钩端螺旋体外膜蛋白LipL32单克隆抗体的制备及鉴定

Monoclonal antibody-based antigen capture immunoassay for detection of LipL32 of Leptospira

LipL32是钩端螺旋体外膜中含量最丰富的蛋白,有很好的免疫原性,且在致病性钩端螺旋体中高度保守。在非变性条件下纯化LipL32重组蛋白,与佐剂混匀后免疫BALB/c小鼠,取脾脏细胞与NS-1骨髓瘤细胞融合,然后用间接酶联免疫吸附试验(ELISA)和有限稀释法分别进行筛选和细胞克隆,获得29株能稳定分泌抗LipL32单克隆抗体的杂交瘤细胞株,它们能识别8个LipL32抗原表位。用这些细胞株制备腹水型抗体,并对特性进行鉴定。用生物素标记这些抗体后两两配对,采用双抗夹心ELISA检测提取自15株致病性钩端螺旋体及其他致病菌的抗原,以评估单克隆抗体的灵敏度和特异度。筛选出1对灵敏度高和特异度好的单克隆抗体,ELISA检测rLipL32的最低浓度为1ng/ml。结果提示,该钩端螺旋体外膜蛋白LipL32单克隆抗体及检测方法有很好的应用前景。

LipL32 is not only the most abundant protein of the outer membrane but also an immunodominant antigen that is highly conserved in pathogenic Leptospira. Recombinant LipL32 （rLipL32） purified under natural conditions was adapted to immunize BALB/c mice for developing monoclonal antibodies （mAbs）. Twenty-nine mAbs against eight epitopes were produced and characterized. The capture and detection of antibodies were selected using a one-by-one pairing method with biotin-conjugated mAbs. To evaluate the sensitivity and specificity of the mAb pairs, antigen capture enzyme-linked immunosorbent assay （ELISA） was applied to detect antigens extracted from 15 pathogenic Leptospira strains and from other pathogenic bacteria. One pair of mAbs was selected and the detection capability of rLipL32 by ELISA was found to be approximately 1 ng/ml, mAbs produced with rLipL32 purified under natural conditions may be promising in the further detection of leptospiral antigens.