ELISpot Technique

The activation and expansion of antigen-specific T lymphocytes is a characteristic of primary viral infections. Upon challenge, CD8+ cytotoxic T lymphocytes (CTL) recognise antigenic peptides in conjunction with MHC class I molecules, leading to the lysis of viral-infected cells as well as the secretion of Interferon-ɣ (IFN-ɣ). In addition, CD4+ T helper lymphocytes recognise antigenic peptides in conjunction with MHC class II molecules, also leading to the secretion of IFN-ɣ, which in turn affects other aspects of the immune system. The number of antigenic-specific precursor T cells available at the time of challenge determines the magnitude of the immune response and may ultimately affect the course of infection.

The ELISpot technique was first reported in 1983 when Czerkinsky et al described a method to enumerate the frequency of B hybridoma cells secreting an antigen specific immunoglobulin [129]. The technique was developed further in 1998 when Czerkinsky outlined the development of an ELISpot assay measuring the frequency of T lymphocytes secreting a specific lymphokine [130]. Further development of the technique included description of a dual-colour ELISpot assay [131] allowing simultaneous assessment of two different types of cell producing antigenically distinct products and the use of computer imaging to detect and analyse spots.

An ELISpot assay capable of detecting IFN-ɣ-producing T cells in a sample of peripheral blood mononuclear cells (PBMCs) can be utilised to estimate precursor frequency. The PBMCs are serially diluted and placed in microplate wells coated with anti-human IFN-ɣ antibody. They are cultured with antigen overnight, resulting in re-stimulation of the precursor cells and secretion of IFN- ɣ. The cells are washed away, leaving the secreted IFN-ɣ bound to the antibody- coated wells in concentrated areas corresponding to the location of the displaced cells. The captured IFN-ɣ is detected with biotinylated anti-human IFN-ɣ

antibody followed by an alkaline phosphatase-conjugated anti-biotin antibody. The addition of insoluble alkaline phosphatase substrate results in dark spots in the wells at the sites where the cells are located, leaving one spot for each T cell that secreted IFN-ɣ. The number of spots per well is directly related to the precursor frequency of antigen-specific T cells.

Equipment

Table 2.3 Table of equipment for ELISpot assay

Biological Safety Cabinet

Humidified CO2 incubator set at 37°C and 5% CO2

Set of micropipettes and corresponding sterile pipette tips Electronic 12-channel pipette or multi-stepper

Sterile individually wrapped 5 mL, 10 mL, 25 mL and 50 mL serological pipettes Sterile polypropylene conical centrifuge tubes, 15 mL and 50 mL volumes

Sterile culture bottles (if required), 250 mL volume Sterile reagent reservoirs

Electric Pipette-Aid

Sterile culture plates or plastic reservoirs

96-well plates with PVDF membrane (Millipore MSIPS4510) Microscope with 20X objective

Materials

Table 2.4 Table of reagents for ELISpot assay

Reagent* Supplier Catalogue No.

Sterile D-PBS (1X) Gibco 14190-136

IFN-ɣ human ELISpot kit MABTECH 3420-2A

RPMI 1640 Gibco 11875-093

200mM L-Glutamine Gibco 25030-024

100X Penicillin/Streptomycin solution Gibco 15140-122

Foetal Bovine Serum (FBS) Sigma F9665

PHA Sigma L1668

Concanavalin A Sigma C5275

Dimethyl sulphoxide (DMSO) Sigma D2650

TWEEN® 20 Sigma P2287

Bovine Serum Albumin Sigma A4919

BCIP/NBT Substrate Solution Pierce 34042

* Use listed reagent or equivalent

Reagent Preparation

See Appendix 2 for details on reagent preparation.

ELISpot plate preparation

The following steps were performed in a biological safety cabinet using aseptic technique and appropriate precautions. The required numbers of 96-well ELISpot plate(s) were labelled appropriately, including the subject ID, visit day and date. Each well of the ELISpot plate was pre-wetted with 15µl of 35% ethanol in H2O. If coating more than one plate, then each plate was pre-wetted and coated separately. The ethanol was discarded into a waste trough immediately. The plate was blotted onto a paper towel gently and washed 4 times with 200µl/well sterile PBS using a multi-channel pipette and sterile pipette tips and trough.

The volume of coating solution required was determined by multiplying the number of plates to be coated by 7.5 ml (this gave the volume of D-PBS required). The required volume of anti-human IFN-ɣ monoclonal antibody clone 1-D1K stock solution was determined by dividing the volume of D-PBS required by 200. The appropriate volume of sterile D-PBS and the appropriate volume of anti-human IFN-ɣ monoclonal antibody clone 1-D1K was dispensed into a 50 ml centrifuge tube. This resulted in a coating solution at 5 µg/ml of the antibody. The tube was capped tightly and mixed by inversion. Using a multi-channel pipette, 75 µl of coating solution per well was dispensed in each well of the labelled ELISpot plates. The lids were placed on the plates and all plates were placed in a refrigerator (2-8°C) overnight (15-24 hours). Plates were stored with coating solution in the refrigerator for up to 3 days. For storage time greater than overnight, the plates were placed in an airtight container within the refrigerator.

ELISpot procedure

The ELISpot plate coating antibody was discarded from each plate. Each coated plate was washed four to six times with 200 µl sterile D-PBS per well using a multi-stepper or electronic multi-channel pipette. The plate was inverted over a reservoir to decant the wash solution after each wash. 200 µl sterile R10 per well was transferred to each washed plate. The lids were placed on the plates and the plates left in a humidified CO2 incubator set at 37°C (±1°C) and 5% CO2 for at least 1 hour.

The blocked plates were then removed from the incubator and inverted to decant the R10 from each well. 50 µl of each antigen/control was transferred from the pre-prepared peptide plate into the appropriate wells of the blocked ELISpot plate (3 wells per antigen tested, including mock, and one or two wells for Con A/PHA control). This step was repeated until all antigen/mock solutions have been plated. A tube containing PMBCs that had been previously isolated, counted and re-suspended at 4 x 106 _{cells/ml in sterile R10 for each test subject} was gently swirled to ensure adequate mixing of the PBMCs. 50 µl PBMC

suspension from each subject was dispensed into the appropriate number of wells of the plate so that each subject has three wells per antigen (at 200,000 cells/well). In addition, at the Week 9 time point only, 25 µl PBMC suspension was dispensed into the appropriate wells of the plate to give the same number of replicates at 100,000 cells/well. The lids were placed on all plates and the plates were placed in a humidified CO2 incubator set at 37°C (± 1°C) and 5% CO2 for 15- 20 hours.

Assay Development

A solution of biotinylated mouse anti-human IFN-ɣ diluted to 0.5 µg/ml (1:2000 dilution of 1 mg/ml stock) in assay diluent was prepared as follows: The volume of assay diluent required was determined by multiplying the number of assay plates by 6 ml. The volume of diluent calculated above was placed into a 50 ml tube. The volume of antibody stock required was determined by dividing the volume of diluent determined above by 2000. The required volume of antibody stock was pipetted into the diluent. The tube or was capped tightly and mixed by inversion.

The ELISpot plates were removed from the incubator and washed with 200 µl PBS/Tween 20 wash buffer (WB) for a minimum of 6 washes. After this step, all cells are washed away, so benchtop manipulation was acceptable.

The plates were blotted dry on a paper towel. 50 µl of diluted biotinylated antibody was transferred to each well of the plate(s). Lids were placed on all plate(s) and the plate(s) placed on the benchtop at room temperature for 3 hours ± 1 hour.

Immediately before the end of the incubation, a solution of alkaline phosphatase (AP)-conjugated anti-biotin antibody, diluted 1:750 in AD, was prepared as follows: The volume of assay diluent required was determined by multiplying the number of assay plates by 6 mL. The volume of diluent calculated above was pipetted into a 50 mL tube. The volume of antibody stock required was determined by dividing the volume of diluent determined by 750. The required

volume of antibody stock was pipetted into the diluent. The tube was capped tightly and mixed by inversion. Each plate was washed a minimum of four times with 200 µL of WB per well. The plate(s) was blotted dry on a paper towel. 50 µL of AP-anti-biotin was transferred to each well of every plate. Lids were placed on all plates and the plate(s) were placed on the benchtop at room temperature for 2 hours.

At the beginning of the 2-hour incubation, the bottle of BCIP/NBT stock solution was removed from the refrigerator and placed at room temperature. At the end of the 2-hour incubation, each plate was washed a minimum of four times with 200 µL WB. The plate(s) was blotted dry on a paper towel. The bottle of BCIP/NBT solution was inverted at least five times to mix. 6 ml BCIP/NBT solution per plate was transferred to an appropriate Falcon tube or bottle with a 0.22 µm filter. Pre-filtered BCIP/NBT solution was dispensed into a sterile reagent reservoir and 50 µl transferred to each well of every plate. The plate(s) was incubated at room temperature for 5 minutes, or until distinct dark spots appeared in the positive control wells. To stop colour development, the BCIP/NBT solution was decanted and each plate rinsed three times with tap water. The rubber bottom was removed from all plates and the bottom side of the membrane rinsed with tap water. Each plate was allowed to air-dry overnight.

Analysis

The spots in each well were counted using an ELISpot plate reader (AID ELISpot Reader classic). To calculate the mean number of spot-forming cells (sfc) per 1 x 106 _{input PBMC, the mean number of spots was multiplied for each set of} replicate wells by x (x = 1 x 106_{/ cells per well input). Example: If 2 x 10}5 cells/well input, multiply mean spot count by 5 (i.e. 5 = 1 x 106_{/2 x 10}5_).

Pass/Fail Criteria:

Positive Controls:

All ELISpot assays performed included positive controls, typically ConA (or PHA). At the concentration used, this should have given a totally blue (saturated) appearance to the well. If there were few to no spots in all positive control wells, an assay reagent problem was suspected. All samples on the assay plate(s) in question were re-tested.

Negative Controls:

If test samples plus mock (no antigen) resulted in more than 45 spots per million cells, these samples were reviewed and discussed within the PEACHI consortium. A plan for re-test was discussed if possible and/or necessary.

If media-only wells (no cells) had more than five apparent spots per well, non- specific assay reagent artefacts were suspected. All samples on the assay plate(s) in question were re-tested.

Re-Tests:

If positive controls were valid upon re-test, the earlier test was deemed invalid and the re-test data was accepted as final. If the mock data was still above 45 spots per million cells upon re-test, this confirmed the earlier test. Therefore, the earlier test was deemed valid and was accepted as final.

If the negative control media-only wells were at or below 5 spots per well upon re-test, the earlier test was deemed invalid and the re-test data was accepted as final. All controls had to be acceptable as described above for data to be accepted as final. All re-tests were reviewed and discussed within the PEACHI consortium.