two values. The assay had no cross reaction with positive sera of classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, foot-and-mouth disease virus, swine influenza virus and porcine pseudorabies virus. The coefficient of variation (CV) of intra- and inter-batch repeated tests were both within 10%. The coincidence rate between the assay and commercial ELISA kit was 98%. In conclusion, the blocking ELISA has ideal specificity, sensitivity and repeatability, and can be used for the antibody detection of ASFV clinical samples.

Key words: African swine fever virus, ASFV, blocking ELISA, p54 protein, mAb

CLC Number:

S852.659.1

XU Lingyu, CAO Chenfu, LI Zhonsheng, CHEN Junhong, LIU Tengfei, WANG Xinkai, JIA Weixin. Preparation of Monoclonal Antibody against African Swine Fever Virus p54 Protein and Development of Blocking ELISA[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 813-821.

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Preparation of Monoclonal Antibody against African Swine Fever Virus p54 Protein and Development of Blocking ELISA

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