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

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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 times 108 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.