Rapid procedure for coupling of protein antigens to red cells to be used in plaque assays by prewashing in chromium chloride

A rapid and efficient procedure is described for the coupling of proteins (protein A, provalbumin, albumin and chicken gamma globulin) to sheep red blood cells (SRBC) to be used in antigen-specific or protein A plaque assays. This modification of the original procedure has three distinct features: prewash of the red cells with a low concentration of essentially freshly prepared CrCl3, use of a relatively high concentration of CrCl3 in the reaction mixture and a coupling time of only 4 min. Protein A plaque assays performed with such target cells have the same sensitivity as those employing red cells coupled with protein A according to the original procedure. Studies with hybridomas secreting antibody specific for a protein antigen showed that antigen-specific plaque assays employing target red cells coupled with the protein antigen according to the modified procedure have the same sensitivity as the protein A plaque assay. The modified procedure greatly facilitates cellular studies on antibody formation after immunization with protein antigens.