庆大霉素人工抗原及多克隆抗体的制备与鉴定

Preparation and Identification of Gentamicin and Its Polyclonal Antibody

摘要:

为研究建立庆大霉素的免疫分析技术，合成庆大霉素人工抗原：采用碳二亚胺法将庆大霉素（gentamicin，GM）偶联到牛血清白蛋白（BSA）和鸡卵清蛋白（OVA）上，分别合成免疫原GM-BSA和包被原GM-OVA。通过紫外扫描和SDS-PAGE垂直电泳鉴定证明人工抗原偶联成功。以GM-BSA为免疫原，采用新的动物免疫方案免疫新西兰大白兔，获得抗庆大霉素抗血清。在本次免疫新方案中，在动物的生命周期内获得了多于普通免疫的血清量，在多抗血清的制备上是一次抗体工程上的尝试。通过间接酶联免疫对抗血清效价和灵敏度进行跟踪检测，结果表明抗血清效价在九免后稳定在1:20000左右，IC50则稳定在3 ng/mL左右。并在八免血清基础上建立了GM间接竞争ELISA法。结果表明，庆大霉素浓度在0.3~243 ng/mL，方法的线性回归方程为y=-0.1009lnx+0.6093和R2=0.9845，方法的检测灵敏度IC50=2.954 ng/mL，最低检测限IC10=0.056 ng/mL。该研究结果为多抗血清制备在量上的突破提供了思路，同时为研制庆大霉素试剂盒提供了良好的基础。

Abstract:

To develop immunoassay for gentamicin, the artificial antigen and polyclonal antibody were prepared in this study. Identified through ultraviolet spectrum (UV) and SDS-PAG, the immunogen (GM-BSA) and coating antigen (gm-ova) of gentamicin were synthesized by the method of carbodiimide successfully. GM-BSA was used to immune New Zealand white rabbits, and the antiserum was obtained. In this study, we utilize a notive method to immune animal and obtained serum volume much more than conventional method’s. One indirect competitive ELISA method has been developed to detect the titer and sensitivity of the antiserum. The results show that the titer of antiserum was continuously improving and the IC50 was nearly 3ng/mL. On this basis, With an concentration range from 0.3 to 243 ng/mL, the IC50 is 2.954 ng/mL, and the IC10 is 0.056 ng/mL. The results of this study provide a basis for the breakthrough of serum volume, and also is a good start for the development of gentamicin ELISA kit.