Purification of nonlipopolysaccharide antigen from Brucella abortus during preparation of antigen used for indirect hemolysis test.

The indirect hemolysis test (IHLT) for the diagnosis of brucellosis uses a lipopolysaccharide (LPS) antigen obtained by dimethyl sulfoxide extraction of Brucella abortus. We showed that a non-LPS antigen can be obtained as a by-product of the IHLT antigen preparation. The antigen was purified to homogeneity by a combination of gel-filtration chromatography and ion-exchange chromatography. The substance contained 8% protein and about 65% carbohydrate. The molecular weight of the primary unit was 19,750, when analyzed by polyacrylamide gel electrophoresis under denaturing conditions. The non-LPS antigen, which is serologically identical to B. abortus smooth LPS O antigen, did not bind to cell membranes. However, it could be used to detect specific antibodies by complement fixation, precipitation in agarose gels, and inhibition of the IHLT.