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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.

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