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Frequency Analysis of HBsAg‐specific B Lymphocytes in High‐responder Individuals to Recombinant Hepatitis B Vaccine: Comparison of LDA and ELISPOT Assays
Author(s) -
Shokrgozar M. A.,
Sam M. R.,
Amirkhani A.,
Shokri F.
Publication year - 2006
Publication title -
scandinavian journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.934
H-Index - 88
eISSN - 1365-3083
pISSN - 0300-9475
DOI - 10.1111/j.1365-3083.2006.01838.x
Subject(s) - elispot , virology , hbsag , medicine , recombinant dna , immunology , hepatitis b vaccine , biology , hepatitis b virus , virus , immune system , t cell , genetics , gene
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.