Isolation and purification of rat acute-phase α2-macroglobulin

Abstract

Acute-phase α 2-macroglobulin was highly purified from the serum of rats in which this protein had been induced 48 h previously by the injection of croton oil, an inflammatory agent. The isolation protocol involved two non-denaturing steps; first, separation according to molecular weight by gel filtration on Ultrogel AcA 22 and second, negative affinity chromatography which bound contaminating proteins to the column while allowing acute-phase α 2-macroglobulin to pass through. Several criteria were used to assess the purity of acute-phase α 2-macroglobulin, after which the protein was standardized by mass determination and by two different protein assays. Pure rat acute-phase α 2-macroglobulin was used to produce a monospecific antiserum and to calibrate a secondary standard of rat acute-phase serum by developing and characterizing a rocket immunoelectrophoresis assay.