title: Development and evaluation of an HIV-1 transfection-neutralization assay.
abstract: We developed a transfection-neutralization assay for human immunodeficiency virus type 1 (HIV-1) infectious molecular clones. In this assay CD4 negative adherent cells, transfected in microtiter plates with fixed amounts of proviral DNA of molecular HIV-1 clones, are cocultivated with CD4 positive T cell lines or primary peripheral blood mononuclear cells (PBMC) in the presence of anti-HIV-1 sera or monoclonal antibodies (MAbs). Results obtained with this technique were reproducible and compared favorably with a conventional cell-free infection inhibition assay. The transfection-neutralization assay obviates the need for virus stock preparation and, therefore, is particularly suitable for the evaluation of HIV-1 clones with slow replication kinetics and of recombinant chimeric HIV-1 clones inclined to undergo additional mutations during stock preparation. The potential value of this assay for the analysis of the specificity of neutralizing sera and MAbs was demonstrated in experiments with V3 chimeric molecular clones.
authors.affiliation: Department of Virology, University of Amsterdam, Academic Medical Center, The Netherlands.
authors.forename: N K
authors.initials: NK
authors.lastname: Back
authors.forename: L
authors.initials: L
authors.lastname: Smit
authors.forename: E
authors.initials: E
authors.lastname: Hogervorst
authors.forename: A C
authors.initials: AC
authors.lastname: van Wijk
authors.forename: J
authors.initials: J
authors.lastname: Goudsmit
authors.forename: M
authors.initials: M
authors.lastname: Tersmette
mesh_terms: D000595:Amino Acid Sequence; D000918:Antibody Specificity; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000502:physiology; D000939:Epitopes / Q000032:analysis; D005069:Evaluation Studies as Topic; D015483:HIV Antibodies / Q000097:blood* / Q000276:immunology; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D015658:HIV Infections / Q000276:immunology; D015497:HIV-1 / Q000235:genetics / Q000276:immunology*; D006367:HeLa Cells; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000382:microbiology; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011533:Proviruses / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D014162:Transfection; D014779:Virus Replication / Q000276:immunology
chemical_list: D004279:DNA, Viral; D000939:Epitopes; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; C059762:HIV envelope protein gp120 (305-321); D007106:Immune Sera; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins