Preliminary Establishment of Indirect ELISA Method Based on African Swine Fever Virus P72 Recombinant Protein

doi:10.11924/j.issn.1000-6850.casb2025-0241

Abstract

Abstract:

This study aimed to develop a rapid indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against African swine fever virus (ASFV) in clinical application, and the recombinant P72 protein was obtained using the recombinant plasmid pET-28a-P72 through IPTG induction and nickel affinity purification. Its reactivity was confirmed, and the purified protein was used as the coating antigen. After optimizing key parameters, an indirect ELISA method for detecting antibodies against ASFV P72 protein was preliminarily established. The ASFV P72 protein was successfully expressed and specifically reacted with ASFV-positive serum antibodies. The optimized assay conditions were as follows: coating antigen concentration at 5 μg/mL, serum dilution at 1:160, coating at 4℃ for 12 h, serum incubation time of 30 min, HRP-labeled secondary antibody dilution at 1:1000, secondary antibody incubation time of 30 min, and substrate reaction time of 10 min. The cut-off values were defined as follows: OD₄₅₀≥0.69 was considered positive, OD₄₅₀≤0.57 was negative, and values between these thresholds were considered suspicious. No cross-reactivity was observed with antisera against six pathogens including classical swine fever virus (CSFV). The sensitivity reached 1:1600, and both intra- and inter-batch coefficients of variation were less than 15%, demonstrating high accuracy and reliability. An indirect ELISA method for detecting antibodies against ASFV P72 protein was successfully established. This assay can be used for ASFV antibody detection and provides technical support for epidemic monitoring and early warning.

Key words: African swine fever virus, recombinant P72 protein, prokaryotic expression, indirect ELISA

XIANG Guoqing, DENG Tianbo, SONG Shuai, YANG Yanqiu, CHEN Yuting, CHEN Meixia, WU Guojing, ZHENG Bobin, NIU Ruihui, WEN Xiaohui, LUO Shengjun. Preliminary Establishment of Indirect ELISA Method Based on African Swine Fever Virus P72 Recombinant Protein[J]. Chinese Agricultural Science Bulletin, 2025, 41(33): 157-164.

Figures/Tables 11

References 22

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血清稀释度	抗原包被浓度/(μg/mL)
	5			10			20			30
	P	N	P/N	P	N	P/N	P	N	P/N	P	N	P/N
1:40	1.519	0.166	9.2	1.394	0.205	6.8	1.329	0.197	6.75	1.33	0.155	11.56
1:80	1.235	0.106	11.7	1.131	0.114	9.92	1.349	0.141	9.57	1.052	0.103	10.21
1:160	1.286	0.056	22.9	0.731	0.081	9.02	1.117	0.079	14.14	1.093	0.07	15.61
1:320	1.127	0.054	20.8	0.921	0.072	12.8	1.286	0.071	18.11	1.086	0.09	12.06
1:640	0.952	0.062	15.3	0.932	0.085	10.9	1.043	0.094	11.09	0.958	0.162	5.91

血清稀释度	抗原包被浓度/(μg/mL)
	5			10			20			30
	P	N	P/N	P	N	P/N	P	N	P/N	P	N	P/N
1:40	1.519	0.166	9.2	1.394	0.205	6.8	1.329	0.197	6.75	1.33	0.155	11.56
1:80	1.235	0.106	11.7	1.131	0.114	9.92	1.349	0.141	9.57	1.052	0.103	10.21
1:160	1.286	0.056	22.9	0.731	0.081	9.02	1.117	0.079	14.14	1.093	0.07	15.61
1:320	1.127	0.054	20.8	0.921	0.072	12.8	1.286	0.071	18.11	1.086	0.09	12.06
1:640	0.952	0.062	15.3	0.932	0.085	10.9	1.043	0.094	11.09	0.958	0.162	5.91

抗原包被条件	阳性样品OD₄₅₀值	阴性样品OD₄₅₀值	P/N值
4℃封闭12 h	1.519	0.166	9.15
37℃封闭2 h	1.369	0.213	6.42

抗原包被条件	阳性样品OD₄₅₀值	阴性样品OD₄₅₀值	P/N值
4℃封闭12 h	1.519	0.166	9.15
37℃封闭2 h	1.369	0.213	6.42

反应时间/min	阳性样品OD₄₅₀值	阴性样品OD₄₅₀值	P/N值
20	0.637	0.147	4.33
30	1.531	0.092	16.64
40	1.124	0.159	7.07
50	0.954	0.257	3.71
60	1.256	0.379	3.31