Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (16): 3237-3246.doi: 10.3864/j.issn.0578-1752.2023.16.015

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Development and Application of a Mab-Based iELISA for the Detection of Antibodies Against African Horse Fever Virus

GUO Kui(), ZHANG ZeNan, WANG YaoXin, LI ShuaiJie, CHU XiaoYu, GUO Wei, HU Zhe(), WANG XiaoJun()   

  1. State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069
  • Received:2022-05-25 Accepted:2022-10-23 Online:2023-08-16 Published:2023-08-18

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Abstract:

【Objective】In order to effectively address the risk of introduction of African horse fever into China, this study aimed to develop an indirect ELISA (iELISA) antibody test specific for African horse fever virus (AHSV) based on monoclonal antibodies, so as to monitor the antibody status of African horse fever in horses in China.【Method】Firstly, Mabs against VP7 antigen were prepared by immunizing mice with the VP7 antigen. Secondly, the ELISA method was developed by optimizing the amount of coating Mab, antigen and secondary antibody concentrations. Negative sera (n=1 000) were used to establish the baseline for a negative population, and then the specificity and sensitivity of it was estimated. The stability of the method was determined by an accelerated test using AHSV-positive reference sera by three independent operators. 10 known positive sera and 400 negative sera for African horse fever were tested using the established method and compared with the results of foreign commercial kits. Finally, the method was tested on 947 clinical samples from 18 provinces or regions in China in 2021 to assess the risk of African horse fever in horses in China.【Result】 Five Mabs were obtained against the VP7 antigen of AHSV, and the 3G9 Mab had the best antigen capture performance. Using the 3G9 Mab as the capture antibody, an ELISA method for the detection of AHSV was established by optimizing different reaction conditions. A cut-off value of 0.25 was selected in AHSV iELISA. The sensitivity of ELISA method was comparable with that of commercial kits and the sensitivity of both methods was consistent. The intra-group coefficients of variation from 3 operators ranged from 3.19% to 7.02%, 0% to 3.11%, and 0.27% to 5.76%, respectively, and the inter-group coefficients of variation for the three operators ranged from 1.17 to 5.03%. The accelerated test at 37 °C demonstrated that the components of the testing kit had good stability over 7 days, thus demonstrating the stability of the method. The results showed that the agreement between the AHSV iELISA and commercial AHSV C ELISA kits were 100%. In this study, a serological survey on AHSV was performed by using 947 serum samples collected from 18 provinces of China in 2021, and the results showed that the positive rates were 0%. 【Conclusion】 A Mab-based AHSV iELISA method was established for the detection of AHSV antibodies, which had the good specificity and sensitivity, and could be a promising candidate tools for use serological surveillance for AHS.

Key words: African horse fever virus, monoclonal antibody, indirect ELISA, application

Fig. 1

SDS-PAGE analysis of recombinant VP7 protein M: Protein marker; 1: Supernatant of VP7 protein after IPTG induction; 2: Sediment of VP7 protein after IPTG induction"

Fig. 2

Western blot analysis of recombinant VP7 protein M: Protein marker; 1: pET32a vector; 2: Purified VP7 protein"

Fig. 3

Sensitivity of AHSV iELISA and commercial cELISA"

Table 1

Results of intergroup and intro-group repeatability of AHSV iELISA"

血清稀释度
Serum dilution		 组内变异系数 Intra-group coefficient of variation (%) 组间变异系数
Coefficient of variation between groups (%)
操作者1 Member 1 操作者2 Member 2 操作者3 Member 3
1× 5.74 2.45 5.76 3.99
2× 7.02 2.03 0.83 5.03
4× 6.44 0.00 0.27 1.17
8× 3.19 0.50 0.67 4.47
16× 4.09 3.11 0.82 1.34
32× 5.41 0.11 1.58 4.22

Fig. 4

Results of S/P values on different day"

Table 2

Comparison of AHSV iELISA with commercial cELISA"

商品化竞争ELISA
Commercial cELISA
+ -
非洲马瘟病毒间接ELISA
AHSV iELISA		 + 10 0
- 0 400
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