Characterization of new murine monoclonal antibodies directed against glycophorins C and D

SUMMARY. Six new murine monoclonal antibodies (mAbs) directed to the erythrocyte membrane glycophorins C (GPC) and D (GPD) were obtained from splenocytes of different BALB/c mice immunized with human red blood cells, and fully characterized. The mAbs were selected by agglutination tests with control and Gerbich-negative cells, and by immunoblotting analysis. They showed specificity for the N-terminal domain(s) of GPC (and GPD) and were classified into three categories by competitive analysis using ¹²⁵Ilabelled antibodies and real-time biospecific interaction. The first group (NaM10-7G11, NaM70-1G4 and NaM77-7B6) compete for epitope(s) located at the N-terminal portion of GPC. Agglutination-inhibition tests revealed that the 7G11 epitope involves the amino group of Met¹ and sialic acid residue(s) whereas the 1G4 and 7B6 epitopes contain O-glycans. NaM89-2G11 belongs to a second group; its epitope is located in a region including Glu¹⁷, Asp¹⁹ and (an) O-glycan(s). The third group comprises mAbs NaM19-3C4 and NaM98-3Cl which bind to both GPC and GPD in proximity of the binding site of human anti-Ge:3 antibodies.
In addition, mAb 3C4 (anti-GPC/GPD) was found to bind to approximately 125 000 sites per red cell. Considering that the ratio of the GPC to GPD is about 3–4 to 1, the number of GPC and GPD molecules was estimated as 95 000 and 35 000, respectively.