X-OVO FLOCKSCREEN ART/TRT Differential Antibody ELISA Kit For Chickens and Turkeys Cat. No.

V180 (2 Plates) Instructions for use (V1)

Introduction Avian Rhinotracheitis (ART) also known as Turkey Rhinotracheitis (TRT), is a rapidly spreading respiratory disease of breeders and layers and may be a sub-clinical disease of broilers. In birds of laying age it has a high morbidity and, unless there are problems of secondary bacterial invasion, a low mortality. Recovery from the uncomplicated disease is rapid (7-10 days) and whilst there may be a significant egg-drop (up to 50%) it returns to normal after 2-4 weeks. Flocks in their first 2-3 weeks of lay are most seriously affected. A, B and C subtypes of ART are found globally. The FLOCKSCREEN ART DIFF ELISA Kit detects the presence of antibodies to subtype A and B, differentiating the results of exposure to each subtype due to the use of highly specific and purified antigens and specific antisera. When to Test The FLOCKSCREEN Avian Rhinotracheitis test can be used: (a) (b) (c) To monitor vaccination response this is especially useful where baseline titre values are known for specific vaccination programmes and breeds of bird (subtype specific) To confirm the presence of antibodies or increasing antibody titres following exposure to the disease (subtype specific). To assist in the application of vaccines in the framework of an eradication strategy when the subtype of the field virus and of the vaccine are known

Note that some degree of cross reactivity may occur between A and B subtype responses may occur dependent upon field conditions, but under these circumstances, the dominant serological response will relate to the dominant antigenic challenge.

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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. Sampling Recommendations As a guide, a 1% sample is usually sufficient for vaccination or disease monitoring. In practice, for ART, about 18-20 birds per house would normally be tested. Assay Description The FLOCKSCREEN Antibody ELISA kit provides a rapid, simple and sensitive method of detecting antibodies to ART in chicken and turkey serum or egg yolk. Microtitre plates are supplied pre-coated with purified ART antigens. Plates are included for detection of both A and B subtype antibodies and are clearly labelled. CARE MUST BE TAKEN TO USE PLATES LABELLED AS A FOR THE DECTECTION OF THE A SUBTYPE ANTIBODY RESPONSE AND PLATES LABELLED AS B FOR THE DETECTION OF THE B SUBTPE ANTIBODY RESPONSE. Diluted samples are incubated in the wells where any antibody specific to ART 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 ART 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

Add Substrate Reagent Incubate 30 mins

Add Stop Sol.

Read at 550nm

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Kit Contents 2 Plate Kit 1 x 96 well plates pre-coated with ART A antigen (supplied as 2 well holders each containing 12 x 8-well strips). 1 x 96 well plates pre-coated with ART B antigen (supplied as 2 well holders each containing 12 x 8-well strips). In a re-sealable foil pouch with silica gel. 2 x Positive Controls (A+B) with antibodies to ART preserved in phosphate buffer with protein stabiliser and ProClin 0.063% v/v. (500l ready to use). 1 x Negative Control with SPF 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 co-factors 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 ltr of wash buffer. Sample Diluent Concentrate, containing phosphate buffer with protein stabiliser and ProClin 0.63% v/v. (50ml) sufficient to make up 50mls of sample diluent.

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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 2.5 ml (or variable 1-5ml). Note pipettes should be calibrated on a routine basis. 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 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.

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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. Plates are coated with purified and 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, DO NOT use the same pipette tips or containers used for other reagents with the Substrate Reagent. The Substrate Reagent should be yellow in colour before addition to the wells. An orange, brown or pink colour indicates 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.

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Reagent Preparation 1 2 Allow all reagents to come to room temperature before use. The Wash Buffer Concentrate and Sample Diluent Concentrate may contain crystals. This is due to the high concentration of salts. Shake the bottle prior to reconstituting. The crystals will dissolve upon mixing. To prepare sample diluent buffer, add the Sample Diluent Concentrate (50ml) to distilled or deionised water and make up to total volume of 500ml. Sample Diluent can be stored at +4C for up to 3 months and can be used for preparing samples for any of the FLOCKSCREEN Kits. To prepare the wash buffer, add the Wash Buffer Concentrate (50ml) to distilled or deionised water and make up to total volume of 1 litre. This is stable at room temperature for 3 months and can be used with any of the FLOCKSCREEN 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. Make a 1:500 dilution of each test sample in sample diluent buffer by adding 2.5ml of reconstituted sample diluent to 5l of serum in a disposable 5ml plastic tube. Invert gently 2 or 3 times to mix. Alternatively a two step dilution can be made using a 96 well deep well multiplate. Yolk Samples: Take 200l of fresh yolk and add to 1.8ml of reconstituted wash buffer. Dilute a further 1:50 in sample diluent buffer (50l in 2.5ml).

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Diluted samples can be kept for several days at +4C for re-testing 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 is not available, mix by gently tapping the side of the plate. Cover the plate with an adhesive cover and incubate at +37C for 60 minutes. Remove the 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. 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.

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Result Interpretation The FLOCKSCREEN ART Antibody ELISA kit can be used for both Turkey and Chicken samples. Samples are considered positive when an S/P ration of 0.45 or greater is achieved. 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. 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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