抗人甘露聚糖结合凝集素(MBL)单克隆抗体的制备、鉴定与检测方法的建立  

Preparation and characterization of monoclonal antibody against human mannan-binding lectin and establishment of its immunoassay

作　　者：骆鹏[1] 孟淑芳[1] 张庶民[1] 贾天军[2] 

机构地区：[1]中国药品生物制品检定所,北京100050 [2]河北北方学院医学检验中心,河北张家口075029

出　　处：《免疫学杂志》2008年第5期579-582,587,共5页Immunological Journal

基　　金：国家863博士基金资助项目(2003AA2Z3509)

摘　　要：目的制备抗甘露聚糖结合凝集素(MBL)的单克隆抗体(McAb),建立免疫学检测方法。方法以纯化MBL为抗原,免疫Balb/c小鼠,制备单克隆抗体,鉴定其特性,建立并验证夹心ELISA定量检测法。结果筛选出2株稳定分泌抗MBL的单抗杂交瘤细胞株,免疫球蛋白亚类均为IgG1。两单抗间抑制率<50%,结合位点不同两单抗特异识别MBL,选择包被抗体B10浓度为8μg/mL,酶标抗体B3稀释度为1∶500,建立夹心ELISA定量检测法。该法检出范围为1～100ng/mL,与其他抗原无交叉反应。重复性试验的批内批间变异均小于10%,回收率在101%～103%之间,cv小于7%。检测结果与国外试剂盒比较无显著差异。结论制备的抗MBL单克隆抗体可用于MBL的免疫学检测。Objective To prepare monoclonal antibodies （McAb） against mannan-binding lectin （MBL） and establish a method for immanoassay. Methods Purified MBL was used as antigen to immane Balb/c mice. Monoclonal antibodies against MBL were prepared by normal hybfidoma technology. By identifying the characters of McAbs, the quantitative ELISA method was established. Results Two hybridomas producing antibodies against MBL were obtained. IgG isotypes of two McAbs were IgG1. The repression ratio in two McAbs was less than 50%, while their binding sites were different. The McAbs were proved to be specific for MBL. The sandwich ELISA method was established by selecting B10 as coast antibody at 8 μg/mL and selecting B3 as HRP-antibody at 1：500 dilution. This method was founded to be able to detect MBL at concentration from 1 to 100 ng/mL and havs no crossing-reaction with other proteins. The repeated test showed all the cv inter-dots and cv intra-dots were less than 10%. The recovery ratio is 101% - 103% and cv was less than 7%. The results of this method and Danmark＇ s kit were in a accord. Conclusion The prepared McAb against MBL can be used for MBL immanoassay.

关 键 词：甘露聚糖结合凝集素 单克隆抗体 ELISA 

分 类 号：R392.11[医药卫生—免疫学]