Isolation and Characterization of Rat α₁ and α₂ Macroglobulin and the Development and Purification of their Specific Antibodies

Abstract

A three step procedure for the isolation of α₁ and α₂ macroglobulin from the serum of turpentine inflamed rats is described. The three steps were; gel filtration on G-150 sephadex, zonal centrifugation using a sucrose gradient, and ion exchange chromatography through DEAE- cellulose. The major improvement in the isolation technique over previous reports was the use of stepwise rather than gradient elution for the separation of α₁ and α₂ macroglobulin on the ion exchange column, which greatly improved the purity and yield of the products. The purified proteins were identified and characterized using acrylamide and SDS gel electrophoresis, thin-layer electrofocusing and immunoelectrophoresis. Differences in the two proteins were noted both in their mobilities and subunit sizes in the electrophoresis gels. A higher pI value than that previously reported was noted for the two α₁ macroglobulin bands formed on electrofocusing plates. This was probably due to improvements in the technique for electrofocusing the macroglobulins. Nitrogen content was determined in order to estimate the actual protein content of the lyophilized material. Amino acid composition of each was determined and compared to levels found by other investigators. Antibody to the purified α₁ and α₂ macroglobulin was developed in goats and purified by ammonium sulfate precipitation and ion exchange chromatography. Titers of antibody in the goat serum were determined by immunodiffusion techniques, and the purity of the isolated antibodies demonstrated by immunoelectrophoresis. Attempts at further purification of the antibodies using affinity chromatography were unsuccessful due to the inability of the α₁ and α₂ macroglobulins which were bound to the support medium to bind their specific antibodies.