Cryptosporidium Antigen Screening ELISA Product Code: VF-P00605

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1 Cryptosporidium Antigen Screening ELISA Product Code: VF-P00605 Introduction Diarrhoea is a major cause of death for the young calves (less than one month old). For cattle, the neonatal gastroenteritis is often due to multiple factors. It may be the consequence of a viral infection (Coronavirus and Rotavirus), bacterial infection (toxin-producing Colibacillus mostly, Salmonella) or protozoan infection (Cryptosporidium parvum). The diagnosis of the cause of diarrhoea must be detected by laboratory tests, since it is not possible to identify the causative agent on the basis of the clinical symptoms. When to Test This kit enables the diagnosis of calf diarrhoea caused by Cryptosporidium parvum (C.parvum), by detecting antigens with the ELISA method. This test should be used to confirm the cause of a case of diarrhoea. Sampling Recommendations Follow veterinary advice in line with a case of diarrhoea. Assay Description Microtitre plates are supplied pre-coated with C.parvum antibodies. The faecal material is diluted in dilution buffer and incubated in the plate where any C.parvum present will bind to the antibody and form a complex. Unbound material is washed from the wells. A monoclonal antibody conjugated to peroxidase and specific to the pathogenic agent is added. This conjugate binds to target antigen if present in the sample added in the first stage of the test. Unbound conjugate is washed away and TMB substrate is added to the wells. The degree of colour intensity (optical density) is directly related to the amount of antigen present in the sample. Assay Procedure Add Sample/ Controls Incubate 30 mins & Wash Add Enzyme Conjugate Incubate 30 mins & Wash Add Substrate Reagent Incubate 10 mins Read by eye For further information or to place an order please contact us at: Guildhay Limited, 6 Riverside Business Centre, Walnut Tree Close, Guildford, Surrey, GU1 4UG England Tel: +44 (0) Fax: +44 (0) sales@guildhay.co.uk Website:

2 Cryptosporidium Antigen Screening ELISA Product Code: VF-P00605 Interpretation of Results Read by eye and interpret results as follows: Colour intensity Colour equal to or lighter than the negative control samples Colour darker than the negative control samples Antigen status to monoclonal added Negative Positive Calf Diarrhoea test range: VF-P00603 Antigen Screening ELISA VF-P00605 Cryptosporidium Antigen Screening ELISA For further information or to place an order please contact us at: Guildhay Limited, 6 Riverside Business Centre, Walnut Tree Close, Guildford, Surrey, GU1 4UG England Tel: +44 (0) Fax: +44 (0) sales@guildhay.co.uk Website:

3 ANTIGENIC DIAGNOSIS OF CRYPTOSPORIDIUM PARVUM IN CALVES FAECES BY ELISA METHOD 94 samples analysed (+ 1 positive and 1 negative control) ELISA CRYPTOSPORIDIUM ANTIGEN SCREENING VERSION : P00605/01

4 INTRODUCTION Neonatal diarrhoea is one of the major causes of death for young calves under one-month-old. For cattle, the neonatal gastroenteritis is often due to several factors. It can be the consequence of a viral infection (Coronavirus and Rotavirus), of a bacterial infection (enterotoxinogenous Colibacillus mostly, Salmonella etc.) or of a protozoan infection (Cryptosporidium parvum). The etiology of the diarrhoea must absolutely be determined by laboratory tests, since it is not possible to identify the causative agent on the base of clinical symptoms. The test proposed in this kit enables the diagnosis of Cryptosporidium parvum, by detecting antigens with the Elisa method. This method is easy to use, fast, reliable and very adequate for the analysis of a large number of samples. The results can be evaluated by a simple «naked eye» reading. PRINCIPLE OF THE TEST The principle of the proposed test is: 1. All the wells microplates were coated with Cryptosporidium parvum antibodies. These antibodies ensure the capture of the agent from the faeces. 2. The faecal material is diluted in the Dilution Buffer and incubated on the microplate. 3. After washing, the conjugate is added. This conjugate is a monoclonal antibody specifically directed to the pathogenic agent, coupled to the peroxidase. 4. After washing, the enzyme substrate (TMB) is added to the wells. In case of presence of Cryptosporidium parvum in the sample, the conjugate remains fixed on the wells and the enzyme catalyses the transformation of the colourless chromogen into a blue product. A positive and a negative control is supplied with the kit in order to validate the obtained results. The results can be read to the «naked eye». The presence of a greater coloration in the sample than in the negative control states the presence of the pathogen agent in the analysed sample. PRECAUTIONS FOR USE 1. Do not place the pipette in the mouth when testing reagents. 2. Avoid contact of the substrate (TMB*) with skin, mucous membranes and eyes. 3. Do not use reagents coming from other kits. 4. Even if the material delivered in the package does not contain any contaminating element, and that the ruminant sera are, in theory, non-infectious, it is nevertheless advised to decontaminate the whole disposable elements used during the handling by using an immersion for at least 1 hour in freshly prepared 5% sodium hypochlorite, before eliminating them, or autoclaving them at 120 C for a minimum of 1 hour or by any other method in accordance with the reglementation in force. * The Pourquier Institute is at your disposal to supply the toxicity form of the product. Elisa Cryptosporidium Antigen screening - Version P00605/01 - Page 1/4

5 KIT CONTENTS and STORAGE It is recommended to work with all the components at +21 C (± 5 C). Thus all reagents must be brought to room temperature at least one hour before the beginning of the test (except the conjugate). COMPONENTS QUANTITY STORAGE AND NOTES Mono-well coated Microplate Concentrated (x 20) wash solution 1 1 x 100 ml May form crystals at, which rapidly disappear at +21 C (± 5 C), a gentle shaking of the solution will accelerate the dissolution of the crystals This solution can also be stored at +21 C (± 5 C) up to 1 month, if the vial is closed in an airtight manner, in order to be immediately available when needed. After dilution, the wash solution can be stored for 3 days at The "Concentrated (X 20) wash solution" is the same for all the kits of the INSTITUT POURQUIER and can be used equally in the different kits. Dilution Buffer 8 1 x 50 ml Positive control (liquid) 1 x 0.5 ml greenish Negative control (liquid) 1 x 0.5 ml brown Anti-cryptosporidium / peroxidase conjugate 1 x 1.2 ml green +5 C (±3 C) The solution of diluted conjugate cannot be stored. Revelation Solution 5 (TMB) Ready to use 1 x 30 ml +5 C (±3 C) It can be left on the draining board at +21 C (± 5 C) from one use to another up to 1 week (if the vial is well closed in an airtight manner), in order to be immediately available when needed. Using Instructions If the kit is used according to instructions, a maximal of 94 reactions can be done in addition to the positive and negative controls. Elisa Cryptosporidium Antigen screening - Version P00605/01 - Page 2/4

6 MATERIALS REQUIRED BUT NOT PROVIDED 1) Vortex or similar type of agitator 2) Microplate washing system that distributes 300 µl per well (optional) 3) Precision Micropipettes and Multi-dispensing micropipettes. (The precision required must be lower or equivalent to 10% for volumes lower or equal to 10µl and to 5% for all the other volumes indicated) 4) Disposable pipette tips 5) Glass marbles 6) Distilled water: the water used for the reconstitution of the wash solution can be produced by a conventional distillation system or any other high-performance water purification system (reverse osmosis, resin or activated charcoal purification...). Do not use water likely to contain oxidising agents (sodium hypochlorite) or heavy metal salts. 7) Microplate covers (lid, aluminium foil or adhesive) 1) DEPOSITING THE SAMPLES INSTRUCTIONS FOR USE a) Dispense: µl of negative control in the well A1-100 µl of positive control in the well B1-50 µl of Dilution buffer 8 in each well following the control wells(see fig.1) - 50 µl of samples in each well containing the Dilution buffer 8 (see note) b) Cover the plate (with a lid, aluminium foil or adhesive) c) Incubate the plate at +21 C (+ 5 C) during 30 minutes (+ 3 min) Note: If the consistency of the sample makes the homogenisation process difficult, it is possible to add glass marbles inside the container and to dilute the faeces by shaking them vigorously. Do not centrifuge. In this case, it is also possible to dilute the sample to 1/4 or up to 1/10 in «Dilution Buffer 8». A N 7 P = Positive Control B P 8 N = Negative Control C 1 1 = Sample n 1 D 2 2 = Sample n 2 E 3 3 =.. F 4 G 5 H Figure 1: distribution of the samples 2) WASHING a) Dilute a vial of "Concentrated (x 20) wash solution" to 1/20 with distilled water or demineralised water. This solution is hereafter called "Wash solution". The dilution can be carried out before the elimination of the crystals which appeared at + 21 C (± 5 C) as long as the whole 100 ml vial is used. b) Empty the content of the plate by «flick-off» or better by a manual or automatic method. c) Fill all the wells of the plate with the wash solution; then empty them again. d) Repeat step c) twice (a total of 3 washes) taking particularly care of avoiding the formation of bubbles in the wells. Elisa Cryptosporidium Antigen screening - Version P00605/01 - Page 3/4

7 3) DEPOSITING THE CONJUGATE a) Dilute the conjugate to 1/10 in the "Dilution buffer 8". b) Dispense 100 µl of diluted conjugate solution in each well. c) Incubate the plate at + 21 C (+5 C) during 30 minutes (+3 mn) preferably while being agitated. 4) WASHING a) Empty the content of the plate by «flick-off» or by some other way or by an automatic method. b) Fill all the wells on the plate with the wash solution; then empty them again. c) Repeat the step b) twice (total of 3 washes) Note: 1. Particular care with the last wash is very important in getting a good test result. 2. If the wash is carried out with a manual method, it is possible after the last wash to drum the microplate on a dry towel in order to empty the wells completely. 5) REVELATION a) Dispense 100 µl of "Revelation Solution 5" per well b) Leave to incubate for 10 minutes at +21 C (±5 C) away from the light. Note: The revelation period given is only indicative. It can be slightly affected by different factors (quality of washes, quality of water used, precision of pipetting, temperature of the reagents...). So, according to the working conditions, the user may stop the reaction several minutes before or after the indicated time. VALIDATION CRITERIA The results can be considered reliable if: the positive control presents a well marked blue colour the negative control presents no colour or a slight blue colour INTERPRETATION Insofar as the plate is validated: is considered as positive any sample, which presents a blue colour that is darker than the colour of the negative control is considered as negative any sample, which presents a slighter or equivalent colour than the negative control Elisa Cryptosporidium Antigen screening - Version P00605/01 - Page 4/4