AEGiS-11IAC: Assaying integration activity of HIV-1 integrase with DNA-coated plates.

11th International AIDS Conference


Vancouver, British Columbia — July 7-12, 1996

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Assaying integration activity of HIV-1 integrase with DNA-coated plates.

Int Conf AIDS 1996 Jul 7-12; 11:58 (abstract no. Mo.A.1019)
Syu W Jr, Chang YC, Ching TT; National Yang-Ming University, Taipei, Taiwan. Fax: 886-2-821-2880. E-mail: wjsyu@ym.edu.tw.

OBJECTIVE: To simplify the measurement of integration reaction, an easy method mimicing enzyme-linked immunosorbent assay (ELISA) procedures was developed.

METHODS: Integration of reverse transcribed viral DNA of HIV into host chromosomes is mediated by the viral enzyme, integrase. This enzymatic activity can be monitored in vitro by integration of a small labeled DNA (donor) into a second unlabeled DNA (target). DNA fragments were adsorbed directly on 96-well plates and used as the target DNA. The donor was a synthetic 21-bp DNA duplex of HIV-1 U5 LTR; biotin was incorporated into the 5' end of one strand whose two nucleotides at the 3' end were specifically removed during the integration.

RESULTS: After integrase reaction, the biotin-labeled donor DNA was joined with the target DNA and became immobilized on plates. These integration products were then measured by binding of avidin-alkaline phosphatase on plates.

CONCLUSIONS: Previously, microplate-based integration assays have been described. However, our new method is relatively simple and straightforward. In particular, no complicated procedures for the immobilization of target DNAs onto plates are involved. Thus, this method should be easily adapted to qualitative screening of integrase inhibitors and quantitative measurement of the potency of different inhibitors.

Keywords: AEGIS, HIV Integrase, DNA, Integrase, HIV Long Terminal Repeat, HIV Integrase Inhibitors, DNA, Viral, DNA Nucleotidyltransferases, Integrase Inhibitors, DNA Footprinting, DNA Primers, HIV, Nucleotides, Enzyme-Linked Immunosorbent Assay, Anti-HIV Agents, In Vitro, analysis, genetics, ICA11

960707
MoA1019

Copyright © 1996 - International AIDS Society (IAS). Reproduction of this abstract (other than one copy for personal reference) must be cleared through the IAS.