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

摘要/Abstract

摘要： 本研究旨在建立定量检测猪瘟病毒（CSFV）E2重组蛋白的双抗体夹心ELISA方法。用 CSFV WH303单克隆抗体包被96孔酶标板，用CSFV 1B6单克隆抗体连接HRP作为酶标抗体，以杆状病毒表达纯化的CSFV E2蛋白作为标准品蛋白，建立双抗体夹心ELISA定量方法。用SDS-PAGE方法检测标准品蛋白的纯度，BCA蛋白定量法定量标准品蛋白的浓度。稀释标准品至线性区间，绘制标准曲线。用棋盘法确定包被单克隆抗体和酶标抗体及标准品蛋白的最适稀释度。用批间重复和批内重复试验验证该方法的重复性和稳定性。SDS-PAGE结果显示，CSFV E2蛋白的标准品纯度>95%，BCA蛋白定量法测定CSFV E2蛋白的标准品浓度为1.553 mg/mL。包被单克隆抗体的浓度为0.1 μg/mL，酶标抗体的最适稀释度为1∶400。CSFV E2蛋白标准品稀释浓度在2.43~77.65 μg/mL范围内有很好的线性关系，相关系数R²值在0.99以上。批间和批内重复性检测试验变异系数均<15%。本试验成功建立了定量检测CSFV E2重组蛋白的双抗体夹心ELISA检测方法，该方法具有良好的重复性和稳定性，为CSFV E2蛋白的定量提供了有效方法。

关键词: 猪瘟病毒(CSFV); E2蛋白; 双抗体夹心ELISA法

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

中图分类号:

S858.28

候玉珍, 王遵宝, 郑侃, 赵兵, 冶莲, 姜智文, 唐慧芬, 候凤, 贺笋. 猪瘟病毒E2蛋白双抗体夹心ELISA定量方法的建立[J]. 中国畜牧兽医, 2020, 47(5): 1523-1530.

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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