Cell surface receptor‐antibody association constants and enumeration of receptor sites for monoclonal antibodies

Abstract

Values of receptors per cell and binding constants obtained by the new method from flow cytometric analyses of mixtures of whole blood with FITC- or RD1-labeled CD4, CD8, and CD3 antibodies compare well with literature values determined by other methods.

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

“…The average number of CD4 receptors on a lymphocyte was about 132,000 ± 11,000 (average of column 9 in Table 3). This value is consistent with the known number of CD4 receptors, 145,000 ± 9000 [9,10]. The large uncertainties in the values of K1 and K2 suggest that the residue function has small curvatures in the K1 and K2 directions in parameter space.…”

“…In the case of CD4 mAb-APC from company 1, the total number of CD4 receptors was found to be 139,000 ± 3000. The number of CD4 receptors found for the two CD4 mAb-APC conjugates is within two standard deviations of the value 145,000 ± 9000 reported previously [9,10]. The difference in the values of the parameter [T0] between mAb labeled with FITC, about 114,000, and those labeled with APC, about 147,000, is significant and may point to problems with producing a "pure" solution of CD4 mAb-FITC conjugates due to the small size difference between CD4 mAb (150 kD) and CD4 mAb-FITC (150.4 kD).…”

“…This value is inconsistent with the results obtained from the analysis of CD4 mAb-FITC conjugates from the two companies. Furthermore, it is also inconsistent with a recently published value of 145,000 ± 9000 CD4 receptors on T lymphocytes [9,10]. Possible sources of the discrepancy and resolution of the discrepancy are discussed in the following two sections.…”

“…Model parameters were adjusted to give a good fit between the measured MFI values and the model predictions. In addition to a good fit, it was necessary to invoke divalent binding for CD4 mAb labeled with APC, and monovalent binding for CD4 mAb labeled with FITC in order to get a total number of CD4 receptors as close as possible to the previously measured value of 145,000 ± 9000 [9,10]. As a result, the number of antibodies bound per cell (ABC) was two times higher for the CD4 mAb with FITC labels compared to CD4 mAb with APC labels.…”

“…Only the physically meaningful roots were considered, where both [ATT] and [ ] are real positive numbers and approached zero as [ 0 ] approached zero. In addition, the predicted MFI at equilibrium had to be associated with a value of [T0] approximately equal to 145,000 ± 9000, the previously measured number of CD4 receptors on the surface of normal lymphocytes [9,10]. The value of [T0] was not constrained during the fit of the model to the observed MFI values.…”