| 牛乳铁蛋白直接竞争ELISA检测法的建立 |
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| 引用本文： | 冯挺财,邵谱,胡珩,杨正涛,张乃生.牛乳铁蛋白直接竞争ELISA检测法的建立[J].安徽农业科学,2008,36(18). |
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| 作者姓名： | 冯挺财 邵谱 胡珩 杨正涛 张乃生 |
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| 作者单位： | 吉林大学畜牧兽医学院动物营养与中毒病研究室,吉林长春,130062 |
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| 摘 要： | 目的]建立一种检测牛乳铁蛋白(LF)含量的竞争ELISA检测方法。方法]制备鼠抗牛LF单克隆抗体,采用直接竞争ELISA测定酶标记物效价,建立牛LF的直接竞争ELISA检测方法。结果]纯化后,制备的鼠抗牛LF单抗效价达1∶1 280 000,且鉴定为IgG1。该单抗对牛乳铁蛋白具有很强的特异性。制备酶标单抗的效价为1∶640 000。抗原包被物和酶标单抗的最适工作浓度分别为1.0μg/ml和1∶40 000。抗原用碳酸盐缓冲液(pH值为9.6)包被先在4℃过夜、再37℃放置2 h的效果最好。采用2%牛血清白蛋白作为封闭剂的效果最好。在31.25~1 000 ng/ml浓度范围内,牛乳铁蛋白的logit(B/B0)与牛乳铁蛋白浓度的对数值呈显著的线性关系,其回归方程为y=-1.899 1x+5.043 3(R2=0.987 3)。结论]该研究为研制用于奶牛乳腺炎诊断的牛LF检测试剂盒奠定基础。
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| 关 键 词： | 牛乳铁蛋白 cELISA 定量检测 |
Establishment of Direct Competitive ELISA for Detecting Bovine Lactoferrin |
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| FENG Ting-cai et al.Establishment of Direct Competitive ELISA for Detecting Bovine Lactoferrin[J].Journal of Anhui Agricultural Sciences,2008,36(18). |
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| Authors: | FENG Ting-cai |
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| Abstract: | Objective] The research aimed to establish a kind of competitive ELISA method for detecting the content of bovine lactoferrin(Lf).Method] Rat anti-bovine LF monoclonal antibody was prepared and the titer of enzyme markers were determined by using direct competitive ELISA.And a direct competitive ELISA for detecting bovine LF was established.Result] After purification,the titer of the prepared rat anti-bovine LF monoclonal antibody reached 1∶1 280 000 and it was ifdentified as IgG1.The monoclonal antibody had stronger specificity to bovine LF.The titer of the prepared enzyme-labelled monoclonal antibody was 1∶640 000.The optimum working concn.of antigen coating substance and enzyme-labelled monoclonal antibody were 1.0 ug/ml and 1:40 000 resp.The effect of using coating antigen with carbonate buffer with pH value of 9.6 to stay over a night at 4 ℃first and then lay at 37 ℃ for 2 h was best.The effect of using 2% bovine serum albumin as sealant was best.In the cocn.range of 31.25～1 000 ng/ml,logit(B/B0) of bovine LF showed a significant linear relationship with the logarithm value of bovine LF concn.and its regression equation was y=-1.899 1x+5.043 3(R2=0.987 3).Conclusion] This research laid the foundation for developing bovine LF detection kit used for the mastitis diagnosis of dairy cattle. |
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| Keywords: | Bovine lactoferrin Direct competitive ELISA Quantitative detection |
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