Establishment and application of an indirect ELISA method for detecting antibodies against fowlpox virus ORF127 protein

doi:10.3969/j.issn.1004-1524.20250139

Acta Agriculturae Zhejiangensis ›› 2025, Vol. 37 ›› Issue (10): 2057-2065.DOI: 10.3969/j.issn.1004-1524.20250139

• Animal Science • Previous Articles Next Articles

Establishment and application of an indirect ELISA method for detecting antibodies against fowlpox virus ORF127 protein

HUA Jionggang¹(), ZHU Yinchu¹, YE Jialin², ZHANG Cun¹, CHEN Liu¹, NI Zheng¹, FU Yuan¹, HUO Suxin¹, YUN Tao¹^,^*()

1. Zhejiang Key Laboratory of Livestock and Poultry Biotech Breeding, Key Laboratory of Livestock and Poultry Resources (Poultry) Evaluation and Utilization, Ministry of Agriculture and Rural Affairs, Zhejiang Engineering Research Center for Poultry Breeding Industry and Green Farming Technology, Institute of Animal Husbandry and Veterinary Sciences, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
2. International Division, Hangzhou High School, Hangzhou 310020, China

Received:2025-02-24 Online:2025-10-25 Published:2025-11-13

Abstract

Abstract:

To establish a rapid and accurate method for detecting fowlpox virus (FPV) antibodies, this study cloned the antigenic epitope region of FPV ORF127 gene into the pET-28a(+) prokaryotic expression vector. The truncated FPV ORF127 protein was successfully expressed and purified. Using this recombinant protein as a coating antigen, an indirect ELISA was developed through systematic optimization of reaction parameters. The method was comprehensively validated, including specificity, sensitivity, repeatability tests, and clinical serum sample detection. The results showed that the optimal reaction conditions for the indirect ELISA were as follows: coating antigen concentration of 10 μg·mL^-1, serum dilution of 1∶100, and enzyme-labeled secondary antibody dilution of 1∶25 000. Specificity tests revealed that except for FPV-positive serum, no cross-reactions were observed with positive sera against six other viruses (Newcastle disease virus, H9 subtype avian influenza virus, infectious bursal disease virus, infectious laryngotracheitis virus, fowl adenovirus type 4, and turkey herpesvirus). Sensitivity tests showed that FPV-positive serum remained detectable at a dilution of 1∶800. Reproducibility tests demonstrated that the intra-assay and inter-assay coefficients of variation were 1.81%-7.07% and 2.12%-7.16%, respectively, both below 10%. Clinical serum sample testing showed a total positivity rate of 79.4% (143/180) among 180 samples. These results indicate that the established indirect ELISA method exhibits high specificity, sensitivity, reproducibility, and stability. It can provides a reliable tool for detecting FPV antibodies in vaccinated flocks and supporting FPV epidemiological investigations.

Key words: fowlpox virus, ORF127 protein, indirect ELISA, antibody

CLC Number:

S852.657

HUA Jionggang, ZHU Yinchu, YE Jialin, ZHANG Cun, CHEN Liu, NI Zheng, FU Yuan, HUO Suxin, YUN Tao. Establishment and application of an indirect ELISA method for detecting antibodies against fowlpox virus ORF127 protein[J]. Acta Agriculturae Zhejiangensis, 2025, 37(10): 2057-2065.

Figures/Tables 8

References 23

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血清稀释度 Serum dilution	项目 Item	不同FPV-127抗原质量浓度的吸光度Absorbance of FPV-127 antigens with different mass concentration
血清稀释度 Serum dilution	项目 Item	20 μg·mL^-1	10 μg·mL^-1	5 μg·mL^-1	2.5 μg·mL^-1	1.25 μg·mL^-1	0.625 μg·mL^-1
1∶25	P	2.220	2.003	1.590	1.138	0.793	0.671
	N	0.673	0.403	0.243	0.167	0.129	0.210
	P/N	3.299	4.970	6.543	6.814	6.117	3.195
1∶50	P	2.146	1.799	1.130	0.753	0.486	0.404
	N	0.389	0.243	0.145	0.101	0.088	0.086
	P/N	5.517	7.403	7.793	7.485	5.523	4.698
1∶100	P	2.117	1.474	0.896	0.497	0.291	0.252
	N	0.257	0.148	0.096	0.074	0.062	0.066
	P/N	8.237	9.959	9.333	6.716	4.694	3.818
1∶200	P	1.896	1.145	0.562	0.311	0.189	0.149
	N	0.215	0.123	0.099	0.065	0.067	0.054
	P/N	8.817	9.309	5.677	4.785	2.821	2.759

血清稀释度 Serum dilution	项目 Item	不同FPV-127抗原质量浓度的吸光度Absorbance of FPV-127 antigens with different mass concentration
血清稀释度 Serum dilution	项目 Item	20 μg·mL^-1	10 μg·mL^-1	5 μg·mL^-1	2.5 μg·mL^-1	1.25 μg·mL^-1	0.625 μg·mL^-1
1∶25	P	2.220	2.003	1.590	1.138	0.793	0.671
	N	0.673	0.403	0.243	0.167	0.129	0.210
	P/N	3.299	4.970	6.543	6.814	6.117	3.195
1∶50	P	2.146	1.799	1.130	0.753	0.486	0.404
	N	0.389	0.243	0.145	0.101	0.088	0.086
	P/N	5.517	7.403	7.793	7.485	5.523	4.698
1∶100	P	2.117	1.474	0.896	0.497	0.291	0.252
	N	0.257	0.148	0.096	0.074	0.062	0.066
	P/N	8.237	9.959	9.333	6.716	4.694	3.818
1∶200	P	1.896	1.145	0.562	0.311	0.189	0.149
	N	0.215	0.123	0.099	0.065	0.067	0.054
	P/N	8.817	9.309	5.677	4.785	2.821	2.759

项目 Item	不同酶标二抗稀释度的吸光度Absorbance with different dilutions of enzyme-labeled second antibody
项目 Item	1∶10 000	1∶15 000	1∶20 000	1∶25 000	1∶30 000	1∶35 000
P	2.227	2.073	1.833	1.693	1.540	1.352
N	0.207	0.133	0.110	0.108	0.103	0.101
P/N	10.758	15.586	16.664	15.676	14.951	13.386

项目 Item	不同酶标二抗稀释度的吸光度Absorbance with different dilutions of enzyme-labeled second antibody
项目 Item	1∶10 000	1∶15 000	1∶20 000	1∶25 000	1∶30 000	1∶35 000
P	2.227	2.073	1.833	1.693	1.540	1.352
N	0.207	0.133	0.110	0.108	0.103	0.101
P/N	10.758	15.586	16.664	15.676	14.951	13.386

血清 Serum	稀释倍数 Dilution ratio	D₄₅₀ D₄₅₀
阳性血清Positive serum	100	1.394
	200	0.988
	400	0.652
	800	0.403
	1 600	0.259
	3 200	0.165
	6 400	0.117
阴性血清Negative serum	100	0.123

Establishment and application of an indirect ELISA method for detecting antibodies against fowlpox virus ORF127 protein

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Figures/Tables 8

References 23

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血清编号 Serum number	批内重复试验Intra batch repeated experiment			批间重复试验Inter batch repeated experiment
血清编号 Serum number	D₄₅₀平均值 Average of D₄₅₀ value	标准差 Standard deviation	变异系数 Coefficient of variation/%	D₄₅₀平均值 Average of D₄₅₀ value	标准差 Standard deviation	变异系数 Coefficient of variation/%
1	1.448	0.029	2.00	1.410	0.041	2.91
2	1.161	0.021	1.81	1.132	0.024	2.12
3	0.779	0.015	1.93	0.768	0.022	2.86
4	0.615	0.019	3.08	0.628	0.045	7.16
5	0.388	0.012	3.09	0.376	0.015	3.99
6	0.341	0.014	4.09	0.352	0.012	3.41
7	0.163	0.009	5.52	0.162	0.010	6.17
8	0.113	0.008	7.07	0.110	0.004	3.63

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