小反刍兽疫病毒H蛋白抗体化学发光免疫分析检测方法的建立

doi:10.13560/j.cnki.biotech.bull.1985.2022-0999

摘要/Abstract

摘要：

本研究旨在建立小反刍兽疫病毒H蛋白抗体的化学发光免疫分析检测方法。以H蛋白4个反应原性较好的B细胞表位串联后为检测抗原，在确定抗原包被量和血清稀释度，优化血清和酶标二抗反应时间的基础上，用ROC曲线分析确定检测临界值，建立了小反刍兽疫病毒H蛋白抗体化学发光免疫分析检测方法，然后对该方法进行敏感性、特异性和重复性评价。结果显示，建立的小反刍兽疫病毒H蛋白抗体化学发光免疫分析检测方法最佳抗原包被浓度为1.5×10^-6µg/孔，待检血清最佳稀释度为1∶100；血清及酶标二抗孵育时间均为10 min，检测方法的临界值为S/P=9.77%，批内及批间变异系数均小于10%；与口蹄疫、羊痘、蓝舌病及山羊传染性胸膜肺炎阳性血清不发生交叉反应；用建立的化学发光法和市售小反刍兽疫抗体cELISA试剂盒平行检测247份田间血清，两者相对符合率为94.33%，但化学发光法敏感性更高。研究结果表明，建立的小反刍兽疫病毒H蛋白抗体化学发光免疫分析方法灵敏、特异、稳定，可用于田间血清样品小反刍兽疫抗体检测。

关键词: 小反刍兽疫病毒, H蛋白, B细胞表位, 化学发光免疫分析法

Abstract:

This study aims to establish a chemiluminescence immunoassay for the detection of anti-PPRV H protein antibody. Four epitope peptides of B cell epitope of H protein, with good reactivity, were used as the detection antigen after synthesized together. After determining the antigen concentration for coating and working dilution for serum, the optimization of the incubation time for serum and enzyme-labeled secondary antibody, the ROC curve analysis was performed to determine the cut-off value of the assay. Thus the chemiluminescence immunoassay for the detection of anti-PPRV H protein was constructed, and the sensitivity, specificity and repeatability of this assay were evaluated. Results showed that the optimal coating concentration of antigen of this assay was 1.5×10^-6 μg/well, the optimal working dilution of the serum to be tested were 1∶100, and the incubation time of serum, and enzyme-labeled secondary antibody were 10 min respectively. The cut-off value of the assay was S/P=9.77%, the intra-assay and inter-assay coefficients of variation were tested to be < 10%. There was no cross-reaction obtained when positive sera of FMD, sheep pox, BT, and contagious caprine pleuropneumonia were used to test the assay, indicating that the assay had reasonable specificity. The detection results of 247 field sera showed that the established assay had a relative coincidence rate of 94.33% and much more sensitivity when compared with commercially available PPRV c-ELISA kit. In conclusion, this study has developed a specific, sensitive and stable chemiluminescence immunoassay method for detecting antibodies of PPRV H protein, which can be used to detect PPRV antibodies in field serum samples.

Key words: peste des petits ruminants virus, H protein, B cell epitope, chemiluminescence immunoassay

钱榜, 刘振东, 赵印, 李静, PRAJAPATI Meera, 李彦敏, 孙跃峰, 窦永喜. 小反刍兽疫病毒H蛋白抗体化学发光免疫分析检测方法的建立[J]. 生物技术通报, 2023, 39(5): 120-129.

QIAN Bang, LIU Zhen-dong, ZHAO Yin, LI Jing, PRAJAPATI Meera, LI Yan-min, SUN Yue-feng, DOU Yong-xi. Establishment of Chemiluminescence Immunoassay for the Detection of Peste des Petits Ruminants Virus H Protein Antibodies[J]. Biotechnology Bulletin, 2023, 39(5): 120-129.

图/表 11

参考文献 37

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表位Epitope	序列Sequence, N-C	长度Length/AA
H123	KFLNPDREYDFRDLR	15
H185	GTGCLGRTVTRA	8
H362	EANWVVPSTDVRDL	14
H487	IRGPRGRCH	9

表位Epitope	序列Sequence, N-C	长度Length/AA
H123	KFLNPDREYDFRDLR	15
H185	GTGCLGRTVTRA	8
H362	EANWVVPSTDVRDL	14
H487	IRGPRGRCH	9

标准Criterion	敏感性Sensitivity	95%置信区间95% CI	特异性Specificity	95%置信区间95% CI	阳性似然比+LR	阴性似然比-LR
> 8.72	96.34	93.7-98.1	89.32	81.7-94.5	9.02	0.041
> 8.88	96.34	93.7-98.1	90.29	82.9-95.2	9.92	0.041
> 9.06	96.34	93.7-98.1	91.26	84.1-95.9	11.03	0.040
> 9.4	96.04	93.3-97.9	91.26	84.1-95.9	10.99	0.043
> 9.52	95.73	92.9-97.6	91.26	84.1-95.9	10.96	0.047
> 9.56	95.43	92.6-97.4	91.26	84.1-95.9	10.92	0.050
> 9.77	95.43	92.6-97.4	92.23	85.3-96.6	12.29	0.050
> 9.93	95.12	92.2-97.2	92.23	85.3-96.6	12.25	0.053
> 10.07	94.82	91.8-97.0	92.23	85.3-96.6	12.21	0.056
> 10.41	94.51	91.5-96.7	92.23	85.3-96.6	12.17	0.059
> 10.45	94.21	91.1-96.5	92.23	85.3-96.6	12.13	0.063
> 10.77	93.90	90.7-96.2	92.23	85.3-96.6	12.09	0.066
> 10.92	93.60	90.4-96.0	92.23	85.3-96.6	12.05	0.069

标准Criterion	敏感性Sensitivity	95%置信区间95% CI	特异性Specificity	95%置信区间95% CI	阳性似然比+LR	阴性似然比-LR
> 8.72	96.34	93.7-98.1	89.32	81.7-94.5	9.02	0.041
> 8.88	96.34	93.7-98.1	90.29	82.9-95.2	9.92	0.041
> 9.06	96.34	93.7-98.1	91.26	84.1-95.9	11.03	0.040
> 9.4	96.04	93.3-97.9	91.26	84.1-95.9	10.99	0.043
> 9.52	95.73	92.9-97.6	91.26	84.1-95.9	10.96	0.047
> 9.56	95.43	92.6-97.4	91.26	84.1-95.9	10.92	0.050
> 9.77	95.43	92.6-97.4	92.23	85.3-96.6	12.29	0.050
> 9.93	95.12	92.2-97.2	92.23	85.3-96.6	12.25	0.053
> 10.07	94.82	91.8-97.0	92.23	85.3-96.6	12.21	0.056
> 10.41	94.51	91.5-96.7	92.23	85.3-96.6	12.17	0.059
> 10.45	94.21	91.1-96.5	92.23	85.3-96.6	12.13	0.063
> 10.77	93.90	90.7-96.2	92.23	85.3-96.6	12.09	0.066
> 10.92	93.60	90.4-96.0	92.23	85.3-96.6	12.05	0.069

血清编号 Serum No.	检测样品S/P值 S/P value of a detection sample/%			结果判定 Result determination
血清编号 Serum No.	A	B	C	结果判定 Result determination
PPRV（+）	18.17	22.13	13.46	+
PPRV（-）	0.27	0.33	0.28	-
FMDV（+）	7.74	9.10	5.85	-
SPV（+）	5.78	6.37	9.45	-
BTV（+）	3.31	6.69	8.48	-
MCCP（+）	5.74	4.72	2.04	-