牛乳铁蛋白直接竞争ELISA检测法的建立

Establishment of Direct Competitive ELISA for Detecting Bovine Lactoferrin

[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1：1280000，且鉴定为IgGl。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1：640000。抗原包被物和酶标单抗的最适工作浓度分别为1．0μg／ml和1：40000。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2h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31．25．1000 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γ=-1．8991x＋5．0433（R^2=0．9873）。[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。

[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 manoclonal 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/Bo） of bovine LF showed a significant linear relationship with the logarithm value of bovine LF concn, and its regression equation was y = - 1. 899 1 x ＋ 5. 043 3 （ R^2 = 0.987 3）. [ Conclusion ] This research laid the foundation for developing bovine LF detection kit used for the mastitis diagnosis of dairy oattle.