A procedure for purification of N. meningitidis lipooligosaccharide (LOS) from cells and outer membrane vesicles (OMV) in spent media was developed. Five LOS-typing strains of group A N. meningitidis were grown in tryptic soy broth with vigorous aeration for 36-48 hours, and centrifuged to collect both cells and supernatants. The OMV in each supernatant were concentrated, pelleted by ultracentrifugation, and treated with 2% sodium deoxycholate (DOC) to dissociate LOS from OMV. The LOS was separated from capsular polysaccharides, proteins and phospholipids by gel filtration on Sephacryl S-300 in buffer containing 1% DOC. In addition, LOS from the cells was also extracted with hot phenol-water, ultracentrifuged once after treatment with nucleases, and purified on Sephacryl S-300. Purification of LOS from OMV was simpler and its yield was higher or equal to that from cells. When compared to the classic phenol-water extraction method, the yield of LOS obtained either from OMV or cells by the above procedure is usually better with a total of 160-300 % increase in yield from both OMV and cells. The LOS purified from the OMV and the cells were indistinguishable by SDS-PAGE analysis.
