A hemolytic plaque assay for the detection of red blood cells carrying abnormal or mutant hemoglobin

Abstract

A simple hemolytic plaque assay for the detection of red blood cells carrying abnormal or mutant hemoglobin is described. The assay is based on the use, as indicator cells, of sheep red blood cells coated with monospecific antibody against the hemoglobin variant, another complement fixing multispecific anti-hemoglobin antibody as developing serum and an antibody against red cell ghosts which can lyse only the red cells of the species being studied and not the indicator sheep red cells. The test red cells are mixed with the indicator sheep red cells, the developing antiserum and the anti-ghost antibody. The mixture is transferred into thin chambers prepared with 2 microscope slides and incubated at room temperature (25°C). The indicator sheep red cells around each test red cell carying abnormal or mutant hemoglobin are lysed resulting in the formation of a plaque. Instead of anti-hemoglobin-coated sheep red blood cells, protein A-coated sheep red blood cells may be used as indicator cells provided the developing antiserum is monospecific for the hemoglobin variant. When sheep red blood cells coated with an antibody specific for DBA/2J mouse hemoglobin were used in the assay, only red cells from DBA/2J mice (carrying d hemoglobin) formed plaques while C57BL/6J red cells (carrying s hemoglobin) did not.