Limiting dilution analysis of virus-specific memory B cells by an ELISPOT assay

Many acute viral infections induce long-term, sometimes even life-long humoral immunity. To characterize this immune response, accurate quantitation of memory B cells and plasma cells is essential. Plasma cells can be quantitated directly ex vivo by virtue of their ability to spontaneously secrete antibody. Memory B cells on the other hand, do not spontaneously secrete antibody but require antigenic stimulation in order to proliferate and differentiate into antibody secreting cells (ASC). In this study, an ELISPOT-based limiting dilution assay (LDA) was developed for quantitating virus-specific B cell memory following acute lymphocytic choriomeningitis virus (LCMV) infection of adult mice. Antiviral memory B cell precursor (MBCp) frequencies were calculated from in vitro stimulated cultures using either a conventional ELISA-based LDA to measure accumulated virus-specific antibody in the culture medium or a new ELISPOT-based LDA that identifies the antibody-secreting daughter cells directly. In terms of sensitivity, the ELISPOT-based LDA and the ELISA-based LDA both calculated LCMV-specific MBCp frequencies to be approximately 1/2 X 10⁴ spleen cells. However, compared to the 12 days of in vitro stimulation required to estimate MBCp frequencies by the ELISA-based LDA, the ELISPOT-based LDA required only 3-6 days of stimulation to quantitate MBCp frequencies. If cell division was blocked by γ-irradiation or treatment with mitomycin C, the MBCp frequency dropped below detection (<1 MBCp/10⁶ cells), indicating that virus-specific B cells quantitated by this assay must both proliferate and differentiate into antibody secreting cells in order to be detected. Naive, uninfected mice did not have LCMV-specific memory B cells, demonstrating that only in vivo-generated antiviral B cells from LCMV-immune mice were detected by this assay. χ² analysis of the ELISPOT-based LDA showed that the MBCp frequency data fit a linear regression model (p = 0.0137), indicating single-hit kinetics in which only one cell type was limiting. These results indicate that the ELISPOT-based LDA provides a rapid and statistically accurate method to quantitate virus-specific B cells.