摘要
硫酸雌酮(estrone-3-sulfate)简称E1S,是一种雌激素的衍生物,目前广泛的应用于畜牧生产上,进行家畜的早期妊娠诊断。本实验合成了硫酸雌酮全抗原物质；制备了硫酸雌酮的多克隆抗体和单克隆抗体,并进行了鉴定。主要内容如下：
1)硫酸雌酮抗原物质的制备
通过雌酮硫酸钠合成了硫酸雌酮半抗原物质并进行了质谱鉴定,将获得的半抗原物质与BSA和OVA通过碳二亚胺法进行偶联,获得了免疫原硫酸雌酮-BSA浓度为9.29mg/ml和包被原硫酸雌酮-OVA浓度为4.68mg/ml,测定了硫酸雌酮-BSA和硫酸雌酮-OVA的偶联比分别为20.4：1和15.2：1,使用紫外方法和SDS-PAGE电泳方法进行了全抗原物质的鉴定,证明全部偶联成功。
2)硫酸雌酮多克隆抗体的制备
用偶联成功的全抗原物质对实验兔进行了免疫,确定了间接ELISA法的最佳反应条件,确定出抗原的最佳工作浓度为8000,甲兔的最佳工作浓度为81000,乙兔的最佳工作浓度为27000。将获得的多克隆抗体使用硫酸铵盐析法进行了纯化,通过电泳证明纯化后纯度得到明显提高。
3)硫酸雌酮单克隆抗体的制备
确定了小鼠ELISA反应最佳条件,血清的最佳稀释倍数为1000,包被原的最佳包被浓度为6ug/ml。筛选出了血清中抗体特异性较好的小鼠,8#作为融合首选,1#和10#作为备用。纯化获得单抗腹水,并通过SDS-PAGE电泳验证纯化纯度得到明显提高。
Estrone sulfate (estrone-3-sulfate) referred to E1S, an estrogen derivative, is widely used in early pregnancy diagnosis of domestic animal. In this study, complete antigens of estrone sulfate was synthesized; polyclonal antibodies and monoclonal antibodies of estrone sulfate were prepared and identified. Following major elements:
1) The synthesis of estrone sulfate antigens
estrone sulfate hapten synthesized by estrone 3-sulfate sodium salt and identified by mass spectrometry,was respectively coupled with BSA and OVA through the carbodiimide method to obtain the complete antigens.Results showed that the concentrations of immunogen estrone sulfate-BSA and the the estrone sulfate-OVA were 9.29mg/ml and 4.68mg/ml.The couping ratios of estrone sulfate-BSA and estrone sulfate-OVA were 20.4:1,and 15.2:1, respectively.Identification of complete antigens by using ultraviolet method and SDS-PAGE electrophoresis method proved the success of coupling.
2) The preparation of estrone sulfate monoclonal antibodies
Complete antigens was used for immunization to rabbits, the optimal reaction conditions of the indirect ELISA method was determined, the optimal concentration of antigen was 8000,optimum working concentration of A rabbit was 81000, best work concentration of B Rabbit was 27000. Polyclonal antibodies were purified by Ammonium sulfate precipitation method, and the purity detected by SDS-PAGE electrophoresis method was improved significantly.
3) The preparation of estrone sulfate polyclonal antibodies
The best conditions of mice ELISA response was determined, and the best dilution of serum was 1:1000, the best package concentration of the package is 6ug/ml.The mice which had the best antibody specificity in serum was selected,8# was choosed as the preferred integration,and 1# and 10# as the backup. Ascites monoclonal antibody obtained was purified and verified by SDS-PAGE,which showed the purity was markedly improved.
引文
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