应用纯化重组外膜脂蛋白LipL32检测钩端螺旋体病抗体

目的 建立以纯化重组外膜脂蛋白LipL32为基础的钩端螺旋体(钩体)病抗体的检测方法 .方法 以摹因重组技术获取重组钩体外膜脂蛋白LipL32,以该蛋白为抗原,分别使用间接法和夹心法ELISA应用于不同人和鼠血清的检测,检测结果 与钩体显微镜凝集试验(MAT)进行比较,同时人血清的检测结果 还与进口试剂盒比较.结果 纯化的重组蛋白检测9例钩体确诊病例双份血清标本,三种ELISA法的检出率与MAT无明显差别.检测45份MAT阳性标本,间接法ELISA敏感性为71.11%(32/45),夹心法ELISA为80.00%(36/45),而进口 ELISA阳性13份,占28.89%(13/45),25份可疑占55.56%(25/45).特异性检测,MAT阴性血清69份,间接和夹心法ELISA特异度均为97.10%(67/69),其中检测门诊体检血清43份,间接和夹心法ELISA均为阴性,进口ELISA检测14份,也为阴性.检测非钩体发热病例血清标本16份,间接和夹心法ELISA共检出2份阳性,进口ELISA则是1份阳性,12份可疑.检测梅毒螺旋体质控阳性血清10份,四种方法 均为阴性.重组蛋白检测鼠血清标本274份,夹心法ELISA的敏感性为86.75%(131/151),特异性为99.19%(122/123),符合率为92.34%(253/274),与MAT检测结果 相符.结论 重组LipL32蛋白具有结合活性,可应用于钩体血清抗体的检测,其中夹心法ELISA对鼠血清钩体抗体的检测显示较好的敏感性和特异性,适用于钩体病现场血清流行病学大样本调查.

Application of purified recombinant outer membrane lipoprotein LipL32 in detecting antibodies among leptospirosis cases

Objective To establish recombinant outer membrane lipoprotein LipL32-based antibody detection assays in identifying leptospirosis. Methods Recombinant leptospiral outer membrane protein LipL32 was obtained by genetic engineering method. This purified protein was used in the indirect and sandwich ELISA assays to test the antibodies in sera of human beings and rats, and the results were compared with those obtained by microscopy agglutination test (MAT) and imported ELISA kit. Results When the acute and convalescent phase specimens from 9 leptospiral patients were tested, the detected rates of three ELISAs were similar to the MAT. Among the 45 probable cases which MAT showed positive, 32 (71.11%) samples were positive by r32-I-ELISA, 36(80.00%) by r32-S-ELISA,while 28.89% (13/45) samples were positive and 55.56% (25/45)were suspicious by D.A.I-ELISA. The specificity of r32-I-ELISA and r32-S-ELISA were 97.10 % (67/69) for 69 specimens. 43 healthy specimens were negative by both r32-I-ELISA and r32-S-ELISA, 14 healthy specimens were negative by D.A.I-ELISA. Among 16 non-leptospirosis patients, two specimens were positive by r32-I-ELISA and r32-S-ELISA, D.A.I-ELISA and identified one positive specimen, while 12 specimens were suspicious by D.A.I-ELISA. For 10 syphilis specimens, data obtained through three ELISAs were in consistent with that by MAT. A sandwiched ELISA, using rLipL32 protein as the antigen was developed to detect rat sera. Considering MAT as standard test, the sensitivity and specificity were 86.75 % (131/151), 99.19 % (122/123) respectively with coincidence rate as 92.34% (253/274). Conclusion The recombinant protein LipL32 had high immunoresctivity and could be used as an antigen for the detection of panthogenic leptospirosis. In summary, the novel sandwiched ELISA with rLipL32 showed similar sensitivity and specificity to that of MAT in the antibody detection of rat leptospirosis. It was suitable for large scales field sero-epidemiological studies.