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Infect Immun. 1972 July; 6(1): 27-31
Copyright © 1972 American Society for Microbiology. All Rights Reserved.

Purification of Bacterial Endotoxins by Zonal Centrifugation

^a Service d'Immunochimie Garches, Institut Pasteur, 92-Garches, France

ABSTRACT

In this paper, we describe a cultivation procedure in large-capacity fermentors (200 liters) and the purification of the Salmonella crude extracts by means of zonal centrifugation in a sucrose gradient. Briefly, the endotoxin has been extracted from salmonellae with hypertonic solutions. The crude endotoxin was shown to contain several antigens, mainly r₁, r₂, r₃, or heterologous antigen. The heavy endotoxin was isolated and purified previously by enzymatic digestion, followed by a series of gel filtrations on Sephadex or Sepharose columns. We report the isolation and purification of heavy endotoxin in sucrose gradients, with the use of a B XIV titanium zonal rotor. From 150 to 300 mg of the crude material resuspended in a solution containing 1 M sodium chloride, 0.1 M sodium citrate, and 2.5% (w/w) sucrose, has been submitted to zonal centrifugation in gradients consisting of 5 to 25% (w/w) sucrose, also containing 1 M sodium chloride and 0.1 M sodium citrate. An overlay of 200 ml of 1 M sodium chloride-0.1 M sodium citrate was introduced after the sample. The separations were obtained after centrifugation for 3.5 hr at 35,000 rev/min (w²dt = 1.66 x 10¹¹) at 4 C; the heavy endotoxin sedimented as heterogeneous material, from the middle toward the distal portions of the gradient. The heterologous antigens (r₁, r₂, r₃), as well as bacterial proteins, remained in the sample zone. The heavy endotoxin recovered from the gradients was quite pure, as revealed by immunodiffusion tests against several antibacterial sera.

¹ Present address: Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, N.J. 08903.