Antitoxin detection

Fig. 26.2. Amperometric immunosensors set-up using a biotinylated copolymer poly(pyrrole-biotin, pyrrole-lactitob-ionamide) coated platinum or glassy carbon electrodes and three enzymatic markers (GOX-B, PPO-B, HRP-Ab) for the detection of cholera antitoxin. (A) HRP-immunosensor, (B) GOX-B-immunosensor, (C) PPO-B-immunosensor. Mred/Mox = hydroquinone/quinone Gox = biotinylated glucose oxidase PPO — biotinylated polyphenol oxidase HRP-Ab = peroxidase-labeled IgG anti-rabbit antibody.

The administration of heterologous antitoxin was one of the first therapeutic approaches developed for botuhsm patients and remains the most effective when initiated in the early stages of intoxication. The primary limitation of antitoxin treatment was established in some of the earhest published reports on experimental botuhsm. One of these reports evaluated the pathogenesis of oral intoxication and the efficacy of antitoxin therapy in monkeys (Back and Wood, 1928). Antitoxin treatment was not effective when administered after symptoms of botulism were already apparent, despite the fact that circulating toxin could still be detected in many of the animals. 

Clostridium difficile can be cultured from the stool, and toxins A and B can be assessed by different techniques (116). The most accurate method is still a cytotoxin tissue culture assay. This detects the cytopathic effect of cytotoxin B, which can be neutralized by Clostridium sordellii antitoxin, but it takes 24 8 hours to show a result. Alternative tests that produce faster results have been developed. A latex agglutination test lacks sensitivity and specificity, and does not distinguish toxigenic from non-toxigenic strains. An enzyme immunoassay for toxin A may be an acceptable alternative to the cell cytotoxin assay and the results are rapidly available. A dot immunobinding assay has not yet been extensively studied (164). 

The mouse bioassay is the standard diagnostic laboratory test for botulism (36). The procedure detects whether a type-specific antitoxin protects the mice against any bomlinum toxin that may be present in the clinical specimen. The bioassay, which takes 1-2 days to complete (range 6-96 h), can detect 0.03 ng of botulinum toxin. In addition to the bioassay, anaerobic cultures of clinical specimens can isolate the organism in 7-10 days (range 5-21) days, but a mouse bioassay is necessary to confirm that the culture isolates produced the toxin (36). 

Although it is commonly believed that circulating BoNT is rapidly cleared from the bloodstream this is not always the case. In the recent Oakland Park, Florida outbreak, detectable levels of toxin were observed in one patient 8 days after receiving a massive overdose of nonapproved BoNT/A during a cosmetic procedure (Chertow et al., 2006). For such severely intoxicated patients, antitoxin administration, even if delayed, may still be effective in limiting the duration of illness, since it would neutralize circulating BoNT and prevent further toxin intemahzation. 

The use of antibody- or aptamer-based approaches to protect against ricin holds potential as a possible pretreatment for armed forces, or as an adjunct to PPE for civilian first-responders and other special populations required to enter contaminated areas. However, extracellular antitoxin would probably be of limited use in a bioterrorist or civilian mass casualty scenario, because the therapeutic window for administration is likely to be short (a few hours), the delay to onset of symptoms is relatively long, and there presently is no method of immediately detecting ricin exposure. It also remains to be determined whether specific combinations of MAbs are required for optimal in vivo protection against the toxin. Moreover, in some cases, MAbs that bind toxin with high avidity and block enzymatic activity of RTA in vitro actually enhance the toxicity of ricin in vivo (Maddaloni et al., 2004). 

Marks RS, Bassis E, Bychenko A, Levine MM (1997) Chemiluminescent optical fiber immunosensor for detecting cholera antitoxin. Opt Engi 36(12) 3258-3264 Mateus CFR, Huang MCY, Chang-Hasnain CJ, Foley JE, Beatty R, Li P, Cunningham BT (2004)... 

The tool kit consisting of carbohydrate synthesizer and carbohydrate microarrays lays the foundation for the discovery and elucidation of new drugs, as studies with the fully synthetic antitoxin malaria vaccine candidate have shown. HIV neutralizing proteins have been identified by studies with carbohydrate microarrays aminoglycoside microarrays were used to test antibacterial resistance. Fluorescent polymers can be utilized to detect small amounts of pathogenic bacteria in a short time. 

Although monoclonal anti-TTX antibody to detect TTX has recently been developed, there are no known antidotes or antitoxins to TTX. 

Enterotoxins. Toxic proteins formed by bacteria with molecular masses in the range from 27000 to 30000 which are usually excreted into the medium ( exotoxins). E. can be taken up with contaminated food or be formed by the bacteria colonizing the intestinal walls. Finally, the bacteria can penetrate the intestinal walls and then start to excrete the E. Some E. are thermally very stable and survive when food is boiled. E. from Salmonella and Staphylococcus species are the most frequent causes of food poisoning. Shortly after uptake, the symptoms of nausea, vomiting, diarrhea, and circulatory complaints occur. Deaths are rare and occur only when the subject is already in a weakened state. The sites of attack by E. vary, e.g., at intestinal epithelial cells or in the vegetative nervous system. For the production of antitoxins, E. are obtained by lysis of bacterial cells or from cell-free culture filtrates. E. have been detected, e. g., in the following bacterial species Bacillus cereus, Clostridium perfringens, Escherichia coli. Vibrio cholerae. Staphylococcus aureus, and Streptococcus faecalis. 

Although the total B-cell population has been low due to the lymphopenia the 7oPBM s recognised as B cells remains normal, as have serum and secretory immunoglobulins. Immunoelectrophoresis has shown no monoclonal band. Sero-conversion to respiratory syncytial virus has been noted (litre 1/10,1/40,1/10 at 6,12, and 16 months respectively. The patient was immunised with diptheria and tetanus toxoid plus inactivated polio vaccine at 4,6 and 12 months. No polio antibodies were detected but following tetanus immunisation 1-2 units/ml tetanus antitoxin were produced. There has been no response to intradermal Candida antigen at 6,12, and 15 months of age. 

The various methods used to detect botulism toxin are listed in Table 2. Some of the assays for toxin can be modified for detection and measurement of antitoxin. Although detection of toxin is often the primary purpose of the test, sometimes it is secondary to identification of toxigenic organisms. In the investigation of botulism, detection of substantial amounts of toxin in the suspected food is confirmatory of the diagnosis. This is also true for detection of any demonstrable amount in the patient s blood or feces. 

Because of the complexity of bioassays, the expenses for animals and facilities, and ethical considerations, many investigators have sought in vitro methods for detection and quantitation of botulism toxins and antitoxins. One of the earliest was the Ramon flocculation test (57), in which the reaction between a toxin and its homologous antitoxin standard was visualized by a precipitation reaction. This was advantageous in vaccine production because the toxoid, which cannot be measured by bioassay can be quantitated in a comparable manner as the toxin by this test. The flocculation unit (Lf) was used as the antigenic unit employed in the formulation of botulism toxoids used for human immunization (7). 

Evancho et al. (12) later attempted to standardize the RPHA using purified type A toxin and toxoid. They found no differences in RPHA results using SRBCs coated with antitoxins produced in response to crystalline toxin or toxin partially purified by ethanol fractionation. The tests were able to detect type A toxin at a minimum concentration of 27 LD o/ml, and no cross reactions were observed with culture filtrates containing toxins of types B, C, D, E or F. 

Many of the early ELISA methods devised for botulism neurotoxin detection, like most of the in vitro tests,suffered from a lack of specificity, due to impurities in the antigen preparation used to produce the antitoxins. More purified toxins are now available for the production of better quality antitoxins. The most sensitive ELISA protocols use an indirect assay sometimes referred to as the sandwich assay. In the basic procedure, a specific antitoxin is first adsorbed to the surface of the wells of a plastic plate. The toxin added to the wells is then bound by these antibodies and detected with a second antitoxin which is conjugated to an enzyme or other labeling molecule. The amount of label is measured by supplying the enzyme substrate, which is converted to a colored product that is measured colorimetrically. Some ELISA protocols use a polyclonal antitoxin on one side of the sandwich and a monoclonal on the other side. Other assays use the same antibody for both sides but label the antibody the second time it is used. A modification of the sandwich assay is the double sandwich ELISA, which employs a third antibody that is conjugated to an enzyme and is directed against the second antitoxin it is an anti-antibody such as rabbit anti-horse IgG. The steps in a typical application of this assay for botulism toxin are shown in Figure 2. 

Antibodies may be conjugated to enzymes such as alkaline phosphatase or horseradish peroxidase, or to other tags such as fluorescein or biotin (55). The fluorescein is measured by antifluorescein antibodies coupled with an enzyme, and biotin is measured by an avidin-enzyme conjugate, which binds with high affinity to biotin. Michalik et al.(47) used human albumin to tag their second antitoxin, and then used an anti-albumin antibody conjugated to peroxidase for detection of botulism toxins Aand B. They reported a sensitivity for this method of 100 and 300 MLD for toxin types A and B, respectively. 

This method has been modified more recently using a solution-phase complexing of the toxin to either biotin-labeled antitoxin or chicken antitoxin prior to binding of the complex to the solid-phase plate (9). Doellgast et al. (11) have also devised an adaptation of the ELISA/ELCA for detecting human antibodies to botulism toxins types A, B, and E. 

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