Botulinum Toxin ELISA A, B, E, F Kits For Clinical Laboratory Investigations Of Human Botulism

Clostridium botulinum is the type of neurotoxin bacterium that generates a particular type of protein which causes serious effects on the functioning of the peripheral or central nervous system. This article is about botulinum toxin ELISA A, B, E, F kits for clinical laboratory investigations of human botulism.

The Clostridium botulinum bacterium can stay alive even in the absence of oxygen and that is the reason it is called an anaerobic bacterium. It is in the shape of rod and form spores which is why it can resist chemicals and physical pressure. Under particular circumstances, these bacteria can produce toxins by developing foods.

The presence of Clostridium botulinum bacteria in foods can lead to serious food poisoning consequences when the food affected by such bacteria is consumed. In the case of the outbreak of botulism in the 962 records in the USA recorded from 1899-1990 included 2320 cases and death of 1036 people. The type of toxin was determined by the researchers which included 384 cases of type A, 106 of type B, 105 of type E and 3 of type F.1 In the other two botulism outbreak there was presence of toxins of both type A and B. However all the studies conducted for Botulinum toxin of type F and G showed that it generally causes the botulism disease in animals.

Clostridium botulinum can be classified into different categories depending on the biochemical, cultural and physiological attributes. The Clostridium botulinum of type C and type D toxins have a metabolous shape that makes them different from the others and they cannot catalyze the splitting of the proteins on the solid egg or meat. The proteolytic enzyme is present in all the type A and few of type B and F of the Clostridium botulinum. All of the strains of the type E and the few leftover type B and F don’t produce proteolytic enzymes and have a metabolic shape formed of carbohydrates which are different from the non-proteolytic classes of the type C and D. The researchers have not yet studied and discussed the type G toxin which is produced by certain strains and couldn’t be characterized effectively.

Let us understand the utility of botulinum toxin ELISA type A, B, E and F kit for clinical laboratory investigations of human botulism. Recent studies have developed faster and alternative ways than in-vitro procedures to detect the organisms that produce a type of Clostridium botulinum and the toxins. With the help of ELISA techniques, one can detect the type of toxins generated through culture media.2 These techniques include amp-ELISA and DIG-ELISA. The ELISA technique takes a day to detect the toxin after it is left for the incubation process of the spores of the Botulinum bacterium for the whole night. As it detects the toxin fetoprotein it can easily detect both biologically mobile and immobile toxins.

The Botulinum toxins of type A, B, E and F can be easily detected by the amplified ELISA procedure, which has a limitation of approx. 10 MLD/ml for the detection. The culture of the toxic presence is moreover antigenic in comparison to the refined toxins and the degree of the detection in case of amplified ELISA is more delicate than the mouse bioassay. The TPGY, as well as the CMM, is examined as there can be a production of more toxins in a single medium in comparison to mouse bioassay or other methods of detection required for the verification of the toxin detection through ELISA technique that also uses the similar media.

Here is the procedure of the botulinum toxin ELISA Type A, B, E and F kit for clinical laboratory investigations of human botulism.

Samples of food are secluded from the plates of agar and are immunized into CMM and TPGY. The TYGA Extract broth and prepared meat acts as a media and cultivated at 26 degrees and 35 degrees Celsius temperature for 5 days. The food is then made to pass a process of ultracentrifuge at 7000 x g and 4 degree Celsius for 30 minutes, pH supernatant is then modified to 7.4 to 7.6 using 1N NaOH or 1N HCL. The samples and their controls are then scrutinized in a duplicate sample for CMM and TPGY. The scrutiny of the neat and the dilution in the ratio of 1:5 for every cultured supernatant is done.

Each of the Microtiter plates should be coated well with the perfect dilution of the goat type A, E or F in 100 microliters or rabbit type B antitoxin dilution within the bicarbonate buffer. Preparation of a particular number of Microtiter plates is required for the examination of the samples. One must work according to the given instructions and dilute the amount of antitoxin as directed. The plate prepared must be stored properly at a 4 degree Celsius and the top must be coated with a plastic seal to protect it from drying.

• The plates should be washed and then blocked. The samples of the toxin and controls should be put and left for 2 hours for incubation.
• The plate should then be washed and soaked in biotinylated IgG’s and left for 1 hour for incubation.
• The plate should be left for 1 hour of incubation after washing and putting it in Extravidin conjugate
• It should be left for 12.5 minutes of incubation after washing and putting it in Gibco substrate and it should again be left for 2 to 10 minutes of incubation after putting on the Gibco amplifier.
• The last but not least the plates should be read on the microplate reader.

The particular method of DIG-ELISA is an advanced version of amplified ELISA. The biotin-labeled IgG is replaced by digoxigenin-labeled antitoxin and the streptavidin-alkaline phosphatize is replaced by anti-digoxigenin horseradish peroxidase conjugate in case of DIG-ELISA. The TMB is considered to be an exact substrate for horseradish peroxidase enzyme and the detection of Botulinum toxin of type A, B, E and F can be done at 10 MLD/ml. The TPGY, as well as the CMM, is examined as there can be the production of more toxins in a single medium in comparison to mouse bioassay which also makes the utility of similar media. The culture of the toxic presence is moreover antigenic in comparison to the refined toxins and the degree of the detection in case of DIG-ELISA is more delicate than the mouse bioassay.

After following the procedure of ultracentrifuge the liquid portion that got separated from the solid part is used for the TPGY and CMM culture for examining the production of toxin by DIG-ELISA. The liquid part that got extracted through the original cultures of CMM and TPGY is mixed with the buffer of similar magnitude of gelatin phosphate. The prepared mixtures are then strained through a syringe filter of 0.22 microliter pore size. This procedure is then accompanied by the mouse bioassay method.

Each of the Microtiter plates should be coated well with the perfect dilution of the goat type A, E or F in 100 microliters or rabbit type B antitoxin dilution within the bicarbonate buffer. Preparation of a particular number of Microtiter plates is required for the examination of the samples. One must work according to the given instructions and dilute the amount of antitoxin as directed. The plate prepared must be stored properly at a 4 degree Celsius and the top must be coated with a plastic seal to protect it from drying.

• After washing the plates they must be blocked and then samples along with controls must be put and left for 2 hours for incubation.
• Digoxigenin-labeled IgG must be put after washing and left for an hour for incubation.
• Anti-digoxigenin horseradish peroxidase conjugate must be put after washing and left for an hour of incubation.
• 20 to 30 mins of the incubation period is required after putting the TMB substrate when washing is done.
• A chemical agent is required for stopping the reaction.
• With the help of a 450-nanometer microplate reader, the absorbance should be measured on the plates.

Clostridium botulinum is widespread in the topsoil of the earth and also in the deposits present on the seas and lakes. This is the very reason why the Clostridium botulinum of type E is mostly found in aquatic animals and fishes or sea-foods are often found to be adulterated by this kind of bacteria. The Clostridium botulinum bacterium of type A and B are mostly found in food products obtained from the soil, which is already contaminated by such a bacterium. In the United States, it is mostly the canned vegetables that are contaminated by such bacterium but in Europe, even meat products are contaminated by such bacteria and cause a serious reason for diseases caused due to the consumption of a particular type of food.

This analysis was mainly done to understand the utility of Botulinum Toxin ELISA Type A, B, E and F Kit for clinical laboratory investigations of human botulism. Previously mouse bioassay was considered to the most-trusted method of detecting the toxins produced by Clostridium botulinum bacterium but in recent studies amplified ELISA and DIG-ELISA is considered to be more effective in finding the toxins produced by the Clostridium Botulinum bacteria of type A, B, E, and F. These methods ensure rapid and exact results on the occasion of an outbreak of the Clostridium Botulinum disease.

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