DISSOCIATION RATE KINETICS IN A SOLID-PHASE FLOW IMMUNOASSAY

Solid-phase displacement assays allow extremely fast analyses when per formed under continuous flow conditions. Continuous dissociation of la beled antigen from the immobilized saturated antibodies occurs even in the absence of competing unlabeled antigen. This spontaneous dissocia tion creates more unoccupied antibody binding sites which affect the m agnitude of the signal generated. In order to evaluate the impact of t his phenomenon in more detail, we extended the law of mass action to s olid-phase binding assays and analyzed the dissociation kinetics of la beled antigen under continuous flow conditions. The effect of the now on the dissociation kinetics was determined by calculation of the appa rent dissociation rate constants (k(d)) which increase with an increas e in the now rate. These dissociation rate constants are approximately 20- to 30-fold lower than those obtained from displacement studies (i .e., in the presence of competing unlabeled antigen). The difference i n the dissociation rate constants obtained in the two studies is most likely a function of the degree of reassociation. The results of this study provide a basis for better understanding antibody kinetics at so lid-liquid interfaces under flow conditions.