Detection and enumeration of immunoglobulin secreting cells

A method is described in which sheep red blood cells (SRBC) are coated with anti-immunoglobulin (Ig) antibodies (Ab) for use in reverse hemolytic plaque assays (RHPA) as follows. The non-complement fixing F(ab')2 fragments of rabbit anti-mouse Ig Ab are derivatized with the N-hydroxysuccinimide ester of palmitate. The hydrophobic palmitate tails spontaneously insert into the SRBC membranes, thus coating the cells with anti-Ig F(ab')2 molecules. The SRBC are lysed by successive additions of mouse Ig, rabbit anti-mouse Ig and complement. When this procedure is carried out in agar gel, Ig-secreting mouse cells produce localized hemolytic areas (plaques). The procedure is more reproducible and more sensitive than RHPA performed with protein A-coated SRBC. In principle, this procedure should be adaptable to the detection of cells secreting any molecule for which specific antibodies are available.