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Objectives:
In the ELISpot assay, IFN-g release by PBMC in the presence of peptide-loaded
or antigen expressing target cells is measured in a modified ELISA assay.
It should be noted that IFN-g can be released by both CD4+ and CD8+cells
as well as other PBMC. To specify the phenotype of IFN-g secreting cells,
it is necessary to perform depletion studies, which are not described
here. PHA can be used to induce the majority of T cells to secrete IFN-g
and is used as a positive control.
Sample:
Fresh or frozen PBMC
Vaccinia
recombinants: The easiest way to obtain vaccinia recombinants is from
the AIDS Research and Reference Reagent program in Rockville MD, USA.
Although the recombinants are provided free of charge, the recombinants
are only shipped to registered users. Application forms for registration
and information on what recombinants are available can be obtained from
http://www.aidsreagent.org
Peptides:
Peptides can be obtained from several commercial sources. For CTL recognition,
it is best to have the C-terminal ends made with a free acid (OH). Solubility
is dependent on the sequence and length of the peptide and each peptide
should be made up according to the manufacturer's recommendation. Caution
is necessary to ensure that the solubilizing agents do not cause cell
toxicity. Peptides should be made up at a concentration of 1-5mg/ml, aliquots
can be stored at -20oC, some peptides loose activity after repeated freeze
thaw cycles. The choice of peptide is entirely dependent on the HIV-1
antigen of interest and of the MHC of the patient. A superb database (frequently
updated) of all published MHC restricted CTL peptides is available from
B. Korber at the Los Alamos National Laboratory, Los Alamos, NM. The HIV
Molecular Immunology Database is also available on-line at http://hiv-web.lanl.gov/immuno/
A strategy for mapping peptides using pools of overlapping peptides will
be discussed at the workshop.
Day1:
Coating Plate with 1 ícapture antibody : anti hu IFN - gamma: Mabtech:
Mab1-DIK (lot#: 3420-3-27 @ 1 mg/ml)
In the hood: until 2 í biotin conjugated Ab is added, all steps should
be done using appopriate sterile technique
- Dilute antibody in sterile DPBS w/o Ca, Mg @ 1:100 = 10 ?g/ml final
- Add 100 ?l/well with sterile combitip : Place @ 4 í C overnight (o/n).
Day
2:
Blocking:
- Remove antibody by flicking contents of plate into a container in the
hood. Wash 4-6x with 200 ?l sterile DPBS.
- Add 150 ?l complete media (CM, see materials below) to each well and
leave for at least 60 mins at room temperature (RT).
Preparation
of PBMC, vaccinia recombinants and peptides.
- Thaw or prepare PBMC. Wash, count and assess viability.
- Resuspend PBMC in CM @ 4, 2 and 1 x 106 cells/ml
- Resuspend vaccinia virus in CM to give a final concentration of 2 PFU/cell
for addition of 50 ml, the virus would be made up at 8, 4 and 2 x 106
PFU/ml
- Make up peptides at 20 mg/ml
Adding
Cells to the Plate
- Remove blocking media by flicking plate into beaker in hood.
- Add 50 ml/well of PBMC effectors. Final 2 x 105, 1 x 105, 5 x 104/well
- Add 50 ml/well of virus. Final vaccinia @ 2 PFU/cell
- Add 50 ml/well of peptides @ 20 mg/ml. Final 10 mg/ml
- Add 50 ml PHA @ 20 mg/ml per well to PHA wells. Final 10 mg/ml
- Add 50 ml of media alone to PBMC only wells
- Place @ 37 í C for 20-24 h
- The plates should not be disturbed during this incubation period.
Day
3:
Removing Cells
- Remove cells into bleach using a vacuum manifold, a NUNC immuno wash
or a multi channel pipette. Removed cells should be discarded in bleach
(HIV and vaccinia).
- Wash wells 8x with DPBS/0.05% Tween-20 (PBS-Tween) using NUNC immuno
wash
- Blot plate on paper towel
Detector
antibody : Biotin anti hu IFN-gamma Mabtech, 7B6-1, lot# 3420-6-15 @ 1mg/ml
- Dilute antibody @ 1:500 in DPBS/0.5% BSA = 2 ?g/ml final
- Add 100 ml/well
- Place @ RT for 2 h
- Wash wells 8x with PBS-Tween
- Blot plate on paper towel
- Prepare Avidin Peroxidase Complex (Vectastain ABC Elite Kit)
...........In
a 50 ml tube: ..20 ml PBS-Tween
.........................................2
drop reagent A. Mix by inverting tube.
.........................................2
drop reagent B. Mix.
- Add 100 ?l/well
- Place @ RT for 1h
- Wash wells 4x with PBS-Tween
- Wash wells 4x with 1x PBS
- Blot plate on paper towel to remove excess liquid.
Preparation of SIGMA AEC substrate
- In a 50 ml tube, dissolve the AEC tablet in 2.5 ml dimethylformamid
(takes around 5 minutes)
Prepare
buffer as follows:
.........46.9 ml distilled water
.........4.6 ml 0.1 N acetic acid
.........11 ml 0.1 M sodium acetate
-
Add 47.5 ml of this buffer to dissolved tablet
- Add 25 ml of 30% H2O2
- Filter through 45 mm filter
- Add 100 ?l/well AEC substrate with multichannel pipetor.
- Place in dark drawer. Spots will develop 4 mins.
- Stop development with distilled water- add with squirt bottle
- Repeat wash 2x. Remove water
- Blot on paper towel.
- Remove bottom part of plate. Blot each well with Kimwipe.
- Allow to air-dry o/n in drawer.
Day
4:
The easiest way to enumerate the ELIspots is to carefully apply the membranes
from the individual wells onto a plastic surface. A method is described
below how to do this, however alternative methods of doing this will be
discussed.
-
Apply adhesive plate sealer onto plate. Place Skatron filter cartridges
on wells and press into wells as far as they will go. With the end of
a pen push each filter further until you hear the membrane come off. May
need to keep a finger over well, on adhesive side, while pushing filter
with the pen.
- The ELIspots can be enumerated using an automated plate reader, a stereomicroscope,
or a simple magnifying glass.
Materials
- 1 í Capture antibody: anti hu IFN - gamma: Mabtech AB, Nacka, Sweden:
Mab1-DIK @ 1 mg/ml; cat# 3420-3-27, 1.0ml $670, .25 ml $195; 46-8-716-2700,
Sweden)
- 2 í Detector antibody : Biotin anti hu IFN-gamma : Mabtech AB, Nacka,
Sweden, 7B6-1 @ 1 mg/ml; cat# 3420-6-15, .25 ml, $230
- Vectastain ABC Kit; PK-6100 Standard; Vector Laboratories, Inc., Burlingame,
CA (650) 697-3600, $160
- AEC tablets: SIGMA 3-Amino-9-Ethylcarbazole, A6926, SIGMA, St.Louis
MI (800- 325-5832), 50 tablets $58.50.
- Plates: Millipore (Bedford, MA, distributed through Fisher Scientific)
multiscreen-HA 96 well; cat# MAHA S4510, 10/pk, $126.00
- Coating Buffer : for 1o capture antibody : sterile DPBS wo Ca, Mg
- Blocking Buffer: sterile complete media (CM/10%NHS: RPMI + 10% NHS HI,
2% L-glut, 2%HEPES, 1% P/S)
- CM: RPMI supplemented with antibiotics, L-glutamine and 10% serum (NHS
or FBS can be used). The serum should be tested in the ELISPOT assay to
ensure low background response of PBMC in the absence of antigen
- Effector /Target/virus/peptide/mitogen dilution media: sterile CM/10%NHS
- Wash Buffer (PBS-Tween): DPBS + .05% Tween - 20 - does not need to be
sterile
- 2o Ab dilution buffer (biotin conjugate): DPBS/0.5% BSA
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