I. PRESENCE OF BOVINE ANTIGENS
D. W. STAINER
Connaught Medical Research Laboratories, University of Toronto, Toronto, Ontario
Received January 31, 1967
Papain digest of beef broth (P.D.B. broth) was used to grow Corynebacterium diphtheriae in submerged culture, and high potency diphtheria toxins were produced. It was found that the resultant toxoids could sensitize animals to beef proteins. Guinea pigs immunized with deep culture toxoid in complete Freund's adjuvant exhibited anaphylaxis when challenged with beef serum. When aluminium phosphate and Bordetella pertussis were used as adjuvant or when the toxoid was given alone (i.e. without adjuvant) the responses to beef serum were reduced but the sensitivity to beef was still observed. Deep culture toxoids from three other manufacturers were tested and showed similar properties. When concentrated P.D.B. broth was used as a challenge dose, anaphylactic reactions were also noted, indicating that P.D.B. broth, as usually prepared, contained bovine antigens.
Introduction
The use of a tryptic digest of beef muscle medium for the production of the toxin of Corynebacterium diphtheriae in surface culture has been described by many workers (8-10). Linggood et al. (3, 4) demonstrated that a similar medium could be used for submerged culture production of diphtheria toxin. Edwards (1) used papain instead of trypsin to hydrolyze the beef muscle and this papain digest of beef broth (P.D.B. broth) yielded high potency toxin (greater than 200 Lf1 per milliliter in 48 h). In these laboratories, production of diphtheria toxin for many years has been carried out in surface culture using a modification of Mueller's formula (6) which contains no meat and consists of an acid digest of casein together with growth factors and maltose. Deep culture fermentation has many advantages and, using P.D.B. broth experimentally, we have prepared large quantities of diphtheria toxin. This has been detoxified to yield highly antigenic, stable toxoids. Since the basis of the original medium was beef muscle, the studies reported here have been undertaken to test for the presence of bovine antigens in these purified toxoids.
Methods
Deep Culture Toxoids Growth conditions.--A modified P.W. 8 strain of C. diphtheriae (M353) was used throughout. The cultures for seeding the fermenters and tanks were ob- tained by growth on Loeffler slopes and subcultured into 100 ml of P.D.B. broth in 500-ml Erlenmeyer flasks, incubated on a shaker for 24 h at 34-35 °C. The P.D.B. broth was prepared by a modification of the method described by Edwards (1). Seven additions of 7.5 g papain (Difco purified) were made to a beef muscle infusion (18 lb of beef in 55,000 ml of water) at half-hourly intervals. Throughout the digestion period the pH was maintained at 7.2-7.4 and the temperature at 60-62 °C. The mixture was acidified with glacial acetic to pH 5.2 and boiled for 5 min. After filtration, the medium was yeast treated [end of text]
1 Lf, Limit flocculation.
Canadian Journal of Microbiology. Volume 13 (1967)