猪瘟病毒E2蛋白双抗体夹心ELISA定量方法的建立

doi:10.16431/j.cnki.1671-7236.2020.05.027

Abstract

Abstract: The purpose of this study was to establish a double antibody sandwich ELISA method for quantitative detection of classical swine fever virus (CSFV) E2 recombinant protein.CSFV WH303 monoclonal antibody was used to coat 96 well enzyme-linked plate,CSFV 1B6 monoclonal antibody was used to connect HRP as enzyme-linked antibody,CSFV-E2 protein expressed and purified by baculovirus was used as standard protein,and a quantitative method of double antibody sandwich ELISA was established.The purity of standard protein was detected by SDS-PAGE and the concentration of standard protein was quantified by BCA protein quantitative method.The standard was diluted to the linear range and draw the standard curve.Chessboard method was used to determine the optimal dilutions of monoclonal antibody,enzyme labeled antibody and standard protein.The repeatability and stability of the method were verified intra-assay and inter-assay.SDS-PAGE result that the purity of CSFV E2 protein standard was more than 95%,and the concentration of CSFV E2 protein standard by BCA protein quantitative method was 1.553 mg/mL.The concentration of the coated monoclonal antibody was 0.1 g/mL,and the optimal dilution of the enzyme labeled antibody was 1:400.The dilution concentration of CSFV E2 protein standard was in the range of 2.43 to 77.65 g/mL,and the correlation coefficient R² was above 0.99.The coefficients of variation of repeatability test of intra-assay and inter-assay were both less than 15%.A double antibody sandwich ELISA method for the quantitative detection of CSFV E2 protein was successfully established.The method had good repeatability and stability,and provided an effective method for the quantitative detection of CSFV E2 protein.

Key words: swine fever virus (CSFV); E2 protein; double antibody sandwich ELISA method

CLC Number:

S858.28

HOU Yuzhen, WANG Zunbao, ZHENG Kan, ZHAO Bing, YE Lian, JIANG Zhiwen, TANG Huifen, HOU Feng, HE Sun. Establishment of Sandwich ELISA Method for Quantitative Analysis of Classical Swine Fever Virus E2 Protein[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(5): 1523-1530.

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Establishment of Sandwich ELISA Method for Quantitative Analysis of Classical Swine Fever Virus E2 Protein

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