Specific detection of antigen-binding cells by localized growth of bacteria

A new method for the enumeration of lymphoid cells with specific surface-receptors for antigen is described, based on the use of beta-D-galactosidase (EC 3.2.1.23), either directly as an antigen or as a conjugated antigen. Binding of beta-D-galactosidase is revealed by its activity in releasing riboflavin from a synthetic substrate, riboflavin-beta-D-galactopyranoside. The riboflavin, inactive as a vitamin in the galactosidic form, becomes active when released by the enzyme, and can be detected by bioassay. Hence, lymphoid cells with receptors for beta-D-galactosidase on their surface can be detected after they have been exposed to the enzyme, washed, and then plated in agar containing riboflavin-beta-D-galactopyranoside, streptomycin, riboflavin-deficient medium, and a streptomycin-resistant strain of Streptococcus faecalis that requires riboflavin. Release of riboflavin is signalled by the growth of characteristic bacterial colonies over the cell that bound beta-D-galactosidase.