Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess.

Conjugates of monoclonal antibodies with radioactive isotopes, drugs or toxins have great potential for specific radiolocalization and inactivation of tumor cells. Because the conjugation procedure may adversely alter the antibody, quality control procedures must be applied to determine important characteristics of the conjugated antibody. One such property is how much of the conjugated antibody is able to bind to the relevant antigen. Based on theoretical considerations, we have developed a binding assay for radiolabeled monoclonal antibodies in which the fraction of immunoreactive antibody is determined by linear extrapolation to conditions representing infinite antigen excess. This ensures that the true value of the immunoreactive fraction is obtained, as opposed to the apparent immunoreactive fraction determined under conditions of limited antigen excess. The described assay is based on a double-inverse plot of the binding data which may be considered a modification of the Lineweaver-Burk plot. We established the method using 125I- and 111In-labeling of the 2 monoclonal antibodies T101 and 9.2.27 which currently are undergoing radioimaging trials at the National Cancer Institute. For properly performed conjugation procedures, immunoreactive fractions of about 0.9 were obtained, but a prolonged chloramine-T reaction for 125I-labeling resulted in an immunoreactive fraction of only 0.6. Due to its principle of determining binding at infinite antigen excess, the present method is quite insensitive to variation in the actual amounts of cells and antibody used, as well as the incubation time. We therefore recommend it as a quality control procedure for radiolabeled antibodies. Under certain conditions, this procedure is also applicable for quality control of drug- and toxin-conjugated monoclonal antibodies.