Quantitative immunofixation of proteins following zone electrophoresis in agarose gel: application to the determination of the stoichiometry of the alpha1-antitrypsin-elastase interaction.

A method for the quantitative determination of proteins by immunofixation-agarose gel electrophoresis is described. The proteins are separated electrophoretically and the resulting circular zone overlaid with specific antibody-impregnated filter paper (10.2 microliter/cm2). Following incubation for 20 h at room temperature the plates were processed, stained and the areas of the precipitin zones determined by planimetry. Intra-plate variation (coefficient of variation) for alpha1-antitrypsin (30 samples) and elastase (30 samples) at concentrations of 0.25, 0.5 and 1.0 mg/ml was 2.2--5.8% and inter-plate variation ranged from 2.9% to 5.2%. Overall, the analyses showed that the method is the statistical equivalent of rocket immunoelectrophoresis. The lowest concentration used was 0.25 mg/ml which corresponds to a sensitivity of 1.25 microgram (5 microliter per sample well). It is conceivable that lower amounts could be successfully determined. Application of the method to the determination of stoichiometry of the alpha1-antitrypsin--elastase interaction permitted the simultaneous quantitative determination of inactivated alpha1-antitrypsin (an acidic protein) and excess elastase (a basic protein) in alpha1-antitrypsin--elastase reaction mixtures (molar ratio elastase/alpha1-antitrypsin = 1.3) and thus by difference, the amount of elastase and and alpha1-antitrypsin in the alpha-antitrypsin--elastase complex. The results showed that the molar combining ratio of inhibitor to enzyme was 1.086 : 1 or 1 : 1. Conventional inhibition experiments (residual elastase activity as a function of increasing amounts of alpha 1-antitrypsin) showed this ratio to be 1.79 : 1 but 1.03 : 1 when the inactivation reaction, as determined by quantitative immunofixation, was taken into account. The quantitative immunofixation method should be applicable to interacting systems which, like elastase and alpha1-antitrypsin, result in complexes with electrophoretic mobilities intermediate to the parent proteins.