To characterize lymphocyte responses in HIV infected patients (cryo-preserved samples) to HIV synthetic peptide pools using an ELISpot assay.

SCOPE:

Cryopreserved peripheral blood mononuclear cells (PBMCs) are thawed and incubated overnight in supplemented media. Two hundred thousand cells are placed in the appropriate number of wells of an ELISpot plate (a 96-well plate containing a 1o antibody to IFN-γ immobilized to a solid support) with or without various stimuli (e.g. control peptides, HIV peptide pools). The cells are stimulated (using 2 µg/ml peptide) for 16 hours (overnight). ELISpot plates are aspirated and washed to remove cells/media. Extracellular IFN-γ is detected by a 2^o antibody to IFN-γ conjugated to biotin. A streptavidin/horseradish peroxidase conjugate is used detect (colourimetric detection) the 2^o antibody. The number of spots per well, or per 2 x 10⁵ cells, is counted using a plate reader.

SAFETY:

If PBMCs are from an HIV-infected source, initial sample prep (up to and including 16 hour stimulation) is to be performed in the BL3. BL3 procedures apply (BL3 room/biosafety hood/PPE/safety glasses) in the initial sample prep. After stimulation cells are aspirated into a 10% bleach-filled container.

ELISPOT ASSAY:

1. Coat ELISPOT Plate: (Day 0)
• Add 50 µl/well of sterile 70% ethanol to moisten membrane
• Dilute 1^oα-IFN-γ Ab (1mg/mL Green tube) in PBS (RT) to 10 µg/mL final.
• Wash 6 times with PBS (200 µl/well)
• Flick plate in sink, tap on gauze to remove excess PBS
• Add 100 µl Ab/well and tap gently to make sure well bottoms are evenly coated, wrap outer edges of plate with parafilm to prevent evaporation
• Let sit on shaker 1 hour at RT
• Incubate plate overnight at 4ºC; use plates the next day

2. Add PBMCs and peptides: (Day 1)
• Remove peptides from –20^oC freezer, let thaw at RT; spin tubes down briefly
• Dilute, into a U-bottom 96-well plate according to Peptide template plate, HIV synthetic peptide pools (env, gag, nef, @ 200 µg/ml) with RH, to 8 µg/ml final, diluted with 57.6 µl RH and 2.4 µl of peptide stock to a final volume of 60 µl (2 x 25 µl reactions).
• Prepare PHA (2.4 µl of 200 µg/ml: 57.6 µl RH for 2 x 25 µl reactions) & CEF (2.4 µl of a 200 µg/ml stock: 57.6 µl RH) for positive controls
• For negative control, use 60 µl RH only
• Dispense HIV peptide pools, positive and negative controls to Peptide template plate (Figure 1) (specific wells defined for each peptide or control, include a no peptide control of media only).

Figure 1.  Peptide template plate

  Each well contains 60 µl of peptide/media, for 2.5<2 x 25 µl aliquots

3. Block IFN-γ-coated Elispot Plate: (Day 1)
• Take BLOCK out of fridge, let get to RT
• Take plates out of fridge, let sit on shaker as above
• Wash plate 6 times with PBS, as above
• Flick plate in sink, tap on gauze to remove excess PBS
• Add 100 γl/well of BLOCK
• Incubate at 37oC/5% CO2 for 45min- 1 hour
• BLOCK from ELISpot plate by emptying plate contents onto gauze, tap well to remove excess BLOCK
• Add 75 µls, using a 12 channel pipettor, of cells (at 2.7 x 106 cells/ml for 200,000 PBMCs/well) to appropriate wells (corresponding to ELISpot plate map) of ELISpot plate (Figure 1
• Add 25 µl of peptide (or controls), using a 12 channel pipettor, to a final concentration 2 µg/ml, from Peptide template plate to ELISpot plate (Figure 1 & 2)
• Incubate plate at 37°C/5% CO2 for 16 hours

4. Secondary Antibody Incubation: (Day 2)
• Dilute secondary biotinylated α-IFN-γ antibody (1 mg/mL) in 2^o Buffer (room temperature) to 2µg/ml final.
• Wash plates 6 times with PBS-T and remove excess liquid by flicking plate and tapping on gauze
• Add 100 µl/well of secondary antibody dilution
• Incubate at RT for 2 hours

5. Enzyme Linkage: (Day 2)
• Prepare Avidin Peroxidase Complex (Vectastain ABC Elite kit): to 11 ml of PBS (room temperature), add 2 drops of reagent A, invert tube to mix, then add 2 drops of reagent B) Incubate solution at RT for 30 minutes prior to use; alternatively add pharmingen stv-HRP (final conc 2ug/ml) to Hrp Buffer (RT) (do not incubate prior to use)
• Wash plates 6 times with PBS-T and remove excess liquid by flicking plate and tapping on gauze
• Add 100 µl/well Pharmingen reagent
• Incubate at RT for 1 hour

6. Substrate/Reaction: (Day 2)
• Wash plate 6 times with PBS-T and incubate 15 minutes in PBS-T
• Prepare Vectastain AEC Substrate: to 10 ml dH2O (RT), add 4 drops of buffer stock, 7 drops of AEC and 5 drops of H2O2, invert tube to mix between each additive, and wrap in tin foil to keep DARK
• Wash plate 4 times with PBS and tap on gauze to remove excess liquid
• Add 100 µl/well of AEC substrate
• Develop 20 minutes in a DARK drawer
• Stop reaction by washing 8 times with dH20. Remove excess liquid and allow plates to air dry in vent on hood (or in drawer overnight)
• When plates are dry they can be read immediately, or wrapped in tin-foil and stored indefinitely

7. Counting Spots using Plate Reader:
• Place ELISpot plate in a plate holder
• Turn on lamp, open CTL software, if not already open
• Under imaging, select plate scan, load plate and scan to CD
• Unload plate, count plate from virtual image (CD)
• Automated counting settings are: 1) Background Balance (ON); Process Diff (ON); Autoacquire (ON); Overlay from Image (ON); Overdeveloped Area Processing (OFF); Sensitivity 160-180

8. MATERIALS NEEDED:
• Pipettaid & disposable 5, 10, 25 ml pipets
• P20 & P200 pipetteman & tips
• 12-(or 8-) channel pipettor
• 15 & 50 ml conical tubes
• clinical centrifuge light microscope & hemocytometer
• 1^o Ab: α-IFN-γ antibody (1-D1K, cat #3420-3 MabTech)
• 2^o Ab: α-IFN-γ biotin antibody (clone 7-B6-1 biotinylated, cat #3420-6 MabTech)
• Multi screen 96 well Filtration Plate (#MAIP S45 10 Millipore)
• AEC Peroxidase Substrate (#5K-4200 Vector Laboratories); alternative Pharmingen streptavidin-HRP (#554066 distributed by Becton-Dickinson)
• Vectastain Standard Elite ABC (#PK-6100 Vector Laboratories)
• Plate washer (ELx 405, Bio-Tek Inc)
• Plate reader (Immunospot Reader, CTL Technologies)

9. Solutions & Buffers:
• RH: RPMI/HEPES with L-glutamine (Gibco cat # 22400-089)
• BLOCK: add 2.5 ml BSA (10% Ultrapure BSA, Panvera P2064, or 10% (in PBS) 0.22 µm filter-sterilized Sigma Fract V BSA A-9674) to 47.5 ml RPMI-HEPES, mix well.
• 2^o Buffer: add 2.5 ml BSA (10% Ultrapure BSA, Panvera P2064, or 10% (in PBS) 0.22 µm filter-sterilized Sigma Fract V BSA A-9674) to 47.5 ml PBS, mix well (alternatively 2.5 ml 10% filter-sterilized BSA to 47.5 ml 1X PBS), add 25 µl Tween-20, mix well (Tween will settle on the bottom if not mixed thoroughly).
• 10X PBS
• Plate washing buffers: PBS: dilute 500 ml 10X PBS: 4.5 L dH2O PBS-T: 1L 10X PBS: 9L dH2O: 5 ml Tween-20 (mix well)
• Trypan blue solution (0.4%) (pre-made Sigma)

Dr. Madhu Vajpayee

Associate Professor
HIV &Immunology Division Department of Microbiology,
All India Institute of Medical Sciences,
New Delhi India 110029.