Analysis of B lymphocyte cross-reactivity at the single cell level.

A method is described for assaying whether individual in vivo activated B cells produce antibodies which cross-react with two distinct antigens. The technique utilizes a modification of the ELISA spot assay to determine the isotype and antigenic specificity(s) of these antibody secreting cells. To perform the assay, two plastic slides are coated with different antigens and secured together to form chambers capable of holding a monolayer of Ig secreting lymphocytes. The immunoglobulin secreted during culture binds to one or both sides of the antigen-coated chamber (depending upon the specificity and cross-reactivity of the antibody product). This technique was used to evaluate the cross-reactivity of hybridomas and freshly isolated IgM and IgG secreting B cells.