Background: The method most commonly used for measuring the affinity of MoAbs specific for the D antigen is a binding assay using 125I-labeled MoAb. Although the method is relatively simple, there are several drawbacks that can lead to inaccurate results. The objective of the present work was to develop a method for the determination of the affinity of anti-D MoAb using unlabelled antibodies.
Study design and methods: Rh-positive RBCs were sensitized with varying amounts of unlabeled anti-D MoAb, and, at equilibrium, the amount of anti-D bound to the RBCs was measured by ELISA. The affinity and the number of antigenic sites were determined with the Scatchard and Langmuir equations.
Results: The method was applied to determine the affinity of several MoAbs specific for the D antigen on RBCs of different Rh-positive phenotypes. Similar results were observed with both equations. The affinity constant (Ka) for 3 MoAbs specific for the D antigen on group O R1r RBCs ranged from 1.3 to 7.4 108 M-1, depending on the antibody. When measured by a binding assay with 125I-labeled anti-D MoAbs, the Ka were significantly lower, indicating that 125I-labeling diminishes anti-D affinity, even when the labeling is at a low level.
Conclusion: A simple ELISA method was developed for the measurement of the affinity of anti-D MoAbs for the D antigen and for the number of antigenic sites per RBC. As a result, the affinity of anti-D can be estimated accurately, thus avoiding the drawbacks inherent in modification of the antibody by 125I-labeling.