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 RIalpha) produced in insect cells. The antibodies secreted by hybridomas were selected for specific binding to ecFc epsilon RIalpha, by enzyme-linked immunosorbent assay (ELISA). The selected clones were further characterized in surface plasmon resonance (SPR) experiments with ecFc epsilon RIalpha 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 epsilon RIalpha in SPR (Group 1). Isotyping revealed that 8A4/G12/F9 and 8H10/F12 were of the IgE/kappa type. Antibodies present in the remaining supernatants were noninhibitory and bound to ecFc epsilon RIalpha 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 RIalpha, 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 epsilon RIalpha. All purified monoclonal antibodies gave positive signals in Western blotting.
Nechansky
A
A
Novartis Forschungsinstitut GmbH, Vienna, Austria.
Pursch
E
E
Effenberger
F
F
Kricek
F
F
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D015374:Biosensing Techniques; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D011487:Protein Conformation; D017455:Receptors, IgE / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D017455:Receptors, IgE; D011994:Recombinant Proteins