Frequency Analysis of HBsAg‐specific B Lymphocytes in High‐responder Individuals to Recombinant Hepatitis B Vaccine: Comparison of LDA and ELISPOT Assays

Abstract

The determination of the frequency of antigen‐specific lymphocytes may provide invaluable information for the evaluation of the immune response to different antigens and pathogens. Different methods are employed to determine the frequency of specific B lymphocytes in peripheral blood. In this study, the frequency of hepatitis B surface antigen (HBsAg)‐specific B lymphocytes was determined by a limiting dilution assay (LDA) and an enzyme‐linked immunospot assay (ELISPOT) in seven healthy adult high responders to recombinant HBsAg. Peripheral blood mononuclear cells isolated at different time intervals (1, 2, 4, 8 and 16 weeks) following administration of a booster dose were either transformed with Epstein–Barr virus (LDA) or stimulated with Pokeweed mitogen (ELISPOT). In an LDA, anti‐HBs positive wells were screened by a sandwich ELISA and the frequency of specific B lymphocytes was estimated based on the Poisson statistical analysis. In an ELISPOT, coloured spots representing specific B lymphocytes were finally enumerated by stereomicroscope. Our results showed a significant increase in the number of specific B lymphocytes in the first week by an ELISPOT compared with an LDA (1:190 versus 1:13,462) (P < 0.001). No significant differences were observed at other time intervals. A significant correlation was observed between the serum titer of anti‐HBs antibody and frequency of HBsAg‐specific B cells obtained by LDA and ELISPOT methods at different time intervals. The highest correlation was found at fourth week in LDA (r = 0.83, P < 0.01) and ELISPOT (r = 0.85, P < 0.01) assays. Furthermore, a significant correlation was observed between an LDA and ELISPOT at different time intervals (highest correlation in second week, r = 0.88, P < 0.008). These findings suggest that in addition to technical advantages, such as speed and simplicity, an ELISPOT is a more sensitive assay, compared with an LDA.

Characterization of the HPV-specific memory B cell and systemic antibody responses in women receiving an unadjuvanted HPV16 L1 VLP vaccine

“…The B cell memory ELISPOT data significantly correlated with the neutralization assay (Spearman ρ = 0.76; p = 0.0002) and with the ELISA assay (Spearman ρ = 0.62; p = 0.0047). Although the correlation between the ELISA and memory B cell ELISPOT results reported here is similar to levels of correlation previously reported in other settings, the reported values cover a wide range of correlations [23–28]. The discrepancies observed between reports could be due to differences in the timing of specimen collection (steady state measurements versus peak responses), vaccination strategy (formulations of live attenuated, recombinant subunit or inactivated immunogens), vaccine regiments (number and timing of doses), antigens studied or age groups.…”

Genetic characterization of human Dsg3-specific B cells isolated by flow cytometry from the peripheral blood of patients with pemphigus vulgaris

“…The frequency of antigen-specific B cells (anti-glycoprotein IIb/IIIa antibody-producing B cells) in patients with immune thrombocytopenic purpura was reported to be 2-30 per 10 5 PBMCs [23], whereas the frequency of Hepatitis B surface antigen (HBsAg)-specific B cells was determined to be 54.9 per 10 5 PBMCs after vaccination using enzyme-linked immunospot (ELISPOT) assay [24]. These figures provide an idea of the frequency of antigen-specific B cells in circulation because they fall within a similar range.…”

Longitudinal Determination of Hepatitis B Surface Antigen-Specific B Lymphocyte Frequency in Healthy High Responder Adults after Booster Vaccination

“…However, the frequency of Ag-specific naïve B cells is very low, much lower than the detection limit of the LDA assay. We have not been able to determine the frequency of naïve HBsAg-specific B cells in nonimmunized individuals in this study and also in previous studies [7,8] due to the low frequency of antigenspecific B cells as well as the sensitivity limit of the assay. Based on this argument, the efficiency of EBV transformation is quite similar in different healthy donors, because (as already mentioned) there is almost no variation between normal people in their B cell potential for EBV transformation.…”

“…In our previous studies, we had determined the frequency of HBsAg-specific B cells in responder and nonresponder vaccines at a single time point after a booster dose using the LDA technique [8] . We have also compared the LDA technique and ELISPOT assay for enumerating of B lymphocytes in high responder adults after booster immunization at specified time intervals [7] . In the present study, we assessed the effect of booster vaccination on frequencies of circulating HBsAg-specific memory B cells at different time intervals using the LDA assay.…”

“…However, immune memory persists beyond the time at which anti-HBs levels may no longer be detectable and protects against clinically relevant disease [5] . To understand the role of memory B lymphocytes following booster vaccination in the regulation of immunoglobin (Ig) secretion and antibody production, it is becoming increasingly important to quantitate the number of B lymphocytes that secrete the specific antibody [6,7] . In humans, the influence of booster vaccination on HBsAg-specific B lymphocytes has not been well characterized.…”