检测抗HIV-1/2型抗体的双抗原夹心方法的建立及其试剂盒的研制

目的 建立更敏感的检测人免疫缺陷病毒（HIV）抗体的方法，并研制检测试剂盒。方法 根据HIV－1／2型的基因序列及其所编码氨基酸结构，采用固相法合成了HIV－1型的gp41.1、gp41.2、gp120、p24和HIV－2型的gp36五条多肽，混合包被酶标板作为固相抗原。用辣根过氧化物酶标记以上多肽抗原作为标记物，建立检测血清中抗HIV－1／2抗体的双抗原夹心ELISA法。同时，应用该方法制备检测HIV抗体的试剂盒，并检测三批中国卫生中药品和生物制品检定所HIV诊断试剂国家参比品。结果 建立了检测HIV－1／2抗体的双抗原夹心法。用检定所参比品检测，该方法特异性、灵敏度均为100％，变异系数小于10％。与间接法相比较其灵敏度、特异性均高于间接法（P＜0．05）。检测210份其他病种患者血清均为阴性。与GBI公司的HIV抗体诊断试剂比较，检测40份卫生部药品和生物制品检定所提供的质控参比品（阳性20份，阴性20份），GBI试剂阴、阳性符合率及总符合率分别为100%(20/20)、85％（17／20）及92．5％（37／40），而应用该方法所研制的诊断试剂盒、阳性符合率及总符合率为100％。该试剂已通过国家卫生部质检。与雅培公司HIV诊断试剂比较检测90份献血员血清和88份HIV－1／2型感染者血清，符合率为100％。试剂盒于37℃放置4d后的检测结果的阴、阳性判定不受影响。结论 本法特异性强、灵敏度高、稳定性好，适用于献血员的筛选和临床HIV感染的检测。

Antigen sandwiched ELISA for detection of total HIV antibodies

OBJECTIVE: To establish a sensitive antigen sandwiched ELISA(AS) for detection of the total antibodies to HIV-1/2. METHODS: Based on the gene sequence of HIV-1/2 type and its coded amino acid structure, 5 polypeptides were synthesized as coated antigens using solid-phase method. These polypeptides were labelled with horseradish peroxidase. And the total antibodies of HIV-1/2 were detected with the same method. RESULTS: These reagents were detected by three batches of HIV panel from The National Institute for the Control of Pharmaceutical and Biological Products (NICPBP). The results indicated that the corresponding rate was 100%. The variant coefficient rate was less than 10%. A comparison of antigen sandwiched ELISA with indirect ELISA in detection of a panel(20 positive sera and 20 negative sera) from the NICPBP showed that the general coincident rate of indirect ELISA was 92.5% and the sandwiched system was 100%. The HIV-AS diagnostic reagent kits have passed the quality examination of NICPBP. A comparison of antigen sandwiched ELISA with Yapei reagents in detection of 90 normal sera and 88 positive sera for HIV-1/2 showed that the coincident rate was 100%. The reagents were stable at 37 degrees C for 4 days. This indicated that our reagents were highly specific, sensitive and stable. CONCLUSION: Our antigen sandwiched ELISA reagent for total antibodies of HIV has a merit of sensitivity specificity and stability. It can be clinically used for detection of HIV-1/2 infection and in blood bank for the screening of blood donors.