PREPARATION AND PROPERTIES OF DIPHTHERIA TOXOIDS IN SUBMERGED CULTURE: I. PRESENCE OF BOVINE ANTIGENS

1967 ◽  
Vol 13 (8) ◽  
pp. 963-968 ◽  
Author(s):  
D. W. Stainer
Keyword(s):  
Bordetella Pertussis ◽  
Guinea Pigs ◽  
Submerged Culture ◽  
Corynebacterium Diphtheriae ◽  
Challenge Dose ◽  
Aluminium Phosphate ◽  
High Potency ◽  
Deep Culture ◽  
Freund’S Adjuvant ◽  
Freund's Adjuvant

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.

SEPARATION OF BOVINE SENSITIZING MATERIAL FROM PAPAIN DIGEST OF BEEF BROTH

1967 ◽  
Vol 13 (8) ◽  
pp. 1001-1008 ◽  
Author(s):  
D. W. Stainer
Keyword(s):  
Ammonium Sulfate ◽  
Bordetella Pertussis ◽  
Aluminium Hydroxide ◽  
Guinea Pigs ◽  
Submerged Culture ◽  
Gel Filtration ◽  
Toxin Production ◽  
Corynebacterium Diphtheriae ◽  
Freund's Adjuvant ◽  
Anaphylactic Response

Papain digest of beef broth (P.D.B. broth), which is routinely used to grow Corynebacterium diphtheriae in submerged culture, was examined for its ability to elicit sensitivity reactions to beef serum in guinea pigs and to induce shock. When ammonium sulfate was added to P.D.B. broth to 45% (w/v) a precipitate was obtained which, when redissolved and combined with Freund's adjuvant, sensitized guinea pigs so that challenge with beef serum produced severe anaphylactic reactions. If aluminium hydroxide and Bordetella pertussis were used as adjuvant, the method of preparation of the broth was shown to have an effect on the anaphylactic response obtained.Sephadex gel filtration of the ammonium sulfate-precipitable material gave an included and excluded ultraviolet-absorbing peak at 278 mμ, and all of the sensitizing properties were shown to reside in the excluded fraction. The amount of sensitizing material could be greatly reduced by either ultrafiltration or by adsorption of the broth with Al(OH)3 gel. These treated media still supported good toxin production.

Preparation and properties of diphtheria toxoids in submerged culture. II. Purification, detoxification, antigenicity, and stability

1968 ◽  
Vol 14 (4) ◽  
pp. 327-330 ◽  
Author(s):  
D. W. Stainer
Keyword(s):  
Guinea Pigs ◽  
Submerged Culture ◽  
Elevated Temperatures ◽  
High Potency ◽  
High Degree

Cornyebacterium diphtheriae was grown in submerged culture using a new semisynthetic medium, and high-potency diphtheria toxins were produced. These toxins were purified before detoxification by formalin–lysine mixtures. The resultant toxoids possessed a high degree of purity (Lf/mg non-dialyzable nitrogen), gave good antigenic responses in animals, and showed no tendency to revert to toxin. Toxins which had been detoxified with formalin alone before purification, however, showed marked reversion when diluted and stored at 25 or 34 °C. When tested in guinea pigs the lysine-treated toxoids were shown to possess good immunizing capacity, and no significant decreases in the antigenic potencies were observed in samples stored for prolonged periods of time at elevated temperatures.

Cellular and Humoral Reactions to Freund’s Adjuvant in Guinea-Pigs

Pathobiology ◽  
1966 ◽  
Vol 29 (6) ◽  
pp. 837-853
Author(s):  
B. Pernis ◽  
A. Bairati ◽  
S. Milanesi
Keyword(s):  
Guinea Pigs ◽  
Freund’S Adjuvant ◽  
Freund's Adjuvant

scholarly journals Induction of T-lymphocyte responses to a small molecular weight antigen. II. specific tolerance induced in azebenzenearsonate (ABA)-specific T cells in Guniea pigs by administration of low doses of an ABA conjugate of chloroacetyl tyrosine in incomplete Freund's adjuvant.

1975 ◽  
Vol 142 (2) ◽  
pp. 261-274 ◽  
Author(s):  
W W Bullock ◽  
D H Katz ◽  
B Benacerraf
Keyword(s):  
T Cells ◽  
Guinea Pigs ◽  
Suppressor Cells ◽  
Small Molecular Weight ◽  
Incomplete Freund’S Adjuvant ◽  
Freund’S Adjuvant ◽  
Freund's Adjuvant ◽  
The One ◽  
Lymphocyte Responses ◽  
Determining Factor

The experiments presented in this paper demonstrate that the induction of tolerance on the one hand and the induction of delayed sensitivity on the other hand can be accomplished by administration of similar doses of azobenzene-arsonate conjugated to N-chloracetyl tyrosine (ABA-T) to guinea pigs with the determining factor being the absence or presence, respectively, of activating bacterial products in the adjuvant mixture used. Thus, complete, persistent ABA-T-specific T-cell tolerance can be induced in adult guinea pigs with 20 mug of ABA-T given intradermally in incomplete Freund's adjuvant (IFA) whereas this same dose of ABA-T induces ABA-specific immunity when administered in complete Freund's adjuvant. This tolerance was not reversible by administration of ABA-T and IFA in the presence of bacterial lipopolysaccharide, was generated before the formation of primed T cells, and persisted for at least 3 mo after initiation. Moreover, cell transfer studies performed herein demonstrate that the unresponsiveness resulting from administration of ABA-T in IFA reflects the activity of suppressor cells to induce and maintain a state of unresponsiveness could only be demonstrated in unprimed animals may indicate a severe limitation on the potential clinical usefulness of such an approach to regulation of the immune system.

EFFECT OF ANTIBODIES ON REMOVAL OF INSULIN FROM THE CIRCULATION

1961 ◽  
Vol 39 (9) ◽  
pp. 1433-1439 ◽  
Author(s):  
Norman Kalant ◽  
Reuben Schucher ◽  
William C. Galley
Keyword(s):  
Guinea Pigs ◽  
Removal Rate ◽  
Test Dose ◽  
Insulin Antibody ◽  
Antibody Complex ◽  
Freund’S Adjuvant ◽  
Freund's Adjuvant ◽  
Delayed Removal

Guinea pigs have been immunized against insulin by the injection of insulin in Freund's adjuvant. All animals developed high titers of antibodies and a decreased hypoglycemic response to insulin; some showed delayed removal of a test dose of insulin from the circulation, while others showed a normal removal rate. These effects were also found in animals passively immunized by the infusion of sera from actively immunized animals. The differences in the rate of removal of insulin from serum in immunized animals may be due to differences in the removal of the insulin–antibody complex.

Preparation and properties of diphtheria toxoids in submerged culture. III. Development of a new semisynthetic medium

1968 ◽  
Vol 14 (10) ◽  
pp. 1155-1160 ◽  
Author(s):  
D. W. Stainer ◽  
J. M. C. Corkill ◽  
M. J. Scholte
Keyword(s):  
Submerged Culture ◽  
Inorganic Phosphate ◽  
Ferrous Sulphate ◽  
Growth Period ◽  
Main Ingredient ◽  
High Potency ◽  
Tryptic Digest ◽  
Deep Culture ◽  
Culture Production ◽  
Phosphate Solutions

A new medium has been developed which does not contain any meat digest products and is suitable for the deep-culture production of high-potency diphtheria toxins. The main ingredient is a commercially available, tryptic digest of casein known as N.Z. Amine type A. It has been found that when the iron, calcium, and phosphate levels in the medium are carefully controlled by suitable additions of ferrous sulphate, calcium chloride, and inorganic phosphate solutions, high levels of diphtheria toxin are regularly produced (200 Lf/ml in a 48-hour growth period).

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