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Equine infectious anemia (EIA) antigen extracted from the spleen of horses infected with EIA virus was purified by pH treatment, (NH4)2SO4 fractionation and affinity chromatography. The homogeneity of the antigen was indicated by sedimentation rate and sedimentation equilibrium experiments. A S20,w of 0.51 was determined and a molecular weight of 7600 was calculated from sedimentation equilibrium analysis. The amino acid composition of the pure antigen indicated the antigen is an acidic protein. Employing radical immunodiffusion (RID) and pure antigen a method for quantitating antigen content of antigen containing preparations was developed.

The extracellular proteinase produced by a depressed strain of Serratia marcescens ATCC 25419 was purified and characterized. This produces more than 10-times the amount of extracellular proteinase produced by other strains of Serratia tested. The purified enzyme was prepared from the culture supernatant by (NH4)2SO4 fractionation and DEAE-cellulose chromatography. The purified enzyme has an so20,w of 3.95 and is a monomer of molecular weight 51,900. The proteinase has a broad pH optimum in the alkaline range with a maximum at pH 9.5. The enzyme will utilize a number of proteins as substrate. The products of digestion are primarily in the size range of tripeptides to hexapeptides. Peptides containing a sensitive bond and a minimum size of size amino acids are hydrolyzed. The proteinase is inhibited by chelating agents but unaffected by sulfhydryl or serine reagents and is devoid of esterase activity.

Immunodiffusion antigen from spleens of horses infected with equine infectious anemia virus was prepared by methods employing freeze-thaw cycles and thiocyanate treatment. Thiocyanate (0.5 M) permitted the recovery of the greatest amount of antigen. Furthermore, it was most effective for recovery of immunodiffusion antigen from spleens which yielded unsatisfactory concentrations of antigen by the conventional freeze-thaw or water-extraction methods. The reactivity of the antigen did not appear to be affected by this chemical treatment.