Production of a New Murine Monoclonal Antibody with Fy6 Specificity and Characterization of the Immunopurified N-Glycosylated Duffy-Active Molecule

Abstract

A murine monoclonal antibody (mAb i3A; IgG1, kappa light chain) was obtained using human red blood cells as immunogen. The antibody showed Fy6 specificity since it agglutinated all but Fy(a-b-)-untreated red cells and failed to agglutinate chymotrypsin-treated cells. An erythrocyte membrane protein of 42-46 kD was revealed as the major component recognized by the antibody on immunoblots. The antibody also bound to 92- to 95- and 200-kD proteins, tentatively identified as oligomers of the 42- to 46-kD monomeric form. The affinity-purified Fy6-active protein was converted to a sharp band of 35 kD after N-glycanase treatment. The molecule appeared as a slightly broadly band after neuraminidase treatment but was not further altered by O-glycanase. The i3A mAb bound to 6,000±1,000 receptor sites on either Fy(a-b+), Fy (a+b+) and Fy(a+b-) red cells with an affinity constant in the range of 3-6 x 10^8 M^-1. No binding was observed to other blood cells nor to several cells (B, T, myelomonocytic and erythro-leukemia cell lines). Also, the bulk of i3A- Fy6 immune complexes could be dissociated from the red cell membrane with as low as 0.2% Triton X-100, showing that the Fy6-active glycoprotein is not tightly associated with the membrane skeleton. Our data obtained with a new monoclonal antibody directed to the Fy6 antigen demonstrate that the blood group Duffy-active component is a red cell-specific glycoprotein carrying one or more N-linked oligosaccharides.

Close Association of the First and Fourth Extracellular Domains of the Duffy Antigen/Receptor for Chemokines by a Disulfide Bond Is Required for Ligand Binding

“…Unlabeled RAN-TES and MGSA were from R&D Systems (Abingdon, United Kingdom). The anti-Fy6 (i3A) mAb was described elsewhere (25). The anti-Fy3 (CRC-512) and anti-Fya mAbs were kindly provided by Dr. Makato Uchikawa (Japanese Red Cross, Tokyo, Japan) and Dr. F. Buffiere (ETS, Bordeaux, France), respectively.…”