Estimation of immunoglobulin protease activity by quantitative rocket immunoelectrophoresis

Previous methods for estimating immunoglobulin protease activity have involved the use of enzyme-linked immunosorbent assays (ELISA) or sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography or Western blotting techniques. An alternative method has been developed to estimate proteolytic activity on human IgA1 and IgG using quantitative rocket immunoelectrophoresis. The method uses agarose containing anti-human IgA or anti-human IgG heavy chain-specific reagent to which protease-digested human immunoglobulin samples are applied to wells and electrophoresed overnight. Because proteolytic activity of immunoglobulins results in many smaller fragments, the optimal antigen-antibody ratio for precipitation changes and migration in an electric field results in a larger rocket. Consequently, the area of the rocket will be larger in a protease-treated immunoglobulin sample than a saline-treated immunoglobulin control. These increased rocket areas are correlated with our ELISA protease results (r greater than or equal to 0.90), as well as with our immunoblot results. The method is sensitive to increasing exposure to proteolysis, as well as to increasing amounts of protease. This technique can be used to quickly estimate the ability of a sample to cleave immunoglobulins.