干血斑用于HIV抗体确证（重组免疫印迹法）方法建立与初步应用

HIV antibody confirmatory method(recombinant immunoblot assay)establishment and application based on dry blood spot samples

目的研究建立干血斑(DBS)样本用于艾滋病病毒(HIV)抗体确证(重组免疫印迹法)的方法,为确证试验提供更加广泛的适用样本。方法制备系列稀释20~210 DBS与配对血浆样本,利用实验室优化的DBS洗脱条件(300μL含0.5‰Tween 20的PBS室温洗脱6小时),研究来自两个公司的人类免疫缺陷病毒(HIV1+2)抗体检测试剂盒(免疫印迹法/重组免疫印迹法)的检测限,选择其中等效性更好且价格低廉的国产(重组免疫印迹法)试剂进一步研究。结果通过310份(110份HIV抗体阳性和200份HIV抗体阴性)已知DBS与血浆配对样本和1 304份临床DBS样本进行该方法验证与应用,两种不同试剂对于血浆样本的检测限均为20~27,但对于来自相同个体的对应DBS样本检测限不同(分别为20~25和20~26);应用与血浆样本等效的国产(重组免疫印迹法)试剂进行方法验证结果表明,阳性样本一致率为100.0%(110/110),阴性样本一致率为98.5%(197/200),总一致率99.0%(307/310),Kappa值0.98(P<0.001);敏感性100.0%(110/110),特异性98.5%(197/200);应用国产(重组免疫印迹法)试剂对1 304份临床DBS样本中HIV抗体初筛阳性132份DBS样本同时进行实验室DBS确证与临床随访确证,实验室DBS确证结果与临床随访确证结果完全一致,确证阳性130份,排除初筛假阳性样本2份。结论 DBS样本用于HIV抗体确证(重组免疫印迹法)与血浆样本等效,具有良好敏感性与特异性,可有效排除初筛假阳性样本。

Objective To establish an accurate and feasible HIV antibody confirmatory method(recombinant immunoblot assay)based on DBS samples.Methods Preparation of a series of diluted 20-210 dry blood spots was made to match plasma samples,and the DBS samples was eluted according to the optimum condition by our laboratory(in 300 ul PBS contains 0.5‰ Tween 20 eluted for 6 hours),to study the detection limit of HIV1+2 antibody detection kit(western blotting/recombinant immunoblot assay)from two different manufacturers.The more equivalent and cheaper reagents were tested by using 310(110 positive and 200 negative)DBS samples of known HIV infection status and 1 304 clinical DBS samples to verify and apply the method.Results The detection limit of the kits from two different manufacturers was the same(both 20-27),but different to match the DBS samples(20-25 and 20-26).The results of domestic reagent(recombinant immunoblot assay)verification showed that the positive coincidence rate was 100.0%(110/110);the negative coincidence rate was 98.5%(197/200);and the total coincidence rate was 99.0%(307/310),with the Kappa value of0.98(P<0.001),sensitivity of 100.0%(110/110),and specificity of 98.5%(197/200).The method was applied to 132 HIV positive samples of 1 304 clinical DBS,showed that the result were completely consistent with the gold standard follow-up.A total of 130 samples were confirmed positive and 2 preliminary screening false positive samples were excluded.Conclusion DBS samples is equivalent to plasma samples in HIV confirmation with good sensitivity and specificity,and can effectively exclude the preliminary screening false positive samples.It can provide more accurate and reliable HIV antibody test results.