Studies of mouse immunoglobulin allotypes by reverse plaque assay: Detection of spleen cells secreting immunoglobulins with specific allotype in C57BL/6J mice

A reverse hemolytic plaque assay for the detection and enumeration of mouse spleen cells secreting immunoglobulins bearing a particular allotypic specificity is described. Sheep red blood cells (SRBC) coated with protein A or anti-mouse gamma globulin antibody were employed as indicator cells and an anti-allotype antibody was used as developer. A comparison of the efficiency of protein A, goat anti-mouse or rabbit anti-mouse gamma globulin antibody-coated SRBC as indicator cells in the plaque assay indicated that the rabbit anti-mouse gamma globulin-coated SRBC gave the best results in terms of number and morphology of the plaques. The number of indicator cells in the assay mixture also significantly affected the quality of the plaques formed. When the mouse spleen cells were assayed with the indicator cells and an anti-allotypic antibody as developer in presence of complement in a liquid medium, only those cells secreting the immunoglobulin of the given allotypic specificity formed hemolytic plaques.