基于H蛋白表位合成肽的小反刍兽疫病毒抗体间接ELISA检测方法的建立

doi:10.11843/j.issn.0366-6964.2021.015

Abstract

Abstract: This study aims to establish an iELISA method for the detection of anti-PPRV H protein antibody. Four epitope peptides with good reactivity were selected and synthesized as the coating antigen based on the B cell epitopes of H protein. Optimization of the reaction conditions of each step and screening various buffers were performed and the cut-off value of detection was determined. After optimization, the optimal coating amount of multi-epitopes antigen peptide was 1.5×10^-6 μg·well^-1, the optimal working dilution of serum and the enzyme-labeled secondary antibody was 1:100 and 1:80 000, respectively. The carbonate-hydrogen buffer was chosen as the coating buffer, and PBST buffer was adopted as dilution buffer for the serum and the secondary antibody. The appropriate blocking buffer was determined to be 2% BSA solution and the cut-off value of the assay was 0.25. The test results of clinical sera showed that the coefficients of variation of intra- and inter-assay were less than 10%, which proved its good repeatability. When the assay was tested with the positive sera of sheep pox, FMD, and BT, there was no cross-reaction obtained, indicating that the assay was specific for the detection of the PPRV antibody. The detection results of 306 sera samples showed that the developed ELISA had a relative sensitivity of 92.81% when compared with the commercially available ID Vet cELISA Kit. This study has developed a specific, repeatable, and sensitive iELISA method for detecting antibodies of PPRV H protein, and it is more convenient, timesaving, and labor-saving than cELISA. Therefore, the assay can be used for the detection of antibodies against PPRV.

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

CLC Number:

S852.659.5

QIAN Bang, LI Yanmin, ZHU Xueliang, ZHANG Xueyan, Niyokwishimira Alfred, DOU Yongxi, ZHANG Zhidong. Establishment of an iELISA Method for Detection of Antibody againist Peste des Petits Ruminants Virus Based on H Protein Epitope Peptide[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(1): 144-153.

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Establishment of an iELISA Method for Detection of Antibody againist Peste des Petits Ruminants Virus Based on H Protein Epitope Peptide

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