A filter immuno-plaque assay for the detection of antibody-secreting cells in vitro

Abstract A plaque assay has been developed that is based on enzyme immunoassay principles and capable of screening several hundred samples in one day. Single cell suspensions of in vivo or in vitro immunized mouse splenocytes are incubated on antigen-coated nitrocellulose membranes in microfilter plates or in petri dishes. The antibody production of individual cells is detected using a horse radish peroxidase-labeled second antibody, and the insoluble products of the enzymatic reaction are visualized as blue plaques on the membranes. The nitrocellulose membrane of the microfilter plates, which readily adsorb a variety of antigens, and the filtration unit used for the washing steps greatly facilitates the plaque assay. Further-more, this procedure only needs small amounts of antigen for the enumeration of isotype-specific antibody-secreting cells in a defined medium containing low protein levels or in a completely serum-free medium. The plaque assay may be used to evaluate the optimal conditions required for in vitro immunizations.