v180 flockscreen art diff instructions for use v1_000

x-OvO Limited, Unit 1, Burnside Business Court, Inverkeithing KY11 1NZ, United Kingdom

Tel: +44 (0) 131 208 3454 Email: [email protected]

Vat No: GB 902 7320 57

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) To monitor vaccination response – this is especially useful where baseline titre values are known

for specific vaccination programmes and breeds of bird (subtype specific)

(b) To confirm the presence of antibodies or increasing antibody titres following exposure to the

disease (subtype specific).

(c) 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.

x-OvO Limited, Unit 1, Burnside Business Court, Inverkeithing KY11 1NZ, United Kingdom Tel: +44 (0) 131 208 3454 Email: [email protected] VAT No: GB 902 7320 57

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

Incubate 60 mins

& wash

Incubate 60 mins

& wash

Incubate 30 mins

Read at 550nm

Add Sample/

Controls

Add Enzyme

Conjugate

Add Substrate

Reagent

Add Stop Sol.


Kit Contents

2 Plate Kit

1 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 2 x Positive Controls (A+B) with antibodies to ART preserved in phosphate buffer with protein

stabiliser and ProClin 0.063% v/v. (500µl ready to use).

3 1 x Negative Control with SPF chicken serum preserved in phosphate buffer with protein stabiliser

and ProClin 0.063% v/v. (500µl ready to use).

4 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)

5 ELISA Substrate Reagent, containing phenolphthalein monophosphate and enzyme co-factors in a

diethanolamine buffer. (11ml)

6 ELISA Stop Solution, containing sodium hydroxide and a chelating agent in a diethanolamine

buffer. (11ml)

WARNING CAUSTIC!

7 Wash Buffer Concentrate, containing phosphate buffer with ProClin 0.63% v/v. (50ml) – sufficient

to make up 1 ltr of wash buffer.

8 Sample Diluent Concentrate, containing phosphate buffer with protein stabiliser and ProClin 0.63%

v/v. (50ml) – sufficient to make up 50mls of sample diluent.

Materials and Equipment Required (Not Supplied):

In order to run the FLOCKSCREEN� assays, the following equipment is ideal:

1. Precision pipettes: 5µl (or variable 1-20µl)

50µl (or variable 10-200µl)

50µl 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. +37ºC incubator

6. Distilled or deionised water

7. Disposable 5ml plastic tubes

It is possible to run the assays without the 50µl 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. This kit is for IN VITRO use only.

2. Optimum results will be obtained by strict adherence to this protocol. Careful pipetting and washing

are necessary to achieve good assay performance.

3. The assay has been developed with incubations at +37oC for more consistent results.

4. Plates are coated with purified and inactivated viral antigens, and control sera have been filtered

with a 0.2µm filter. However, because your sample sera may be infected with bacteria or viruses, all

reagents should be treated as potential biohazards and handled appropriately.

5. Do not intermix reagents from different Lot numbers with the exceptions of wash buffer and sample

diluent.

6. 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.

7. Caution should be exercised in the handling of alkali or other hazardous chemicals in accordance

with Good Laboratory Practice.

8. Never pipette by mouth.

9. Wash solution and waste should be properly decontaminated with bleach or other strong oxidising

agents before disposal.

10. 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.

Reagent Preparation

1 Allow all reagents to come to room temperature before use.

2 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.

3 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 +4ºC for up to 3 months and

can be used for preparing samples for any of the FLOCKSCREEN™ Kits.

4 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.

5 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 +4ºC (up to 2 days) or at

- 20ºC 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 5µl 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 200µl of fresh yolk and add to 1.8ml of reconstituted wash buffer. Dilute a further 1:50 in

sample diluent buffer (50µl in 2.5ml).


Diluted samples can be kept for several days at +4ºC for re-testing or at –20ºC 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.

2. Add 50µl of the diluted samples and undiluted controls to the appropriate wells. Diluted samples

should be retained at +4ºC 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 +37ºC for 60 minutes.

3. Remove the adhesive cover and wash the plate 4 times with wash buffer (300µl 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.

4. Add 50µl of Enzyme Conjugate Reagent to each well. Mix on a plate shaker or by gently tapping

the side of the plate.

5. Cover the plate with adhesive cover and incubate at +37ºC for 60 minutes.

6. Remove adhesive cover and wash the plate 4 times with wash buffer (300µl per well), invert and

tap firmly on absorbent paper.

7. Add 50µl 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.

8. Cover the plate with adhesive cover and incubate at +37ºC for 30 minutes. Colour development is

pale pink, which deepens on addition of Stop Solution.

9. Remove adhesive cover and add 50µl Stop Solution to each well. Mix on a plate shaker to obtain

full colour development.

10. 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.

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 +4ºC 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

v180 flockscreen art diff instructions for use v1_000

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