V080 V084 V085 FLOCKSCREEN CAV Instructions for Use _v2__000

7/29/2019 V080 V084 V085 FLOCKSCREEN CAV Instructions for Use _v2__000

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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

Chicken Anaemia Virus (CAV)

Antibody ELISA Kit

Cat. No. V080 (2 Plates), V084 (4 Plates) & V085 (5 Plates)

Instructions for use (V2)

Introduction

Chicken Anaemia Virus (CAV) formerly known as Chicken Infectious Anaemia, is a serious disease of young

chickens. Chicks infected in ovo (i.e. where the breeder stock first became infected during lay) or chicksinfected during their first week will have a mortality rate of 10-60% at 2-3 weeks of age.

Normally chicks are infected at about 5 weeks of age from the faeces of vertically infected birds. Although

occasionally 'Bluewing' may be seen, there are not usually clinical signs in birds infected during the rearing

period. Once a bird has been infected, antibody will protect it and (with breeder flocks) maternal antibody willprotect its progeny during the risk period.

Where the disease occurs most of the signs (anaemia, subcutaneous haemorrhages, muscular haemorrhages,gangrenous dermatitis, and generalised lymphoid atrophy) are due to a plastic anaemia and the virus's immuno-

suppressive effects.

Applications of the Test

The FLOCKSCREEN CAV test can be used:

(a) To monitor vaccination response in the parent flock this is especially useful where baseline titre

values are known for specific vaccination programmes and breeds of bird.

(b) To confirm the presence of antibodies or increasing antibody titres following natural exposure tothe disease.

(c) To confirm protective status in day-old chicks.(d) It may also be used to monitor CAV-free status in a flock if that is desired.

Antibody responses are detectable 7-10 days after infection and peak at about two weeks post infection. Theresponse to natural exposure is variable but should not be tested for sooner than 14 days post exposure. The

antibody response will usually be detectable within 7 days of boosting with live or killed vaccine. Vaccine


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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

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.

Assay Description

The FLOCKSCREEN CAV Antibody ELISA Kit provides a rapid, simple and sensitive method of detecting

antibodies to CAV in chicken serum or egg yolk.

Microtitre plates are supplied pre-coated with purified viral antigens. Diluted samples are incubated in the

wells where any antibody specific to CAV binds and forms a complex. Unbound material is washed from thewells and an alkaline phosphatase labelled rabbit anti-chicken IgG conjugate reagent is added which binds to

the chicken antibodies attached to CAV 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 ofantibody to CAV present in the sample.

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-coated

with inactivated CAV antigen(supplied as 2 well holders

each containing 12 x 8-well

4 x 96 well plates pre-coated with

inactivated CAV antigen(supplied as 4 well holders each

containing 12 x 8-well strips). In

5 x 96 well plates pre-coated with

inactivated CAV antigen(supplied as 5 well holders each

containing 12 x 8-well strips). In

Add Sample/

Controls

Add EnzymeConjugate

Add Substrate

Reagent

Add Stop Sol.


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VAT No: GB 902 7320 57

strips). In a re-sealable foil

pouch with silica gel.a re-sealable foil pouch with

silica gel.

a re-sealable foil pouch with silica

gel.

2 Positive Control withantibodies to CAV preserved

in phosphate buffer withprotein stabiliser and ProClin

0.063% v/v. (500l ready to

use).

Positive Control with antibodiesto CAV preserved in phosphate

buffer with protein stabiliser andProClin 0.063% v/v. (2 x 500l

ready to use).

Positive Control with antibodiesto CAV preserved in phosphate

buffer with protein stabiliser andProClin 0.063% v/v. (2 x 600l

ready to use).

3 Negative Control with SPF

chicken serum preserved in

phosphate buffer with proteinstabiliser and ProClin 0.063%

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

Negative Control with SPF


phosphate buffer with proteinstabiliser and ProClin 0.063%

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

Negative Control with SPF


phosphate buffer with proteinstabiliser and ProClin 0.063% v/v.

(2 x 600l ready to use).

4 Enzyme Conjugate Reagent,containing alkaline

phosphatase labelled rabbit

anti-chicken IgG in tris bufferwith an inert blue dye and

sodium azide 0.1% w/v.

(11ml)

Enzyme Conjugate Reagent,containing alkaline

phosphatase labelled rabbit anti-

chicken IgG in tris buffer with aninert blue dye and Sodium Azide

0.1% v/v. (2 x 11ml)

Enzyme Conjugate Reagent,containing alkaline phosphatase

labelled rabbit anti-chicken IgG in

tris buffer with an inert blue dyeand sodium azide 0.1% w/v.

(28ml)

5 ELISA Substrate Reagent,

containing phenolphthalein

monophosphate and enzymeco-factors in a diethanolamine

buffer. (11ml)

ELISA Substrate Reagent,


monophosphate and enzyme co-factors in a diethanolamine

buffer. (2 x 11ml)

ELISA Substrate Reagent,


monophosphate and enzyme co-factors in a diethanolamine

buffer. (28ml)

6 ELISA Stop Solution,

containing sodium hydroxide

and a chelating agent in adiethanolamine buffer. (11ml)

WARNING CAUSTIC!

ELISA Stop Solution, containing

sodium hydroxide and a chelating

agent in a diethanolamine buffer.(22ml)

WARNING CAUSTIC!

ELISA Stop Solution, containing

sodium hydroxide and a chelating

agent in a diethanolamine buffer.(27.5ml)

WARNING CAUSTIC!

7 Wash Buffer Concentrate,containing phosphate buffer

with ProClin 0.63% v/v.

(50ml) - sufficient to make up1 litre of wash buffer.

Wash Buffer Concentrate,containing phosphate buffer with

ProClin 0.63% v/v. (100ml) -

sufficient to make up 2 litres ofwash buffer.

Wash Buffer Concentrate,containing phosphate buffer with

ProClin 0.63% v/v. (125ml) -

sufficient to make up 2.5 litres ofwash buffer

8 Sample Diluent Concentrate,containing phosphate buffer

with protein stabiliser and

ProClin 0.63% v/v. (50ml) -sufficient to make up 500ml of

sample diluent.

Sample Diluent Concentrate,containing phosphate buffer with

protein stabiliser and ProClin

0.63% v/v. (100ml) - sufficient tomake up 1 litre.

Sample Diluent Concentrate,containing phosphate buffer with

protein stabiliser and ProClin

0.63% v/v. (100ml) - sufficient tomake up 1 litre of sample diluent.


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VAT No: GB 902 7320 57

Materials and Equipment Required (Not Supplied):

In order to run the FLOCKSCREEN assays, the following equipment is recommended: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 Washer5. +37oC 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. It is also possible to use a

wash bottle for plate washing instead of an automated plate washer. The results will however, be lessconsistent. 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 andwashing are necessary to achieve good assay performance.

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

eliminates problems associated with varying room temperature conditions.

4. Plates are coated with purified inactivated viral antigens and control sera have been filtered with a0.2m 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 & sample

diluent.6. The Substrate Reagent is very sensitive and under no circumstances should the same pipette tips or

containers 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 indicatesdeterioration or contamination and the reagent should not be used.

7. Caution should be exercised in the handling of alkaline 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.


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Reagent Preparation

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

2 The Wash Buffer Concentrate and Sample Diluent Concentrate may partly recrystallise. This is due to thehigh 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

3 To prepare sample diluent

buffer, add the Sample Diluent

Concentrate (50ml) to distilledor deionised water and make up

to total volume of 500ml. This

sample diluent can be stored at+4C for up to 3 months and can

be used for preparing samplesfor any of theFLOCKSCREEN Kits.

To prepare sample diluent

buffer, add the Sample Diluent

Concentrate (100ml) to distilledor deionised water and make up

to total volume of 1 litre. This

sample diluent can be stored at+4C for up to 3 months and

can be used for preparingsamples for any of theFLOCKSCREEN Kits.

To prepare sample diluent buffer,

add the Sample Diluent

Concentrate (100ml) to distilledor deionised water and make up to

a total volume of 1 litre. This

sample diluent can be stored at+4oC for up to 3 months and can

be used for preparing samples forany of the FLOCKSCREENKits.

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 stableat room temperature for 3

months and can be used with

any of the FLOCKSCREENKits.

To prepare the wash buffer, add

the Wash Buffer Concentrate(100ml) to distilled or

deionised water and make up to

total volume of 2 litres. This isstable at room temperature for

3 months and can be used with

any of the FLOCKSCREENKits.

To prepare the wash buffer, add

the Wash Buffer Concentrate(125ml) to distilled or deionised

water and make up to a total

volume of 2.5 litres. This isstable 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.

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 ofsample diluent to achieve the 1:500 final dilution directly. Invert gently 2 or 3 times to mix.

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 andnegative 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 not available, mix by gently tapping the side of the plate. Cover the plate with

adhesive 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 andtap firmly on absorbent paper. N.B. To reduce the possibility of sample carryover, it is


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VAT No: GB 902 7320 57

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 tapping

the side of the plate.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 achievemaximum 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 is

pale 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 aMicrotitre 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

Generally, differentiation between negative and positive samples will be very clear.

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 CAV titre can be calculated using the following equation:

Log10 Titre = 1.24 x (Log10 S/P) + 3.70Titre = Antilog of Log10 Titre

The CAV S/P ratio values and/or ELISA titre values of the samples should be interpreted using the followingguide:


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S/P Ratio CAV Titre CAV

Antibody StatusLess than or equal to 0.10 0-289 Negative

Greater than 0.10 & less than or equal to 0.25 290-899 SuspectGreater than 0.25 900 and above 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 +4oC on delivery. Do not freezeAvoid exposure to sunlight.

Do not use after the stated expiry date.

Do not use if silica gel desiccant in the pouches 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

Documents

V080 V084 V085 FLOCKSCREEN CAV Instructions for Use _v2__000