v140 v144 v145 Flockscreen Art Instructions for Use v1

7/29/2019 v140 v144 v145 Flockscreen Art Instructions for Use v1

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x-OvO Limited, Unit 1, Burnside Business Court, Inverkeithing, ScotlandKY11 1NZ, United Kingdom

Tel: +44 (0) 131 208 3454 Email: [email protected] No: GB 902 7320 57

X-OVO FLOCKSCREEN

ART/TRT Antibody ELISA KitFor Chickens and Turkeys

Cat. No. V140 (2 Plates), V144 (4 Plates) & V145 (5 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 highmorbidity 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.

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 knownfor specific vaccination programmes and breeds of bird.

(b) 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. Vaccineresponses 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.


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x-OvO Limited, Unit 1, Burnside Business Court, Inverkeithing, ScotlandKY11 1NZ, United KingdomTel: +44 (0) 131 208 3454 Email: [email protected]

VAT No: GB 902 7320 57

Assay Description

The FLOCKSCREEN Antibody ELISA kit provides a rapid, simple and sensitive method of detectingantibodies to ART in chicken and turkey serum or egg yolk.

Microtitre plates are supplied pre-coated with purified ART antigens. Diluted samples are incubated in thewells 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 isadded to the wells.

The degree of colour developed (optical density) is directly related to the amount of antibody present in thesample.

Assay Procedure

Incubate 60 mins

& wash

Incubate 60 mins& wash

Incubate 30 mins

Read at 550nmKit Contents

2 Plate Kit 4 Plate Kit 5 Plate Kit

1 2 x 96 well plates pre-coatedwith ART antigen (supplied as

2 well holders each containing

12 x 8-well strips). In a re-sealable foil pouch with silica

gel.

4 x 96 well plates pre-coatedwith ART antigen (supplied as

4 well holders each containing

12 x 8-well strips). In a re-sealable foil pouch with silica

gel.

5 x 96 well plates pre-coatedwith inactivated ART antigen

(supplied as 5 well holders each

containing 12 x 8-well strips).In a re-sealable foil pouch with

silica gel.

2 Positive Control with Positive Control with Positive Control with

Add Sample/

Controls

Add Enzyme

Conjugate

Add SubstrateReagent

Add Stop Sol.


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antibodies to ART preserved inphosphate buffer with protein

stabiliser and ProClin 0.063%

v/v. (500l ready to use).



v/v. (2 x 500l ready to use).




3 Negative Control with SPF

chicken serum preserved inphosphate buffer with protein


v/v. (500l ready to use).

Negative Control with SPF




Negative Control with SPF




4 Enzyme Conjugate Reagent,

containing alkaline

phosphatase labelled rabbitanti-chicken IgG in tris buffer

with an inert blue dye and

Sodium azide 0.1% w/v.(11ml)

Enzyme Conjugate Reagent,

containing alkaline

phosphatase labelled rabbitanti-chicken IgG in tris buffer

with an inert blue dye and

Sodium azide 0.1% w/v. (2 x11ml)

Enzyme Conjugate Reagent,

containing alkaline phosphatase

labelled rabbit anti-chicken IgGin tris buffer with an inert blue

dye and sodium azide 0.1%

w/v. (28ml)

5 ELISA Substrate Reagent,

containing phenolphthaleinmonophosphate and enzyme

co-factors in a diethanolamine

buffer. (11ml)

ELISA Substrate Reagent,



buffer. (2 x 11ml)

ELISA Substrate Reagent,



buffer. (28ml)

6 ELISA Stop Solution,

containing sodium hydroxideand a chelating agent in a

diethanolamine buffer. (11ml)WARNING CAUSTIC!

ELISA Stop Solution,


diethanolamine buffer. (22ml)WARNING CAUSTIC!

ELISA Stop Solution,


diethanolamine buffer (27.5ml)WARNING CAUSTIC!

7 Wash Buffer Concentrate,

containing phosphate buffer

with ProClin 0.63% v/v.(50ml) sufficient to make up

1 litre of wash buffer.

Wash Buffer Concentrate,


with ProClin 0.63% v/v.(100ml) sufficient to make up

2 litres of wash buffer.

Wash Buffer Concentrate,


with ProClin 0.63% v/v.(125ml) - sufficient to make up

2.5 litres of wash buffer

8 Sample Diluent Concentrate,


with protein stabiliser andProClin 0.63% v/v. (50ml)

sufficient to make up 500ml of

sample diluent.

Sample Diluent Concentrate,


with protein stabiliser andProClin 0.63% v/v. (100ml)

sufficient to make up 1 litre of

sample diluent.

Sample Diluent Concentrate,


with protein stabiliser andProClin 0.63% v/v. (100ml) -

sufficient to make up 1 litre of

sample diluent.

Materials and Equipment Required (Not Supplied):

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


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

50l (or variable 10-200l)

50l repeater or an 8 or 12 channel2.5 ml (or variable 1-5ml). Note pipettes should be calibrated on a

routine basis.

2. Disposable tips for pipettes3. Microtitre Plate Reader with 550nm filter (a 540nm filter will give lower ODs, but accurate results)

4. Microtitre Plate Washer

5. +37C incubator6. 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.

Warnings and Precautions

1. This kit is forIN 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 0.2m filter. However, because your sample sera may be infected with bacteria or viruses, allreagents 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 reagentshould 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.

2 Plate Kit 4 Plate Kit 5 Plate Kit

3 To prepare sample diluent To prepare sample diluent buffer, To prepare sample diluent buffer,


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buffer, add the Sample DiluentConcentrate (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 preparingsamples for any of the

FLOCKSCREEN Kits.

add the Sample DiluentConcentrate (100ml) to distilled

or deionised water and make up

to total volume of 1 litre SampleDiluent can be stored at +4C for

up to 3 months and can be used

for preparing samples for any ofthe FLOCKSCREEN Kits.

add the Sample DiluentConcentrate (100ml) to distilled

or deionised water and make up

to a total volume of 1 litre. Thissample diluent can be stored at

+4oC for up to 3 months, and can

be used for preparing samples forany of the FLOCKSCREEN

kits.

4 To prepare the wash buffer,add the Wash Buffer

Concentrate (50ml) to distilled

or deionised water and makeup to total volume of 1 litre.

This is stable at room

temperature for 3 months and

can be used with any of theFLOCKSCREEN Kits.

To prepare the wash buffer, addthe Wash Buffer Concentrate

(100ml) to distilled or deionised

water and make up to totalvolume of 2 litres. This is stable

at room temperature for 3 months

and can be used with any of the

FLOCKSCREEN Kits.

To prepare the wash buffer, addthe Wash Buffer Concentrate

(125ml) to distilled or deionised

water and make up to a totalvolume of 2.5 litres. 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 +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 samplediluent, 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.Yolk Samples: Take 200l of fresh yolk and add to 1.8ml of reconstituted wash buffer. Dilute a further 1:50 insample diluent buffer (50l in 2.5ml).

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.2. 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 ifplate shaker is not available, mix by gently tapping the side of the plate. Cover the plate withadhesive cover and incubate at +37C for 60 minutes.

3. 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.

4. Add 50l of Enzyme Conjugate Reagent to each well. Mix on a plate shaker or by gently tappingthe side of the plate.


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

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

tap firmly on absorbent paper.7. 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.

8. Cover the plate with adhesive cover and incubate at +37C for 30 minutes. Colour development ispale pink, which deepens on addition of Stop Solution.

9. Remove adhesive cover and add 50l 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, however,the assay validity criteria and titre calculations are different for each species. Set out below is the informationfor each different type of sample. If running the sample on the SoftSCREEN data acquisition and analysis

software from x-OvO please ensure that the correct kit is selected.

Interpretation of results for chicken samples:

For the test to be valid:

1. Mean Negative control absorbance must be


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Less than orequal to 0.123

0 - 575 Negative Less than orequal to 0.083

0 876 Negative

Greater than

0.123 and lessthan 0.181

576 - 824 Suspect Greater than

0.083 and lessthan 0.126

877 1209 Suspect

Greater than or

equal to 0.181

825 or greater Positive Greater than or

equal to 0.126

1210 or

greater

Positive

Where samples fall within the suspect range the flock should be retested within 10-14 days.

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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