强力霉素人工抗原的制备及鼠源多抗的ELISA鉴定

Preparation of Artificial Antigen and ELISA Identification of Mouse Polyclonal Antiserum for the Detection of Doxycycline

摘要:

为制备强力霉素（Doxycycline，DC）人工抗原并获得免疫学特性良好的DC鼠源多抗血清。本试验采用琥珀酸酣法在DC分子上引入羧基基团，然后采用改进碳二亚胺法（EDC）将其与载体蛋白偶联制备人工抗原，经紫外扫描（UV）和凝胶电泳（SDS-PAGE）鉴定其偶联效果，以30 μg/只的剂量（人工抗原）免疫4只BALB/c小鼠，并对所获鼠源多抗血清采用间接ELISA和间接竞争ELISA鉴定其免疫学特性。结果表明，人工抗原偶联效果良好，免疫的4只小鼠多抗血清效价均达到1:12800，且均有抑制效果，其中4号小鼠产生的多抗血清效果最佳，半数抑制浓度（IC50）为24.75 ng/mL，与土霉素、金霉素、四环素的交叉反应率分别为3.4%、3.8%、3.0%，与其他兽药及载体蛋白的交叉反应率均小于0.3%，特异性良好。本试验成功合成DC人工抗原并获得免疫学特性良好的DC鼠源多抗血清，为后期单克隆抗体的制备及免疫学快速检测方法的建立奠定良好基础。

Abstract:

To prepare complete antigen of doxycycline (DC) and obtain mouse polyclonal antiserum with good immunological characteristics, carboxyl groups were introduced into DC molecules by succinate method in this work. A Complete antigens were prepared by coupling with carrier proteins using an improved succinic acid method. The coupling effect of complete antigen was identified by UV and SDS-PAGE. Four BALB/c mouse were immunized with 30 μg artificial antigen, and the antiserum immunological characteristics were identified by indirect ELISA and indirect competitive ELISA. The results showed that the coupling effect of artificial antigen was good, the titer of antiserum of all four mouse were higher than 1:12800, the serum of all four mouse had inhibitory effect. The antiserum produced by mouse number 4 was the best. The inhibitory concentration (IC50) was 24.75 ng/mL. The cross-reaction rates of OTC, CTC and TC of their homologues were 3.4%, 3.8% and 3.0%, respectively. The cross-reaction rate with other veterinary drugs and carrier protein was less than 0.3%, and had a good specificity. This study successfully synthesized DC artificial antigens and prepared DC antiserum with good immunological properties, which provided a good foundation for the preparation of monoclonal antibody and the establishment of immunology rapid detection method.