Improvement of the protein A plaque assay for immunoglobulin secreting cells by using immunoglobulin-depleted guinea pig serum as a source of complement.
This paper describes a modification of the protein A hemolytic plaque assay for the enumeration of immunoglobulin (Ig)-secreting cells independent of antibody specificity of the Ig. This assay was originally developed by Gronowicz et al. (1976), and is based upon binding of the Fc portion of IgG to protein A. Ig-secreting cells are mixed with protein A-coated sheep erythrocytes, developing rabbit anti-Ig antiserum and guinea pig serum as a source of complement. This mixture is either pipetted between two microscope slides, or added to agarose and plated on a petri dish or microscope slide. The hemolytic plaques are enumerated after incubation at 37 degrees C. Here we show that purification of the guinea pig complement over a Sepharose-protein A column in order to eliminate the IgG fraction facilitates plaque formation. This modification reduces the incubation period required for plaque formation, and yields a higher number of, and more discrete plaques, than the original method.
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D000921:Antibody-Producing Cells / Q000276:immunology*; D001769:Blood; D002478:Cells, Cultured; D003165:Complement System Proteins; D003470:Culture Media; D006168:Guinea Pigs; D006462:Hemolytic Plaque Technique; D013205:Staphylococcal Protein A; D013997:Time Factors
D000906:Antibodies; D003470:Culture Media; D013205:Staphylococcal Protein A; D003165:Complement System Proteins