INSTRUCTIONS FOR USE

Primagam™ Non‑Human Primate M. tuberculosis
Interferon‑Gamma Kit
ELISA for detection of tuberculosis in non‑human primates
Catalog Number 63311
Pub. No. MAN0018635 Rev. A.0

Technology

Species

Sample type

ELISA

Non‑human primates

Whole blood

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clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.

Product description™

™

The Applied Biosystems Primagam Non-Human Primate M. tuberculosis Interferon-Gamma Kit is an ELISA-based diagnostic test thatmeasures interferon-γ (IFN-γ) in whole blood samples to detect tuberculosis (TB). Release of IFN-γ in response to stimulation withtuberculin purified protein derivative (PPD) antigens is a highly predictive biomarker of TB infection. The kit detects IFN-γ in thefollowing species: Aotus (night/owl monkey), Ateles (spider monkey), Colobus (guerezas), gibbon/siamang, guenon/De Brazza’s, langur,
lemur, macaque, mandrill, marmoset, squirrel monkey, tamarin, and vervet.
Tuberculosis is a progressive disease that affects the respiratory systems of primates. It can be transmitted from non-human primates totheir caretakers in zoos and research laboratories, which presents a public health hazard. The Intradermal Eyelid Skin Test is the traditional
method that is used to test for TB in non-human primates. This test requires immobilization and detects TB based on inflammation and™
swelling at the site of injection. The Primagam Non-Human Primate M. tuberculosis Interferon-Gamma Kit can be used for the detectionof TB in addition to or as an alternative to the Intradermal Eyelid Skin Test.
The kit follows a two-day laboratory procedure:• Day 1—Whole blood samples are collected in heparin tubes, then aliquots of each sample are incubated with a single antigen: Bovine
PPD, Avian PPD, or Nil Antigen Control.
• Day 2—IFN-γ that is produced by antigen-stimulated lymphocytes is measured using ELISA technology.
The kit was evaluated during a tuberculosis outbreak in 54 cynomolgus (Macaca fascicularis) and 22 rhesus (Macaca mulatta) macaquemonkeys. The results of the kit revealed 92% sensitivity with 100% specificity. An extra evaluation in a separate colony, presumed free of
TB based on history, and a regular skin testing regime, also resulted in 100% specificity.

For Veterinary Use Only. For In Vitro Use Only.

Contents and storage
Reagents and plates for 30 tests are supplied.
Note: For reconstitution and dilution instructions, see “Before you begin“ on page 3.
Table 1 Primagam™ Non‑Human Primate M. tuberculosis Interferon‑Gamma Kit (Cat. No. 63311)
Contents

Description

Storage [1]AmountMicroplate Strip[2]Ready‑to‑use.

Bovine PPD

Dilute before use.

5 mL

Avian PPD

Dilute before use.

5 mL

2 × 96‑well microplates• Ready‑to‑use.
Nil Antigen Control• Contains 0.01% weight by volume(w/v) thimerosal.
• Reconstitute before use.

Positive Primate IFN-γ Control• Contains 0.01% w/v thimerosal.
• Reconstitute before use.

Negative Primate IFN-γ Control• Contains 0.01% w/v thimerosal.
• Ready‑to‑use.

Green Diluent (sample and Conjugate diluent buffer)• Contains 0.01% w/v thimerosal.
• Reconstitute before use.

Conjugate‑100x Concentrate (horseradish
peroxidase‑labeled anti‑primate IFN‑γ antibody)• Contains 0.01% w/v thimerosal.
• Dilute before use.

Wash Buffer‑20x Concentrate• Contains 0.01% w/v thimerosal.
• Ready‑to‑use.

Enzyme Substrate Buffer• Contains H2O2.
• Dilute before use.

Chromogen Solution‑100x Concentrate• Contains TMB in DMSO.
• Ready‑to‑use.

Enzyme Stopping Solution• Contains 0.5M H2SO4.

5 mL

1 vial of lyophilized solids
1 vial of lyophilized solids
2–8°C

40 mL
1 vial of lyophilized solids
2 × 50 mL
30 mL
0.5 mL
15 mL[1] See label for expiration date.

[2] Wells are coated with antibodies directed against primate IFN‑γ.

Required materials not supplied
Unless otherwise indicated, all materials are available through thermofisher.com.
Item

Source[1]Instruments
Multiskan™ FC Microplate Photometer, or equivalent plate reader[2]51119000

Wellwash Microplate Washer, or equivalent

5165000™

(Optional) Microplate shaker

MLS(Optional) Tube rocker

MLS

2

Primagam™ Non‑Human Primate M. tuberculosis Interferon‑Gamma Kit Instructions for UseSource[1]ItemEquipment
Biosafety cabinet (BSC, level II)

MLSSingle channel pipette (10–1000 µL)

MLSMultichannel pipette (50–500 µL)

MLS

Laboratory mixer (vortex or equivalent)

MLS

Needle holders, 2–3 per collector

MLS

Humidified incubator set to 37°C

MLS

Measuring cylinders (100 mL, 1 L, and 2 L)

MLS

Tubes, plates, and other consumables
Lithium heparin blood‑collection tubes, 1 tube per animal

MLS

23G–18G 1.5‑inch Vacutainer needles, 1 needle per animal

MLS

Sterile, graduated 1‑ or 5‑mL pipettes

MLS

24‑well tissue‑culture plate, 1 plate per 8 animals

MLS

1‑mL microtubes in 96‑well format with rack and caps, 1 rack per 30 animals

MLS

Polypropylene tubes

MLS

Pipette tips (as recommended by pipette manufacturer)

thermofisher.com/pipettetips

Solution reservoirs

MLS

Reagents
Deionized or distilled water (2 L)

MLS

RPMI medium or PBS (1X), pH 7.4

MLS[1] MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.

[2] Plate reader must be capable of measurement at 450 nm with a reference filter at 620–650 nm.

Procedural guidelines• Strictly follow National Safety Regulations.
• Perform the kit protocol in laboratories that are suited for its purpose.
• Consider samples as potentially infectious and all items that contact the samples as potentially contaminated.
• Handle all primate blood samples in a biosafety cabinet (BSC, level II).
• Assay all samples and controls in duplicate.
• Dispose of containers and unused reagents in accordance with local biomedical waste requirements.
• Change pipette tips after every pipetting step.
• Maintain separate solution reservoirs for each reagent.
• Do not use kit components after their expiry date or if you observe changes in their appearance.
• Do not use kit components that have different kit lot numbers.
• Use deionized or distilled water, or water of equivalent quality.
• Use a microplate washer for all washing steps.

Before you begin
Determine the maximum plate shaker setting
If a plate shaker is used, determine the maximum setting.
1. Verify that the plate fits securely on your shaker.
2. Add 1.6 mL of water to each well of the plate, then cover with sealing foil.
3. Determine the maximum setting that you can use on your shaker without any of the water splashing onto the sealing foil.
Primagam™ Non‑Human Primate M. tuberculosis Interferon‑Gamma Kit Instructions for Use

3

Dilute the antigens
1. Add 160 µL of Bovine PPD to 840 µL of RPMI medium or PBS for a final assay concentration of 300 IU/mL.
2. Add 160 µL of Avian PPD to 840 µL of RPMI medium or PBS for a final assay concentration of 250 IU/mL.
Store the diluted antigens at 2–8°C for up to 1 month.

Reconstitute the controls
1. Reconstitute Positive Primate IFN-γ Control and Negative Primate IFN-γ Control in 1.0 mL of deionized or distilled water.
2. Allow the contents to sit for at least 15 minutes until dissolved.
3. Gently invert each tube 4–5 times to mix.
Store the reconstituted controls at 2–8°C for up to 3 months.

Reconstitute Conjugate‑100x Concentrate
IMPORTANT! Keep Conjugate-100x Concentrate at 2–8°C always, even during reconstitution.
1. Reconstitute Conjugate-100x Concentrate in 0.5 mL deionized or distilled water.
2. Allow the contents to sit for at least 15 minutes until dissolved.
3. Gently invert the tube 4–5 times to mix, then keep at 2–8°C until use.
Store the reconstituted Conjugate-100x Concentrate at 2–8°C for up to 3 months.

Prepare Wash Buffer‑1x
Note: One 96-well microplate requires 1 L of Wash Buffer‑1x.
1. (Optional) If Wash Buffer‑20x Concentrate shows a precipitate, warm the bottle in a 37°C water bath until the precipitate is dissolved.
2. Mix Wash Buffer‑20x Concentrate thoroughly.
3. Combine 1 part Wash Buffer‑20x Concentrate with 19 parts deionized or distilled water.
For example, add 50 mL of Wash Buffer‑20x Concentrate to 950 mL of deionized or distilled water.
4. Mix until a clear solution is obtained.
Store Wash Buffer‑1x at room temperature (22±3°C) for up to 2 weeks, or at 2–8°C for up to 1 month.

Day 1—Collect whole blood samples, then incubate with the antigens
Collect whole blood samples
1. Collect at least 5 mL of blood from each animal in heparin tubes, then transfer the samples to the laboratory at room temperature.
2. Culture the blood samples within 12 hours of collection to maintain lymphocyte viability (next section).

Aliquot the blood samples, then incubate with the antigens
1. Mix each blood sample to ensure that the heparin is dissolved, according to your mixing method.
• Using a roller shaker—Rock gently for 1–2 minutes.
• By inverting—Gently invert each tube 10 times.
2. Transfer three, 1.5-mL aliquots of each blood sample to separate wells of a 24-well tissue-culture plate (see Table 2).

4

Primagam™ Non‑Human Primate M. tuberculosis Interferon‑Gamma Kit Instructions for Use

3. Add 100 µL of Nil Antigen Control (N), diluted Bovine PPD (B), or diluted Avian PPD (A) to the appropriate wells containing blood,according to the following table.
Table 2 Recommended Sample Plate layout
1

2

3

4

5

6

A

Animal 1
N

Animal 1
B

Animal 1
A

Animal 2
N

Animal 2
B

Animal 2
A

B

Animal 3
N

Animal 3
B

Animal 3
A

Animal 4
N

Animal 4
B

Animal 4
A

C

Animal 5
N

Animal 5
B

Animal 5
A

Animal 6
N

Animal 6
B

Animal 6
A

D

Animal 7
N

Animal 7
B

Animal 7
A

Animal 8
N

Animal 8
B

Animal 8
A

4. Using a plate shaker, thoroughly mix the samples with the antigens for 1 minute (see “Determine the maximum plate shaker
setting“ on page 3).
Alternatively, if a plate shaker is not available, swirl the tissue-culture plate to mix. Hold the lid and plate firmly together, swirl 10
times in a clockwise 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 toover mix.

5. Transfer the tissue-culture plate to a humidified incubator set to 37°C, then incubate for 16–20 hours.

Day 2—Harvest plasma, then perform the ELISA
Before you begin
Equilibrate all kit components (except Conjugate-100x Concentrate) to room temperature, then gently mix reagents, samples, and controls.
Note: Some reagents can require several hours to equilibrate. If a shorter equilibration time is required, warm the reagents in a 30°C waterbath.

Harvest plasma, then store samples (optional)
1. Remove the tissue-culture plate from the incubator.
2. (Optional) Centrifuge the plate at 500 × g at room temperature for 5 minutes.
3. Avoiding the bottom layer of red blood cells, carefully pipette to transfer 150–300 µL of plasma to a clean 1-mL microtube in a 96-wellstorage rack (see Table 3).
Note: It is important to avoid harvesting red blood cells with the plasma during this step. However, a few red blood cells will notaffect the assay.
The following storage layout allows for efficient transfer of samples from the storage rack to the microplate strips, using a
multichannel pipette.
Table 3 Recommended plasma storage layout
1

2

3

4

5

6

7

8

9

10

11

12

A

1N[1]

1B

1A

2N

2B

2A

3N

3B

3A

4N

4B

4A

B

5N

5B

5A

6N

6B

6A

7N

7B

7A

8N

8B

8A

C

9N

9B

9A

X

X

X

10N

10B

10A

11N

11B

11A

D

12N

12B

12A

13N

13B

13A

14N

14B

14A

15N

15B

15A

E

16N

16B

16A

17N

17B

17A

18N

18B

18A

19N

19B

19A

F

20N

20B

20A

21N

21B

21A

22N

22B

22A

23N

23B

23A

G

24N

24B

24A

X

X

X

25N

25B

25A

26N

26B

26A

H

27N

27B

27A

28N

28B

28A

29N

29B

29A

30N

30B

30A

[1] Each well indicates the animal number followed by the antigen (N—Nil Antigen Control, B—Bovine PPD, A—Avian PPD, X—empty).

Primagam™ Non‑Human Primate M. tuberculosis Interferon‑Gamma Kit Instructions for Use

5Note: After the samples are transferred to the microplate strips, use the empty wells (X) for the controls that are supplied with the kit.
4. (Optional) Store plasma samples in sealed microtubes at 2–8°C for up to 28 days, or at −20°C for up to 5 years.
Ensure that sample racks are dated and include the initials of the operator, tube contents, and animal information.

Perform the ELISA• Equilibrate all kit components (except Conjugate-100x Concentrate) to room temperature, then gently mix reagents, samples, andcontrols.
• Determine the number of 8-well microplate strips that are required for the assay, then place the strips in the microplate frames. Return
any unused strips and frames to the bag, then store at 2°C to 8°C for future use.
= Anti-IFN-γ antibody |1= IFN-γ |Incubate the samples with
Green Diluent= Conjugate |= Substratea. Add 50 µL of Green Diluent to each well of the assembled microplate.
b. Transfer 50 µL of each plasma sample to the appropriate well containing Green Diluent.
The plasma samples for each animal must be added to the wells at the same time. We recommend
using a multichannel pipette.
Note: Plasma samples can clot when thawed. Pipette carefully to ensure that the correct volumeof plasma is added to each well.
c. Add 50 µL of each control to the appropriate well containing Green Diluent.
d. Mix the samples and controls with Green Diluent using a plate shaker set at moderate-high speed
for 1 minute.
Alternatively, if a plate shaker is not available, pipet up and down 5 times to mix.
e. Cover the plate with a lid, then incubate for 60 minutes at room temperature.

2

Wash the wells

a. Wash the wells of the microplate 6 times with 400 µL of Wash Buffer‑1x, allowing the wells tosoak for 10 seconds between each wash.
Note: Carefully fill the wells with Wash Buffer‑1x to avoid cross-contamination with adjacentwells.
b. Tap the plate over a paper towel to remove all Wash Buffer‑1x from the wells.

3

Dilute Conjugate‑100x
Concentrate

IMPORTANT! Prepare Conjugate-1x no longer than 15 minutes before use.
a. For each 8-well microplate strip, combine 10 µL of Conjugate-100x Concentrate with 1 mL ofGreen Diluent.
b. Mix the Conjugate-1x thoroughly. Avoid frothing, which can cause denaturation of the
Conjugate.
c. Return the unused portion of Conjugate-100x Concentrate to 2–8°C storage.

6

Primagam™ Non‑Human Primate M. tuberculosis Interferon‑Gamma Kit Instructions for Use

4a. Add 100 µL of Conjugate-1x to each well of the microplate.

Add Conjugate‑1x

b. Mix thoroughly using a plate shaker set at moderate-high speed for 1 minute.
Alternatively, if a plate shaker is not available, pipet up and down 5 times to mix.
c. Cover the plate with a lid, then incubate for 60 minutes at room temperature.
d. Aspirate the solution from the wells, then wash the wells 6 times with Wash Buffer‑1x (see “Wash
the wells“ on page 6).

5Prepare the enzyme
substrate solution

IMPORTANT!

· Prepare the enzyme substrate solution immediately before use.
· Use sterile, plastic polypropylene containers. Do not use polystyrene containers or pipettes.
a. For each 8-well microplate strip, combine 10 µL of Chromogen Solution-100x Concentrate with
1 mL of Enzyme Substrate Buffer.
Example: For six microplate strips, combine 60 µL of Chromogen Solution-100x Concentrate with
6 mL of Enzyme Substrate Buffer.
b. Mix the enzyme substrate solution thoroughly.
Note: If blue discoloration occurs, discard the solution.

6

Add the enzyme substrate Note: Start the 30-minute incubation time when the enzyme substrate solution is added to the firstsolution
well.
a. Add 100 µL of enzyme substrate solution to each well of the microplate.
b. Mix thoroughly using a plate shaker set at moderate-high speed for 1 minute.
Alternatively, if a plate shaker is not available, pipet up and down 5 times to mix.
c. Cover the plate with a lid, then incubate for 30 minutes at room temperature in the dark.

7

Add Enzyme Stopping
Solution, then read theplatea. Add 50 µL of Enzyme Stopping Solution to each well in the same order that the enzyme substratesolution was dispensed.
b. Tap the side of the plate to gently mix.
The solution in the wells changes from blue to yellow.
c. Read the plate at 450 nm (reference filter at 620–650 nm) within 5 minutes.

Analyze the results
Calculate the mean absorbance
1. Calculate the mean OD450 value of all samples and controls.
2. If IFN-γ is detected in Nil Antigen Control-stimulated samples, subtract the mean OD450 value from the results for that animal.
Note: Detection of IFN-γ in Nil Antigen Control-stimulated samples can indicate coinfection or another disorder.

Primagam™ Non‑Human Primate M. tuberculosis Interferon‑Gamma Kit Instructions for Use

7

Validation criteria
If the following criteria are not met, the results are invalid and the samples must be retested.
Reaction typemean OD450

Coefficient of variation (%)

Negative Primate IFN-γ Control<0.15030

Positive Primate IFN-γ Control

>1.00

15

Interpretation of results
Interpret the results according to the following table:
Mean OD450

Interpretation

Bovine PPD − Avian PPD > 0.050 and
Bovine PPD − Nil Antigen Control > 0.050

Positive

Avian PPD > Bovine PPD and
Avian PPD > Nil Antigen Control

Negative[1][1] The animal is considered an avian reactor.

• For absorbance readings that are outside of the limit of the plate reader, see “Retest samples with invalid results“ on page 8.
• This test can give a false-positive or false-negative result due to local conditions. Interpret the results in the context of all available
clinical, historical, and epidemiological information relevant to the animal being tested. Further confirmatory testing can be requiredin specific circumstances.
• Test interpretation and consequent animal husbandry decisions are the responsibility of the user, any consulting veterinarian, animal
health advisor, or authority.

Retest samples with invalid results
1. Dilute the plasma samples 1:4 in Green Diluent.
For example, add 25 µL of test plasma to 75 µL of Green Diluent.
2. Vortex at a moderate-high speed 3–5 times, then repeat the ELISA procedure.

Customer and technical support
Visit thermofisher.com/support for the latest service and support information.
• Worldwide contact telephone numbers• Product support information– Product FAQs
– Software, patches, and updates– Training for many applications and instruments• Order and web support• Product documentation– User guides, manuals, and protocols– Certificates of Analysis– Safety Data Sheets (SDSs; also known as MSDSs)
Note: For SDSs for reagents and chemicals from other manufacturers, contact the manufacturer.

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Primagam™ Non‑Human Primate M. tuberculosis Interferon‑Gamma Kit Instructions for UseThis page is intentionally blank.

Primagam™ Non‑Human Primate M. tuberculosis Interferon‑Gamma Kit Instructions for Use

9Prionics Lelystad B.V. | Platinastraat 33 | 8211 AR Lelystad | The Netherlands
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Revision history: Pub. No. MAN0018635
Revision

Date

A.0

25 November 2019

Description• Converted the legacy document (Primagam_PI_V1.4e_140512.pdf) to the current document template, with associatedupdates to the publication number, limited license information, warranty, trademarks, and logos.
• Updated the procedures for antigen preparation and blood stimulation.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all
applicable Limited Use Label Licenses.
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