沙丁胺醇多克隆抗体的制备与鉴定

Preparation and Identification of Polyclonal Antibodies against Salbutamol

摘要:

为了对沙丁胺醇进行酶联免疫吸附法（ELISA）测定，通过四种方法制备沙丁胺醇衍生物，分别将其与牛血清蛋白（BSA）、卵清蛋白（OVA）偶联，制备人工完全抗原，采用紫外分光光度法测定蛋白质浓度并计算偶联比，用所制备的四种免疫原免疫新西兰白兔获得抗沙丁胺醇（SAL）抗体，每一种抗体都用四种包被抗原进行配对筛选，据此得到最佳的抗体-包被抗原的组合，对筛选出的抗体用正辛酸-硫酸铵法进行纯化，采用间接ELISA法测定多克隆抗体（pAb）的效价并进行特异性鉴定。结果表明：成功偶联了完全抗原，四种免疫原的偶联比分别为9:1、6:1、6:1、12:1，得到最佳的抗体-包被抗原的组合，筛选出的抗体效价达到1:64000，此抗体对克伦特罗和特布他林的交叉反应率分别为144%和169%，对其他类似物几乎没有交叉反应。本研究为沙丁胺醇、克伦特罗和特布他林多残留酶联免疫检测法的建立奠定了基础。

Abstract:

In order to prepare the polyclonal antibody against Salbutamol(SAL), SAL derivatives were prepared by four kinds of reactions, then they were coupled to BSA and OVA, synthesizing complete antigen. The protein concentration was determined by Ultraviolet spectrophotography and the coupling ratios of four immunogens were calculated. New Zealand white rabbits were immunized by four immunogens to develop anti-SAL ant?ibodies. All the antibodies and coating antigenes were screened and then selected the best combination. The polyclonal antiserum was purified by caprylic acid-ammonium sulfate precipitation. Antibody titer and specificity were analyzed by indirect ELISA. It turned out that the study successfully got the coupling of the SAL complete antigen and the coupling ratios of four immunogens were 9:1, 6:1, 6:1, 12:1, respectively. The optimal antibody-coated antigen combination was obtained and antibody titer was 1:64000. The antibody showed high cross-reactivity with clenbuterol(144%) and terbutaline (169%), without notable cross-reactivity with other structural analogues of SAL. This study lays the foundation for the establishment of Salbutamol, Clenbuterol and Terbutaline multi-residue enzyme-linked immunosorbent assay.