抗富组氨酸糖蛋白单克隆抗体的制备及鉴定

PREPARATION AND CHARACTERIZATION OF A McAb AGAINST HISTIDINE-RICH GLYCOPROTEIN

目的 :制备抗富组氨酸糖蛋白 (HRG)单克隆抗体 ,用于建立夹心ELISA方法检测血浆HRG浓度。方法 :用淋巴细胞杂交瘤技术 ,制备单克隆抗体 ,并对其免疫学特性进行了研究。结果 :建立了二株稳定分泌抗HRG单克隆抗体的杂交瘤细胞株 (9E7C9和 3C6 F3) ,染色体数分别为 10 2和 96条 ,免疫球蛋白亚类均为IgG1。以羟基磷灰石法从腹水纯化McAb 9E7C9,SDS -PAGE检测其纯度 ,Westernblotting分析表明其可特异性地识别分子量为 670 0 0的抗原分子。用辣根过氧化物酶标记hcAb ,建立双抗夹心ELISA法 ,测定正常人血浆HRG含量为 110 .3± 9.7mg/L。结论 :所得McAbs特异性强 ,用其建立的夹心ELISA检测HRG的方法线性较好 ,灵敏度达 1ng/ml。

Objective:To generate monoclonal antibody (McAb) against histidine - rich glycoprotein (HRG),which is used to establish ELISA method and determine plasma HRG concentration. Methods: By lymphocyte hybridoma technique, McAbs were generated. The immunological characteristics were investigated, too. Results:Two hybridoma cell lines 9E 7C 9 and 3C 6F 3 were raised. They are subclassed into IgG 1. The mode value of their chromosome are 102 and 96 respectively. The McAb 9E 7C 9 was purified from ascites by hydroxylapatite (HA) method.Immunoblotting indic ated that the McAb recognized a single hand of HRG with a molecular weight of 67?000. The purified McAb was conjugated with horseradish peroxidase (HRP) by a modified periodate oxidation procedure. The MoAb was proved to increase the thrombin coagulation time. The results of Sandwich ELISA using the HRP Labeled McAb showed the mean HRG in normal human plasma is 110.3 ± 9.7mg/L.Conclusions:ELISA kit established with McAbs showed high sensitive and specific. It may be a useful kit for HRG research.