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PubMed2006

Botulinum type A toxin neutralisation by specific IgG and its fragments: a comparison of mouse systemic toxicity and local flaccid paralysis assays.

In this study, we have compared two in vivo assay methods to measure the type A botulinum toxin neutralising activity of specific immunoglobulin G (IgG) and its fragments (F(ab')(2), Fab', Fab) purified from pentavalent botulinum antisera raised in goats. Each assay method was repeated on three separate occasions in mice and relative potencies calculated with respect to a type A equine reference antitoxin. The conventional assay, which measures the number of mice surviving typically after 72 or 96 h following the intraperitoneal administration of a mixture of toxin and antitoxin, gave the following order of potency IgG>F(ab')(2)>Fab'>Fab (6.8>4.7>3.5>2.6 IU/mg). Differences in potency are likely to be due to differences in the pharmacokinetics of the antitoxins, which are related to their molecular weight. The alternative local flaccid paralysis assay, where toxin and antitoxin are injected subcutaneously into the left inguinocrural region, gave results with a narrower range of activities: IgG>Fab'>F(ab')(2)>Fab (6.0>5.9>5.5>4.6 IU/mg). Comparison of the two assay methods showed no significant differences for IgG, F(ab')(2) or Fab', although the Fab fragment was significantly more potent in the non-lethal assay probably because of the reduced influence of antitoxin pharmacokinetics in this localised assay. These findings show that a local flaccid paralysis assay provides a less time consuming and more humane alternative to the lethal assay for the potency testing of botulinum IgG and F(ab')(2) antitoxins.

Animals↗
PubMed1964

AGGLUTINATING, LETHAL TOXIN AND E1 TOR HAEMOLYSIN NEUTRALIZING POTENCY OF SERA IN CHOLERA.

There is a lack of agreement as to the significance of agglutination test results in cholera, and recent epidemics of "classic" Vibrio comma and El Tor infections have provided an opportunity to compare this test with two other serological procedures recently introduced in cholera work. These are determination of the circulating antitoxin expressed as the lethal toxin neutralizing power of a serum, and determination of the haemolysin neutralizing power through serial test-tube dilutions.Applying these tests to sera from cholera patients, convalescents, carriers and contacts and from immunized persons either exposed or not exposed to cholera, the authors found the agglutination test to be a less sensitive indicator of the immunological status than the other two procedures. They therefore recommend antitoxin and antihaemolysin titration for the study of cholera patients, convalescents and carriers. In the conditions of the experiments described, the results with exposed and non-exposed vaccinated individuals were less conclusive, but nevertheless suggest that antitoxin and antihaemolysin tests may be helpful in the follow-up of vaccination experiments.

Agglutination Tests↗
PubMed1962

Effect of cooling, local anaesthetic compounds and botulinum toxin on the responses of and the acetylcholine output from the electrically transmurally stimulated isolated guinea-pig ileum.

Electrical transmural stimulation of the isolated guinea-pig ileum preparation which was distended by an intraluminal pressure insufficient to elicit peristalsis produced two effects, one a longitudinal contraction and the other an emptying response brought about by a co-ordinated propagated contraction of the circular muscle. The parameters of the electrical stimulus used were such that these effects were probably produced by stimulation of the nervous plexuses in the wall of the ileum. In the presence of the anticholinesterase NN-diisopropylphosphodiamidic fluoride (Mipafox), acetylcholine was detected in the fluid passing through the lumen and also in the fluid in the organ bath. The amount was increased after a period of electrical stimulation. As variation of the frequency of stimulation from 5/min to 25/sec did not alter statistically significantly the acetylcholine output, a frequency of 5/min was used. Cooling to 13 degrees C, the effect of local anaesthetic compounds and botulinum toxin reduced both the emptying response and longitudinal contractions of the ileum which followed transmural stimulation, with a concurrent reduction in acetylcholine output. It was concluded that the acetylcholine was released by electrical transmural stimulation from the intramural nerve plexuses in the wall of the guinea-pig ileum.

Acetylcholine↗
PubMed1994

Chemiluminescence immunosorbent assay (CLISA) and a possibility of the specific detection of soluble antigens of Clostridium botulinum type A.

A double antibody version of CLISA was demonstrated to be a rapid method (1 h) for detection and quantitative determination of Clostridium botulinum toxin antigens in biological samples. The sensitivity of this assay is about ten-fold higher than both ELISA and passive hemagglutination test. Thus, the double antibody version of CLISA appeared to be useful for the control of food products contaminated with Cl. botulinum type A bacteria.

Antigens, Bacterial↗
PubMed1976

[Standardization of the control technics of Clostridium perfringens vaccine. Use of a reference vaccine].

Control of Cl perfringens toxins. It is possible to employ a standardized technique for the control of the toxins of Cl. perfringens. Titres of LD50 per ml for the mouse and L+/50 per ml may be expressed with a confidence limit of 1.5. The assay of toxins by L+/50 (relating to their antigenic value) permits the establishment of standards by which it is possible to obtain a satisfactory vaccine with a high degree of certainty. 10 L+/50 per dose for Cl. perfringens A 20 L+/50 per dose for Cl. perfringens B 180 L+/50 per dose for Cl. perfringens C 50 L+/50 per dose for Cl. perfringens D Control of vaccine values. Technique adopted : Determination of antibodies in the rabbit following vaccination. The confidence limit of the antitoxin titre is similarly equal to 1.5 when P = 0.05. It would not appear to be necessary to establish minimum levels of antibodies to confer protection; it seems nevertheless that the results are influenced by considerable variations attributable to the animals used for the most part and that a reference vaccine is required. Freeze-dried vaccine. There is a natural antigenic activity in the rabbit which retains a considerable part of its antigenic properties after maintenance for one week at +37 degrees C. In our view this would be suitable for a reference vaccine.

Animals↗
PubMedDate not supplied

[Use of serum to thermostable toxin for identification of Yersinia pseudotuberculosis].

Serum to thermostable Y. pseudotuberculosis toxin is shown to induce agglutination of bacteria belonging to all 8 serologic variants of this species. No cross reactions with other enterobacterial species, including four Yersinia species, were detected. The results permit us recommend the antitoxic serum for identification of Y. pseudotuberculosis.

Agglutination Tests↗
PubMed1979

Infant botulism.

Infant botulism is a unique neuromuscular disease affecting infants less than six months old. It is the result of intraintestinal toxin production by C. botulinum (toxi-infection). Characteristic symptoms include constipation, lethargy, and decreased feeding. Physical examination often reveals generalized hypotonia with cranial nerve impairment. Recovery is dependent on supportive care in an intensive care setting. The relationship of this disease to the sudden infant death syndrome requires further study.

Botulinum Antitoxin↗
PubMed1996

Diphtheria antitoxin levels in US blood and plasma donors.

Plasma samples from 500 blood donors were titrated for diphtheria antitoxin (DAT) by the toxin neutralization (TN) test. Only 1.6% of donors had <0.01 IU/mL DAT, the minimum protective level against diphtheria; 15% had levels between 0.01 and <0.1 IU/mL, indicating basic protection, and 83.4% had levels > or =0.1 IU, indicating full protection. Three hundred fifty samples were studied by ELISA for diphtheria toxoid IgG antibodies to assess the utility of the assay as a quick, convenient method for evaluating diphtheria immunity. Although the correlation between TN and ELISA titers for the 350 samples was high (r = .80), there was no correlation (r = .07) for samples with antitoxin titers <0.1 IU/mL, the level of special interest for serosurveys for protection. Titration of 62 immune globulin samples (prepared from 1957 to 1994) showed that DAT levels in Massachusetts blood donors increased concurrently with increased use of tetanus-diphtheria vaccine in the state.

Animals↗
PubMed1984

Analysis of the immune response to papain digestion products of tetanus toxin.

Tetanus toxin was degraded by papain and the previously characterized Fragment B and Fragment C were isolated. The induction of protective immunity against tetanus toxin was subsequently investigated in two animal models using Fragment B and Fragment C, as well as conventional tetanus toxoid for comparison. In guinea pigs, Fragment B showed an immunizing potency similar to that recorded for an equal amount of tetanus toxoid, whereas Fragment C was considerably less efficient, on an equal weight basis. In contrast, mice immunized with Fragment C reached a protection level similar to that achieved after immunization with the complete tetanus toxoid antigen. In both animal models, Fragments B and C acted in a synergistic fashion when injected together. Analysis of sera from guinea pigs using ELISA, after administration of a low dose of tetanus immunogen corroborated these results. The data indicate that antigenic determinants in distinct regions of the toxin molecule independently may induce antibodies neutralizing the lethal action of tetanus toxin. The immune response to the complete toxoid antigen may be subject to influence by intramolecular competition between antigenic determinants. The immunogenicity of the fragments as measured by the protection rate after active immunization may depend upon the animal species used.

Animals↗
PubMed1977

Cultural and physiological characteristics of Clostridium botulinum type G and the susceptibility of certain animals to its toxin.

Strain 89 of Clostridium botulinum type G, isolated by Gimenez and Ciccarelli in 1969, was characterized culturally, biochemically, and toxigenically. It was motile, hemolytic asaccharolytic, weakly proteolytic, lipase and lecithinase negative, and it produced acetic, isobutyric, butyric, and isovaleric acids in peptone-yeast extract-glucose broth. No spores were seen in smears from solid or liquid media. Very low levels of toxin were produced in regular broth cultures, but dialysis cultures yielded 30,000 mouse 50% mean lethal doses (LD50 per kg, orally and subcutaneously, respectively; and for guinea pigs, 10,000 to 20,000 and 100 mouse LD50 per kig, intragastrically and intraperitoneally, respectively.

Animals↗
PubMed2006

Analysis of anti-protective antigen IgG subclass distribution in recipients of anthrax vaccine adsorbed (AVA) and patients with cutaneous and inhalation anthrax.

The anti-PA IgG1, IgG2, IgG3, and IgG4 subclass responses to clinical anthrax and to different numbers of anthrax vaccine adsorbed (AVA, BioThrax) injections were determined in a cross-sectional study of sera from 63 vaccinees and 13 clinical anthrax patients. The data show that both vaccination with three AVA injections and clinical anthrax elicit anti-PA IgG1, IgG2, and IgG3 subclass responses. An anti-PA IgG4 response was detected in AVA recipients after the fourth injection. The anthrax lethal toxin (LTx) neutralization efficacy of sera from recipients who received 4 to > or =10 AVA injections did not vary significantly in relation to changes in distribution of anti-PA IgG1 and IgG4 subclasses.

Adult↗
PubMed1986

Dimeric form of diphtheria toxin: purification and characterization.

Many preparations of diphtheria toxin were found to contain dimeric and multimeric toxin forms. The monomeric and dimeric forms were fractionated to greater than 98% purity, and their properties were compared. Dimeric toxin slowly dissociated to native monomers in solution at neutral pH and could be rapidly dissociated with dimethyl sulfoxide. In cell culture assays and rabbit skin tests, the dimer exhibited no significant toxic activity, except for that attributable to trace contamination by monomer, or partial dissociation to monomer during the incubation period. In guinea pig lethality tests, however, toxic activity varied depending upon the dose. At least 7-fold greater amounts of dimer than monomer (161 ng vs. 22 ng, respectively) were required to cause death at 18 h, whereas similar weights of the two toxin forms (22 ng) caused death at 120 h. This variability probably reflected slow dissociation of dimer to monomer in the animal. The dimer was unable to bind toxin receptors on the surface of susceptible cells, whereas it retained full activity in the ADP-ribosyltransferase, NAD-glycohydrolase, or ligand-binding assays. Thus, the lack of toxicity of the dimeric toxin may have resulted from distortion or occlusion of the receptor binding site on the B moiety. We propose that the dimer contains two monomeric units bound by hydrophobic interactions and that the points of contact involve regions of the B moieties that are normally buried in the native monomer.

Animals↗
PubMed1971

Park-Williams number 8 strain of Corynebacterium diphtheriae.

Five clones of the Park-Williams number 8 strain of Corynebacterium diphtheriae, previously maintained in separate laboratories, were examined for their colonial and biochemical properties, for the restriction and modification system which operates to obscure their lysogeny, and for their capacity to produce large amounts of toxin under ordinary laboratory conditions. The phenotypes of their phage, P, produced in strain 603 and C7 (P.603 and P.C7) differ both as to stability to storage in the cold and to inactivation by antiphage serum. Evidence for a high degree of stability in the integration of P prophage in the PW8 genome is presented.

Animals↗
PubMed1979

Role of clostridial toxin in the pathogenesis of clindamycin colitis in rabbits.

The pathophysiology of antibiotic-associated colitis was studied in rabbits with severe ileocolitis induced by oral administration of clindamycin. Cell-free, sterile filtrates of cecal contents of rabbits with clindamycin colitis contained a toxin that was lethal for mice and cytotoxic for HeLa-cell monolayers. The toxin was heat labile, was inactivated by pronase but not trypsin, and had a mol wt by gel filtration on Sephadex G-100 of 45,000. The toxin was neutralized by antiserum to Clostridium perfringens type E, but not by other clostridial antisera. The toxin also caused severe necrosis of rabbit rectal epithelium during 18-hr organ culture, which could be completely reversed by neutralization with C. perfringens type E antiserum. These studies indicate that clindamycin colitis in rabbits is caused by overgrowth of a clostridial species, which releases a heat-labile toxic protein of mol wt of 45,000 capable of necrosing colonic epithelial cells.

Administration, Oral↗
PubMed1986

A sensitive method for the detection of specific antibody production in different isotypes from single lamina propria plasma cells.

A sensitive and reproducible method for the detection of specific antibody production (or total immunoglobulin secretion) at the single cell level from isolated lamina propria lymphocytes was developed. The cells were prepared from mouse intestinal mucosa by enzyme extraction with collagenase, and antibody secretion was demonstrated with a solid phase enzyme-linked immunospot (ELISPOT) assay. Oral immunizations with cholera toxin or keyhole limpet haemocyanin to mice gave high numbers of highly antigen-specific spot-forming cells (SFC) among isolated lamina propria lymphocytes. Spots were shown to result from active synthesis of immunoglobulin in vitro. The variation in SFC numbers between individual animals after a given protocol of oral immunizations was found to be 25% and between equal groups analysed on different occasions, 12%. Kinetics of primary as well as secondary immune responses after oral immunizations with cholera toxin were easily monitored. A single dose of cholera toxin gave rise to 230 antitoxin SFC/10(7) isolated lamina propria lymphocytes. Each additional dose stimulated to increasing numbers of specific SFC with roughly 7000 antitoxin SFC/10(7) cells after five immunizations. Monitoring of day-by-day responses after oral booster immunizations demonstrated peak SFC numbers on day 8 after antigen administration. The total number of immunoglobulin-secreting (Ig) cells and the isotype distribution of specific SFC could also be determined. In the peak antitoxin response, 8% of the isolated total Ig-secreting lamina propria cells were active against cholera toxin, and of these 80% were producing IgA. This method has also been successfully used in humans and rabbits to demonstrate specific antibody production by single lamina propria plasma cells.

Animals↗
PubMed1997

Preventing ptosis after botulinum treatment.

In a series of 33 blepharospasm patients who had the side effect of ptosis following therapeutic botulinum toxin type A (Botox: Allergan, Inc., Irvine, CA, U.S.A.) injection, we administered 41 injections of human botulinum immune globulin (IG) following injections of the toxin to test the dosage and timing of IG injection and its effectiveness in limiting or avoiding ptosis. An IG dose of 3.2 x 10(-3) international units (IU) per unit of Botox was effective in blocking toxin effect when injected into the same tissue site within 4 hours. An IG dose of 1.6 x 10(-2) to 3.2 x 10(-2) into the levator of the eye having more frequent ptosis in 19 patients reduced the incidence of ptosis to 11%. The fellow (control) eye had a ptosis incidence of 37%. No orbital hemorrhage or other adverse effect occurred from the IG or its injection.

Animals↗
PubMed1979

Evaluation of Pseudomonas aeruginosa toxin A in experimental rat burn wound sepsis.

The search for methods to achieve control of Pseudomonas aeruginosa infection continues with the introduction of aluminum-absorbed toxoid developed from P. aeruginosa exotoxin. This toxoid induces significant titers of neutralizing and precipitating antibodies for toxin A when given with appropriate adjuvants. These experiments show that immunization with aluminum phosphate-absorbed toxoid failed to protect burned rats infected with P. aeruginosa. These and previous experiments show that active immunization with live P. aeruginosa provides good strain-specific protection in the same model. No cross-protection was demonstrated between strains of P. aeruginosa in these experiments.

Animals↗