Frequency analysis of HBsAg-specific B lymphocytes in high-responder individuals to recombinant hepatitis B vaccine: comparison of LDA and ELISPOT assays.
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.
Shokrgozar
M A
MA
National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran.
Sam
M R
MR
Amirkhani
A
A
Shokri
F
F
D000293:Adolescent; D000328:Adult; D000917:Antibody Formation; D001402:B-Lymphocytes / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006510:Hepatitis B Antibodies / Q000276:immunology; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D017325:Hepatitis B Vaccines / Q000276:immunology*; D006801:Humans; D007117:Immunization, Secondary; D007201:Indicator Dilution Techniques; D008137:Longitudinal Studies; D008297:Male; D008875:Middle Aged
D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D017325:Hepatitis B Vaccines