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目的制备高特异性的抗雌三醇(E3)单克隆抗体(mAb),建立操作简便、敏感、快速适用于基层使用的E3检测方法。方法采用活化酯法制备偶联物E3-HRP、E3-BSA、3-OVA。应用mAb技术制备抗E3的mAb;以硝酸纤维素膜为固相载体,抗E3 mAb为包被抗体,建立竞争抑制模式的斑点酶免疫渗滤法。结果筛选出5株可分泌特异性抗E3 mAb的杂交瘤细胞株。mAb效价为1×105~5×105,亲和常数为5.1×108~5.2×109L/mol。检测敏感度为2 ng/mL,检测范围2~200 ng/mL。结论建立了检测速度快、灵敏度高、应用方便的斑点酶免疫渗滤法。
Objective To prepare highly specific anti-estriol (E3) monoclonal antibody (mAb) and establish a simple, sensitive and rapid E3 detection method for grassroots use. Methods The conjugates E3-HRP, E3-BSA and 3-OVA were prepared by activated ester method. The anti-E3 mAb was prepared by mAb technology. The nitrocellulose membrane was used as the solid phase carrier and the anti-E3 mAb was coated antibody to establish a competitive inhibition mode of dot blot immunofiltration. Results Five hybridoma cell lines secreting specific anti-E3 mAb were screened. The titer of mAb was 1 × 105 ~ 5 × 105, the affinity constant was 5.1 × 108 ~ 5.2 × 109L / mol. The detection sensitivity is 2 ng / mL and the detection range is 2 ~ 200 ng / mL. Conclusion The method of dot enzyme immunofiltration was established to detect the speed, sensitivity and convenience of application.