A procedure for purification of Neisseria meningitidis lipooligosaccharide (LOS) from outer membrane vesicles (OMV) in spent growth media was developed. Five different strains of group A N. meningitidis having different LOS types were grown in tryptic soy broth with vigorous aeration for 36-48 hours, and centrifuged to collect both cells and supernatants. The amount of LOS in the OMV in the supernatants was higher or at least equal to that in the cells. The OMV in each supernatant were concentrated, pelleted by ultracentrifugation, and treated with 2% sodium deoxycholate to dissociate LOS from OMV. The LOS was then separated from capsular polysaccharides, proteins and phospholipids by gel filtration on Sephacryl S-300 column in 1% sodium deoxycholate, and precipitated from the column fractions in 70% ethanol. In addition, LOS was also extracted from cells with hot phenol-water, ultracentrifuged once after treatment with ribonuclease, and purified on Sephacryl S-300. When compared with an improved phenol-water extraction method, the LOS obtained from either OMV or cells by the above methods gave a 40-180% increase in yield. The LOS also had much higher activities in limulus amebocyte lysate assay, rabbit pyrogenic test, and ELISA assay. The LOS purified from cells and from OMV were indistinguishable by SDS-PAGE analysis.
