Experimental allergic encephalomyelitis: An encephalitogenic basic protein from bovine myelin

A homogeneous encephalitogenic protein was prepared in high yield (35–40%) from bovine spinal cord and myelin. This protein, referred to as A1 protein, was found to be susceptible to proteolysis in situ at pH 6–7; this effect was arrested by prompt freezing of tissue upon removal from the animal. The purification procedure consisted of (1) defatting with chloroform-methanol (2:1); (2) acid extraction at pH 1.7; (3) precipitation of contaminating protein and dialysis at pH 7; (4) DEAE-cellulose chromatography; and finally, (5) gel filtration. CM-cellulose or Cellex-P chromatography was also used for final purification in place of gel filtration. The DEAE eluate contained the basic protein fraction of which the A1 protein comprised 80–95%. The data suggest that the A1 protein is very likely the only encephalitogenic basic protein of native myelin, constituting approximately 30% of the total myelin protein. It has been obtained in homogeneous form as demonstrated by a single sharp band on polyacrylamide gel electrophoresis at pH 4.5 and 8.6, and by immunoelectrophoresis and immuno-double diffusion. As little as 0.1 μg induced histologic lesions in the CNS and 10–100 μg induced clinical disease in 80–100% of guinea pigs. In addition to its encephalitogenic activity the A1 protein was found to induce in rabbits and guinea pigs the formation of precipitating antibody which was detected by immuno-double diffusion and assayed in a passive hemagglutination test. Antibody-combining activity of the A1 protein was also demonstrated by immuno-double diffusion and was measured by a passive hemagglutination-inhibition test. The delayed hypersensitive response developed in guinea pigs after injection of 20 μg of the A1 protein and was demonstrated using 5 μg as skin test antigen. The positive skin test and histologic lesions characterizing EAE both appeared on days 4–5 after sensitization and developed simultaneously. The principal findings here reported, on the in situ proteolysis of the encephalitogenic basic protein, provide a partial explanation for the reports from different laboratories of encephalitogens possessing widely differing properties.