A simple method for quantifying high density antigens in erythrocyte membrane by flow cytometry

Journal of Biochemical and Biophysical Methods
Bibiana D RiquelmeJean-François Stoltz

Abstract

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 enab...Continue Reading

References

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Citations

Sep 12, 2008·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·María Belén Jiménez-DíazIñigo Angulo-Barturen
Feb 8, 2011·Journal of Immunological Methods·Brian T Grimberg
Dec 11, 2007·Langmuir : the ACS Journal of Surfaces and Colloids·Darby KozakMatt Trau

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