CAJ | 학술논문

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.
목적 건립이순화중조외막지단백LipL32위기출적구단라선체(구체)병항체적검측방법 .방법 이모인중조기술획취중조구체외막지단백LipL32,이해단백위항원,분별사용간접법화협심법ELISA응용우불동인화서혈청적검측,검측결과 여구체현미경응집시험(MAT)진행비교,동시인혈청적검측결과 환여진구시제합비교.결과 순화적중조단백검측9례구체학진병례쌍빈혈청표본,삼충ELISA법적검출솔여MAT무명현차별.검측45빈MAT양성표본,간접법ELISA민감성위71.11%(32/45),협심법ELISA위80.00%(36/45),이진구 ELISA양성13빈,점28.89%(13/45),25빈가의점55.56%(25/45).특이성검측,MAT음성혈청69빈,간접화협심법ELISA특이도균위97.10%(67/69),기중검측문진체검혈청43빈,간접화협심법ELISA균위음성,진구ELISA검측14빈,야위음성.검측비구체발열병례혈청표본16빈,간접화협심법ELISA공검출2빈양성,진구ELISA칙시1빈양성,12빈가의.검측매독라선체질공양성혈청10빈,사충방법 균위음성.중조단백검측서혈청표본274빈,협심법ELISA적민감성위86.75%(131/151),특이성위99.19%(122/123),부합솔위92.34%(253/274),여MAT검측결과 상부.결론 중조LipL32단백구유결합활성,가응용우구체혈청항체적검측,기중협심법ELISA대서혈청구체항체적검측현시교호적민감성화특이성,괄용우구체병현장혈청류행병학대양본조사.
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.