title: Assaying the activity of HIV-1 integrase with DNA-coated plates.
abstract: 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). The methodology usually involves isotope labeling and gel electrophoresis. To simplify the measurement, a method mimicking enzyme-linked immunosorbent assay (ELISA) procedures was developed. Fragments of DNA 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. As a result of integration, 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. The method is simple and straightforward and can easily be adapted for high throughput screening of integrase inhibitors.
authors.affiliation: Institute of Microbiology and Immunology, National Yang-Ming University, Taiwan, ROC.
authors.forename: Y C
authors.initials: YC
authors.lastname: Chang
authors.forename: T T
authors.initials: TT
authors.lastname: Ching
authors.forename: W J
authors.initials: WJ
authors.lastname: Syu
mesh_terms: D004247:DNA / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D019427:HIV Integrase / Q000032:analysis* / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D004247:DNA; D019427:HIV Integrase