X-OVO FLOCKSCREEN Infectious Bronchitis Virus (IBV) Antibody ELISA Kit Cat. No.

V130 (2 Plates), V134 (4 Plates) & V135 (5 Plates) Instructions for use (V2)
Introduction Infectious Bronchitis (IB) is a common respiratory disease of all types of poultry and frequently forms part of a respiratory disease complex. There are a number of variants that cause disease, even in vaccinated birds. Where post vaccination titres have been monitored and an infection due to an IB variant is suspected, it should be possible to detect a significant secondary rise in antibody titre. When to Test The FLOCKSCREEN Infectious Bronchitis test can be used: (a) (b) To monitor vaccination response this is especially useful where baseline titre values are known for specific vaccination programmes and breeds of bird. To confirm the presence of antibodies or increasing antibody titres following exposure to the disease.

Antibody responses are detectable 7-10 days after infection and peak at about two weeks post infection. The response to live vaccination is variable but should not be tested for sooner than 14 days post vaccination. The antibody response will usually be detectable within 7 days of boosting with live or killed vaccine. Vaccine responses should take the form of a normal distribution curve when vaccination has been effectively administered. It is recommended that flocks should not be tested for at least three weeks after immunisation with oiladjuvinated vaccines as these are often known to cause short term interference with serological tests. Sampling Recommendations As a guide, a 1% sample is usually sufficient for vaccination or disease monitoring. In practice, for IBV, about 18-20 birds per house would normally be tested.

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Assay Description The FLOCKSCREEN IBV Antibody ELISA kit provides a rapid, simple and sensitive method of detecting antibodies to IBV in serum or egg yolk. Microtitre plates are supplied pre-coated with purified IBV antigens. Diluted samples are incubated in the wells where any antibody specific to IBV binds and forms a complex. Unbound material is washed from the wells and an alkaline phosphatase labelled rabbit anti-chicken IgG conjugate reagent is added which binds to the chicken antibodies attached to IBV antigens. Unbound conjugate is washed away and PMP substrate is added to the wells. The degree of colour developed (optical density) is directly related to the amount of antibody present in the sample.

Assay Procedure

Add Sample/ Controls Add Enzyme Conjugate Incubate 60 mins & wash Incubate 60 mins & wash Incubate 30 mins Read at 550nm Kit Contents 2 Plate Kit 1 2 x 96 well plates pre-coated with IBV antigen (supplied as 2 well holders each containing 12 x 8-well strips). In a re-sealable foil pouch with silica gel. Positive Control with antibodies to IBV preserved in phosphate buffer with protein stabiliser and ProClin 0.063% v/v. (500l ready to use). Negative Control with SPF 4 Plate Kit 4 x 96 well plates pre-coated with IBV antigen (supplied as 4 well holders each containing 12 x 8well strips). In a re-sealable foil pouch with silica gel. Positive Control with antibodies to IBV preserved in phosphate buffer with protein stabiliser and ProClin 0.063% v/v.. (2 x 500l ready to use). Negative Control with SPF 5 Plate Kit 5 x 96 well plates pre-coated with IBV antigen (supplied as 5 well holders each containing 12 x 8-well strips). In a re-sealable foil pouch with silica gel. Positive Control with antibodies to IBV preserved in phosphate buffer with protein stabiliser and ProClin 0.063% v/v.. (2 x 500l ready to use). Negative Control with SPF Add Substrate Reagent

Add Stop Sol.

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chicken serum preserved in phosphate buffer with protein stabiliser and ProClin 0.063% v/v. (500l ready to use). Enzyme Conjugate Reagent, containing alkaline phosphatase labelled rabbit anti-chicken IgG in tris buffer with an inert blue dye and sodium azide 0.1% w/v. (11ml) ELISA Substrate Reagent, containing phenolphthalein monophosphate and enzyme cofactors in a diethanolamine buffer. (11ml) ELISA Stop Solution, containing sodium hydroxide and a chelating agent in a diethanolamine buffer. (11ml) WARNING CAUSTIC! Wash Buffer Concentrate, containing phosphate buffer with ProClin 0.63% v/v. (50ml) - sufficient to make up 1 litre of wash buffer. Sample Diluent Concentrate, containing phosphate buffer with protein stabiliser and ProClin 0.63% v/v. (50ml) sufficient to make up 500ml of sample diluent.

chicken serum preserved in phosphate buffer with protein stabiliser and ProClin 0.063% v/v. (2 x 500l ready to use). Enzyme Conjugate Reagent, containing alkaline phosphatase labelled rabbit anti-chicken IgG in tris buffer with an inert blue dye and Sodium Azide 0.1% v/v. (2 x 11ml) ELISA Substrate Reagent, containing phenolphthalein monophosphate and enzyme cofactors in a diethanolamine buffer. (2 x 11ml) ELISA Stop Solution, containing sodium hydroxide and a chelating agent in a diethanolamine buffer. (22ml) WARNING CAUSTIC! Wash Buffer Concentrate, containing phosphate buffer with ProClin 0.63% v/v. (100ml) sufficient to make up 2 litres of wash buffer. Sample Diluent Concentrate, containing phosphate buffer with protein stabiliser and ProClin 0.63% v/v. (100ml) - sufficient to make up 1 litre.

chicken serum preserved in phosphate buffer with protein stabiliser and ProClin 0.063% v/v. (2 x 500l ready to use). Enzyme Conjugate Reagent, containing alkaline phosphatase labelled rabbit anti-chicken IgG in tris buffer with an inert blue dye and Sodium Azide 0.1% v/v. (2 x 14ml) ELISA Substrate Reagent, containing phenolphthalein monophosphate and enzyme co-factors in a diethanolamine buffer. (2 x 14ml) ELISA Stop Solution, containing sodium hydroxide and a chelating agent in a diethanolamine buffer. (27.5ml) WARNING CAUSTIC! Wash Buffer Concentrate, containing phosphate buffer with ProClin 0.63% v/v. (125ml) - sufficient to make up 2.5 litres of wash buffer. Sample Diluent Concentrate, containing phosphate buffer with protein stabiliser and ProClin 0.63% v/v. (100ml) sufficient to make up 1 litre.

Materials and Equipment Required (Not Supplied): In order to run the FLOCKSCREEN assays, the following equipment is ideal: 1. Precision pipettes: 5l (or variable 1-20l) 50l (or variable 10-200l) 50l repeater or an 8 or 12 channel 2.5ml (or variable 1-5ml) 2. Disposable tips for pipettes 3. Microtitre Plate Reader with 550nm filter 4. Microtitre Plate Washer 5. 37C incubator 6. Distilled or deionised water 7. Disposable 5ml plastic tubes x-OvO Limited, Unit 1, Burnside Business Court, Inverkeithing, KY11 1NZ, United Kingdom Tel: +44 (0) 131 208 3454 Email: sales@x-ovo.com VAT No: GB 902 7320 57

It is possible to run the assays without the 50l repeater, or an 8 channel pipette and to use a wash bottle for plate washing instead of a plate washer. The results will be less consistent however. Note pipettes should be calibrated on a routine basis. Warnings and Precautions 1. 2. 3. 4. This kit is for IN VITRO use only. Optimum results will be obtained by strict adherence to this protocol. Careful pipetting and washing are necessary to achieve good assay performance. The assay has been developed with incubations at +37oC for more consistent results. This eliminates problems associated with varying room temperature conditions. Plates are coated with purified inactivated viral antigens, and control sera have been filtered with a 0.2m filter. However, because your sample sera may be infected with bacteria or viruses, all reagents should be treated as potential biohazards and handled appropriately. Do not intermix reagents from different Lot numbers with the exceptions of wash buffer and sample diluent. The Substrate Reagent is very sensitive and under no circumstances should the same pipette tips or containers be used for other reagents, be used with the Substrate Reagent. The Substrate Reagent should be yellow in colour before addition to the wells. An orange, brown or pink colour indicates deterioration or contamination and the reagent should not be used. Caution should be exercised in the handling of alkali or other hazardous chemicals in accordance with Good Laboratory Practice. Never pipette by mouth. Wash solution and waste should be properly decontaminated with bleach or other strong oxidising agents before disposal. Some kit components contain low levels of sodium azide. Disposal should include flushing plumbing installations with large quantities of water to prevent the formation of copper azides which can be explosive on impact.

5. 6.

7. 8. 9. 10.

Reagent Preparation 1 2 Allow all reagents to come to room temperature before use. The Wash Buffer Concentrate and Sample Diluent Concentrate may partly recrystallise. This is due to the high concentration of salts. Simply shake the bottle prior to reconstituting as described in the next two steps. The crystals will dissolve readily upon mixing. 2 Plate Kit 4 Plate Kit 5 Plate Kit To prepare sample diluent To prepare sample diluent To prepare sample diluent buffer, buffer, add the Sample Diluent buffer, add the Sample Diluent add the Sample Diluent Concentrate (50ml) to distilled Concentrate (100ml) to distilled Concentrate (100ml) to distilled or deionised water and make up or deionised water and make up or deionised water and make up to to total volume of 500ml. This to total volume of 1 litre. This total volume of 1 litre. This sample diluent can be stored at sample diluent can be stored at sample diluent can be stored at +4C for up to 3 months and can +4C for up to 3 months and +4C for up to 3 months and can be used for preparing samples can be used for preparing be used for preparing samples for x-OvO Limited, Unit 1, Burnside Business Court, Inverkeithing, KY11 1NZ, United Kingdom Tel: +44 (0) 131 208 3454 Email: sales@x-ovo.com VAT No: GB 902 7320 57

for any of the samples for any of the any of the FLOCKSCREEN FLOCKSCREEN Kits. FLOCKSCREEN Kits. Kits. To prepare the wash buffer, add To prepare the wash buffer, add To prepare the wash buffer, add the Wash Buffer Concentrate the Wash Buffer Concentrate the Wash Buffer Concentrate (50ml) to distilled or deionised (100ml) to distilled or (125ml) to distilled or deionised water and make up to total deionised water and make up to water and make up to total volume of 1 litre. This is stable total volume of 2 litres. This is volume of 2.5 litres. This is at room temperature for 3 stable at room temperature for stable at room temperature for 3 months and can be used with 3 months and can be used with months and can be used with any any of the FLOCKSCREEN any of the FLOCKSCREEN of the FLOCKSCREEN Kits. Kits. Kits. DO NOT DILUTE THE POSITIVE AND NEGATIVE CONTROLS.

Sample Preparation Serum Samples: These should be as fresh and clean as possible and may be stored at +4C (up to 2 days) or at - 20C for long term storage. Dilute 5 microlitres of sample into 1.25ml of sample diluent then double dilute an appropriate volume of this dilution e.g. 100 microlitres diluted in a further 100 microlitres of sample diluent, to give a 1:500 final dilution. Alternatively, dilute 1 microlitre of sample into 500 microlitres of sample diluent to achieve the 1:500 final dilution directly. Invert gently 2 or 3 times to mix. Diluted samples can be kept for several days at +4C for retesting or at -20C for longer term storage. Assay Procedure 1. Remove the pre-coated plates from their sealed bags and record sample and control locations on a 12 x 8 template sheet. Each sample should be run in duplicate for optimal results. The positive and negative controls must always be run in duplicate. Add 50l of the diluted samples and undiluted controls to the appropriate wells. Diluted samples should be retained at +4C until successful results are confirmed. Mix plate on a plate shaker or if plate shaker not available, mix by gently tapping the side of the plate. Cover the plate with adhesive cover and incubate at +37C for 60 minutes. Remove adhesive cover and wash the plate 4 times with wash buffer (300l per well), invert and tap firmly on absorbent paper. N.B. To reduce the possibility of sample carryover, it is recommended where possible, that the washer is programmed to wash each strip individually four times before washing the next strip. Add 50l of Enzyme Conjugate Reagent to each well. Mix on a plate shaker or by gently tapping the side of the plate. Cover the plate with adhesive cover and incubate at +37C for 60 minutes. Remove adhesive cover and wash the plate 4 times with wash buffer (300l per well), invert and tap firmly on absorbent paper. Add 50l Substrate Reagent to each well. The reagent must be at room temperature to achieve maximum colour development. Mix on a plate shaker or by gently tapping the side of the plate. Cover the plate with adhesive cover and incubate at +37C for 30 minutes. Colour development is pale pink, which deepens on addition of Stop Solution.

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9. 10.

Remove adhesive cover and add 50l Stop Solution to each well. Mix on a plate shaker to obtain full colour development. Wipe the under surface of the plate free of dust etc. with a soft tissue. Read the plate using a Microtitre Plate Reader at 550nm. In order to obtain optimum results the plate should be read immediately after adding the stop solution.

Results For the test to be valid: a) Mean Negative control absorbance must be < 0.2 b) Mean Positive control absorbance must be at least 2 times the optical density of the negative control absorbance with a minimum difference of 0.2 OD. It is important that the results fall within these parameters in order to prove that the components of the kit are all in good condition and that there have been no operator errors. Interpretation of Results For calculation of results, an S/P ratio is required (Sample value related to Positive Control value). The following formula is applied (using mean absorbance values for controls and paired samples): SAMPLE ABSORBANCE - NEGATIVE CONTROL ABSORBANCE = S/P POSITIVE CONTROL ABSORBANCE - NEGATIVE CONTROL ABSORBANCE A titre can be calculated using the following equation: Log10 Titre = Titre =

0.650 x (Log10 S/P) + 3.691 Antilog of Log10 Titre

The S/P ratio values and/or ELISA titre values of the samples should be interpreted using the following values: S/P Ratio Less than or equal to 0.102 Greater than 0.102 and less than 0.166 Greater than or equal to 0.166 IBV Titre 0-1113 1114-1525 1526 or greater IBV Antibody Status Non-significant Marginal positive Positive

Results where S/P ratios are 0.102 or below (titres of 1113 or lower) indicate non-significant antibody levels to Infectious Bronchitis. S/P ratios between 0.102 and 0.166 (titres of 1114-1525) may be regarded as marginal. Protective levels must be established for each set of circumstances (vaccine used, type of flock etc.) and on a flock basis, looking at pre- as well as post-vaccination titres. Storage and Stability All reagents should be stored at +4C on delivery. Do not freeze. Avoid exposure to sunlight. Do not use after the stated expiry date.

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Do not use if silica gel desiccant in the pouch containing the microtitre plates is pink. Any unused strips should be resealed in the re-sealable foil pouch together with the silica gel. ONCE A KIT HAS BEEN OPENED IT HAS A MAXIMUM SHELF-LIFE OF 3 MONTHS

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