A simple method for quantifying high density antigens in erythrocyte membrane by flow cytometry.
Fiche publication
Date publication
juillet 2006
Journal
Journal of biochemical and biophysical methods
Auteurs
Membres identifiés du Cancéropôle Est :
Dr DE ISLA Natalia
Tous les auteurs :
Riquelme BD, de Isla NG, Valverde JR, Stoltz JF
Lien Pubmed
Résumé
RBC flow cytometric analysis is usually used to quantify antigen content. Calibration systems enable antigen content determination by relating mean fluorescence intensity with the number of bound antibody molecules (equivalent to the number of antigen molecules). For that reason, antibodies must be used at saturating concentration, which may lead to agglutination when working with high density antigens. Then, forward scattering, side scattering and fluorescence will be increased, thus obtaining wrong results. In this work, the simple Langmuir adhesion model was applied. Flow cytometry was used to quantify GPA, a transmembrane protein present at high density on RBC. The fluorescence intensity of samples at different anti-GPA sub-saturating concentrations was measured. Sometimes, agglutinates were present and two peaks of fluorescence were observed, the principal one corresponding to isolated cells and the secondary one corresponding to agglutinated cells. In those cases, the principal peak was taken into account for the analysis. The GPA antigen content obtained for nine analyzed samples ranged from 3 to 13 x 10(5) sites per cell, which is similar to those values found in literature. Therefore, the Langmuir adsorption model enables us to determine the antigen content for the anti-GPA/GPA system on RBC membrane. This model could be used to quantify high density antigens in RBC and in other cells.
Mots clés
Antibodies, Monoclonal, immunology, Antigens, analysis, Calibration, Erythrocyte Membrane, immunology, Flow Cytometry, methods, Glycophorin, immunology, Humans, Microscopy, Fluorescence
Référence
J. Biochem. Biophys. Methods. 2006 Jul 31;68(1):31-42