Production of a new murine monoclonal antibody with Fy6 specificity and characterization of the immunopurified N-glycosylated Duffy-active molecule.
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.
Riwom
S
S
Centre Régional de Transfusion Sanguine de Nantes, France.
Janvier
D
D
Navenot
J M
JM
Benbunan
M
M
Muller
J Y
JY
Blanchard
D
D
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity; D004375:Duffy Blood-Group System / Q000276:immunology* / Q000302:isolation & purification; D004910:Erythrocyte Membrane / Q000276:immunology; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D007145:Immunoglobulin kappa-Chains / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D004375:Duffy Blood-Group System; D007074:Immunoglobulin G; D007145:Immunoglobulin kappa-Chains