Chromatographic assessment of polyvalent antivenom activity

While new technologies for the rapid isolation and purification of antibodies using HPLC methods have been coupled with current data acquisition and analysis systems, additional technologies have been required to measure antibody activity. A chromatographic approach for rapid measurement of antibody reactivity is shown with two systems. First, to test the suppositions of a model in which reactivity was assessed by conducting the reaction in solution followed by chromatographic analysis of the changes in amounts of reactants and products, monoclonal antibody to human serum albumen (HSA) and HSA were used. Measured parameters of free and reacted/free antigen illustrated ideality of behavior to classical reaction modeling by the Law of Mass Action. A simple titration based on the reduction of the free (unbound) antigen chromatogram peak reveals reactions that are linearly dependent on antibody concentration. In a second system, the polyvalent antibody activity in Antivenin (Crotalidae) Polyvalent (Wyeth) reacting to Crotalus atrox venom was studied. A concentration dependent appearance of a high molecular weight peak (immune complexes) could be identified from the elution profiles providing an indication of overall antibody binding activity; while the reduction of specific antigen peaks demonstrates individual reactivities. An overlay plot of elution profiles of venom visualized antibody specificity and overall reactivity. Thus, the effectiveness of the peak reduction approach in demonstrating the in vitro binding of a component in an antigen mixture is shown using C. atrox venom. The method is straightforward, rapid, and the ideal nature of the modeling allows easily interpreted information to be gained from the data describing the reactions. Further, simultaneous measurements of activity could be made for each resolvable chromatographic entity. In an approach which is rapid, simply implemented by most biotechnology laboratories, and holds great promise for antigen-antibody systems not amenable to assay by ELISA, a single dilution series serves to both visualize the overall selectivity of the antisera toward the antigens in a mixture, and rapidly titrate antibody activity to as many antigens as one can resolve by the chromatographic method.