CHARACTERIZATION OF MONOCLONAL-ANTIBODIES DIRECTED AGAINST THE ALPHA-SUBUNIT OF THE HUMAN IGE HIGH-AFFINITY RECEPTOR

A panel of monoclonal antibodies (8H10/D11, 6F9/H8, 6F9/G9, 5F2/F8/H11 , 5F2/F8/G10, 8A4/G12/F9, and 8H10/F12) was raised in mice against the recombinant 20-kDa extracellular part of the alpha-chain of the human IgE high affinity receptors (ecFc epsilon RI alpha) produced in insec t cells. The antibodies secreted by hybridomas were selected for speci fic binding to ecFc epsilon RI alpha, by enzyme-linked immunosorbent a ssay (ELISA). The selected clones were further characterized in surfac e plasmon resonance (SPR) experiments with ecFc epsilon RI alpha coval ently immobilized on the surface of a sensor chip. The generated hybri domas can be divided into three groups. Hybridoma supernatants 8A4/G12 /F9 and 8H10/F12 inhibited binding of human IgE to immobilized ecFc ep silon RI alpha in SPR (Group 1). Isotyping revealed that 8A4/G12/F9 an d 8H10/F12 were of the IgE/kappa type. Antibodies present in the remai ning supernatants were noninhibitory and bound to ecFc epsilon RI alph a in ELISA with intensities comparable to each other. Isotype analysis of antibodies secreted by these hybridomas showed that the antibodies 6F9/H8, 6F9/G9, 5F2/F8/H11, 5F2/F8/G10, and 8H10/D11 were IgG1/kappa. The hybridoma supernatants were purified via protein A chromatography . In a SPR experiment, ecFc epsilon RI alpha displayed by immobilized human IgE, was still recognized by 6F9/H8 and 6F9/G9 (Group 2) as expe cted for noninhibitory antibodies. Surprisingly, 8H10/D11, 5F2/F8/H11, and 5F2/F8/G10 (Group 3) did not bind to this complex although they d o not inhibit the binding of human IgE to ecFc epsilon RI alpha. All p urified monoclonal antibodies gave positive signals in Western blottin g.