Activation of the human classical complement pathway by a mouse monoclonal hybrid IgG1-2a monovalent anti-TNP antibody bound to TNP-conjugated cells.
A mouse hybridoma selected and cloned for anti-TNP specificity produced three distinct monoclonal antibody species that were separated on protein A-Sepharose by stepwise acid elution. The IgG1 kappa product of the parental myeloma was eluted at pH 6.0. An IgG2a kappa bivalent anti-TNP antibody was eluted at pH 4.5, whereas elution at pH 5.0 yielded a hybrid IgG1-2a kappa monovalent anti-TNP antibody. The IgG2a molecules agglutinated TNP-conjugated sheep erythrocytes (TNP-ES) and lysed TNP-ES in the presence of normal human serum (NHS). Hybrid IgG1-2a antibody was also capable of lysing the cells in NHS, although it did not agglutinate TNP-ES. A threshold in monovalent antibody input was necessary for the lysis of TNP-ES, indicating a requirement for a minimal density of bound monovalent IgG to trigger complement activation. Lysis occurred in NHS-VBS++ but not in NHS-MgEGTA, and it was associated with a dose-dependent consumption of C1, C4, and C2 hemolytic activities. Quantitation of the antibody bound to TNP-ES when using radiolabeled rabbit anti-mouse Fab antibody demonstrated that for similar inputs, 5.4 times as much bivalent as monovalent antibody bound to TNP-ES. When similar amounts of antibody were effectively bound to TNP-ES, monovalent hybrid IgG1-2a was five times less efficient than bivalent IgG2a to yield 50% cell lysis in the presence of NHS. These results indicate that neither bivalent binding nor the presence of two identical heavy chains are necessary requirements for antibody-dependent activation of the classical complement pathway.
D000371:Agglutination; D000818:Animals; D000906:Antibodies / Q000032:analysis; D000911:Antibodies, Monoclonal; D003167:Complement Activation; D003171:Complement Pathway, Classical; D003165:Complement System Proteins / Q000032:analysis / Q000276:immunology; D004912:Erythrocytes / Q000276:immunology; D006461:Hemolysis; D006801:Humans; D005779:Immunodiffusion; D007074:Immunoglobulin G / Q000276:immunology*; D007525:Isoelectric Focusing; D051379:Mice; D008815:Mice, Inbred Strains; D011863:Radioimmunoassay; D012756:Sheep; D014301:Trinitrobenzenes
D000906:Antibodies; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D014301:Trinitrobenzenes; D003165:Complement System Proteins