Isolation of the capsular polysaccharide from culture supernatant of Haemophilus influenzae type b

Abstract

The capsular polysaccharide (CP) of Haemophilus influenzae type b is known to be spontaneously released from the cells in culture. The CP is precipitable from culture supernatant by the cationic detergent hexadecyltrimethylammonium. Most of the nucleic acid and some of the protein, but almost none of the endotoxin, in the supernatant are co-precipitated. Extraction of the precipitate with progressively stronger NaCl solutions separates nucleic acid and protein from the CP and also effects a molecular size fractionation. Residual endotoxin and protein can be reduced by extraction with cold phenol and ultracentrifugation. The resulting preparation has ribose, ribitol, and phosphate as principal components and contains less than 1% other sugars, protein, or nucleic acid; it elutes on Sepharose 2B as a symmetrical peak with Kav 0.51.