A new method for reduction of endotoxin contamination from protein solutions.
A method of reducing endotoxin contamination in protein-containing solutions is described here using a combination of polymyxin B-Sepharose 4B (PB-Seph 4B) affinity binding and endotoxin-protein dissociation with the dialyzable surfactant, octyl-beta-D-glucopyranoside (OBDG). Using the limulus amoebocyte lysate (LAL) assay to detect endotoxin, greater than 1000-fold reduction of endotoxin reactivity could be accomplished from a contaminated commercial preparation of bovine catalase. Importantly, this occurred with only a 24% protein loss and an 11% loss of catalase enzymatic activity after treatment. The treated catalase appeared to be largely endotoxin-free since it no longer elicited a pyrogenic response in rabbits or primed for intravascular coagulation of the generalized Shwartzman reaction. Of interest, OBDG treatment of Salmonella minnesota Re595 lipopolysaccharide enhanced its ability to bind to serum high density lipoproteins which might contribute to decreased in vivo toxicity. In quantitative studies using radiolabeled endotoxin, the OBDG was shown to be capable of dissociating protein-bound endotoxin thereby facilitating its binding to the PB-Seph 4B adduct. The technique was also useful in removing radiolabeled endotoxin added to human IgG. The methodology described here would be expected to have general usefulness in reducing endotoxin contamination of macromolecular solutions that can bind and retain endotoxin.
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
T E
TE
Karplus
R J
RJ
Ulevitch
C B
CB
Wilson
D002374:Catalase / Q000032:analysis; D002846:Chromatography, Affinity; D003956:Dialysis; D004731:Endotoxins / Q000032:analysis*; D008070:Lipopolysaccharides / Q000032:analysis; D011112:Polymyxin B; D011506:Proteins; D011749:Pyrogens / Q000032:analysis*; D012996:Solutions
D004731:Endotoxins; D008070:Lipopolysaccharides; D011506:Proteins; D011749:Pyrogens; D012996:Solutions; D002374:Catalase; D011112:Polymyxin B