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Studies for practical use of novel ultrasensitive methods to detect HIV antigens and antibodies, improving the diagnosis of HIV infection

研究实际使用新型超灵敏方法检测 HIV 抗原和抗体，改善 HIV 感染的诊断

基本信息

批准号：
08557016
负责人：
ISHIKAWA Eiji
金额：
$ 8.13万
依托单位：
Miyazaki Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

In order to make more reliable the diagnosis of HIV infection, studies were made to facilitate the practical use of ultrasensitive enzyme immunoassays (immune complex transfer enzyme immunoassays) for HIV-1 p24 antigen and antibody IgGs to HIV-1 antigens with emphasis on their automation. Firstly, the use of magnetic beads as solid phase in the immune complex transfer enzyme immunoassays was tested, since the period of time used for performing the immune complex transfer enzyme immunoassays had to be shortened for their automation, and magnetic beads have been considered to be the most useful for performing solid phase immunoassays within shorter periods of time in general. However, high nonspecific signals were observed, ruling out their use for obtaining high sensitivities. In parallel with this, the immune complex transfer enzyme immunoassays using polystirene beads were tested under various conditions. As a results, the period of time required for performing the immune complex transfer enzyme immunoassays was markedly shortened with considerable improvement of the sensitivity by incubations with shaking and using larger volumes of serum samples. Secondly, the time courses of binding of the immune complexes comprising 2,4-dinitrophenyl-reactants, antigens or antibodies and beta-D-galactosidase-labeled reactants to microplates and polystirene sticks coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG and the capacities of those solid phases to trap the immune complexes were examined using 2,4-dinitrophenyl-beta-D-galactosidase. The bindings to the microplates and polystirene sticks, which were shown to have larger binding capacities than polystirene beads, were faster than that to polystirene beads, ruling out the use of polystirene beads for automation of the immune complex transfer enzyme immunoassays. On the basis of these and other results, the immune complex transfer enzyme immunoassays will be fully automated with further improvements.

为了使HIV感染的诊断更加可靠，研究了促进HIV-1 p24抗原和HIV-1抗原抗体IgS的超灵敏酶免疫分析(免疫复合物转移酶免疫分析)的实际应用，并着重于其自动化。首先，对免疫复合物转移酶免疫分析中使用磁珠作为固相进行了测试，因为进行免疫复合物转移酶免疫分析所用的时间必须缩短以实现自动化，而且通常认为磁珠在较短的时间内进行固相免疫分析是最有用的。然而，观察到了高度的非特异性信号，排除了它们用于获得高灵敏度的可能性。同时，对聚苯乙烯小球免疫复合物转移酶免疫测定法在不同条件下进行了测试。结果表明，通过振荡孵育和使用更多的血清样品，进行免疫复合物转移酶免疫分析所需的时间显著缩短，灵敏度显著提高。其次，用2，4-二硝基苯基-β-D-半乳糖苷酶检测了由2，4-二硝基苯基反应物、抗原或抗体和β-D-半乳糖苷酶标记的免疫复合物与包被亲和纯化的(抗2，4-二硝基苯基)免疫球蛋白的微孔板和聚苯乙烯棒结合的时间过程以及固相捕获免疫复合物的能力。与微板和聚苯乙烯棒的结合能力被证明比聚苯乙烯小球更大，结合速度比聚苯乙烯小球更快，排除了使用聚苯乙烯小球进行免疫复合物转移酶免疫测定自动化的可能性。在这些和其他结果的基础上，免疫复合物转移酶免疫分析将完全自动化，并有进一步的改进。