HYBRIDOMA Volume 16, Number 6, 1997 Mary Ann Liebert, Inc. A New Set of Monoclonal Antibodies Against Human FcyRII (CD32) and FcyRIII (CD16): Characterization and Use in Various Assays FREDERIC VELY,1,3,4 NADEGE GRUEL,2,4 JANINE MONCUIT,1 OLIVIER COCHET,2 HELENE ROUARD,1 SOPHIE DARE,2 JEROME GALON,1 CATHERINE SAUTES,1 WOLF-HERMAN FRIDMAN,1 and JEAN-LUC TEILLAUD1,2 ABSTRACT Four mouse anti-human FcyRII (CD32) (6C4, 2B2, 3D3, 93.4) (IgGl, k) and one anti-human FcyRIII (CD16) (7.5.4) (IgGl, #c) MAbs were raised. An in vitro switch variant, 7.5.4Sw50 (IgG2b, k), was also derived from the 7.5.4 MAb. 6C4, 2B2, and 3D3 MAbs bind both FcyRIIa and FcyRIIb isoforms. Two of them (6C4 and 2B2 MAbs) allow a complete blockade of the binding of immune complexes to FcyRII. All three MAbs im- munoprecipitate the receptor and bind both its glycosylated and nonglycosylated forms. The fourth anti- FcyRII MAb, 93.4, directed against the intracellular region of FcyRIIai/2, allows its detection by Western blotting only when it is not phosphorylated. The 7.5.4 MAb binds both FcyRIIIa and FcyRIIIb, can be used in Western blotting and does not inhibit aggregated IgG binding. ELISA using IV.3 (anti-FcyRIIai/2)/6C4 and 3G8 (anti-FcyRIIIa/b)/7.5.4Sw50 MAb pairs make it possible to detect soluble FcyRIIai/2 and FcyRIII, with a sensitivity of 200 pg/mL and 1 ng/mL, respectively. Surface plasmon resonance analyses indicated that the KD of two of the three anti-FcyRII and of the anti-FcyRIII are in the same order of magnitude (6C4: 0.78 nM, 2B2: 0.28 nM, 7.5.4: 0.47 nM). The anti-FcyRII 3D3 MAb exhibits an off-rate constant higher than the 6C4 and 2B2 MAbs and a KD of 2.19 nM. I INTRODUCTION scribed,'7' but MAbs recognizing only FcyRIIIa are not avail- able. A soluble form of the low-affinity FcyRIII (CD 16), termed N man, FcyRII (CD32) and FcyRIII (CD16) are Low-affin- sCD16, derived from proteolytic cleavage of the membrane ity receptors that bind IgG-containing immune complexes.'1' FcyRIIIa and FcyRIIIb is also present in human serum.'8-10' It FcyRIII exists in multiple isoforms. It is encoded by two dis- has been detected in human sera by various assays including tinct genes, FcyRIII-A and FcyRIII-B, each producing a single ELISA using 3G8 anti-CD16 MAb and polyclonal antibodies.'8' transcript that encodes the FcyRIIIa and FcyRIIIb isoforms, re- FcyRII (CD32) is a 40-kDa protein expressed on granulo- spectively. The GPI-anchored FcyRIIIb is expressed on neu- cytes, B cells, monocytes, macrophages, platelets, and epider- trophils under two allelic forms (NA1/NA2),'2' whereas the mal Langerhans cells (LC). At least three genes (FcyRIIA, IIB, transmembrane FcyRIIIa is expressed on macrophages and NK IIC) encode FcyRII. Alternative splicing of FcyRIIA and cells under three allelic forms.'3' Several anti-FcyRIII MAbs FcyRIIB transcripts gives rise to different isoforms (ai and a2, have been described.'2,4,5' Some of them block the binding of bi, b2, and b3, respectively). Two allelic forms of FcyRIIA IgG to the receptor. Some others bind FcyRIII even when the (High Responder; HR, and Low-Responder; LR) have also been receptor is occupied with IgG.'4,6' Several FcyRIII MAbs also defined.'1,2' MAbs raised against human FcyRII have shown show differential reactivity with the NA1/NA2 polymorphic two patterns of reactivity. Group I MAbs bind well to Fc- forms.'4' One MAb that is specific for FcyRIIIb has been de- yRIIai+ cells but are only marginally reactive with FcyRIIb+ 'Unité INSERM 255, and 2Laboratoire de Biotechnologie des Anticorps, Institut Curie, 26, Rue d'Ulm, 75248 Paris Cedex 05, France. 3Present address: Centre d'Immunologie de Marseille-Luminy, Case 906, 13288 Marseille cedex 09, France. 4Work for which these authors equally contributed. 519