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...
Gespeichert in:
Veröffentlicht in: | AIDS research and human retroviruses 2005-09, Vol.21 (9), p.784-790 |
---|---|
Hauptverfasser: | , |
Format: | Artikel |
Sprache: | eng |
Schlagworte: | |
Online-Zugang: | Volltext |
Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
Zusammenfassung: | 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. |
---|---|
ISSN: | 0889-2229 1931-8405 |
DOI: | 10.1089/aid.2005.21.784 |