Thermo Fisher Scientific BOVIGAM Information Manual

Catalog Number 63320, 63326

Pub. No. MAN0019490 Rev. A.0

Registered by the Spanish Ministry of Agriculture, Fisheries, and Food (MAPA) No. 0531-RD

WARNING!

gloves. Safety Data Sheets (SDSs) are available from

WARNING! POTENTIAL BIOHAZARD.

Ten (10) Microplate Tes t Kit

(150 Maximum test samples)

Thirty (30) Microplate Test Kit

(450 Maximum test samples)

Contains 0.01% w/v thimerosal.

deionized or distilled water.

Contains 0.01% w/v thimerosal.

deionized or distilled water.

Contains 0.01% w/v thimerosal.

deionized or distilled water.

7A: Blue Diluent (Conjugate diluent buffer – 5x Concentrate)

Contains 0.01% w/v thimerosal. Dilute with deionized or distilled water.

Contains 0.01% w/v thimerosal. 8: Enzyme Substrate Buffer

1 × 125 mL

2 × 175 mL

Contains H2O2. Ready for use.

Contains TMB in DMSO. Dilute in Enzyme Substrate Buffer.

10: Enzyme Stopping Solution (0.5M H2SO4)

1 × 75 mL

1 × 175 mL

Ready for use.

Use

Description

(1)

Blood collection

• Lithium heparin Vacutainer

tubes: 1 per animal

• Needle holders: 2–3 per blood collector

Blood culture

• Sterile, graduated 5–10 mL pipettes: 1 per animal

Cat. No. 7600065): 10X concentrate, 10 µL per animal

Plasma

• Single-use tips to fit 100–1000 µL pipette: 3 per animal

plasma storage): 1 rack per 30 animals

Bovine IFN-γ EIA

• Tips to fit 12-channel pipette: 3 per animal

General

• 37°C humidified incubator (5% CO

optional)

INSTRU C TION S FOR U S E

BOVIGAM™ TB Kit

Enzyme-linked immunosorbent assay for the detection of bovine gamma interferon in plasma samples

appropriate protective eyewear, clothing, and gloves.

Contents and storage

Store the Applied Biosystems™ BOVIGAM™ TB Kit at 2–8°C. Bring all reagents (except the Conjugate – 100x Concentrate) to room temperature (22±3°C) before use, then return to 2–8°C immediately after use.

Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and

thermofisher.com/support.

Read the biological hazard safety information at this product’s page at thermofisher.com. Wear

Component

1: Microplates coated with bovine IFN-γ antibodies 10 × 96 well plates with lids 30 × 96 well plates with lids Ready for use.

2: Positive Bovine IFN-γ Control 2 × 1 mL 3 × 2 mL

3: Negative Bovine IFN-γ Control 2 × 1 mL 3 × 2 mL

4: Green Diluent (Plasma diluent buffer) 1 × 60 mL 1 × 175 mL

5: Wash Buffer – 20x Concentrate 3 × 125 mL 2 × 500 mL

6: Conjugate – 100x Concentrate. (Horseradish peroxidase-labele d anti-bovine IFN-γ)

7B: Blue Diluent (Conjugate diluent buffer) - 2 × 175 mL

9: Chromogen Solution – 100x Concentrate 1 × 1.5 mL 2 × 2 mL

Required materials not supplied

(Cat. No. 63320)

1 × 1.5 mL 2 × 2 mL

1 × 25 mL -

(Cat. No. 63326)

Procedural guidelines

• National Safety Regulations must be strictly followed.

• Equ ilibrate all plas ma test samp les and reagents, except the

Conjugate - 100x Concentrate, to room temperature (22±3°C) before use. Do not warm above 37°C.

• 18G Vacutainer

™

needles — 1 inch: 1 per animal

™

Note: Some reagents can require several hours to equilibrate. If a

• Sterile, 24-well tissue culture plates: 1/8 per animal

• Tips for PPD dispenser (5 mL): 3 per herd

• Sterile phosphate buffered saline (PBS): 100 µL per

animal (0.01M, pH 7.2)

• Bovine Tuberculin PPD 3000 (0.3 mg/mL;

(Cat. No. 7600060): 10X concentrate, 10 µL per animal

• Avian Tuberculin PPD 2500 (0.3 mg/mL;

shorter equilibration t ime is required, an ambient-temperature water bath can be used.

• Store all kit components at 2-8°C, then return to 2-8°C immediate ly

after use. The working strength wash buffer can be stored at room temperature (22±3°C) for up to 2 weeks.

• Keep the Conjugate - 100x Concentrate at 2-8°C always, even during

reconstitut ion.

• Complete reconstitution of the lyo philized components is essential to

ensure valid assay results.

• Use high quality deionize d or distilled water to reconstitute and dilute

harvesting

• 1-mL microtubes and caps in 96-well storage rack (for

the reagents.

• Once the assay is started, complete the assay without interruption.

• Use a separate disposable tip for each sample to prevent

• Various polypropylene tubes, EIA reagent reservoirs,

and pipette tips

• Calibrated micropipettes (to deliver up to 1 mL)

• Graduated 1, 5, and 10 mL pipettes

• Measuring cylinders - 100 mL, 1 L, and 2 L

• Deionized or distilled water - 6 L

• 12-channel pipette (to deliver 50 µL and 100 µL)

• Plate stirrer (optional)

(1)

Unless otherwise indicated, all materials are available through thermofisher.com.

• Microplate washer (optional)

• Microplate reader with a 450 nm and 620-650 nm filter

cross-contamination.

• Plas ma test samples from individual animals should be adde d to the

EIA wells at the same time. We recommend using a 12-channel pipette.

• Elevate the EIA test plate on an inverted test-tube rack (or similar) to

minimize intraplate variations. Do not incubate the test plate directly on the bench.

• Assay all plasma test sample s in duplicate in adjacent wells.

• Assay the Positive and Negat ive Bo vine IFN-γ Controls in triplicate in

serial wells of Columns 4, 5, and 6 (for example, row G for positive and row C for negative controls).

Description

Lyophilized. Reconstitute with

Ready for use.

Dilute with deionized or distilled water.

Lyophilized. Reconstitute with

Ready for use.

For Veterinary Use Only. For In Vitro Use Only.

Test procedure

Table 1

Animal 1 NIL

AvPPD

BoPPD

NIL

AvPPD

BoPPD

Animal

Animal 3 NIL

AvPPD

BoPPD

NIL

AvPPD

BoPPD

Animal

Animal 5 NIL

AvPPD

BoPPD

NIL

AvPPD

BoPPD

Animal

Animal 7 NIL

AvPPD

BoPPD

NIL

AvPPD

BoPPD

Animal

Table 2

Row 1 2 3 4 5 6 7 8 9 10

11 12 A 1N

4B B 5N

8B C 9N

9B X X X 10N

10A

10B

11N

11A

11B D 12N

12A

12B

13N

13A

13B

14N

14A

14B

15N

15A

15B E 16N

16A

16B

17N

17A

17B

18N

18A

18B

19N

19A

19B F 20N

20A

20B

21N

21A

21B

22N

22A

22B

23N

23A

23B G 24N

24A

24B X X X 25N

25A

25B

26N

26A

26B H 27N

27A

27B

28N

28A

28B

29N

29A

29B

30N

30A

30B

Stage one – Aliquot the blood samples, then incubate with the stimulating antigens

1. Dispense the blood

Thoroughly mix each blood sample before aliquoting. Note: It is important to keep cell damage to an absolute minimum as the test requires viable lymphocytes.

Transfer three, 1.5-mL aliquots of each blood sample to separate wells of a 24-well tissue-cu lture plate (see Table 1 for the recommended layout). Perform this procedure under aseptic conditions, using either sterile sing le-use pipettes, or sterile disposable pipettes with an automatic pipette filler.

Recommended layout for dispensing blood and antigens into a

24-well tissue-culture plate

NIL = Nil antigen control (PBS); AvPPD = Avian PPD; BoPPD = Bovine PPD

2. Add the stimulating antigens

The antigens used must be free of bacterial endotoxins (lipopo lysaccharides). Before use, dilute the antigens 1:10 with sterile PBS (for example, add 10 µL PPD to 90 µL of PBS). Using aseptic techniques, add 100 µL of either PBS (nil antigen control), avian PPD, or bovine PPD to the appropriate 3 wells containing bloo d. We recommend using a repeater pipette, such as the Eppendorf Combitip system, fitted with sterile 5-mL tips. Thoroughly mix the samples with the antigens. We recommend using a plate stirrer that is optimized for mix ing blood. Alternatively, if a plate stirrer is not available, manually swirl each plate ten times in a clockw ise motion, then repeat in a counter-clockwise motion.

IMPORTANT! For optimal assay performance, ensure that the samples are thoroughly mixed with the antigens. It is not possible to over mix.

3. Inc ubate the blood with the antigens

Incubate the plate, containing the blood and antigens, at 37°C in a humidifie d atmo sphere for 16–24 hours. An environment with 5% CO is optional.

4. Collect the plasma, then store the samples (optional)

(Optional) Centrifuge the 24-well tissue-culture plate at 500 × g at room temperature (22±3°C) for 10 minutes. After the incubation, carefully remove approximately 500 µL of plasma from each well. We recommend transferring the collected plasma to 1-mL microtubes that can be stored in a 96-well storage rack. Use a new pipette tip for each plasma sample. See Table 2 for the recommended plasma storage layout.

Note: It is important to minimize harvesting any cell ular material along with the plasma. However, contamination of the plasma with a small amount of erythrocytes will not affect the EIA results. Similarly, slight hemoly sis o f blood samples has little effect on the EIA.

The following storage layout allows for efficient transfer of samples from the storage rack to the test plate, using a 12-channel pipette. After the samples are transferred to the test plate, use the empty wells for the controls that are supplied with the kit.

Assay all test samples in duplicate. One full storage rack requires two EIA test plates.

Recommended plasma storage layout

N = Nil antigen control (PBS); A = Avian PPD; B = Bovine PPD; X = Empty

™

Stage two – Perform th e bovine IFN-γ EIA

Prepare the reagents

1. Plates

Equilibrate the plastic pouch containing the plate(s) to room temperature before unsealing. Allow at least 30 minutes.

2. Positive and negative controls

Reconstitute the appropriate vials with 1 mL (10 -plate kit) or 2 mL (30-plate kit) of disti lled wa ter. Ensure complete resolubilization. Store the reconstituted controls at 2-8°C for up to 3 months.

3. Green Dilue nt

Equilibrate the Green Diluent to room temperature, then mix thoroughly before use.

4. Conjugate – 100x Concentrate

IMPORTANT! Keep the Conjugate - 100x Concentrate at 2–8°C always, even during reconstitution.

Reconstitute the lyophilized Conjugate - 100x Concentrate with 1.5 mL (10-plate kit) or 2 mL (30-plate kit) of distilled water. Ensure complete resolubilization. Avoid frothing. Store the reconstituted Conjuga te - 100x Concentrate at 2–8°C for up to 3 months.

5. Blue Diluent

- For the 10-plate kit (Cat. No. 63320): Equilibrate the Blue Diluent

(5x concentrate) to room temperature, then mix thoroughly. Prepare workin g strength Blue Diluent by mixing one part Blue Diluent (5x concentrate) with 4 parts of dist illed water. Store the working strength Blue Diluent at 2–8°C for up to 3 months. Before use, bring to room temperature, then mix thoroughly.

- For the 30-plate kit (Cat. No. 63326): The Blue Diluent is supplie d

pre-diluted and ready for use. Do not dilute.

6. Conjugate working solution

IMPORTANT! Prepare the conjugate working solution no longer than 5 minutes before use.

Prepare the conjugate working solution by diluting the reconstituted Conjugate – 100x Concentrate 1:100 in Blue Diluent. Use the conjugate workin g solution within 5 minutes of preparation, then immediately discar d any unused reagent. We recommend only preparing the quantity required for each assay: 110 µL of Conjugate - 100x Concentrate in 11 mL of Blue Diluent is su fficient for one plate.

7. Wash buffer

Prepare working strength wash buffer by mixing one part Wash Buffer 20x Concentrate with 19 parts of distilled water. Mix thoroughly.

Store the working strength wash buffer at room temperature for up to 2 weeks. Note: If th e Wash Buffer 20x Concentrate contains precipitated salts, warm the bottle in a 37°C water bath until the precipitates dissolve.

8. Enzyme substrate solution

IMPORTANT! Prepare the enzyme substrate solution im mediately before use.

Equilibrate the Enzyme Substrate Buffer and Chromogen Solution 100x Concentrate to room temperature, then mix each reagent thoroughly. Prepare the enzyme substrate solut ion just before use by combining the appropriate volumes of Chromogen Solution 100x Concentrate and Enzyme Substrate Buffer. Ensure the solution is thoroughly mixed and colorless. We recommend only preparing the quantity required for each assay: 110 µL of Enzyme Substrate Buffer in 11 mL of Chromogen Solution 100x Concentrate is sufficient for one plate. Use within 10 minutes of preparation.

Perform the bovine IFN-γ EIA

1. Equilibrate all kit components (except the Conjugate – 100x

Concentrate) to room temperature, then gently mix the reagents, samples, and controls.

2. Add 50 µL of Green Diluent to each well of the test plate.

3. Transfer 50 µL of each test sample and control to the appropriate wells

containing Green Diluent. Add the controls to the plate after the test samples. Carefully mix the contents of the wells using a microplate shaker. Alternatively, if a microplate shaker is not available, mix by pipetting up and down at least 5 times.

4. Cover the plate with a lid, then incubate for 60±5 minutes at room

temperature (22±5°C).

5. Empty the plate, then wash the wells at least 6 times at room

temperature. Carefully fill the wells with wash buffer to avoid cross-contamination with adjacent wells. Empty the plate, then repeat the process 5 more times. After the sixth wash, place the plate(s) face down on clean filter paper, then shake the plate repeatedly to remove all wash buffer from the wells.

Note: If available, an automatic microplate washer can be used.

6. Add 100 µL of the conjugate working solution to each well, then mix

thoroughly as described in step 2.

2 BOVIGAM™ TB Kit Instructions for Use

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