Generation of monoclonal murine anti-DNP-IgE, IgM and IgG1 antibodies: biochemical and biological characterization

Abstract

Monoclonal DNP-specific IgG (lambda 2 epsilon 2), IgM (kappa 2 mu2) and IgG [kappa 2 (gamma 1)2] were isolated fom the culture supernatant of hybridomas by affinity chromatography with 2,4-dinitrophenol bovine serum albumin (DNP-BSA) sepharose and characterized by biochemical and biological methods. The molecular weights were 84,200 for the epsilon chain, 55,400 for the gamma chain and 77,500 for the mu chain as determined by sodium dodecylsulphate polyacrylamide del electrophoresis (SDS-PAGE). The association constants for [3H]-DNP-lysine determined by equilibrium dialysis were 0 . 87 X 10(7) l/mol for IgE and 1 . 91 X 10(8) 1/mol for IgG1. The isoelectric focusing of the purified monoclonal antibodies revealed for IgG1 seven bands at a pH range of 6 . 3 - 7 . 2 and for IgE sixteen bands at a pH range of 4 . 5 to 6 . 8. the binding of 125I-anti-IgE to rat basophilic leukaemia (RBL) and rat mast cells which had been preincubated with various amounts of monoclonal IgE was studied. At saturation conditions of IgE, about 2 . 14 X 10(5) molecules of anti-IgE were bound per rat mast cell. Rat mast cells coated with monoclonal anti-DNP IgE were triggered for the release of histamine in the presence of either the antigen or guinea-pig anti-mouse IgE. A mutual inhibition of the passive cutaneous anaphylaxis (PCA) reaction in the rat by either mixing mouse reaginic serum directed against ovalbumin or rat reaginic serum directed against Nippostrongylus brasiliensis with monoclonal mouse anti-DNP IgE was demonstrated.