间接ELISA法在注射用矛头蝮蛇血凝酶蛇毒种属鉴别中的应用

The application of indirect ELISA on snake venom species identification in Hemocoagulase Bothrops atroxfor Injection

目的建立间接ELISA法定性检测矛头蝮蛇血凝酶,用于巴曲亭蛇毒种属来源检测。方法将矛头蝮蛇血凝酶为抗原免疫小鼠,与尖吻蝮蛇血凝酶、白眉蝮蛇血凝酶交叉筛选获得特异性单克隆抗体。优化矛头蝮蛇血凝酶单抗(一抗)、HRP标记的羊抗鼠(二抗)IgG、矛头蝮蛇血凝酶(抗原)工作浓度,建立间接ELISA检测方法。考察方法的重复性、中间精密度,确定方法的适用性。采用膜超滤法将巴曲亭中辅料去除,按照建立的ELISA法检测,判定种属来源。结果矛头蝮蛇血凝酶单抗具有强特异性,一抗的最佳工作浓度1∶4000(250ng),二抗的最佳工作浓度1∶4000(250ng),抗原浓度2μg/孔,该法重复性和中间精密度较好。巴曲亭与矛头蝮蛇血凝酶单抗有特异性反应,与其它蛇毒来源血凝酶单抗无交叉反应,确定巴曲亭来源于矛头蝮蛇蛇毒。结论本研究建立的ELISA法具有较好的特异性,可作为巴曲亭蛇毒种属来源判断的方法。

Objective The indirect ELISA for qualitative analysis of hemocoagulase from the snake venom of Bothrops atroxwas established,applied in testing Baquting snake venom species. Methods The mice were injected the hemocoagulase from the snake venom of Bothrops atrox,and the specifically monoclonal antibodies had been obtained by cross selecting that involved in the haemocoagulase from the snake venom of Agkistrodon acutus（Guenther）and Agkistrodon halys（ussriensis Emelianor）.The hemocoagulasemonoclonal antibodies（primary antibody）from the snake venom of Bothrops atroxconcentration had been optimized,as well as the HRP-conjugated goat anti-mouse IgG（secondary antibody）and the hemocoagulase from the snake venom of the Bothropsatrox（antigen）.The repeatability and intermediate precision of indirect ELISA were studied.The excipients of Baquting were removed by ultrafiltration,and the snake venom species was tested by indirect ELISA. Results The monoclonal antibody of Bothrops atrox snake venom was specific,and the primary antibody optimum concentration was 1∶4000（250ng）,as well as the secondary antibody,and the antigen is 2μgper well.Its repeatability and intermediate precision werewell.Baquting was specifically detected bythe hemocoagulasemonoclonal antibody from the snake venom of Bothrops atroxonly,and no cross reaction with other snake venom hemocoagulase monoclonal antibody. Conclusion The snake species of Baquting is Bothrops atrox.The indirect ELISA method specificity is well and can be appliedto detect thesnake venom species of Baquting.