Hanekom: Immunol. outcomes of novel TB vaccine trials: WHO panel recommendations. 07-PLME-BP0785. Supplementary File S3. Seven day whole blood assay. Page 1 of 6
Whole Blood Assay STANDARD OPERATING PROCEDURE
(Last date of modification 22/01/08 by Maeve Lalor, London School of Hygiene &
Tropical Medicine)
1. PRINCIPLE: The analysis of immune responses to mycobacterial antigens. The
cellular production of cytokines is determined following antigen stimulation for 7 days
in a 1 in 10 diluted whole blood assay.
All steps of the WBA must be done under sterile conditions using a Class II
laminar flow hood.
2. HARDWARE:
2.1
Disposable pipettes, 1ml, 2ml, 5ml and 10ml
2.2
Na Heparin Vacutainer tubes/polypropylene tubes with added heparin
2.3
96 well (round bottomed) tissue culture plates
2.4
96 well (flat bottomed) microtitre plates with lids
2.5
Microtitre plate sealers
2.6
Reagent reservoirs (50mls)
2.7
P20, P200, P1000 Gilson Pipettes, Pipette man
2.8
Multi-channel pipette (12 channel)
2.9
Sterile universal tubes (20ml)
2.10
Eppendorf tubes
2.11
Microtubes and lids
2.12
Micropore Tape
2.13
Water-bath
2.14
CO2 incubator
2.15
Minus 800 C freezers
3. REAGENTS:
1. RPMI 1640 Culture Medium (Invitrogen cat: 42201-042) containing 1% LGlutamine (Invitrogen cat: 25030-024)
Store at 4C and discard after expiry date is reached.
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Hanekom: Immunol. outcomes of novel TB vaccine trials: WHO panel recommendations. 07-PLME-BP0785. Supplementary File S3. Seven day whole blood assay. Page 2 of 6
2. Phytohemaglutinin (PHA-M; positive mitogen control)
Sigma Cat No L8902
Make up a stock solution of 1mg/ml by adding 5ml sterile 1 X PBS directly to the vial.
Store stock at 1mg/ml and aliquot in 20l aliquots. Store at -80oC until use.
OR
2. Staphylococcus enterotoxin B (SEB; positive antigen control)
Sigma Cat No S4881 (1mg vial)
Make up a stock solution of 1mg/ml by adding 1ml of sterile 1 X PBS directly to the
vial.
Make up 9 x 100ul aliquots at 1mg/ml.
Take the remaining 100ul and make up to 1ml total volume (add 900ul) with RPMI
containing 1% L-glutamine (100ug/ml) - Make up 50 x 20ul aliquots at 100ug/ml.
Store at -80oC until use.
NB: There is no need to use both of the positive controls.
3. Mycobacterium tuberculosis purified protein derivative (PPD).
Statens Serum Institute – Denmark 1ml vials at 1mg/ml, PPD for in vitro use
(preservative-free).
Divide the 1ml stock into 10 x 100ul aliquots.
Keep all stock aliquots at 1mg/ml at 4oC prior to use.
4. FROZEN ANTIGEN PLATES
4.1 Antigens and Controls
Plan the panel of antigens and controls, and decide how many replicates are
required (eg 10 antigens in quadruplicate or 20 antigens in duplicate/triplicate)
1. RPMI 1640 Medium containing 1% L-Glutamine (negative control)
2. PHA at 5ug/ml or SEB (positive control)
3. M.tb PPD for in vitro use from Statens Serum Institute at 5ug/ml
4. Mycobacterial antigens eg ESAT6/CFP10 at 10ug/ml
4.2 Re-constitution and dilution of antigens:
Re-constitute the mycobacterial antigens by adding sterile PBS to make up to a
concentration of 1mg/ml, and aliquot into multiple sterile ependorfs for storage at 80oC. (Make aliquots of the size you will need for a batch of plates).
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Hanekom: Immunol. outcomes of novel TB vaccine trials: WHO panel recommendations. 07-PLME-BP0785. Supplementary File S3. Seven day whole blood assay. Page 3 of 6
Calculate how many plates are to be prepared in a batch (eg 25 plates per batch).
Prepare diluted antigen at a concentration of twice the final concentration (eg for
PPD at 10ug/ml, for other mycobacterial antigens at 20ug/ml) in sterile universals.
Calculate as follows: 25 plates x 2 replicates x 100ul volume per well = 5000ul
Antigen stock (1mg/ml), final concentration desired (10ug/ml), 2x final concentration
(20ug/ml), therefore dilute 1/50. Use 100ul antigen (1mg/ml) diluted in 4900ul RPMI
with L-glutamine.
4.3 Making up the frozen antigen plates
The 25 x 96 well tissue culture plates should be clearly labelled with the date the
plates have been prepared, the antigen template code (which changes if any of the
antigens or batches change) and the study name.
According to the 96 well plate template below, and using a well calibrated multichannel micropipette, transfer all antigens and controls in 100ul volumes (at twice the
final concentration as described above) to duplicate wells of 25 x 96 well tissue
culture plates.
PHA or SEB are added to the wells only on the day of the WBA (i.e. are not prefrozen in the plates).
Add 200ul of HBSS or sterile water to the outer wells of the plate to prevent drying.
96 well plate template – 1 plate can fit 1, 2 or 3 study participants if using 10 antigens
in duplicate. Make some plates with 1, 2 and 3 participants per plate. (Example below
shows plate with 3 participants in duplicate). If using triplicate wells, then 2 study
participants can be tested on one plate.
A
B
C
D
E
F
G
H
HBSS
HBSS
HBSS
HBSS
HBSS
HBSS
HBSS
HBSS
HBSS
Neg
Neg
Neg
Neg
Neg
Neg
HBSS
HBSS
Ag 1
Ag 1
Ag 1
Ag 1
Ag 1
Ag 1
HBSS
HBSS
Ag 2
Ag 2
Ag 2
Ag 2
Ag 2
Ag 2
HBSS
HBSS
Ag 3
Ag 3
Ag 3
Ag 3
Ag 3
Ag 3
HBSS
HBSS
Ag 4
Ag 4
Ag 4
Ag 4
Ag 4
Ag 4
HBSS
HBSS
Ag 5
Ag 5
Ag 5
Ag 5
Ag 5
Ag 5
HBSS
HBSS
Ag 6
Ag 6
Ag 6
Ag 6
Ag 6
Ag 6
HBSS
HBSS
Ag 7
Ag 7
Ag 7
Ag 7
Ag 7
Ag 7
HBSS
HBSS
PPD
PPD
PPD
PPD
PPD
PPD
HBSS
Label the lid of each tissue culture plate with the following information
- Name of study
3
HBSS
PHA
PHA
PHA
PHA
PHA
PHA
HBSS
HBSS
HBSS
HBSS
HBSS
HBSS
HBSS
HBSS
HBSS
Hanekom: Immunol. outcomes of novel TB vaccine trials: WHO panel recommendations. 07-PLME-BP0785. Supplementary File S3. Seven day whole blood assay. Page 4 of 6
- Date of antigen plate preparation
- Antigen template code
Place lids on plates and store plates at -80oC until needed.
4.4 CONTROLS
NB: PHA or SEB are added fresh to the tissue culture plates on the day the assay is
done i.e. they are not pre-frozen.
PHA: stock antigen at 1mg/ml
Prepare PHA at 2x final concentration (2x5ug/ml=10ug/ml). Add 100ul directly to the
desired well just prior to use.
OR
SEB: working stock at 100µg/ml
Prepare SEB at 2x final concentration (2x1ug/ml=2ug/ml). Add 100ul directly to the
desired well just prior to use.
5. THE WHOLE BLOOD ASSAY
5.1 Remove pre-made frozen antigen plates (appropriate number of plates for
number of study participants bled that day).
5.2 Label 20ml universal tubes with participant/study number (for blood dilution). One
tube will be required per study participant.
5.3 Place RPMI with 1% glutamine in 37C waterbath.
In the Laminar flow-hood:
5.4 Invert all heparinised tubes of blood very gently 3 times
5.5 Make a 1 in 5 dilution of whole blood samples with RPMI (pre-warmed in 37C
water-bath) in a labelled universal tube, ie 1 ml blood added to 4 mls RPMI.
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Hanekom: Immunol. outcomes of novel TB vaccine trials: WHO panel recommendations. 07-PLME-BP0785. Supplementary File S3. Seven day whole blood assay. Page 5 of 6
5.6 Invert the 1 in 5 diluted blood.
5.7 Pour diluted blood into a reagent reservoir for pipetting.
5.8 Draw up diluted blood into the multi-channel pipette, add 100l of 1 in 5 diluted
blood to each antigen well (total final volume = 200l) in the following order: a)
negative control b) antigen wells c) PHA.
5.9 Put on the tissue culture plate lid and surround plate with micropore tape.
5.10 Immediately place tissue culture plates in a 5% CO2 incubator at 37C for 7
days.
5.11 Record the time the blood was diluted, the culture plate was put into culture in
the incubator, operator, antigen plate batch and date, and date of assay.
5.12 Check gauge on CO2 cylinder at least twice per week, and always on the
morning before a weekend. Ideally a switch-over valve and second CO2 cylinder
should be attached to prevent possible loss of CO2 during culture.
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HARVESTING WBA SUPERNATANTS.
Per condition (i.e. antigen or control) 3 sets of supernatants will be generated
2 sets of sups are to be stored in 96 well plates. One storage plate will contain a
volume of 110ul of each sup,
1 set of sups (~100ul) are stored in microtubes with lids.
In a single 96 well storage plate the supernatants of maximum 8 study subjects can
be stored.
6.1 Label the lids of the storage plates, and the storage plates themselves in
advance of the harvest with:
the date that the supernatants are being removed (i.e. harvest date), the initials of the
lab staff taking off the supernatants and the study subjects number
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Hanekom: Immunol. outcomes of novel TB vaccine trials: WHO panel recommendations. 07-PLME-BP0785. Supplementary File S3. Seven day whole blood assay. Page 6 of 6
Supernatants for longer term storage should be stored in microtubes in boxes.
6.2 Label the tubes with:
the date that the supernatants are being removed (i.e. harvest date), the initials of the
lab staff taking off the supernatants and the study subjects number
Removing and pooling the supernatants
Process the blood of one patient or subject at a time.
6.3 For pooling duplicate supernatants, arrange 10 microtubes per sample into a 96
well microtube box
6.4 Harvest 162ul of supernatant from replicate 1 (row B of culture plate) and transfer
to microtubes. Harvest 162ul of supernatant from replicate 2 (row C of culture plate)
and transfer to the same microtubes. Avoid harvesting any red blood cells from the
pellet at the bottom.
6.5 Mix the 320ul of pooled supernatant in the tubes by pipetting 3 times.
Transferring the harvested supernatants to storage plates
6.6 Transfer 110ul of supernatant to the first pre-labelled storage plate.
6.7 Transfer 110ul of supernatant to the second pre-labelled storage plate.
6.8 Place a plate sealer on the supernatant plate before putting on the lid.
6.9 Place lids on the microtubes containing the remaining ~100ul.
6.10 Store both storage plates and microtubes at –80C.
6.11 Record the time of harvest and any comments.
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