Hashida S, Ishikawa S, Hashinaka K, Nishikata I, Oka S, Shimada K, Saito A, Takamizawa A, Shinagawa H, Ishikawa E
Department of Biochemistry, Miyazaki Medical College, Japan.
J Clin Lab Anal. 1998;12(2):98-107. doi: 10.1002/(SICI)1098-2825(1998)12:2<98::AID-JCLA5>3.0.CO;2-F.
The immune complex transfer enzyme immunoassays for antibody IgGs to p17, p24, and reverse transcriptase (RT) of HIV-1 were tested under various conditions. Antibody IgGs to HIV-1 were reacted for up to 20 hr with 2,4-dinitrophenyl-bovine serum albumin-recombinant HIV-1 protein conjugates and recombinant HIV-1 protein-beta-D-galactosidase conjugates, and the immune complexes formed, comprising the three components, were trapped onto polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG by incubation at 4-30 degrees C for up to 2 hr with shaking and were transferred onto polystyrene beads coated with (antihuman IgG gamma-chain) IgG in the presence of excess of epsilon N-2,4-dinitrophenyl-L-lysine by incubation at 4-30 degrees C for up to 2 hr with shaking. When serum randomly collected from an HIV-1 seropositive subject and serum included in an Western blot kit were tested, the formation of the immune complex was almost completed within 1 hr for antibody IgG to p17, within 1-2 hr for antibody IgG to p24 and within 4 hr for antibody IgG to RT. Even for antibody IgG to p17, however, the immune complex continued to be formed for at least 2 hr, when serum samples at early stages of HIV-1 infection were tested. Trapping and transferring of the immune complexes were faster at higher temperatures and were almost completed within 0.5-1.5 hr, although the amount of the immune complexes trapped and transferred at 25 and/or 30 degrees C increased for 0.5-1 hr, but subsequently tended to decline. When the formation, trapping, and transferring of the immune complexes were performed for 0.5, 1, and 1 hr, respectively, with shaking followed by 1 hr assay of bound beta-D-galactosidase activity, the sensitivities for antibody IgGs to p17, p24, and RT using 10 microliters of serum samples were similar to or significantly higher than those of the corresponding previous immune complex transfer enzyme immunoassays using 10 microliters of serum samples, in which the formation, trapping, and transferring of the immune complexes were performed for 3, 16, and 3 hr, respectively, without shaking, followed by 2.5 hr assay of bound beta-D-galactosidase activity, and the sensitivities for antibody IgGs to p17, p24, and RT using 100 microliters of serum samples were 21-22-fold, 5.5-6.3-fold, and 5.3-6.0-fold, respectively, higher. When each period of time for the formation, trapping, and transferring of the immune complexes was prolonged to up to 4 hr, the sensitivities for antibody IgGs to p17, p24, and RT using 100 microliters of serum samples were improved 88-93-fold, 15-17 fold and 20-24-fold, respectively, as compared with those of the previous ones.
在各种条件下对用于检测针对HIV-1的p17、p24和逆转录酶（RT）的抗体IgG的免疫复合物转移酶免疫测定法进行了测试。将针对HIV-1的抗体IgG与2,4-二硝基苯基-牛血清白蛋白-重组HIV-1蛋白缀合物和重组HIV-1蛋白-β-D-半乳糖苷酶缀合物反应长达20小时，形成的包含这三种成分的免疫复合物，通过在4至30℃下振荡孵育长达2小时，捕获到包被有（抗2,4-二硝基苯基基团）IgG的聚苯乙烯珠上，然后在过量ε-N-2,4-二硝基苯基-L-赖氨酸存在下，在4至30℃下振荡孵育长达2小时，转移到包被有（抗人IgGγ链）IgG的聚苯乙烯珠上。当检测从一名HIV-1血清阳性受试者随机采集的血清以及包含在蛋白质印迹试剂盒中的血清时，针对p17的抗体IgG在1小时内免疫复合物的形成几乎完成，针对p24的抗体IgG在1至2小时内完成，针对RT的抗体IgG在4小时内完成。然而，当检测HIV-1感染早期阶段的血清样本时，即使针对p17的抗体IgG，免疫复合物也会持续形成至少2小时。免疫复合物的捕获和转移在较高温度下更快，并且在0.5至1.5小时内几乎完成，尽管在25℃和/或30℃下捕获和转移的免疫复合物的量在0.5至1小时内增加，但随后趋于下降。当免疫复合物的形成、捕获和转移分别进行0.5小时、1小时和1小时，振荡后接着检测结合的β-D-半乳糖苷酶活性1小时时，使用10微升血清样本检测针对p17、p24和RT的抗体IgG的灵敏度与相应的先前免疫复合物转移酶免疫测定法（其中免疫复合物的形成、捕获和转移分别在不振荡的情况下进行3小时、16小时和3小时，接着检测结合的β-D-半乳糖苷酶活性2.5小时）使用10微升血清样本的灵敏度相似或显著更高，并且使用100微升血清样本检测针对p17、p24和RT的抗体IgG的灵敏度分别高21至22倍、5.5至6.3倍和5.3至6.0倍。当免疫复合物的形成、捕获和转移的每个时间段延长至长达4小时时，与先前的方法相比，使用100微升血清样本检测针对p17、p24和RT的抗体IgG的灵敏度分别提高了88至93倍、15至17倍和20至24倍。
