China Animal Husbandry and Veterinary Medicine ›› 2024, Vol. 51 ›› Issue (4): 1362-1371.doi: 10.16431/j.cnki.1671-7236.2024.04.004

• Biotechnology • Previous Articles    

Establishment and Application of a Chemiluminescent Detection Method for African Swine Fever Virus Antibody

XIANG Guoqing1,2, SUN Jinghan1,2, SONG Shuai2, WEN Xiaohui2, LYU Dianhong2, JIA Chunling2, NIU Ruihui2, GU Youfang1, LUO Shengjun2   

  1. 1. Anhui Provincial Key Laboratory of Animal Nutrition Regulation and Health, College of Animal Science, Anhui Science and Technology University, Fengyang 233100, China;
    2. Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangdong Provincial Key Laboratory of Livestock and Poultry Disease Prevention, Guangdong Provincial Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Technology of the Ministry of Agriculture and Rural Affairs, Guangzhou 510640, China
  • Received:2023-09-25 Published:2024-03-27

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Abstract: 【Objective】 The study was conducted to establish a chemiluminescent antibody detection method for African swine fever virus (ASFV) with high specificity,high sensitivity,simple operation and high throughput,which could be used for the early monitoring of the prevalence of ASFV.【Method】 In this study,we used carboxylated magnetic beads as a solid-phase carrier to couple ASFV recombinant p72 protein,which was closed with an appropriate amount of bovine serum albumin (BSA),and then added to the test sample for the reaction.Rabbit anti-porcine IgG-AP antibody was used as the enzyme-labeled antibody,and the corresponding substrate was added for the chromatography to optimize the conditions of each reaction, establish the chemiluminescent detection method of the ASFV antibody,and validate the specificity,sensitivity,reproducibility and detection rate of the method.【Result】 The chemiluminescence detection method for ASFV antibody was established,the optimal pH for protein coupling with carboxyl beads was 7.0,the optimal protein coupling concentration was 5 μg/mL,the best effect was achieved by using 10% BSA solution for closure,the final concentration of the reaction beads was 1.00 mg/mL,and the optimal dilution of enzyme-labeled secondary antibody was 1∶40 000.At the same time,the reaction time of this method was 30 min,and the serum dilution was 1∶512.The sensitivity of this method was higher than that of the commercial ASFV antibody ELISA detection kit,and there was no cross-reaction with the antibody-positive standard sera of Classical swine fever virus,Foot-and-mouth disease virus type A,Foot-and-mouth disease virus type O,Porcine reproductive and respiratory syndrome virus,Porcine circovirus type 2,Porcine pseudorabies virus gE,Porcine pseudorabies virus gB.In the reproducibility test,the coefficients of variation ranged from 4.41% to 8.70% in intra-assay and from 2.43% to 8.07% in inter-assay,which were both ＜10%.By testing 120 serum samples and analyzing them in comparison with commercial ASFV antibody ELISA detection kit,the detection rate was 96.7% for commercial ELISA kit and 100% for the chemiluminescent detection method established in this study.【Conclusion】 The ASFV chemiluminescent antibody detection method established in this study could be used to detect the antibody level after ASFV infection and provide a reference for the development of subsequent kits.

Key words: African swine fever virus (ASFV); recombinant p72 protein; chemiluminescence; antibody

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