Purification of meningococcal lipo-oligosaccharide by FPLC techniques.
A rapid and efficient method for the preparation of highly pure meningococcal lipo-oligosaccharide (LOS) was developed. This used a Superose 6 column on a FPLC system to purify LOS from phenol-water extracts of cell lysates of Neisseria meningitidis. The purest LOS preparations, with no detectable protein contamination and less than 0.5% (w/w) residual RNA, were obtained when cell lysates had been treated with RNase ONE before phenol extraction and chromatographic separation. Preparations that had received no ribonuclease treatment had 2-3% residual RNA contamination and predigestion of samples with RNase A, which only partially degraded the RNA present in the crude extracts, resulted in LOS samples contaminated with 15-20% residual RNA. The LOS purified from RNase ONE-treated extracts was highly endotoxic, and showed no reduction in antibody binding or specific endotoxin activity compared to unpurified material. Approximately 80% of the LOS applied to the chromatography column was recovered as purified material.
Evans
J S
JS
Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK.
Maiden
M C J
MCJ
Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK.
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000032:analysis; D001427:Bacterial Toxins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004731:Endotoxins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay; D015151:Immunoblotting; D008033:Limulus Test; D008070:Lipopolysaccharides / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D009345:Neisseria meningitidis / Q000737:chemistry* / Q000276:immunology; D012329:RNA, Bacterial / Q000032:analysis
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D008070:Lipopolysaccharides; D012329:RNA, Bacterial; C023023:lipid-linked oligosaccharides