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 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.
Patiris
Peter
P
California Department of Health Services, Richmond, California 94804, USA. ppatiris@dhs.ca.gov
Hanson
Carl
C
D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006046:Gold; D015483:HIV Antibodies / Q000097:blood; D016655:HIV Core Protein p24 / Q000276:immunology; D015658:HIV Infections / Q000097:blood; D017320:HIV Protease Inhibitors; D006801:Humans; D007963:Leukocytes, Mononuclear; D009500:Neutralization Tests / Q000379:methods*; D012680:Sensitivity and Specificity; D012685:Sepharose; D013194:Staining and Labeling
D015483:HIV Antibodies; D016655:HIV Core Protein p24; D017320:HIV Protease Inhibitors; D006046:Gold; D012685:Sepharose