v130 v134 v135 flockscreen ibv instructions for use _v3__000

7/29/2019 V130 V134 V135 FLOCKSCREEN IBV Instructions for Use _v3__000

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

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

X-OVO FLOCKSCREEN

Infectious Bronchitis Virus (IBV)Antibody ELISA Kit

Cat. No. V130 (2 Plates), V134 (4 Plates) & V135 (5 Plates)

Instructions for use (V2)

Introduction

Infectious Bronchitis (IB) is a common respiratory disease of all types of poultry and frequently forms part of a

respiratory disease complex. There are a number of variants that cause disease, even in vaccinated birds.

Where post vaccination titres have been monitored and an infection due to an IB variant is suspected, it should

be possible to detect a significant secondary rise in antibody titre.

When to Test

The FLOCKSCREEN Infectious Bronchitis 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. Theantibody 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.

It is recommended that flocks should not be tested for at least three weeks after immunisation with

oiladjuvinated vaccines as these are often known to cause short term interference with serological tests.

Sampling Recommendations

As a guide, a 1% sample is usually sufficient for vaccination or disease monitoring. In practice, for IBV, about

18-20 birds per house would normally be tested.


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x-OvO Limited, Unit 1, Burnside Business Court, Inverkeithing,

KY11 1NZ, United Kingdom


Assay Description

The FLOCKSCREEN IBV Antibody ELISA kit provides a rapid, simple and sensitive method of detecting

antibodies to IBV in serum or egg yolk.

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

wells where any antibody specific to IBV 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 tothe chicken antibodies attached to IBV 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

Kit Contents

2 Plate Kit 4 Plate Kit 5 Plate Kit

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

well holders each containing 12

x 8-well strips). In a re-sealablefoil pouch with silica gel.

4 x 96 well plates pre-coated withIBV antigen (supplied as 4 well

holders each containing 12 x 8-

well strips). In a re-sealable foilpouch with silica gel.

5 x 96 well plates pre-coatedwith IBV antigen (supplied as 5

well holders each containing 12

x 8-well strips). In a re-sealablefoil pouch with silica gel.

2 Positive Control with antibodies

to IBV preserved in phosphatebuffer with protein stabiliser

and ProClin 0.063% v/v. (500l

ready to use).

Positive Control with antibodies

to IBV preserved in phosphatebuffer with protein stabiliser and

ProClin 0.063% v/v.. (2 x 500l

ready to use).

Positive Control with

antibodies to IBV preserved inphosphate buffer with protein

stabiliser and ProClin 0.063%

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

3 Negative Control with SPF Negative Control with SPF Negative Control with SPF

Add Sample/

Controls

Add Enzyme

Con u ate

Add Substrate

Reagent

Add Stop Sol.


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chicken serum preserved in

phosphate buffer with proteinstabiliser and ProClin 0.063%

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



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



v/v. (2 x 500l ready to use).4 Enzyme Conjugate Reagent,

containing alkaline phosphataselabelled rabbit anti-chicken IgG

in tris buffer with an inert blue

dye and sodium azide 0.1% w/v.(11ml)

Enzyme Conjugate Reagent,



dye and Sodium Azide 0.1% v/v.(2 x 11ml)

Enzyme Conjugate Reagent,



dye and Sodium Azide 0.1%v/v. (2 x 14ml)

5 ELISA Substrate Reagent,

containing phenolphthaleinmonophosphate and enzyme co-

factors in a diethanolamine

buffer. (11ml)

ELISA Substrate Reagent,

containing phenolphthaleinmonophosphate and enzyme co-

factors in a diethanolamine

buffer. (2 x 11ml)

ELISA Substrate Reagent,

containing phenolphthaleinmonophosphate and enzyme

co-factors in a diethanolamine

buffer. (2 x 14ml)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,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 up 1 litre ofwash 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 up2.5 litres of wash 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.

Sample Diluent Concentrate,

containing phosphate bufferwith protein stabiliser and

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

sufficient to make up 1 litre.

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

2.5ml (or variable 1-5ml)

2. Disposable tips for pipettes3. Microtitre Plate Reader with 550nm filter

4. Microtitre Plate Washer

5. 37C incubator6. Distilled or deionised water

7. Disposable 5ml plastic tubes


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It is possible to run the assays without the 50l repeater, or an 8 channel pipette and to use a wash bottle forplate 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. Thiseliminates problems associated with varying room temperature conditions.

4. Plates are coated with purified 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.

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 be 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 alkali or other hazardous chemicals in accordancewith 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 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 Kit3 To prepare sample diluent

buffer, add the Sample Diluent

Concentrate (50ml) to distilled

or deionised water and make upto total volume of 500ml. This

sample diluent can be stored at

+4C for up to 3 months and canbe used for preparing samples

To prepare sample diluentbuffer, add the Sample Diluent


or deionised water and make upto total volume of 1 litre. This


+4C for up to 3 months andcan be used for preparing

To prepare sample diluent buffer,add the Sample Diluent


or deionised water and make up tototal volume of 1 litre. This


+4C for up to 3 months and canbe used for preparing samples for


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for any of the

FLOCKSCREEN Kits.samples for any of the

FLOCKSCREEN Kits.

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 totalvolume of 1 litre. This is stable

at room temperature for 3

months and can be used withany of the FLOCKSCREEN

Kits.

To prepare the wash buffer, add

the Wash Buffer Concentrate(100ml) to distilled or

deionised water and make up tototal volume of 2 litres. This is

stable at room temperature for

3 months and can be used withany of the FLOCKSCREEN

Kits.

To prepare the wash buffer, add

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

water and make up to totalvolume of 2.5 litres. This is

stable at room temperature for 3

months and can be used with anyof 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 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.

Diluted samples can be kept for several days at +4C for retesting 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 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

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.


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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 readimmediately 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 controlabsorbance 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 areall in good condition and that there have been no operator errors.

Interpretation of Results

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 titre can be calculated using the following equation:

Log10 Titre = 0.650 x (Log10 S/P) + 3.691

Titre = Antilog of Log10 Titre

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

values:

S/P Ratio IBV Titre IBV Antibody Status

Less than or equal to 0.102 0-1113 Non-significant

Greater than 0.102 and less than 0.166 1114-1525 Marginal positiveGreater than or equal to 0.166 1526 or greater Positive

Results where S/P ratios are 0.102 or below (titres of 1113 or lower) indicate non-significant antibody levels to

Infectious Bronchitis. S/P ratios between 0.102 and 0.166 (titres of 1114-1525) may be regarded as marginal.Protective levels must be established for each set of circumstances (vaccine used, type of flock etc.) and on a

flock basis, looking at pre- as well as post-vaccination titres.

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.


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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

v130 v134 v135 flockscreen ibv instructions for use _v3__000

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