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J Immunol Methods. 2013 Aug 30;394(1-2):84-93. doi: 10.1016/j.jim.2013.05.007. Epub 2013 May 23.

Optimization and qualification of a memory B-cell ELISpot for the detection of vaccine-induced memory responses in HIV vaccine trials.

Author information

1
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

Abstract

Various aspects of the human immune system can be analyzed to determine the efficacy of a vaccine. We have developed a B-cell ELISpot to measure HIV-specific antibody-secreting B cells in the peripheral blood as a result of vaccination or natural infection. Our method includes stimulating peripheral blood mononuclear cells with interleukin-2 and a polyclonal activator, R848, to induce memory B cells to differentiate into antibody-secreting cells. Total immunoglobulin-secreting as well as antigen-specific B cells are then quantified. We have tested several HIV Env gp120 and gp140 proteins from different HIV subtypes, as well as a sensitive consensus group M Env gp140. Our findings indicate that the B-cell ELISpot provides a sensitive and specific tool to detect antigen-specific memory B-cell responses, and it is equally suited to detect antibody-secreting plasmablasts present in the circulation shortly after infection or vaccination.

KEYWORDS:

ASC; Ag; Antibody-secreting cells; B-cell ELISpot; HBSS; HIV; Hanks balanced salt solution; Ig; KLH; Keyhole Limpet Hemocyanin; Memory B-cell responses; Mucosal responses; PBMC; PVDF; SFC; TMB; Vaccine; antibody-secreting cell; antigen; immunoglobulin; peripheral blood mononuclear cells; polyvinylidene fluoride; spot-forming cells; tetramethylbenzidine

PMID:
23707324
PMCID:
PMC3736720
DOI:
10.1016/j.jim.2013.05.007
[Indexed for MEDLINE]
Free PMC Article
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