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ANTIGENIC ELISA KIT FOR DETECTION
OF CLOSTRIDIUM BOTULINUM TYPES C AND D
NEUROTOXINS

Sandwich Indirect ELISA test
Test of culture supernatants
Diagnostic test for all species


I INTRODUCTION

Clostridium botulinum spores are found in the ground and sediments, as well as in plants growing in
contaminated soil (Sugiyama, 1986). Clostridium botulinum is ubiquitous, occurring all over the world. The
Clostridium botulinum species that produce the neurotoxins of types A to G are divided into four groups (I to IV)
according to the type of toxin produced. The species in Group III produce C or D toxin and their mosaic
isoforms: C/D or D/C (Moriishi et al., 1996). The species in Group III are typically responsible for botulism in
animals, including in livestock (Sunagawa et al., 1992; Tsukamoto et al., 2005). Biological testing in mice is the
reference test for diagnosing botulism in livestock. This test is expensive and highly contested on ethical
grounds due to animal welfare issues. The BIO K 300 sandwich ELISA test detects C and D neurotoxin
complexes (Brooks et al. 2010). The gut contents of deceased animals with suspected botulism are heated at
80C for 10 minutes, and then incubated under anaerobic conditions for five days so as to obtain maximum C
and D neurotoxin yields (Brooks et al., 2011)


II - PRINCIPLE OF THE TEST

The test uses 96-well microtitration plates sensitised by specific monoclonal antibodies for Clostridium
botulinum types C and D toxins. These antibodies allow specific capture of the corresponding antigen which is
present in the samples. Rows A, C, E, G have been sensitised with these antibodies and rows B, D, F, H contain
non-specific antibodies. These control rows allow differentiation between a specific immunological reaction and
non-specific binding.
The culture supernatants (five-day cultures of the gut contents of animals with suspected botulism) are incubated
on the microplate at 21C +/- 3C for one hour. After this first incubation step, the plate is washed and
incubated for 1 hour with the first conjugate (a specific monoclonal antibody against Clostridium botulinum
types C and D toxins coupled to biotin), then the plate is incubated at 21C +/- 3C for 1 hour. The plate is then
washed, the second conjugate a peroxidase-coupled avidine specific to biotin is applied, and the plate is
incubated at 21C +/- 3C for another hour. After this second incubation, the plate is washed again and the
chromogen (tetramethylbenzidine) is added. This chromogen has the advantages of being more sensitive than
the other peroxidase chromogens and not being carcinogenic.
If Clostridium botulinum types C and D toxins are present in the tested samples, the conjugate remains bound to
the corresponding microwells and the enzyme catalyses the transformation of the colourless chromogen into a
pigmented compound. The intensity of the resulting blue colour is proportionate to the titre of Clostridium
botulinum types C and D toxins in the sample. The enzymatic reaction can be stopped by acidification and the
resulting optical density at 450 nm can be recorded using a photometer. The signals recorded for the negative
control microwells are subtracted from the corresponding positive microwells.
A positive antigen is supplied with the kit.




Bio-X Diagnostics Site du Complexe des postes 49, rue J. Wauters 5580 Jemelle Belgique
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III - COMPOSITION OF THE KIT

- Microplates: 96-well microtitration plates (12 strips x 8 wells). Rows A, C, E, G are sensitised by anti-
Clostridium botulinum types C and D toxin-specific antibodies, while rows B, D, F, H are sensitised by non-
specific antibodies.
- Washing solution: Bottle of 20x concentrated washing solution. The solution crystallises spontaneously
when cold. If only part of the solution is to be used, bring the bottle to 21C +/- 3C until disappearance of
all crystals. Mix the solution well and remove the necessary volume. Dilute the buffer twentyfold with
distilled or demineralised water.
- Dilution buffer: One 50-ml bottle of 5x colored and concentrated buffer for diluting samples and conjugate.
Dilute this concentrated dilution buffer fivefold with distilled or demineralised water. If a deposit forms at
the bottom of the container filter the solution on Whatman filter paper.
- Conjugate: One vial of a 50-fold concentrate of biotinylated conjugated anti- Clostridium botulinum types C
and D toxins antibody. The reagent must be diluted fiftyfold in the dilution buffer.
- Avidine: One vial of a 50-fold concentrate of peroxidase-coupled avidine. The reagent must be diluted
fiftyfold in the dilution buffer.
- Control antigen: Bottle containing control antigen. This reagent is ready to use.
- Single component TMB: Bottle of the chromogen tetramethylbenzidine (TMB). Store at + 2C and + 8C
protected from light. This solution is ready to use.
- Stopping solution: Bottle of the 1 M phosphoric acid stop solution.


BIO K 300/1 BIO K 300/2
Microplates 1 2
Washing solution 1 X 100 ml (20 X) 1 X 100 ml (20 X)
Colored Dilution buffer 1 X 50 ml (5 X) 1 X 50 ml (5 X)
Biotinylated Conjugate 1 x 0.3 ml (50 X) 1 X 0.5 ml (50 X)
Avidine peroxidase 1 x 0.3 ml (50 X) 1 X 0.5 ml (50 X)
Control antigen 1 X 2 ml (1 X) 1 X 4 ml (1 X)
Single component TMB 1 X 12 ml (1 X) 1 X 25 ml (1 X)
Stopping solution 1 X 6 ml (1 X) 1 X 15 ml (1 X)



IV - ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED

Distilled water, graduated cylinders, beakers, heating water bath; glass tubes, plastic tubes, tube rack, dispenser
tips, reagent reservoir for multichannel pipettes, lid, adhesive for microplates, graduated automatic (mono- and
multichannel) pipettes, incubator 40C, microplate reader, and microplate washer and shaker (optional).

Gelatin-phosphate buffer
0.2% gelatin
80 mM Na
2
HPO
4

12 mM KH
2
PO
4

Adjust the pH to 6.5

Anaerobic culture medium
Brain Heart Infusion (BHI) (Becton Dinkinson Biosciences: 237500)
Cooked Meat Medium (CMM) (Oxoid: CM0081)
Dissolve 37 gm of the BHI in 1 litre of water
Add 1 gm of CMM per 20 ml of BHI per tube
Autoclave.

It is preferable to use freshly reconstituted and sterile medium which is inoculated as soon as it has cooled to
approximately 35C.
Tubes which are not used on the day of preparation should be placed in a boiling water bath or steamer for about
15 minutes to remove dissolved oxygen. They should be allowed to cool without agitation and then inoculated.
Inoculation should be made near the bottom of the tube in the meat particles.


Bio-X Diagnostics Site du Complexe des postes 49, rue J. Wauters 5580 Jemelle Belgique
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V - PRECAUTIONS FOR USE

- This test may be used for in vitro diagnosis only. It is strictly for veterinary use.
- The reagents must be kept between +2C and +8C. The reagents cannot be guaranteed if the shelf-life dates
have expired or if they have not been kept under the conditions described in this insert.
- The concentrated wash solution and dilution buffer may be stored at room temperature. Once diluted, these
solutions remain stable for six weeks if kept between +2C and +8C.
- Unused strips must be stored immediately in the aluminium envelope, taking care to keep the desiccant dry
and the envelopes seal airtight. If these precautions are taken, the strips activity can be conserved up to the
kits shelf-life date.
- Do not use reagents from other kits.
- The quality of the water used to prepare the various solutions is of the utmost importance. Do not use water
that may contain oxidants (e.g., sodium hypochlorite) or heavy metal salts, as these substances can react with
the chromogen.
- Discard all solutions contaminated with bacteria or fungi.
- The stop solution contains 1 M phosphoric acid. Handle it carefully.
- All materials and disposable equipment that come in contact with the samples must be considered infectious
and be disposed of in compliance with the legislation in force in the country.
- The neurotoxins produced by Clostridium botulinum are extremely active and very dangerous. All
biological products, and especially the cultures, must be handled with the greatest caution. All of these
samples and the materials that come in contact with them must be destroyed by incineration before
being disposed of in accordance with the laws in force in the country.
- To guarantee the reliability of the results, one must follow the protocol to the letter. Special care must be
taken in observing the incubation times and temperatures, as well as measuring the volumes and dilutions
accurately.


VI PROCEDURE

1- Samples from suspect cases may be taken from the following digestive tract organs and tissues: rumen,
abomasum, small intestine, and faeces.
Add 1 gr of sample into 5 ml of gelatin buffer. Heat this preparation at 80C for 10 minutes. Take care to
observe these temperature and time requirements. Take up 100 l of the preparation and add it to 20 ml of
anaerobic culture medium. (See chapter IV)
Incubate under anaerobic conditions at 40C for five days.
2- Bring all the reagents to 21C +/- 3C before use.
3- Take the microplate out of its wrapper.
4- Add 100-l aliquots of the non diluted supernatants to the wells as follows: Sample 1 in wells A1 and B1,
sample 2 in wells C1 and D1, etc. Proceed in the same manner for the positive reference(ex : G1 and H1)
5- Incubate the plate at 21C +/- 3C for 1 hour. Cover the plate with a lid.
6- Rinse the plate with the washing solution, prepared as instructed in chapter composition of the kit, as
follows: Empty the microplate of its contents by flipping it over sharply over a receptacle containing an
inactivating agent (an oxidant or other agent). Tap the microplate upside down against a piece of clean
absorbent paper to remove all the liquid. Fill all the used wells with the washing solution using a spray bottle
or by plunging the plate in a vessel of the right dimensions, then empty the wells once more by turning the
plate over above a receptacle containing an inactivating agent (oxidant or other agent). Repeat the entire
operation two more times, taking care to avoid the formation of bubbles in the microwells. After the plate
has been washed three times proceed to the next step.
7- Dilute the necessary amount of the biotinylated conjugate anti-Clostridium botulinum types C and D toxins
fiftyfold in the reagent dilution buffer (20 l of conjugate + 980 l of the reagent dilution buffer per strip).
8- Add 100 l of the diluted anti-Clostridium botulinum types C and D toxins conjugate solution to each well.
9- Incubate at 21C +/- 3C for one hour. Cover with a lid.
10- Wash the plate as described in Step 6.
11- The avidine-peroxidase conjugate is liquid and must be diluted fiftyfold in the reagent dilution buffer (20 l
of conjugate + 980 l of the reagent dilution buffer per strip).
12- Add 100 l of the diluted peroxidase-linked conjugate solution to each well.
13- Incubate at 21C +/- 3C for one hour. Cover with a lid.
14- Wash the plate as described in Step 6.
Bio-X Diagnostics Site du Complexe des postes 49, rue J. Wauters 5580 Jemelle Belgique
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15- Add 100 l of the chromogen solution to each well on the plate. The chromogen solution must be absolutely
colourless when it is pipetted into the wells. If a blue colour is visible, this means that the solution in the
pipette has been contaminated.
Incubate at 21C +/- 3C in the absence of light for 10 minutes. Do not cover. This time interval is given as
a guideline only, for in some circumstances it may be useful to lengthen or shorten the incubation time.
16- Add 50 l of stop solution to each well. The blue colour will change into a yellow colour.
17- Read the optical densities by means of a microplate spectrophotometer with a 450 nm filter. The results must
be read as quickly as possible after the stop solution has been applied, for in the case of a strong signal the
chromogen can crystallise and lead to incorrect measurements


VII INTERPRETING THE RESULTS

Calculate the net optical density of each sample by subtracting from the reading for each sample well the optical
density of the corresponding negative control.
Proceed in the same way for the positive control antigen.
The test is validated only if the positive control antigen yields a difference in the optical density at 10 minutes
that is greater than the value given on the QC data sheet.

Divide the signal read for each sample well by the corresponding positive control signal and multiply this result
by 100 to express it as a percentage. Using the first table in the quality control procedure, determine each
samples status (positive, negative).

Delta DO spl * 100
Val(ue) = ---------------------------
Delta DO pos

Determine each samples positive or negative status by referring to the first table in the quality control
procedure.



VIII ORDERING INFORMATION


BIO-X ELISA KIT CLOSTRIDIUM BOTULINUM TOXIN TYPES C and D: 1 X 48 tests BIO K 300/1
2 X 48 tests BIO K 300/2




IX BIBLIOGRAPHY


Brooks, C. E., Clarke, H. J., Finlay, D. A., McConnell, W., Graham, D. A., and Ball, H. J. (2010) Culture
enrichment assists the diagnosis of cattle botulism by a monoclonal antibody based sandwich ELISA.
Veterinary Microbiology 144, pp. 226-230

Brooks, C. E., Clarke, H. J., Graham, D. A., & Ball, H. J. (2011) Diagnosis of botulism types C and D in cattle
by a monoclonal antibody-based sandwich ELISA. Vet Rec. 2011 Apr 30;168(17):455. Epub 2011 Apr 11

Moriishi, K., Koura, M., Abe, N., Fujinaga, Y., Inoue, K., and Oguma, K. (1996) Mosaic structures of
neurotoxins produced from Clostridium botulinum types C and D organisms. Biochimica Biophysica Acta 1307,
pp 123-126

Sugiyama, H. (1986) Clostridium botulinum. Pathogenesis of bacterial infections in Animals. in Carlton L.
Gyles and Charles O. Thoen (Eds.). Published by The Iowa State University Press. Chapter 8, pp 60-68

Sunagawa, H., Ohyama, T., Watanabe, T., and Inoue, K. (1992) The complete amino acid 480 sequence of the
Clostridium botulinum type D neurotoxin, deduced by nucleotide 481 sequence analysis of the encoding phage
d-16 phi genome. Journal of Veterinary Medical Science 54, pp. 905-913

Bio-X Diagnostics Site du Complexe des postes 49, rue J. Wauters 5580 Jemelle Belgique
Tl : 0032(0)84.32.23.77 - Fax : 0032(0)84.31.52.63 - E-mail : a.ginter@biox.com (26/11/2012)V2.0
5
Tsukamoto, K., Kohda, T., Mukamoto, M., Takeuchi, K., Ihara, H., Saito, M., and Kozaki, S. (2005) Binding of
Clostridium botulinum type C and D neurotoxins to 500 ganglioside and phospholipid. Journal of Biological
Chemistry 280, pp 35164-35171