Analysis of anti body-antigen interactions in mixtures containing reactiveand nonreactive components using size-exclusion high-performance (pressure) liquid chromatography

Analysis of antibody-antigen interactions using size-exclusion high-perform ance (pressure) liquid chromatography was applied to a polyvalent system co mposed of both reactive and nonreactive components. Mixtures containing var ying concentrations of antivenin (Crotalidae) polyvalent (equine origin) an d either Crotalus atrox (Western diamondback rattlesnake) venom (CV) or iso lated C. atrox phospholipase A(2) (PLA(2)) were separated using a Bio-Sil S EC 250-5 size-exclusion column (300 x 7.8 mm). Major regions containing hig h-molecular-weight aggregates, antivenin IgG, and CV components or PLA(2) c ould be identified from the elution profiles. Changes in elution profile ar eas could be modeled by equations derived from the law of mass action that included values for the maximum fraction of reactivity, antigen valence, ap parent binding constants, profile area proportionality constants, and nonre active profile area. The analysis was simple, fast, and readily interpretab le and may be applicable to a variety of situations in which stable antigen -antibody complexes are formed in the presence of nonreactive components. ( C) 2001 Academic Press.