Optimization and qualification of a memory B-cell ELISpot for the detection of vaccine-induced memory responses in HIV vaccine trials
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- 作者：Paula N. Walsh ; David P. Friedrich ; Julie A. Williams ; Rebecca J. Smith ; Terri L. Stewart ; Donald K. Carter ; Hua-Xin Liao ; M. Juliana McElrath ; Nicole Frahm
- 关键词：PBMC ; peripheral blood mononuclear cells ; Ag ; antigen ; Ig ; immunoglobulin ; ASC ; antibody-secreting cell ; TMB ; tetramethylbenzidine ; KLH ; Keyhole Limpet Hemocyanin ; HBSS ; Hanks balanced salt solution ; PVDF ; polyvinylidene fluoride ; SFC ; spot-forming cells
- 刊名：Journal of Immunological Methods
- 出版年：2013
- 出版时间：30 August, 2013
- 年：2013
- 卷：394
- 期：1-2
- 页码：84-93
- 全文大小：1488 K
文摘
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.