摘要
【目的】建立促乳素单克隆抗体制备中筛选阳性分泌细胞的间接ELISA检测方法。【方法】用促乳素纯品与Freund佐剂免疫6--8周龄雌性BALB／c小鼠，初次免疫采用抗原与完全Freund佐剂，每2周免疫1次，从第2次开始采用抗原与不完全Freund佐剂，免疫4次后尾静脉采血，收集血清制备阳性对照抗体，以未免疫的BALB／c小鼠血清为阴性对照，筛选间接ELISA法检测促乳素抗体的最佳反应条件，并对单克隆抗体制备中的阳性细胞进行筛选。【结果】间接ELISA法的最佳反应条件：血清稀释倍数为1：200，抗原包被质量浓度为100ng／mL，封闭液为1％牛血清白蛋白，酶标抗体的工作浓度为1：10000，酶标抗体作用时间为37℃90min，底物作用时间37℃30min。用该方法检测融合的杂交瘤细胞，最终获得OD450为0．875和0．460的阳性细胞株。
[Objective] The research was to filter the optimal conditions of enzyme-linked immunosorbent assay to detect the antibody of prolactin,then establish an indirect enzyme-linked immunosorbent assay to screen the the positive cells in the production of monoclonal antibody. [Method] Female BALB/c mice of eight weeks old were immunized with prolactin and Freund adjuvant. The mice were immunized every two weeks,using prolaetin and complete Freund adjuvant for the first time, and prolactin and incomplete Freund adjuvant for the others. After four times of immunization,the serum was used as posotive control. Serum of unimmunized BALB/c mice was used as negative control to filter the optimal condition of indirect enzyme-linked immunosorbent assay. [Result] The optimal conditions of this experiment were as follows：coating concentration of antigen was 100 ng/mL, confini enzyme labelled antibody 1 ： 10000,action time of enzyme la substrate 30 min at 37 ℃. [Conclution] Improved reaction nosorbent assay was constituted and this way can be used to ng liquid 1% BSA,optimal dilution of enzyme beled antibody 90 min at 37 ℃ ,action time of conditions of indirect enzyme-linked immuselect the antibody of prolactin.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2009年第2期47-51,共5页
Journal of Northwest A&F University(Natural Science Edition)
基金
农业部产业结构调整项目(05-07-03B)
陕西省科学技术研究发展计划项目(2005K02-G4-1)
关键词
促乳素
抗体
间接ELISA法
prolactin
antibody
indirect enzyme-linked immunosorbent assay
