Establishment of Chemiluminescence Immunoassay for the Detection of Peste des Petits Ruminants Virus H Protein Antibodies

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

Abstract

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

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.

Figures/Tables 11

References 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	-