Single-round HIV type 1 neutralization measured by ELISPOT technique in primary human cells

To accurately quantify HIV-1 neutralizing antibody titers in primary human cells, we developed a single round, focus-forming unit (FFU) reduction assay in human peripheral blood mononuclear cells (PBMC). Infected PBMC were enumerated by a reverse ELISPOT technique in which they were incubated under...

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Veröffentlicht in:AIDS research and human retroviruses 2005-09, Vol.21 (9), p.784-790
Hauptverfasser: PATIRIS, Peter, HANSON, Carl
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HANSON, Carl
description To accurately quantify HIV-1 neutralizing antibody titers in primary human cells, we developed a single round, focus-forming unit (FFU) reduction assay in human peripheral blood mononuclear cells (PBMC). Infected PBMC were enumerated by a reverse ELISPOT technique in which they were incubated under agarose in the presence of a protease inhibitor in anti-p24 antibody-coated microtiter plates. Viral p24, secreted in the immediate vicinity of infected cells and captured by immobilized antibodies, was subsequently stained using gold-labeled anti-p24-antibody and a precipitating silver substrate. The resulting spots were counted visually, without the aid of a microscope, and percent neutralization titers were determined using curve-fitting software. Results of this ELISPOT neutralization assay (ENA) for 15 HIV-positive human specimens were compared with results from a standard PBMC neutralization assay (standard assay) that measured neutralization as a function of p24 concentration by enzyme immunoassay (EIA). The ENA measures FFU reduction of both syncytium-inducing (SI) and non-syncytium-inducing (NSI) primary isolates. Completed assay plates may be retained as a physical record of results or saved as an image using a flat-bed computer scanner.
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source Mary Ann Liebert Online Subscription; MEDLINE; Alma/SFX Local Collection
subjects AIDS/HIV
Biological and medical sciences
Cells, Cultured
Enzyme-Linked Immunosorbent Assay - methods
Fundamental and applied biological sciences. Psychology
Gold
HIV Antibodies - blood
HIV Core Protein p24 - immunology
HIV Infections - blood
HIV Protease Inhibitors
Human immunodeficiency virus 1
Human viral diseases
Humans
Infectious diseases
Leukocytes, Mononuclear
Medical sciences
Microbiology
Miscellaneous
Neutralization Tests - methods
Sensitivity and Specificity
Sepharose
Staining and Labeling
Viral diseases
Virology
title Single-round HIV type 1 neutralization measured by ELISPOT technique in primary human cells
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