摘要
用自制并纯化的马立克病肿瘤相关表面抗原(MATSA)免疫SPF级BALB／c小鼠制备阳性对照抗体,以SPF级BALB／c小鼠血清为阴性对照抗体,筛选间接ELISA检测MATSA抗体的最佳反应条件。结果,包被液为0．1 mol／L pH 9．6的碳酸盐缓冲液,抗原包被浓度为4μg／mL,封闭液为含10 g／L BSA的PBST,抗血清稀释比例为1:800,酶标抗体稀释比例为1:1200,酶标抗体作用时间为37℃温育45 min,底物作用时间为37℃温育15 min,终止液为2 mol／L H2SO4,洗涤4次,每次1min为最佳反应条件。将建立的方法应用于MATSA单克隆抗体研制过程中阳性克隆的筛选,成功获得了MATSA的单克隆抗体,表明该优化条件为间接ELISA检测MATSA抗体的最佳反应条件。
BALB/c mice were immunized with Marek＇s disease tumor-associated surface antigen（MAT- SA） and the serum was used as positive control. Serum of SPF grade BALB/c mice without immunization was used as negative control. Detection conditions of MATSA antibody by indirect ELISA were optimized as following： coating liquid was buffer bicarbonate of 0.1 mol/L （pH 9.6）, optimal coating concentration of antigen was 4μg/mL, confining liquid was PBST containing 1% of BSA, optimal dilution of antiserum was 1 ＂ 800, optimal dilution of enzyme labelled antibody was 1 ＂ 1 200, action time of enzyme labelled antibody was 45 min at 37℃, action time of substrate was 15 min in 37℃, stop buffer was 2 mol/L of sulfuric acid, and washing protocol was 1 min per wash for four times. The developed assay was used in the screening of positive clones of monoclonal antibodies against MATSA and the monoclonal antibodies were obtained successfully.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2006年第7期538-542,共5页
Chinese Veterinary Science
基金
国家自然科学基金资助项目(30371067)霍英东教育基金资助项目(91033)
