重组结核分支杆菌16000蛋白抗原(rPA16)的免疫原性研究

IMMUNOLOGICAL CHARACTERIZATION OF THE RECOMBINANT 16 000 ANTIGEN OF MYCOBACTERIUM TUBERCULOSIS

目的 :研究结核分枝杆菌重组 16 0 0 0蛋白 (rPA16 )诱发家兔产生抗体的能力并评价其血清学诊断的价值。方法 :家兔按注射的抗原及佐剂的不同分组 ,皮下多点注射分别进行免疫。 6次免疫后采血 ,双向琼脂扩散试验和酶联免疫吸附试验 (ELISA)跟踪兔血清抗体效价的时间效应并检测兔血清与所试各类分支杆菌PPD有无交叉反应 ;同时检测rPA16与各分支杆菌PPD的免疫羊血清的反应 ,并用rPA16抗原检测人血清标本 ,分析该抗原的临床检测的敏感性和特异性。结果 :兔血清抗体效价结果显示 :5个月后 ,加佐剂的抗原组的兔免疫血清仍能检测到抗体 ,不加佐剂组 2个月后血清抗体检测即为阴性。ELISA检测兔免疫血清比双向琼脂扩散方法要更敏感 ,rPA16抗原加佐剂引起的兔血清抗体滴度最高可达 1∶6 4 0 0 ,不加佐剂组的血清抗体滴度仅为 1∶4 0 0。ELISA法分析抗rPA16的兔血清的特异性表明其特异性较好 ,仅与牛、人型结核分支杆菌的PPD呈阳性反应 ,与其他所试分支杆菌PPD反应均为阴性。同时又分析了rPA16抗原的特异性 ,与人型PPD相比 ,该抗原只与所试各类分支杆菌、BCG、牛分支杆菌免疫的血清呈阳性反应 ,而人型PPD则基本上与结核分支杆菌免疫的血清均呈阳性反应。ELISA检测血清标本结果 :肺结核病组中rPA16和PPD检测敏感性?

Objective:We studied the ability of inducing antibodies by recombinant 16?000 protein of Mycobacterium tuberculosis(rPT16) and valued rPT16 as serological diagnostic reagent.Methods:The rabbits were classed into five groups, according to the different antigen injected and the incomplete Freud's adjuvant.The rabbits were injected subcutaneously. Blood samples were collected one week following the sixth immunization.The sera from the immuned rabbits were evaluated with both enzyme-linked immunosorbent assay (ELISA) and bilateral agar diffusion assay(BADA).328 human sera,including 145 pulmonary tuberculosis patients,105 healthy controls,78 BCG-vaccinated healthy controls, were detected with ELISA.Results:Sera titer of all rabbits decreased with time. After two months, sera titer from rabbits rejected only by rPA 16 was negative.However,antibodies were still found in rabbits rejected by rPA16 and adjuvant after five months, and the highest sera titer of this group was 1∶6?400.The sensitivity of ELISA was higher than that of BADA.Detected with ELISA,sera of anti-rPA16 had only reaction with PPD from the M.tuberculosis and M.bovis.About the test of the specificity of rPA16, there was positive reaction sera immuned by M.tuberculosis, BCG,and M.bovis to rPA16.But most sera immuned by the tested Mycobacteria except M.intracellular could react with PPD.The sensitivity of rPA16 and PPD in detecting the pulmonary tuberculosis was 64.8% and 75.2%,respectively.The specificity of rPA16 and PPD in detecting the healthy controls,BCG-vaccinated healthy controls was 96.2%, 88.5% and 94.3%, 66.7%,respectively.Conclusion:rPA16 has stronger immunologen and may become one of the immunodiagnostic reagents.