v170 v174 v175 flockscreen ai instruction for use v1

7/29/2019 v170 v174 v175 Flockscreen Ai Instruction 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

Avian Influenza Virus (AI)Antibody ELISA Kit

Cat.No. V170 (2 Plates), V174 (4 Plates) & V175 (5 Plates)

Instructions for use (V1)Introduction

Avian Influenza (AI) is a highly infectious and usually fatal disease of birds and is caused by type A influenza

virus. AI can be classified into low pathogenic (LPAI) and highly pathogenic (HPAI) forms depending on theseverity of the disease.

In poultry the first clinical symptoms may go unrecognised but the course of the disease can manifest itself

rapidly into depression, drop in egg production, coughing, sneezing, swollen heads. In some outbreaks birds

die rapidly without any clinical signs.

When to Test

The FLOCKSCREEN Avian Influenza 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

antibody response will usually be detectable within 7 days. Vaccine responses should take the form of a

normal distribution curve when vaccination has been effectively administered.

Sample Recommendation

As a guide, a 1% sample is usually sufficient for vaccination or disease monitoring. In practice 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 Avian Influenza Antibody ELISA kit provides a rapid, simple and sensitive method

of detecting antibodies to AI in chicken or turkey serum. Microtitre plates are supplied pre-coated withpurified, inactivated AI antigen. Diluted samples are incubated in the wells where any antibody specific to AIbinds and forms a complex. Unbound material is washed from the wells and alkaline phosphatase labelled

rabbit anti-chicken IgG conjugate reagent is added which binds to the chicken antibodies attached to the AI

antigen.

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 30 mins

& wash

Incubate 30 mins

& wash

Incubate 15 mins

Read at 550nm

Kit Contents

2 Plate Kit 4 Plate Kit 5 Plate Kit

1 2 x 96 well plates pre-coated

with inactivated AI antigen

(supplied as 2 well holderseach containing 12 x 8-well

strips). In a re-sealable foil

pouch with silica gel.

4 x 96 well plates pre-coated with

inactivated AI antigen (supplied

as 4 well holders each containing12 x 8-well strips). In a re-

sealable foil pouch with silica gel.

5 x 96 well plates pre-coated with

inactivated AI antigen (supplied

as 5 well holders each containing12 x 8-well strips). In a re-

sealable foil pouch with silica gel.

2 Positive Control with Positive Control with antibodies Positive Control with antibodies

Add Sample/

Controls

Add Enzyme

Conjugate

Add Substrate

Reagent

Add Stop Sol.


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VAT No: GB 902 7320 57

antibodies to AI preserved in

phosphate buffer with proteinstabiliser and ProClin 0.063%

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

to AI preserved in phosphate

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

ready to use).

to AI preserved in phosphate

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

ready to use).

3 Negative Control with SPFchicken serum preserved in

phosphate buffer with protein

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

Negative Control with SPFchicken serum preserved in


stabiliser and ProClin 0.063%v/v. (2 x 500l ready to use).

Negative Control with SPFchicken serum preserved in


stabiliser 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 an inert bluedye and sodium azide 0.1% w/v.

(2 x 11ml).

Enzyme Conjugate Reagent,containing alkaline phosphatase

labelled rabbit anti-chicken IgG

in tris buffer with an inert bluedye and sodium azide 0.1% w/v

(28ml).

5 ELISA Substrate Reagent,containing phenolphthalein

monophosphate and enzyme

co-factors in a diethanolaminebuffer. (11ml).

ELISA Substrate Reagent,containing phenolphthalein

monophosphate and enzyme co-

factors in a diethanolaminebuffer. (2 x 11ml).

ELISA Substrate Reagent,containing phenolphthalein

monophosphate and enzyme co-

factors in a diethanolamine buffer(28ml).

6 ELISA Stop Solution,containing sodium hydroxide

and a chelating agent in a

diethanolamine buffer. (11ml).WARNING CAUSTIC!

ELISA Stop Solution, containingsodium hydroxide and a chelating

agent in a diethanolamine buffer.

(22ml)WARNING CAUSTIC!

ELISA Stop Solution, containingsodium 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 up one litreof wash buffer

Wash Buffer Concentrate,containing phosphate buffer with

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

sufficient to make up two 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 bufferwith protein stabiliser and

ProClin 0.63% v/v. (50ml) -

sufficient to make up 500ml ofsample diluent.

Sample Diluent Concentrate,

containing phosphate buffer withprotein stabiliser and ProClin

0.63% v/v. (100ml) - sufficient to

make up 1 litre of sample diluent.

Sample Diluent Concentrate,

containing phosphate buffer withprotein stabiliser and ProClin

0.63% v/v. (100ml) - sufficient to

make 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 a plate washer. The results will however be less consistent. Notepipettes 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 washingare 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 bacterial antigens and control sera have been filteredwith 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.

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

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 Reagentshould 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.

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 oxidisingagents before disposal.


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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 contain crystals. This is due to the

high concentration of salts. Shake the bottle prior to reconstituting. The crystals will dissolve uponmixing.

2 Plate Kit 4 Plate Kit 5 Plate Kit

3 To prepare sample diluent

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 andcan be used for preparingsamples for any of the

FLOCKSCREEN Kits.

To prepare sample diluent buffer,

add the Sample DiluentConcentrate (100ml) to distilled


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

up to 3 months and can be usedfor preparing samples for any ofthe FLOCKSCREEN Kits.

To prepare sample diluent

buffer, add the Sample DiluentConcentrate (100ml) to distilled


to total volume of 1 litre.Sample Diluent can be stored at

+4C for up to 3 months andcan be used for preparingsamples for any of the

FLOCKSCREEN Kits.

4 To prepare the wash buffer, add

the Wash Buffer Concentrate

(50ml) to distilled or deionisedwater and make up to total

volume of 1 litre. This is stable

at room temperature for 3months and can be used with

any of the FLOCKSCREEN

Kits.

To prepare the wash buffer, add



volume of 2 litres. This is stable

at room temperature for 3 monthsand can be used with any of the

FLOCKSCREEN Kits.

To prepare the wash buffer, add



volume of 2.5 litres. This is

stable at room temperature for 3months 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 practicable and stored at +4oC (up to 2 days) or at -20oC for longer 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 2-step dilution protocol using deepwell multiplates or dilution plates may befollowed using a minimum of 5l of sample.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). Diluted samples can be kept for several days at +4oC for retesting or at -20oC for longer term storage.


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VAT No: GB 902 7320 57

Assay Procedure

1. Remove the pre-coated plates from their sealed bags and record sample and control locations on a12 x 8 template sheet. Each sample should be run in duplicate for optimum results. The positive andnegative controls should always be run in duplicate.

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

should be retained at +4oC until successful results are confirmed. Cover the plate with an adhesivecover and incubate at +37oC for 30 minutes. Mix on a plate shaker or by gently tapping the side of

the plate.

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

recommended where possible, that the plate 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 the adhesive cover and incubate at +37oC for 30 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 ELISA Substrate Reagent to each well. The reagent must be at room temperature toachieve maximum colour development. Mix on a plate shaker or by gently tapping the side of the

plate.

8. Cover the plate with the adhesive cover and incubate at +37oC for 15 minutes. Colourdevelopment is pale pink, which deepens on addition of ELISA Stop Solution.

9. Remove adhesive cover and add 50l ELISA 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 having first blanked on air. In order to obtain optimum resultsthe plate should be read immediately after adding the ELISA Stop Solution.

Results

For the test to be valid:

a) Mean Negative control absorbance must be < 0.2b) Mean Positive control absorbance must be at least 5 times the Mean Negative Absorbance.

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):


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SAMPLE ABSORBANCE - NEGATIVE CONTROL ABSORBANCE = S/P

POSITIVE CONTROL ABSORBANCE - NEGATIVE CONTROL ABSORBANCEAn AI titre can be calculated using the following equation:

Log10 Titre = 0.604 x (Log10 S/P) + 4.169Titre = Antilog of Log10 Titre

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

S/P Ratio AI Antibody StatusLess than or equal to 0.249 Negative

Greater than 0.250 and less than 0.399 Suspect

Greater or equal to than 0.4 Positive

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

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 plate 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

v170 v174 v175 flockscreen ai instruction for use v1

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