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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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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
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
phosphate buffer with protein
stabiliser and ProClin 0.063%v/v. (2 x 500l ready to use).
Negative Control with SPFchicken serum preserved in
phosphate buffer with protein
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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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
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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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
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
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 usedfor preparing samples for any ofthe FLOCKSCREEN Kits.
To prepare sample diluent
buffer, add the Sample DiluentConcentrate (100ml) to distilled
or deionised water and make up
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
the Wash Buffer Concentrate
(100ml) to distilled or deionisedwater and make up to total
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
the Wash Buffer Concentrate
(125ml) to distilled or deionisedwater and make up to total
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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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 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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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
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