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: 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.

scholarly journals The influence of mineral carriers on the simultaneous active and passive immunization of guinea-pigs against tetanus

1965 ◽  
Vol 63 (2) ◽  
pp. 243-262 ◽  
Author(s):  
A. J. Fulthorpe
Keyword(s):  
Calcium Phosphate ◽  
Tetanus Toxoid ◽  
Aluminium Hydroxide ◽  
Guinea Pigs ◽  
Calcium Salt ◽  
Primary Response ◽  
Tissue Response ◽  
Aluminium Phosphate ◽  
Tetanus Antitoxin ◽  
Significant Difference

Guinea-pigs given two doses of 2–25 Lf of fluid tetanus toxoid at 28 days interval had very satisfactory antitoxin titres 10 days after the second dose of toxoid (g.m. 28–2 units/ml.). Similar groups of animals given 150 units of horse tetanus antitoxin simultaneously with the first dose of toxoid responded very badly (g.m. < 0·016).Interference by passive antitoxin occurred even when the antitoxin was given as late as 4 days after the first dose of toxoid.Interference by passively administered antitoxin was minimal when aluminium hydroxide-adsorbed toxoid was used. It was necessary to increase the dose of antitoxin from 150 to 2400 units before significant interference occurred.The route of administration of antitoxin did not significantly affect the results except when the antitoxin was given intravenously.When guinea-pigs were immunized and bled at regular intervals it was found that with both fluid and aluminium hydroxide-adsorbed preparations, titratable antitoxin was present on the 14th day. The increase in titre thereafter was more rapid with the adsorbed preparation, but after a second dose of toxoid there was no significant difference in titre.Passively administered antitoxin virtually abolished the active response to fluid toxoid, but with aluminium hydroxide-adsorbed preparations the primary response was not abolished but reduced and delayed and there was much individual variation.Horse serum-sensitive guinea-pigs given adsorbed toxoid with simultaneous passive horse antitoxin gave a better primary response to the toxoid than did unsensitive animals.The effectiveness of adsorbed tetanus toxoid in the simultaneous immunization procedure was directly related to the concentration of aluminium hydroxide or phosphate used: this concentration was critical and amounts below a certain level were ineffective. Calcium phosphate used as an adsorbent was unsatisfactory in this way, although it was an excellent adsorbent.Investigation of the adsorbent characteristics of aluminium hydroxide and phosphate and of calcium phosphate, showed that the calcium salt on a molar basis was the most effective and that aluminium phosphate was the least effective.Elution of toxoid from centrifuged precipitates of the three types of adsorbent showed that only 5% of toxoid was removed from the aluminium hydroxide, 13–18% from the calcium phosphate and 31–33% from the aluminium phosphate preparation when incubated with normal serum at 37° C.Aluminium hydroxide adsorption in vitro interfered with the ability of antitoxin to combine with toxoid and to a lesser extent calcium phosphate had the same effect; aluminium phosphate, however, did not appear to interfere at all in this wayHistological observations on the tissue response to aluminium phosphate and calcium phosphate indicated that the typical alum granuloma produced by aluminium phosphate was not produced by the calcium salt.

Purification and characterization of tuberculin-active components from BCG

1975 ◽  
Vol 21 (12) ◽  
pp. 2019-2027
Author(s):  
M. Laguerre ◽  
R. Turcotte
Keyword(s):  
Physicochemical Properties ◽  
Guinea Pigs ◽  
Gel Filtration ◽  
Biologically Active ◽  
Polyacrylamide Gels ◽  
Active Components ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Antigenic Activity

The tuberculin activity of protoplasmic extracts isolated from living BCG was purified successively by gel filtration on Sephadex G-100 and G-75, and by electrophoresis on 7.5% and on gradient (6–18%) polyacrylamide gels. The tuberculin-active fractions, as determined in BCG-sensitized guinea pigs, were used as the starting material for each of the following fractionation steps.The physicochemical properties and the antigenic activity of the biologically active fractions have shown that a single component, or only a few ones with similar properties, possessed high tuberculin activity. These active components were proteins having relatively high molecular weights (about 72 000) and could behave as antigens.

scholarly journals Adsorption of F VIII on Aluminium Hydroxide

1977 ◽  
Author(s):  
M. J. Seghatchian ◽  
T. Barrowcliffe ◽  
M. Miller-Andersson
Keyword(s):  
Molecular Weight ◽  
High Purity ◽  
Aluminium Hydroxide ◽  
Gel Filtration ◽  
Procoagulant Activity ◽  
Void Volume ◽  
Low Molecular Weight ◽  
Two Stage ◽  
Volume Fractions ◽  
High Purification

Adsorption of plasma by A1(OH)3 is a requirement for the two stage assay of F VIII. It is generally accepted that factors II, VII, IX and X are removed by the procedure, while factors V and VIII are unaffected. Following gel filtration of a F VIII concentrat on Sepharose 4 B F VIII:c was found in the low molecular weight area, as well as in the void volume as expected. This activity was found with both one and two stage techniques. After adsorption of the fractions with Al(OH)3 to eliminate the non F VIII procoagulant activity F VIII:c disappeared from the void volume fractions and was much reduced in the low molecular region. F VIII: R Ag was also removed from these fractions by A1(OH)3 adsorption. After adsorption of fractions in the presence of hemophilia plasma clotting activity remained in both regions suggesting the presence of true F VIII activity. Thus at concentration of 1 IU of F VIII:c per ml, a low purity preparation was unaffected by A1(OH)3 adsorption whereas both antigen and clotting activity of a high purity concentrate were conciderably reduced. Addition of 5 % albumin to the high purity preparation prevented this adsorption. It is concluded that under conditions of high purification F VIII:c can be adsorbed preferentially on A1(OH)3 and this appears to be due to removal of F VIII:R Ag.

scholarly journals Population genomics and antimicrobial resistance in Corynebacterium diphtheriae

2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Melanie Hennart ◽  
Leonardo G. Panunzi ◽  
Carla Rodrigues ◽  
Quentin Gaday ◽  
Sarah L. Baines ◽  
...  
Keyword(s):  
Soviet Union ◽  
Antimicrobial Resistance ◽  
Diphtheria Toxin ◽  
Population Genomics ◽  
Bacterial Species ◽  
Toxin Production ◽  
Multidrug Resistant ◽  
Corynebacterium Diphtheriae ◽  
Toxin Gene ◽  
Phylogenetic Structure

Abstract Background Corynebacterium diphtheriae, the agent of diphtheria, is a genetically diverse bacterial species. Although antimicrobial resistance has emerged against several drugs including first-line penicillin, the genomic determinants and population dynamics of resistance are largely unknown for this neglected human pathogen. Methods Here, we analyzed the associations of antimicrobial susceptibility phenotypes, diphtheria toxin production, and genomic features in C. diphtheriae. We used 247 strains collected over several decades in multiple world regions, including the 163 clinical isolates collected prospectively from 2008 to 2017 in France mainland and overseas territories. Results Phylogenetic analysis revealed multiple deep-branching sublineages, grouped into a Mitis lineage strongly associated with diphtheria toxin production and a largely toxin gene-negative Gravis lineage with few toxin-producing isolates including the 1990s ex-Soviet Union outbreak strain. The distribution of susceptibility phenotypes allowed proposing ecological cutoffs for most of the 19 agents tested, thereby defining acquired antimicrobial resistance. Penicillin resistance was found in 17.2% of prospective isolates. Seventeen (10.4%) prospective isolates were multidrug-resistant (≥ 3 antimicrobial categories), including four isolates resistant to penicillin and macrolides. Homologous recombination was frequent (r/m = 5), and horizontal gene transfer contributed to the emergence of antimicrobial resistance in multiple sublineages. Genome-wide association mapping uncovered genetic factors of resistance, including an accessory penicillin-binding protein (PBP2m) located in diverse genomic contexts. Gene pbp2m is widespread in other Corynebacterium species, and its expression in C. glutamicum demonstrated its effect against several beta-lactams. A novel 73-kb C. diphtheriae multiresistance plasmid was discovered. Conclusions This work uncovers the dynamics of antimicrobial resistance in C. diphtheriae in the context of phylogenetic structure, biovar, and diphtheria toxin production and provides a blueprint to analyze re-emerging diphtheria.

Purification and some properties of the glutamate dehydrogenase of Salmonella typhimurium

1973 ◽  
Vol 19 (4) ◽  
pp. 427-438 ◽  
Author(s):  
J. W. Coulton ◽  
M. Kapoor
Keyword(s):  
Salmonella Typhimurium ◽  
Glutamate Dehydrogenase ◽  
Ammonium Sulfate ◽  
Gradient Centrifugation ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient Centrifugation ◽  
Ammonium Sulfate Fractionation ◽  
Sulfate Fractionation

NADP-specific glutamate dehydrogenase (GDH) from Salmonella typhimurium was purified 190-fold by heat treatment, ammonium sulfate fractionation, DEAE-Sephadex chromatography, reverse ammonium sulfate fractionation, and gel filtration. The enzyme proved to be stable to 55 °C, and displayed a pH optimum at 8.6 in the amination reaction. The sedimentation coefficient of GDH, as determined by sucrose density gradient centrifugation, was about 10.3 S. From gel filtration chromatography, the molecular weight and Stokes' radius for the enzyme were estimated at 280 000 daltons and 54 × 10−8 cm, respectively. Unusual resistance was displayed by the enzyme to high concentrations of the protein denaturants, urea, SDS, and guanidine hydrochloride.

scholarly journals ASPERMATOGENESIS, ANAPHYLAXIS, AND CUTANEOUS SENSITIZATION INDUCED IN THE GUINEA PIG BY HOMOLOGOUS TESTICULAR EXTRACT

1955 ◽  
Vol 101 (6) ◽  
pp. 591-604 ◽  
Author(s):  
Jules Freund ◽  
George E. Thompson ◽  
Murray M. Lipton
Keyword(s):  
Acetic Acid ◽  
Guinea Pig ◽  
Ammonium Sulfate ◽  
Guinea Pigs ◽  
Anaphylactic Shock ◽  
Proteolytic Enzymes ◽  
Trichloracetic Acid ◽  
Oil Emulsion ◽  
Highly Active ◽  
Water In Oil Emulsion

Guinea pig testicles were extracted with acetic acid; the extract was purified by removing material in consecutive precipitations with 30 per cent saturated ammonium-sulfate, trichloracetic acid, and chloroform. The solution so purified, when administered with complete adjuvants, was highly active in inducing impairment of spermatogenesis in guinea pigs. The activity resisted autoclaving at 15 pounds' pressure for 20 minutes, proteolytic enzymes, and formamide. Anaphylactic shock and cutaneous reaction to the purified homologous extract occurred in guinea pigs sensitized by the extract combined with adjuvants. For the production of aspermatogenesis it was essential to incorporate killed mycobacteria into the water-in-oil emulsion containing the antigen; but anaphylactic sensitization did not require the presence of mycobacteria.

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