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Studies on Vi Antigen

II. Purification of Vi Antigen from Escherichia Coli 5396/38

From the Department of Biochemistry and the Immunology Division, Army Medical Service Graduate School, Washington 12, D. C.

Abstract

A simple ethanol-salt fractionation procedure followed by acetic acid hydrolysis is described which yields a purified Vi antigen from saline extracts of acetone dried Escherichia coli, strain 5396/38. The various stages in the purification were accompanied by increased serological activity. The final product was the only fraction that was free from contamination with E. coli somatic antigen. This fraction showed a high degree of serological activity and specificity.

The residue left after extracting the bacterial cells with saline contained only 1% of the original activity as determined by mouse protective potency tests. On a dry weight basis, however, the saline extract was consistently less active than the intact bacteria. It is suggested that the presence of other antigens in the whole organisms may have influenced the response to the specific Vi antigen. Alternatively, the insoluble cellular components may have functioned as a depot substance which indirectly stimulated the production of Vi antibody.

The stages up to and including ethanol-salt concentration effected a 10-fold purification of the Vi antigen on a dry weight basis and a 25-fold purification on the basis of total nitrogen content. Chemical analysis at this stage revealed the presence of phosphorus, protein and carbohydrate. No differences were observed in the antibody response of rabbits to the various fractions up to this stage.

The next stage, shaking with chloroform-ethanol, removed only trace amounts of inactive material, yet the antigenicity of the product for rabbits was significantly reduced. No comparable reduction in antigenicity for mice was observed. Acetic acid hydrolysis liberated the Vi antigen from its complex without further impairing its antigenicity.

Chemical analysis of the purified Vi antigen showed that it is a fairly strong acid but that it does not contain uronic, sulphuric or phosphoric acid residues. Both nitrogen and acetyl groups are present, but apparently not in the usual form of N-acetylhexosamine.

Footnotes

^* Abstracted in part from a thesis presented to the Graduate School, Georgetown University, in partial fulfillment of requirements for the degree of Doctor of Philosophy.