Characterization of Monoclonal Antibodies Directed Against the α-Subunit of the Human IgE High-Affinity Receptor

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Abstract
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 α-chain of the human IgE high affinity receptors (ecFc∈RIα) produced in insect cells. The antibodies secreted by hybridomas were selected for specific binding to ecFc∈RIα, by enzyme-linked immunosorbent assay (ELISA). The selected clones were further characterized in surface plasmon resonance (SPR) experiments with ecFc∈RIα covalently immobilized on the surface of a sensor chip. The generated hybridomas can be divided into three groups. Hybridoma supernatants 8A4/G12/F9 and 8H10/F12 inhibited binding of human IgE to immobilized ecFc∈RIα in SPR (Group 1). Isotyping revealed that 8A4/G12/F9 and 8H10/F12 were of the IgE/κ type. Antibodies present in the remaining supernatants were noninhibitory and bound to ecFc∈RIα 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/κ. The hybridoma supernatants were purified via protein A chromatography. In a SPR experiment, ecFc∈RIα, displayed by immobilized human IgE, was still recognized by 6F9/H8 and 6F9/G9 (Group 2) as expected for noninhibitory antibodies. Surprisingly, 8H10/D11, 5F2/F8/H11, and 5F2/F8/G10 (Group 3) did not bind to this complex although they do not inhibit the binding of human IgE to ecFc∈RIα. All purified monoclonal antibodies gave positive signals in Western blotting.