Determination of immunoreactive fraction and kinetic parameters of a radiolabeled monoclonal antibody in the absence of antigen excess

Abstract

Monoclonal antibodies (Mabs) directed against cell surface determinants and conjugated to fluochromes, radionuclids or drugs are of increasing importance in cell and tumor biology as well as in clinical oncology. Many of the applications of Mab require precise and quantitative information regarding the molecular interactions of labeled antibody with the respective antigen expressed on the cell surface. These interactions are characterized by the association rate constant (ka), the dissociation rate constant (kd) and the antibody affinity constant (K). The immunoreactive fraction (IRF) of the labeled antibody molecules directly influences these parameters. IRF is usually reduced below 100% by antibody purification and labeling procedures and, in case of radiolabeled antibodies, by radiation damage during antibody storage. Besides the calculation of kinetic parameters, IRF should, therefore, be determined for the quality control of any antibody preparation before experimental or clinical application. Commonly used methods for measuring IRF are based on radioimmunoassays (RIA) on intact cells performed under antigen excess. However, especially with Mabs directed against cell surface antigens expressed in small numbers per cell and for displaying low affinity constants, these assays often give unsatisfactory results. We have, therefore, established a method which permits us to determine IRF, ka, kd and K for an 125I-labeled Mab with precision even in the absence of antigen excess.