Optimization of a human IgG B-cell ELISpot assay for the analysis of vaccine-induced B-cell responses

Abstract

B-cell responses after infection or vaccination are often measured as serum titers of antigen-specific antibodies. Since this does not address the aspect of memory B-cell activity, it may not give a complete picture of the B-cell response. Analysis of memory B cells by ELISpot is therefore an important complement to conventional serology. B-cell ELISpot was developed more than 25 years ago and many assay protocols/reagents would benefit from optimization. We therefore aimed at developing an optimized B-cell ELISpot for the analysis of vaccine-induced human IgG-secreting memory B cells. A protocol was developed based on new monoclonal antibodies to human IgG and biotin-avidin amplification to increase the sensitivity. After comparison of various compounds commonly used to in vitro-activate memory B cells for ELISpot analysis, the TLR agonist R848 plus interleukin (IL)-2 was selected as the most efficient activator combination. The new protocol was subsequently compared to an established protocol, previously used in vaccine studies, based on polyclonal antibodies without biotin avidin amplification and activation of memory B-cells using a mix of antigen, CpG, IL-2 and IL-10. The new protocol displayed significantly better detection sensitivity, shortened the incubation time needed for the activation of memory B cells and reduced the amount of antigen required for the assay. The functionality of the new protocol was confirmed by analyzing specific memory B cells to five different antigens, induced in a limited number of subjects vaccinated against tetanus, diphtheria and pertussis. The limited number of subjects did not allow for a direct comparison with other vaccine studies. Optimization of the B-cell ELISpot will facilitate an improved analysis of IgG-secreting B cells in vaccine studies.

Detection of Influenza A/H1N1-Specific Human IgG-Secreting B Cells in Older Adults by ELISPOT Assay

“…We selected an elderly population and specifically focused on influenza A/H1N1-specific memory B cells. Our data support the conclusion that the B cell ELISPOT assay is a highly effective method to detect influenza A/H1N1-specific B cells after vaccination (1,12,23). Several strains of the influenza virus exist due to the known variances in the HA and NA surface antigens.…”

“…Jahnmatz et al recently utilized the B cell ELISPOT assay to detect the presence of memory B cells in a small population of subjects (n = 8) with vaccineinduced immunity to five different viral antigens (12). However, no studies based on influenza A/H1N1 vaccineinduced detection of antigen-specific memory B cells in a large elderly population have been performed.…”

“…The recognition of the influenza virus by B cells and production of influenza-specific antibodies are crucial for the amplification of the immune response and neutralization of the influenza virus. While the VN and HAI assays provide important data regarding humoral protection from infection, influenza-specific B cell counts provide an alternative perspective for assessing humoral immunity preparedness for a subsequent influenza infection (4,9,12). Therefore, influenzaspecific B cells could serve as important markers of active humoral immunity, and these data can be used along with other measures of immunity to predict influenza vaccine efficacy.…”

The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation

“…Thus, PBMCs were collected, treated with IdeS, and stimulated with rIL-2 and R848 to activate memory B cells and differentiate them into Ig-producing cells (14). After 72-96 h, the cells were washed extensively to remove IdeS and then analyzed for the frequency of Ig-producing cells.…”

Suppression of innate and adaptive B cell activation pathways by antibody coengagement of FcγRIIb and CD19

“…10 6 cells/ml were cultured in RPMI 1640 containing 10% FCS in the presence of 10 ng/ml rhIL-2 and 1 μg/ml R848 (Sigma-Aldrich, SML0196) polyclonal activator with or without XmAb5871. 54 Cells were harvested on the third day, washed and counted, and then were transferred into the wells of ELISPOT plates precoated with neutravidin and the biotinylated citrulline-containing 19mer filaggrin peptide (SHQESTXGXSXGRSGRSGS, X stands for citrulline). 37 The spots were developed after 20 h.…”