Problems encountered with the capillary tube immunodiffusion method for detection of botulinal toxin.

By using antitoxin specific for the neurotoxin molecule, the capillary tube immunodiffusion method did not detect low levels of crystalline toxin. Reactions described earlier with crude toxin and less specific antitoxin were probably due to nontoxigenic botulinal antigens.     

Toxicity of pseudomonas toxin for mouse LM cell fibroblasts

Pseudomonas toxin inhibited protein synthesis in mouse LM fibroblast monolayers. Incubation of toxin with LM cell monolayers resulted in a depletion of functional elongation factor 2. The initial interaction of pseudomonas toxin with mouse LM cells was rapid; within 2.5 min, toxin was rendered inaccessible to neutralization with specific pseudomonas antitoxin. At 4°C toxin adsorbed to the cell surface, but remained at a site where it could be neutralized with antitoxin. Ammonium chloride (20 mM) rendered LM cells insensitive to the action of toxin. The ammonium salt did not prevent adsorption of toxin to the cell membrane; rather, it appeared to maintain toxin at a site amenable to antitoxin neutralization.     

Quantitation of commercial equine tetanus antitoxin by competitive enzyme-linked immunosorbent assay

In the USA, the potency of commercially prepared equine tetanus antitoxin is determined by the method outlined in the Code of Federal Regulations, Title 9, Part 113.451. In the current test, commercial equine tetanus antitoxin is tested by a toxin neutralization test in guinea pigs. The in vivo test measures antitoxin content through effectiveness of protection of guinea pigs injected with diluted mixtures of antitoxin and a standard toxin. A competitive enzyme-linked immunosorbent assay, designed as an in vitro alternative to the in vivo test, measures antitoxin content based on a competitive reaction between standard or unknown serum and murine monoclonal antibody specific for tetanus toxin. The monoclonal antibody used in the assay delayed death in mouse passive protection studies and reacted with the C fragment of tetanus toxin. No cross-reaction was observed when the antibody was tested with the toxins of Clostridium chauvoei, C. novyi, C. perfringens, or C. sordellii. The in vitro test will measure the antitoxin content of serum samples containing 100-1500 units of antitoxin. Tetanus antitoxin titers obtained by the competitive enzyme-linked immunosorbent assay compared favorably with the toxin neutralization test conducted in guinea pigs. The in vitro assay serves as a feasible alternative to the in vivo test because it can be completed in less time, is reproducible, and eliminates the use of test animals.     

Affinity purification of tetanus toxin using polyclonal and monoclonal antibody immunoadsorbents

Tetanus toxin has been immunopurified on immunoadsorbent columns derived from equine polyclonal antitoxin coupled to cyanogen bromide-activated Sepharose CL4B. Desorption of bound toxin in active form was achieved only when the immunoadsorbent was mixed with Sephadex G15 and this mixture overlaid on a further volume of Sephadex G15. With equine antibody, 64% of adsorbed toxin was recovered with a specific activity of 2400 limiting flocculation units (Lf)/mg protein N (1.2 × 10⁸ minimum lethal doses (MLD)/mg protein N). Similarly prepared immunoadsorbent derived from murine monoclonal antitoxin of low affinity had improved desorption with less acidic desorbents, without the requirement for Sephadex G15; greater than 80% of adsorbed toxin was recovered with a specific activity of 3000 Lf/mg protein N (1.6 × 10⁸ MLD/mg protein N).     

Affinity purification of tetanus toxin using polyclonal and monoclonal antibody immunoadsorbents

Tetanus toxin has been immunopurified on immunoadsorbent columns derived from equine polyclonal antitoxin coupled to cyanogen bromide-activated Sepharose CL4B. Desorption of bound toxin in active form was achieved only when the immunoadsorbent was mixed with Sephadex G15 and this mixture overlaid on a further volume of Sephadex G15. With equine antibody, 64% of adsorbed toxin was recovered with a specific activity of 2400 limiting flocculation units (Lf)/mg protein N (1.2 X 10(8) minimum lethal doses (MLD)/mg protein N). Similarly prepared immunoadsorbent derived from murine monoclonal antitoxin of low affinity had improved desorption with less acidic desorbents, without the requirement for Sephadex G15; greater than 80% of adsorbed toxin was recovered with a specific activity of 3000 Lf/mg protein N (1.6 X 10(8) MLD/mg protein N).     

In vitro acetylcholinesterase inhibition by type A botulinum toxin

Type A botulinum toxin was studied for its ability to inhibit the action of acetyl-cholinesterase. The chromogenic substrate, indophenyl acetate, was used for assay of enzyme activity. Inhibition of enzyme function was detected through use of both 6.6 x 10(-6) mg (20 ld(50)) and 6.6 x 10(-10) mg (2 x 10(-3)ld(50)) of type A botulinal toxin. Control assays were performed by use of both homologous antitoxin and heterologous antitoxins (types B and E). Enzyme inhibition was effectively prevented by use of homologous antitoxin only. The inhibition noted was specific and reproducible for given substrate, enzyme, and toxin concentrations.     

An alternative to the toxin neutralization assay in mice for the potency testing of the Clostridium tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium perfringens Type D epsilon components of multivalent sheep vaccines

Potency testing of veterinary vaccines containing clostridial antigens currently requires the vaccination of laboratory rabbits followed by the determination of specific antitoxin concentration in the rabbit sera by toxin neutralization test in mice. ELISAs are described as an alternative method to toxin neutralization for the determination of Clostridium tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium perfringens Type D epsilon antitoxins. The assays were found to be rapid, specific and economical and showed good correlation with the toxin neutralization test.     

Specificity of the precipitation reaction of one-zone diphtheria antitoxin for toxicity determination ofCorynebacterium diphtheriae strains

In immunodiffusion analysis of crude diphtheria toxin, one-zone diphtheria antitoxin may give one or two subsidiary lines in addition to the main precipitation line. The subsidiary lines belong to antigenic fragments of the toxin molecule. These fragments are formed from the complete molecule, probably by proteolytic degradation by bacterial enzymes. Other forms of fragment production were not demonstrated. When testing the toxicity of strains ofCorynebacterium diphtheriae by means of one-zone antitoxin, any precipitation reaction observed can thus be regarded as specific evidence of the toxicity of the test strain.     

Antitoxin in human pertussis immune globulins

The level of antitoxin i.e. neutralizing antibodies to pertussis toxin, or lymphocytosis promoting factor, was determined in six pertussis immune globulin preparations from different manufactures. A comparison with antitoxin levels after natural pertussis disease in adults showed that pertussis immune globulins did not contain more antitoxin than convalescent phase sera, i.e. they had very low antitoxin content for specific immune globulins. Agglutinin and anti-FHA titres were relatively higher in immune globulins, probably reflecting a difference between the antibody response elicited by whole cell vaccines used for hyperimmunization in immune globulin production and by natural disease. The low antitoxin content of currently available pertussis immune globulin preparations could explain the inefficacy or conflicting results obtained with these products in prophylaxis and therapy of whooping cough.     

Significance of circulating toxin and antitoxin in unimmunized tetanus cases of neonates and infants

The level of circulating tetanus toxin, antitoxin and their individual influence on the outcome of tetanus cases were determined in unimmunized 125 neonatal and 39 infant cases of tetanus. PHA (passive haemagglutination) test showed 40% positive cases for toxin while its absence in the remaining cases indicated of either toxin fixation to the central nervous system (CNS) or it got neutralized by antitoxin. TN (toxin neutralization) and PHA test carried out in 46 sera samples revealed a strong positive correlation (r = 0.9) showing that 35/46 (76%) and 38/46 (82.6%) samples were positive for antitoxin, respectively. 25.4% of the neonate and infant cases and 34% of the control group had a protective serum tetanus antitoxin level. 42.5% of the paired sera from unimmunized mothers and their neonates showing nonprotective antitoxin levels suggested that a high level of antitoxin is needed for transplacental transfer, although transfer may not play a decisive role in the resistance against the disease. The presence of toxin or antitoxin in the clinical cases did not affect the outcome of the disease, although in neonates, presence of toxin was found to be a bad prognostic sign. This study explicitly advocates for the need to improve the vaccination coverage strategy.     

Assembly Dynamics and Stability of the Pneumococcal Epsilon Zeta Antitoxin Toxin (PezAT) System from Streptococcus pneumoniae

The pneumococcal epsilon zeta antitoxin toxin (PezAT) system is a chromosomally encoded, class II toxin antitoxin system from the human pathogen Streptococcus pneumnoniae. Neutralization of the bacteriotoxic protein PezT is carried out by complex formation with its cognate antitoxin PezA. Here we study the stability of the inhibitory complex in vivo and in vitro. We found that toxin release is impeded in Escherichia coli and Bacillus subtilis due to the proteolytic resistance of PezA once bound to PezT. These findings are supported by in vitro experiments demonstrating a strong thermodynamic stabilization of both proteins upon binding. A detailed kinetic analysis of PezAT assembly revealed that these particular features of PezAT are based on a strong, electrostatically guided binding mechanism leading to a stable toxin antitoxin complex with femtomolar affinity. Our data show that PezAT complex formation is distinct to all other conventional toxin antitoxin modules and a controlled mode of toxin release is required for activation.     

Titration of botulinum antitoxin of low levels by the score method

Botulinum antitoxin is commonly titrated by injecting a mixture of toxin and antitoxin into mice and by utilizing deaths as a marker to measure the amount of unneutralized toxin. We attempted to titrate antitoxin by converting the severity of symptoms (notably palsy) and time-to-death in days into scores. In neutralization tests with toxin levels at 5.9 LD50 and 23.5 LD50, a linear relationship was obtained for antitoxin dose in a range between 0.03 to 0.003 IU/ml. Statistical analysis showed that homogeneity of variance or slope was not denied for the scores obtained on any day from the first to the fourth days after injection, demonstrating that this method can titrate accurately antitoxin of such a low level as 0.003 IU/ml within 4 days after injection.     

Bacterial toxin–antitoxin systems: Properties,functional significance,and possibility of use (Review)

Toxin–antitoxin systems are genetic modules usually consisting of two genes encoding a stable toxin and labile antidote (antitoxin). These systems are localized on plasmids, phages, and chromosomes and are widespread in bacteria and archaea. The review summarizes recent data regarding the classifications of toxin–antitoxin systems, their mechanisms of action and toxin targets, as well as their functional significance for bacterial cells and possibility of use.     

Detection of Type E Botulinal Toxin in Cultures by Fluorescent-Antibody Microscopy

Vegetative cells of toxigenic Clostridium botulinum type E cultures were stained with fluorescent antitoxin prepared against purified toxin. The staining seems to be specific.     

Evaluation of Type A Botulinal Toxin Assays that Use Antitoxin to Crystalline Toxin

The type A botulinal toxin assay by the reverse passive hemagglutination procedure which uses antitoxin to crystalline toxin was examined for specificity. The analysis was based on the fact that crystalline type A toxin is a complex of neurotoxic protein (Aalpha) and a nontoxic protein (Abeta). By using these components, obtained in essentially pure forms, it was shown that the antitoxin to crystalline toxin has a significantly higher titer to Abeta than to Aalpha. When Formalin-treated red blood cells were sensitized with this antitoxin, the antibodies coupled to the cells were, for practical results, only anti-Abeta. When the suspension is reacted with dilutions of type A toxic solutions, the limiting dilutions are determined by Abeta and not by the neurotoxin, which should be the determinant if the assay is to measure toxicity. These observations may be pertinent to the development of serological assays for other botulinal toxin types.     

A comparison of enzyme-linked immunosorbent assay (ELISA) with the toxin neutralization test in mice as a method for the estimation of tetanus antitoxin in human sera

An enzyme-linked immunosorbent assay (ELISA) has been developed for the measurement of tetanus antitoxin in human sera as an alternative to the toxin neutralization test in mice, the currently accepted method of assay. The ELISA was found to be simple and quick to perform and required only small amounts of materials. In addition, the assay was found to give reproducible estimates of antitoxin levels and to measure antitoxin at levels as low as 0.01 IU per ml, a sensitivity similar to that of the neutralization test. Furthermore, a comparison of the results of the ELISA and the neutralization test involving 80 human sera, including sera with both high and low antitoxin levels, showed close agreement in antitoxin levels obtained by the two methods. It was concluded that ELISA was an acceptable alternative to the toxin neutralization test in mice for the measurement of tetanus antitoxin levels in human sera.