| 摘要: |
| 【目的】研究并建立大豆抗原蛋白血清抗体间接ELISA检测方法。【方法】首先用大豆抗原蛋白对仔猪进行致敏，然后采用棋盘滴定法确定最佳抗原包被质量浓度及血清稀释倍数，并对其他条件进行优化，最终建立检测血清大豆抗原蛋白11S、7S抗体的间接 ELISA 方法，确立待测血清判定标准，并对建立的ELISA方法进行重复性、准确性和敏感性检测。【结果】建立的ELISA方法的最佳反应条件为：11S抗原蛋白最适包被质量浓度为2.0 μg/mL，包被作用时间37 ℃下2 h；将10 g/L BSA作为封闭液，37 ℃封闭1 h；待测血清37 ℃作用1 h；酶标二抗最适稀释度为1∶1 500，37 ℃作用1 h；显色剂最佳显色时间15 min。7S抗原蛋白的最适包被质量浓度为2.5 μg/mL，包被作用时间37 ℃下2 h；将10 g/L BSA 作为封闭液，37 ℃封闭1 h；待测血清37 ℃作用1 h；酶标二抗最适稀释度为1∶1 500，37 ℃作用1 h；显色剂最佳显色时间25 min。11S和7S抗原蛋白间接ELISA检测方法的批内、批间变异系数均小于10%，重复性较好，灵敏度较高。【结论】建立了仔猪血清大豆抗原蛋白抗体间接ELISA检测方法，该法具有很强的特异性和敏感性，可用于大豆抗原蛋白11S、7S过敏反应的临床诊断。 |
| 关键词: 大豆抗原蛋白 仔猪 血清抗体 间接ELISA |
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| 基金项目:科技部科技富民强县专项行动计划项目（国科发农【2012】745号） |
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| Establishment of an indirect enzyme-linked immune sorbent assay for detection of serum antibody of soybean antigen protein in piglets |
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KOU Ya-nan,MA Liang-you,LUO Ying,et al
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| Abstract: |
| 【Objective】The experiment was conducted to establish an indirect enzyme-linked immune sorbent assay (ELISA) to detect the serum antibody levels in piglets sensitized by soybean antigen protein.【Method】After the piglets were sensitized with 11S or 7S,the serum samples were collected and the reactive conditions were optimized.The optimal coating mass concentrations of antigens and dilution times of sera were determined using board titration method.An indirect enzyme-linked immune sorbent assay to detect the serum contents of 11S and 7S in piglets was then established and its repeatability,accuracy and sensitivity were detected.【Result】The optimal conditions of ELISA 11S were:the coating mass concentration was 2.0 μg/mL,the action time of the coating at 37 ℃ was 2 h;using 10 g/L BSA as sealing fluid for 1 h at 37 ℃;the test serum worked for 1 h at 37 ℃,the enzyme labeled antibody was diluted into 1∶1 500 and the reactive time was 1 h at 37 ℃,and the reaction time of substrate was 15 min.The optimal conditions of ELISA 7S were:the coating mass concentration was 2.5 μg/mL and the action time of the coating at 37 ℃was 2 h,using 10 g/L BSA as sealing fluid for 1 h at 37 ℃;the test serum worked for 1 h at 37 ℃,the enzyme labeled antibody was diluted into 1∶1 500 and the reaction time was 1 h at 37 ℃,and the reaction time of substrate was 25 min.Both intra-assay and inter-assay variation coefficients were less than 10%.【Conclusion】The established indirect ELISA for detection of serum antibody levels in piglets sensitized by soybean antigen protein had good repeatability and high sensitivity,and could be used in clinical diagnosis of allergy induced by soybean protein antigen. |
| Key words: soybean antigen protein piglets serum antibody indirect ELISA |
