Application of optical properties of the Vi capsular polysaccharide for quantitation of the Vi antigen in vaccines for typhoid fever

Abstract

The capsular polysaccharide of Salmonella typhi and of Citrobacter freundii (Vi) is a linear homopolymer of alpha 1,4-linked N-acetylgalactosaminuronic acid, variably O-acetylated at the C-3 position. Vaccines composed of Vi confer protection against typhoid fever with an efficacy of about 70%; Vi has recently been conjugated to proteins to increase its immunogenicity and effectiveness (I.L. Acharya, R. Tapa, V.L. Gurubacharya, M.B. Shrestha, C.U. Lowe, D.D. Bryla, R. Schneerson, J.B. Robbins, T. Crampton, B. Trollfors, M. Cadoz, D. Schulz, and J. Armand, N. Engl. J. Med. 317:1101-1104, 1987; K.P. Klugman, I. Gilbertson, H.J. Kornhof, J.B. Robbins, R. Schneerson, D. Schulz, M. Cadoz, and J. Armand, Lancet ii:1165-1169, 1987; S.C. Szu, A.L. Stone, J.D. Robbins, R. Schneerson, and J.B. Robbins, J. Exp. Med. 166:1510-1524, 1987). Vi, however, cannot be measured by conventional colorimetric methods. Two optical techniques were adapted to quantitate Vi in vaccines. The first, Fourier-transformed infrared spectroscopy, was performed on salt-free, freeze-dried samples. The intensities of the absorbance peaks of Vi were proportional to the amount of Vi within the range of 0.25 to 2.0 mg. The amount of Vi was determined from integrated absorptions at the 1,235- or 1,417-cm-1 band. The second technique, spectrophotometric titration, was more sensitive than the Fourier-transformed infrared spectroscopy and could be performed on dilute solutions. The metachromatic effect of the reaction between the aromatic cationic dye acridine orange and the carboxyl groups of Vi was quantitative within +/- 2% in the range of 20 to 700 micrograms of Vi per ml. The accuracy of the titration of Vi in the vaccines was within +/- 8%. These two methods may be applicable to measure other capsular polysaccharides in vaccines.