Method for producing a vaccine against bacterial infections caused by pseudomonas aeruginosa

Method for isolating a nontoxic, nonpyrogenic polysaccharide antigen from the crude slime of Pseudomonas aeruginosa comprising the steps of:

• a. adding about 1% by volume of a 10% cetyltrimethylammonium bromide solution to said slime to form a mixture which is allowed to stand for about 30 minutes to permit precipitation of nucleic acids;
• b. removing the nucleic acids precipitated in step a. by centrifugation;
• c. adding about 80% by volume of a 95% ethanol to the product of step b. to cause precipitation;
• d. collecting the precipitate formed in step c. by centrifugation and dissolving it in phosphate buffered saline;
• e. subjecting the product formed in step d. to the same treatment given to said slime by repeating steps a., b. and c. then collecting a second precipitate in the manner provided for in step d.;
• f. subjecting the precipitate collected in step e. to acetic acid hydrolysis followed by heating at around 90.degree. C. for about 18 hours and allowing to cool to room temperature;
• g. centrifuging the cooled product formed in step f. to separate the lipid polysaccharides from the acetic acid supernate;
• h. extruding the acetic acid supernate formed in step g. with chloroform to remove residual lipid polysaccharides;
• i. subjecting the polysaccharides products formed in step h. to alcoholysis to precipitate the polysaccharides from the aqueous layer and separating said polysaccharides by centrifugation;
• j. dissolving the polysaccharide products in phosphate buffered saline, digesting with nucleases to remove nucleic acids and extracting the remaining protein therefrom with phenol;
• k. mixing the polysaccharide product of step j., cooling and maintaining at a temperature of 4.degree. C. for about 12 hours;
• l. centrifuging the product of step k. to separate and remove the aqueous layer from the phenol layer;
• m. subjecting the water layer formed in step l. to alcoholysis and separating the polysaccharides from the aqueous layer by centrifugation;
• n. dissolving the polysaccharides of step m. in phosphate buffered saline and applied to a 2.6.times.100 cm column of Sephadex G-100 equilibrated in phosphate buffered saline;
• o. combining the void volume fractions and subjecting to alcoholysis to precipitate the polysaccharides;
• and
• p. isolating the polysaccharides having a molecular weight of from 100,000 to 300,000 daltons in the void volume by dissolving the polysaccharides of step o. in water and desalting on a 1.5.times.60 cm column of Sephadex G-50 equilibrated with a liquid selected from the group of distilled water and phosphate buffered saline having a pH of about 7.2.