A Model for the Binding of Fluorescently Labeled Anti-Human CD4 Monoclonal Antibodies to CD4 Receptors on Human...

The CD4 glycoprotein is a component of the T cell receptor complex which plays an important role in the human immune response. This manuscript describes the measurement and modeling of the binding of fluorescently labeled anti-human CD4 monoclonal antibodies (mAb; SK3 clone) to CD4 receptors on the surface of human peripheral blood mononuclear cells (PBMC). CD4 mAb fluorescein isothiocyanate (FITC) and CD4 mAb allophycoerythrin (APC) conjugates were obtained from commercial sources. Four binding conditions were performed, each with the same PBMC sample and different CD4 mAb conjugate. Each binding condition consisted of the PBMC sample incubated for 30 min in labeling solutions containing progressively larger concentrations of the CD4 mAb-label conjugate. After the incubation period, the cells were re-suspended in PBS-based buffer and analyzed using a flow cytometer to measure the mean fluorescence intensity (MFI) of the labeled cell populations. A model was developed to estimate the equilibrium concentration of bound CD4 mAb-label conjugates to CD4 receptors on PBMC. A set of parameters was obtained from the best fit of the model to the measured MFI data and the known number of CD4 receptors on PBMC surface. Divalent and monovalent binding had to be invoked for the APC and FITC CD4 mAb conjugates, respectively. This suggests that the mAb binding depends on the size of the label, which has significant implications for quantitative flow cytometry. The study supports the National Institute of Standards and Technology program to develop quantitative flow cytometry measurements.