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.
Schlueter
A J
AJ
Department of Veterinary Pathobiology, University of Illinois, Urbana 61801.
Segre
M
M
Segre
D
D
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D000937:Antigen-Antibody Reactions; D004912:Erythrocytes / Q000276:immunology*; D005260:Female; D006386:Hemagglutination Tests; D006462:Hemolytic Plaque Technique; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007136:Immunoglobulins / Q000378:metabolism*; D051379:Mice; D013205:Staphylococcal Protein A / Q000276:immunology; D005719:gamma-Globulins / Q000276:immunology
D000888:Antibodies, Anti-Idiotypic; D007140:Immunoglobulin Fab Fragments; D007136:Immunoglobulins; D013205:Staphylococcal Protein A; D005719:gamma-Globulins