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Measurement of anti‐D intrinsic affinity with unlabeled antibodies
Author(s) -
Debbia Martine,
Lambin Patrick
Publication year - 2004
Publication title -
transfusion
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.045
H-Index - 132
eISSN - 1537-2995
pISSN - 0041-1132
DOI - 10.1111/j.1537-2995.2004.00651.x
Subject(s) - antibody , chemistry , chromatography , medicine , immunology
BACKGROUND: The method most commonly used for measuring the affinity of MoAbs specific for the D antigen is a binding assay using 125 I‐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 deter‐mination 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 (K a ) for 3 MoAbs specific for the D antigen on group O R 1 r RBCs ranged from 1.3 to 7.4 10 8 M –1 , depending on the antibody. When measured by a binding assay with 125 I‐labeled anti‐D MoAbs, the K a were significantly lower, indicating that 125 I‐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 125 I‐labeling.