Coupling of protein antigens to erythrocytes through disulfide bond formation: preparation of stable and sensitive target cells for immune hemolysis.
An efficient technique has been developed for coupling protein antigens to erythrocyte membranes. The procedure involves three steps. First, 3-(2-pyridyldithio)propionyl residues are introduced into the protein by reaction with a heterobifunctional reagent, N-succinimidyl 3-(pyridyldithio) propionate. Second, the addition of disulfide groups to sheep erythrocytes (SRBC) is achieved by coupling dithiodiglycolic acid to SRBC with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The disulfide bonds of the dithiodiglycolyl-SRBC conjugate are then reduced with dithiothreitol. Finally, the 3-(2-pyridyldithio)propionyl-protein conjugate is covalently coupled to the thiolated SRBC through thiol/disulfide exchange to form the disulfide-linked antigen-SRBC conjugate. The procedure requires only 10-500 microgram of protein antigen for the preparation of 50 microliter of packed protein-coupled SRBC. Antibodies binding to antigen on the erythrocyte initiate a complement-dependent immune lysis of the target cells. Target cells prepared by this method are stable for at least 4 wk at 4 degrees C in phosphate buffer (pH 7.2) and are capable of detecting as little as 40 pg of antibody in a hemolytic assay without noticeable nonspecific lysis.
D000937:Antigen-Antibody Reactions; D000941:Antigens; D003165:Complement System Proteins / Q000276:immunology; D004220:Disulfides; D004910:Erythrocyte Membrane / Q000276:immunology*; D004912:Erythrocytes / Q000276:immunology*; D006462:Hemolytic Plaque Technique
D000941:Antigens; D004220:Disulfides; D003165:Complement System Proteins