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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.

Original publication

DOI

10.1099/13500872-142-1-57

Type

Journal article

Journal

Microbiology

Publication Date

01/1996

Volume

142 ( Pt 1)

Pages

57 - 62

Keywords

Antibodies, Bacterial, Antibodies, Monoclonal, Antigens, Bacterial, Bacterial Proteins, Bacterial Toxins, Chromatography, Gel, Chromatography, High Pressure Liquid, Endotoxins, Enzyme-Linked Immunosorbent Assay, Immunoblotting, Limulus Test, Lipopolysaccharides, Neisseria meningitidis, RNA, Bacterial