Analysis of antibody-antigen interactions in mixtures containing reactive and nonreactive components using size-exclusion high-performance (pressure) liquid chromatography

Analysis of antibody-antigen interactions using size-exclusion high-performance (pressure) liquid chromatography was applied to a polyvalent system composed of both reactive and nonreactive components. Mixtures containing varying concentrations of antivenin (Crotalidae) polyvalent (equine origin) and either Crotalus atrox (Western diamondback rattlesnake) venom (CV) or isolated C. atrox phospholipase A2 (PLA2) were separated using a Bio-Sil SEC 250-5 size-exclusion column (300 x 7.8 mm). Major regions containing high-molecular-weight aggregates, antivenin IgG, and CV components or PLA2 could be identified from the elution profiles. Changes in elution profile areas could be modeled by equations derived from the law of mass action that included values for the maximum fraction of reactivity, antigen valence, apparent binding constants, profile area proportionality constants, and nonreactive profile area. The analysis was simple, fast, and readily interpretable and may be applicable to a variety of situations in which stable antigen-antibody complexes are formed in the presence of nonreactive components.