Quantification of glycophorin A and glycophorin B on normal human RBCs by flow cytometry.
BACKGROUND
The quantification of antigens and proteins on RBCs has been achieved by different approaches. Flow cytometry allows the results of the earliest studies to be to reappraised because it offers the possibility of measuring the immunofluorescence intensity of single cells and integrating the individual data of a large number of cells within a very short time.


METHODS
Flow cytometry was used in this work to analyze the binding of four MoAbs to glycophorin A (GPA) and glycophorin B (GPB). RBCs in their native state (nonfixed) were utilized. To avoid the agglutination problem, cells were disaggregated before measurements, dates were taken on 20,000 events on the single-cell region, and the fluorescence intensity of the principal peak present in the fluorescence histograms was used for the analysis. The quantification of sites per RBC was estimated by applying the Langmuir adhesion model.


RESULTS
The numbers of GPA and GPB sites obtained for samples from healthy donors were similar to those found in the literature (1.86-4.9) x 10(5) and (0.48-1.61) x 10(5) for GPA and (0.21-1.14) x 10(5) and (0.47-0.88) x 10(5) for GPB. Differences between antibodies were found that depend on the binding site of each one.


CONCLUSIONS
A simple method to quantify antigen sites on RBCs was developed. It could be applied whenever one antibody is assumed to bind exactly one antigen.
de Isla
Natalia G
NG
Cellular and Tissue Mechanics and Engineering, UMR CNRS 7563, Vandoeuvre-lès-Nancy, France. Natalia.De-Isla@hemato.u-nancy.fr
Riquelme
Bibiana D
BD
Rasia
Rodolfo J
RJ
Valverde
Juana R
JR
Stoltz
Jean F
JF
D000371:Agglutination; D000911:Antibodies, Monoclonal / Q000276:immunology; D000941:Antigens / Q000032:analysis; D001666:Binding Sites, Antibody; D004912:Erythrocytes / Q000276:immunology / Q000378:metabolism*; D005434:Flow Cytometry; D006021:Glycophorins / Q000032:analysis*; D006801:Humans; D018448:Models, Immunological; D020033:Protein Isoforms / Q000097:blood; D012016:Reference Values; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; D000941:Antigens; D006021:Glycophorins; D020033:Protein Isoforms