Discrimination between innate and cytophilic immunoglobulin on human peripheral blood lymphocytes: analysis by the direct antiglobulin rosette-forming reaction.
Bovine red blood cells linked to polyclonal or monoclonal anti-immunoglobulin antibodies are used in the direct antiglobulin rosetting reaction to detect surface-Ig on human lymphocytes. The sensitivity of this test is markedly increased by pretreating the red cells with trypsin. Enzyme-treated red cells, coupled to anti-human Fab or anti-light chain antibodies, react not only with innate Ig on B lymphocytes but also with smaller amounts of passively adsorbed, cytophilic Ig on up to 25% of freshly prepared peripheral blood (non-B) lymphocytes. In contrast, trypsinized red cells carrying anti-Ig isotype-specific antibodies react exclusively with B cell surface-Ig. Cytophilic Ig is abnormally firmly bound to lymphocytes separated on Ficoll-Hypaque at 20 degrees C or below, and is released very slowly during 3 h or more at 37 degrees C in vitro. Lymphocytes are free of detectable cytophilic Ig when isolated on Ficoll-Hypaque at 37 degrees C, and very little Ig is retained by non-B cells in suspensions purified on Percoll which, unlike Ficoll, does not increase Ig binding affinity. These lymphocyte separation procedures are recommended as a preliminary to B cell assays by sensitive antiglobulin techniques.
D000371:Agglutination; D000372:Agglutination Tests; D000936:Antigen-Antibody Complex; D005455:Fluorescent Antibody Technique; D006801:Humans; D008214:Lymphocytes / Q000276:immunology*; D011947:Receptors, Antigen, B-Cell / Q000032:analysis*; D012397:Rosette Formation / Q000379:methods; D014357:Trypsin
D000936:Antigen-Antibody Complex; D011947:Receptors, Antigen, B-Cell; D014357:Trypsin