| Budget Amount *help |
¥12,700,000 (Direct Cost: ¥12,700,000)
Fiscal Year 1997: ¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1996: ¥9,400,000 (Direct Cost: ¥9,400,000)
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| Research Abstract |
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.
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