Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

Podsumowanie

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b molecules. Immunization with Qa-1-binding epitopes has been shown to augment tissue-specific immune regulation and ameliorate several autoimmune diseases. Herein we describe an overlapping peptide library strategy for the identification of Qa-1 epitopes in a protein.

Streszczenie

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules play important roles in surveying cells for structural and functional integrity, inducing immune regulation, and limiting immune responses to viral infections. Additionally, functional augmentation of Qa-1-restricted CD8⁺ T cells through epitope immunization has shown therapeutic effects in several autoimmune disease animal models, e.g. experimental allergic encephalomyelitis, collagen-induced arthritis, and non-obese diabetes. Therefore, there is an urgent need for a method that can efficiently and quickly identify functional Qa-1 epitopes in a protein. Here, we describe a protocol that utilizes Qa-1-restricted CD8⁺ T cell lines specific for an overlapping peptide (OLP) library for determining Qa-1 epitopes in a protein. This OLP library contains 15-mer overlapping peptides that cover the whole length of a protein, and adjacent peptides overlap by 11 amino acids. Using this protocol, we recently identified a 9-mer Qa-1 epitope in myelin oligodendrocyte glycoprotein (MOG). This newly mapped MOG Qa-1 epitope was shown to induce epitope-specific, Qa-1-restricted CD8⁺ T cells that enhanced myelin-specific immune regulation. Therefore, this protocol is useful for future investigation of novel targets and functions of Qa-1-restricted CD8⁺ T cells.

Wprowadzenie

Qa-1 belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules in mice. Its human homolog is HLA-E. Previous evidence has demonstrated that Qa-1 molecules have important biological functions. Firstly, Qa-1 molecules play an important role in surveying cells for structural and functional integrity. In this regard, Qa-1 molecules have evolved several strategies to monitor the normal function of a cell. One such strategy enables Qa-1 molecules to form complexes with a processed leader peptide (epitope), i.e. the Qa-1 determinant modifier (Qdm) that is processed from classical MHC-Ia molecules in the endoplasmic reticulum¹. These Qa-1/Qdm complexes later display on the surface of a cell and bind to inhibitory NKG2A receptors on NK cells to inhibit NK killing activity². If the expression of MHC-Ia molecules is lost, a cell (e.g. a malignant cell) becomes sensitive to NK killing². The other strategy enables Qa-1 molecules to form new Qa-1/epitope complexes on the surface of a cell that is deficient in TAP (transporter associated with antigen processing)³ and/or ERAAP (endoplasmic reticulum aminopeptidase associated with antigen processing)⁴ (both deficiencies often occur in malignant cells). The cell that expresses these new Qa-1/epitope complexes can then be recognized and eliminated by the epitope-specific Qa-1-restricted CD8⁺ T cells. Secondly, Qa-1 molecules induce immune regulation⁵. In this regard, Qa-1/epitope complexes have been shown to stimulate CD8⁺ regulatory T (Treg) cells that are important for the prevention of immune-mediated damage of self-tissues^6,7,8,9,10. Thirdly, Qa-1-restricted CD8⁺ Treg cells have been shown to limit immune responses against viral infection¹¹.

Therefore, specific augmentation of epitope-specific Qa-1-restrictred CD8⁺ T cells is a potentially promising strategy for the elimination of abnormal cells, for the enhancement of immune regulation, and for the control of the magnitude of virus-induced immune responses. While it has not been determined whether augmentation of epitope-specific Qa-1-restricted CD8⁺ T cells can enhance immune surveillance and limit virus-induced immune responses, our laboratories and others have clearly demonstrated that immunization with Qa-1 epitopes can augment the function of Qa-1-restricted CD8⁺ Treg cells specific for pathogenic autoimmune CD4⁺ T cells, leading to efficient control of CD4⁺ T cell-mediated autoimmune diseases in a variety of animal models such as experimental allergic encephalomyelitis (an animal model of human multiple sclerosis)^6,10, collagen-induced arthritis (an animal model of human rheumatoid arthritis)⁷, and non-obese diabetes (an animal model of human type 1 diabetes)⁸. Additionally, we have discovered that immunization with a tissue-specific Qa-1 epitope leads to specific control of immune-mediated inflammation in that tissue through augmentation of CD8⁺ Treg cells¹². The above successes of preclinical studies indicate a need for a full evaluation of Qa-1 epitope immunization for the treatment of tissue-specific immune-mediated diseases and potentially for the therapy of other diseases associated with deficiencies in TAP and ERAAP.

Accordingly, there is a demand for a technology that can reliably and quickly analyze Qa-1 epitopes in a protein. In this regard, a limited number of biologically important Qa-1 epitopes has been described. Most of these Qa-1 epitopes were identified serendipitously during the study of CD8⁺ T cell responses to bacteria¹³, cells deficient in TAP³, cells deficient in ERAAP⁴, and cells that cause EAE^6,9. Therefore, a high throughput technique is desirable for the identification of biologically important Qa-1 epitopes in a defined protein. In the following, we describe an overlapping peptide (OLP) library strategy that maps functional Qa-1 epitopes in a protein using Qa-1-resrticted CD8⁺ T cell lines specific for the OLP pool (OLP_pool) of a protein.

Protokół

All experiments were done in compliance with an Institutional Animal Care and Use Protocol approved by Animal Care and Use Committee at the University of Texas at El Paso and Loma Linda University.

1. Generation of an OLP Library Covering the Whole Length of a Protein

1. Design an OLP library in which all peptides are 15-mer in length, and adjacent peptides overlap by 11 amino acids.
   NOTE: In the MOG OLP study, sequence of the MOG precursor [Mus musculus] was retrieved from NCBI protein database by following this link: https://www.ncbi.nlm.nih.gov/protein/NP_034944. The MOG precursor (247 amino acids), which contained both signal and mature peptides, was used because most of reported Qa-1 (HLA-E) epitopes were located in signal peptides (e.g. Qdm¹). Beginning at its N-terminus, the sequences of 15-mer peptides were identified such that two adjacent peptides overlapped by 11 amino acids (Figure 1). Hence, exactly 59 OLPs were identified in the 247 amino acid MOG precursor. However, the last OLP can range from 12 to 15-mer depending on the length of the protein. Additionally, we choose 15-mer library because we and others have shown that 15-mer peptides, when added into dendritic cells (DCs) or macrophages, can be efficiently processed into epitopes for recognition by CD8⁺ T cells^14,15.
2. Purchase each individual peptide commercially. 5 mg per peptide should be enough for the screening. OLPs and truncated peptides (Step 6.1) can be desalted peptides (purity is approximately 50 - 70%). The purity of optimal peptide (Step 6.2) that will be used for the generation of tetramer and for future biological analyses should be >90%. Reconstitute the peptides under sterile condition.
3. Make 50 mg/mL individual peptide stocks in 100% DMSO. For 5 mg of each peptide, add 100 μL of DMSO into each tube, mix, and store the peptides at -20 °C. These stocks will be used to make an OLP_pool stock for the generation of CD8⁺ T cell lines reactive to the OLP_pool. In addition, these stocks will also be used to make 10 mg/mL individual peptide stocks for determining the ability of each individual peptide to stimulate a Qa-1-restricted response in an OLP_pool-reactive CD8⁺ T cell line.
4. Make OLP_pool stock by adding an equal volume of each peptide into a fresh tube. This OLP_pool stock contains 100% DMSO. In the MOG OLP study, there were 59 OLPs¹². Hence, concentration of each peptide in the OLP_pool was 847.46 μg/mL (50 mg/mL ÷ 59).
5. Make 10 mg/mL individual peptide stocks by diluting (5x) the 50 mg/mL stock in sterile H₂O in a V-bottom 96-well plate (this stock contains 20% DMSO). Make these stocks in a 96-well plate because the determination of the Qa-1-restricted response of each individual peptide will be performed in a 96-well plate using an 8- or 12-channel multichannel pipette.
   NOTE: All peptides, including OLPs and truncated peptides, should be diluted in either a V-bottom 96-well plate or a 96-well sample rack filled with 1 mL tubes since the peptide library screening is performed in 96-well plates using a multichannel pipette. If it is difficult to dilute a peptide, add 1 N NaOH drop wise to help dissolve the peptide.

2. Priming of K^b-/- D^b-/- CD8⁺ T Cells with the OLP_pool-pulsed K^b-/- D^b-/- Dendritic cells (DCs).

NOTE: There are two major Qa-1 alleles: one is Qa-1^a, and the other is Qa-1^b. Since the animals commonly used for academic research, e.g. C57BL/6 and Balb/c mice, carry Qa-1^b, this protocol describes the procedure for mapping Qa-1^b epitopes in a protein. CD8⁺ T cells used in this protocol are purified from K^b-/-D^b-/- mice (C57BL/6 background) in which CD8⁺ T cells are restricted mostly by non-classical MHC-Ib molecules including Qa-1.

1. Produce bone marrow-derived DCs as previously described¹².
   1. Briefly, culture bone marrow single cell suspensions (1 x 10⁶ cells/mL) in RPMI-10 medium (RPMI 1640 supplemented with 10% fetal bovine serum, 5.5 x 10^-5 M 2-mercaptoethanol, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acid) containing 10 U/mL IL-4 and 100 U/mL GM-CSF in a 6-well plate (4 mL/well) at 37 °C, 5% CO₂.
   2. Two days later, remove non-adherent cells carefully and add fresh media and cytokines.
   3. After culturing the cells for another two days, transfer non-adherent cells containing fresh media and cytokines into a new 6-well plate.
   4. Culture the cells for another two days and replenished with fresh media and cytokines containing LPS (0.1 µg/mL) to activate the DCs.
   5. 24 h later, collect the DCs for experiments.
2. Irradiate the DCs with 3000 Rads.
   1. Alternatively, treat the DCs (5 x 10⁷ cell/mL) with mitomycin C (50 μg/mL) in PBS at 37 °C for 20 min. Add RPMI-0 (RPMI 1640 without serum) to fill the tube (~12 mL) and spin the cells for 10 min in a table-top centrifuge at 300 x g. Discard supernatants and repeat the washing procedure two more times. These three washes are critical because any trace amount of mitomycin C may inhibit the response of CD8⁺ T cells during the DC-T cell co-culture.
3. Adjust the DC concentration to 5 x 10⁶ cells/mL in a serum-free medium (AIM-V serum-free medium supplemented with 5.5 x 10^-5 M 2-mercaptoethanol, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acid).
4. Add the OLP_pool stock solution, which contains 100% DMSO, to the DCs such that final DMSO concentration will be 0.5% (200x). In the MOG OLP study, the concentration of each OLP in the OLP_pool was 847.46 μg/mL; hence the final concentration of each OLP in the DC culture was 4.2 μg/mL (847.46 μg/mL ÷ 200). However, this concentration can be scaled up to 100 μg/mL as long as the DMSO concentration in the cell culture remains less than 1%.
5. Incubate the DCs at room temperature for 3 h and shake the cells gently every 15 min.
6. During this incubation period, purify CD8⁺ T cells from the harvested spleen or lymph nodes of K^b-/-D^b-/- mice using a commercial CD8⁺ T cell purification kit, adjust the cell concentration to 10 x 10⁶ cells/mL in the serum-free medium containing 50 U/mL interleukin 2 (IL-2) and 100 U/mL of IL-7.
7. Now, add the CD8⁺ T cells into a 48-well plate as 0.5 mL/well (after the addition of 0.5 mL/well of the OLP_pool-pulsed DCs in Step 2.8, the final concentration of the CD8⁺ T cells will be 5 x 10⁶ cells/mL).
   NOTE: CD8⁺ T cells from mouse spleen and lymph nodes can be purified using CD8 Positive Isolation Kit by following the instruction provided by the manufacturer. CD8⁺ T cells obtained are free of beads.
8. Spin down the OLP_pool-pulsed DCs (300 x g, 10 min) at RT. Reconstitute the OLP_pool-pulsed DCs to 2 x 10⁶ cells/mL in the serum-free medium and add 0.5 mL/well into the 48-well plate that contains the CD8⁺ T cells (final concentration of the OLP_pool-pulsed DCs is 1 x 10⁶ cells/mL, final concentration of CD8⁺ T cells is 5 x 10⁶ cells/mL, final concentration of IL-2 is 25 U/mL, and final concentration of IL-7 is 50 U/mL). Culture the cells at 37 °C and 5% CO₂.
9. On day 4, remove and discard about 400 μL of the culture medium and add 500 μL of the fresh serum-free medium containing 100 U/mL IL-2 and 100U/mL of IL-7 to each well. Incubate the cells at 37 °C and 5% CO₂.
10. On day 7 or 8, re-stimulate the OLP_pool-primed CD8⁺ T cells with the OLP_pool.

3. Restimulation of the Primed CD8⁺ T Cells with Macrophages Pulsed with the OLP_pool

1. Four days before re-stimulation of the OLP_pool-primed CD8⁺ T cells, prepare 2% (v/v) polyacrylamide bead solution: wash 2 g of polyacrylamide beads twice in 20 mL endotoxin-free H₂O or PBS. Pellet the polyacrylamide beads by centrifugation (400 x g) for 5 min and resuspend in 100 mL of PBS. Autoclave at 15 lb/m for 20 min. Store at room temperature.
2. Inject mice intraperitoneally with 1 mL/mouse of the sterile 2% polyacrylamide bead solution to attract the migration of monocytes/macrophages into the peritoneal cavity¹⁶.
3. Four days later, sacrifice the animals by CO₂ overdose.
   1. Under sterile conditions, cut a small opening at the center of the abdomen such that the opening is just enough for passing a 5 mL transfer pipette. Fill the transfer pipette with RPMI-0 and insert the pipette into the abdomen cavity through the opening.
   2. Rinse the abdomen cavity by pipetting. Pipette out as much liquid as possible from the abdomen cavity into a sterile tube (these are peritoneal macrophages). Repeat the rinse step 4 - 5 times.
4. Irradiate the peritoneal macrophages with 3000 Rads.
   1. Alternatively, treat the peritoneal macrophages with mitomycin C (follow the procedure described in Step 2.2).
5. Adjust the concentration of the peritoneal macrophages to 5 x 10⁶ cells/mL in the serum-free medium. Add OLP_pool stock (the final DMSO concentration is less than 1%) and M-CSF (final concentration=100 U/mL).
   NOTE: In this MOG OLP study, we used 0.5% DMSO. Thus, MOG OLP_pool stock was diluted 200x (e.g. 1 μL of MOG OLP_Pool stock was added into 199 μL of peritoneal macrophages). Hence, the final concentration of each OLP was 4.2 μg/mL).
6. Add 200 μL/well (1 x 10⁶ cells/well) in a 48-well tissue culture plate and incubate the plate at 37 °C for 4 h.
7. Remove the non-adherent cells and the polyacrylamide beads by gentle washing with 200 μL/well of the pre-warmed RPMI-0.
8. Collect and pool the OLP_pool primed CD8⁺ T cells from Step 2.10. Adjust the OLP_pool primed CD8⁺ T cell concentration to 1 x 10⁶ cells/mL in the serum-free medium containing 25 U/mL IL-2 and 50 U/mL of IL-7. Add 1 mL/well to the 48-well plate that contains the OLP_pool-pulsed peritoneal macrophages.
9. Four days later, replenish the medium in the 48-well plate. Remove and discard about 400 μL of the culture medium in the 48-well culture plates. Add 500 μL of fresh serum-free medium containing 100 U/mL IL-2 and 100 U/mL of IL-7 into each well. Culture the cells at 37 °C and 5% CO₂.
10. Three or four days later, examine the OLP_pool-restimulated CD8⁺ T cells for OLP_pool-specific, Qa-1-restricted response by enzyme-linked immunospot assay (ELISPOT). After this point, re-stimulate the CD8⁺ T cells every 7 - 10 days.
    NOTE: Macrophages are preferred for the re-stimulation of the OLP_pool-primed CD8⁺ T cells. We noticed that CD8⁺ T cells re-stimulated by macrophage grew better than DCs in vitro.

4. Determination of OLP_pool-specific, Qa-1-restricted Response in an OLP_pool-restimulated CD8⁺ T Cell Line

NOTE: OLP_pool-specific, Qa-1-restricted response in an OLP_pool-restimulated CD8⁺ T cell line is determined by IFNγ secretion following stimulation by the OLP_pool in the presence of C1R or C1R.Qa-1^b cells using an IFNγ ELISPOT assay. C1R cells can be obtained commercially. C1R.Qa-1^b cells can be generated by transducing the C1R cells with the Qa-1 lentiviral vector.

1. Add 100 μL/well of a capture anti-IFN-γ antibody diluted in coating buffer (PBS) into the wells of an ELISPOT plate. Seal and incubate the plate at 4 °C overnight.
2. On the second day, discard the coating buffer containing the capture anti-IFN-γ antibody and add 200 μL/well-blocking solution (serum-free medium) and incubate the plate for 2 h at room temperature.
3. Irradiate the C1R and C1R.Qa-1^b cells with 9,600 Rads.
   1. Alternatively, treat the C1R and C1R.Qa-1^b cells with mitomycin C as described in the Step 2.2 except that the treatment time will be 30 min.
4. Adjust the C1R and C1R.Qa-1^b cells in the serum-free medium to 4 x 10⁶ cells/mL.
5. Discard the blocking solution in the plate and add the C1R and C1R.Qa-1^b cells at 50 μL/well (200,000 cells/well) and mix.
6. Add 50 μL/well of 100x diluted OLP_pool stock (in the serum-free medium) and mix properly. Incubate the plate at room temperature for 2 - 3 h.
7. Adjust the OLP_pool-restimulated CD8⁺ T cells to 1 - 2 x 10⁶ cells/mL in the serum-free medium containing 150 U/mL of IL-7 and add 50 μL/well (50,000 - 100,000 cells/well) to the plate. Centrifuge the plate (58 x g) for 5 min.
8. Incubate the plate at 37 °C and 5% CO₂ overnight.
9. Aspirate cell suspension. Wash wells 2 times with deionized (DI) water (250 μL/well). Allow wells to soak for 5 min at each wash step.
10. Wash wells 3 times with Wash Buffer I (PBS containing 0.05% Tween20). Allow wells to soak for 1 min at each wash step. Discard wash buffer.
11. Add 100 μL of detection antibody diluted in a dilution buffer (PBS containing 10% fetal bovine serum (FBS)).
12. Incubate the plates at room temperature for 2 h.
13. Discard detection antibody solution. Wash wells 3 times with 250 µL/well Wash Buffer I. Allow wells to soak for 1 min at each wash step.
14. Add 100 μL/well of enzyme conjugate (Streptavidin-HRP) diluted in the dilution buffer at 1:100 dilution.
15. Incubate the plates for 1 h at room temperature.
16. Discard enzyme conjugate solution. Wash wells 4 times with 250 µL/well Wash Buffer I. Allow wells to soak for 1 min at each wash step.
17. Wash wells 2 times with 250 µL/well Wash Buffer II (PBS).
18. Add 100 µL of Substrate Solution (mix 1 drop=20 µL of AEC Chromogen with 1 mL of AEC substrate) to each well. Monitor spot development for 5 to 60 min.
19. When desired results start to appear, stop the substrate reaction by washing wells with distilled water.
20. Air-dry the plates at room temperature for 2 h or overnight until it is completely dry. Removal of the plastic tray under the plates will facilitate drying. Store plates in a sealed plastic bag in the dark until it is analyzed.
21. Enumerate spots manually by inspecting under a dissecting microscope or using an ELISPOT plate reader. See the representative result in Figure 2.

5. Determination of Individual Peptides in the OLP_pool which Stimulate to the Qa-1 Restricted IFN-γ Secretion in an OLP_pool-specific CD8⁺ T Cell Line

1. Determine the individual peptides that stimulate the OLP_pool-specific, Qa-1-restrictred IFN-γ secretion in an OLP_pool-specific CD8⁺ T cell line using the above described IFN-γ ELISPOT assay (final concentration of each individual peptide used for the ELISOPT assay is 10 μg/mL). See representative results in Figure 3.
   NOTE: An OLP-specific, Qa-1-restircted response is defined as a response that is at least three times of the response in the presence of C1R.Qa-1^b cells but without the OLP. In the MOG OLP study, OLP68, OLP96, and OLP105 all met this criterion. Therefore, all the three OLPs potentially contain Qa-1 epitopes¹² (Figure 3). However, the OLP105 consistently gave the strongest response; we hence performed a detailed analysis of the OLP105 (Figure 4 and Figure 5)¹².

6. Identification of the Optimal Qa-1 Epitope in a 15-mer OLP which Stimulates the Epitope-specific, Qa-1-restricted Response in an OLP_pool-specific CD8⁺ T Cell Line

1. Synthesize C- and N-terminally truncated peptides of a 15-mer OLP as shown in Figure 4.
2. Examine IFN-γ secretion in an OLP-specific CD8⁺ T cell line by stimulating the CD8⁺ T cells with each of the truncated peptides as well as the parental 15-mer OLP in the presence of C1R or C1R.Qa-1^b cells using the above described IFN-γ ELISPOT assay. The final concentration of each individual peptide used for the ELISPOT assay is 10 μg/mL. A peptide is defined as the optimal Qa-1 epitope if the peptide: 1) consistently gives similar or stronger IFN-γ response, as compared to the original 15-mer peptide, in the presence of C1R.Qa-1^b cells; 2) is between 8 - 10-mer (9-mer peptide preferred) which are the peptide lengths bound to MHC-I molecules^15,17. See Representative Results in Figure 5.

Wyniki

Design of an OLP library covering the whole length of a protein

Beginning at the N-terminus of a protein, each peptide is 15 amino acids (15-mer). Hence, the first peptide spans position 1 to position 15. The N-terminus of the second peptide overlaps with the C-terminus of the first peptide by 11 amino acid. Hence, the second peptide spans the position 5 to position 19. Design the rest of the peptides to the end of C-terminus of th...

Dyskusje

Here, we have described a protocol for analyzing Qa-1 epitopes in a protein. In relation to this protocol, several other strategies were also reported previously. First, allogeneic CD8⁺ T cell lines and clones were used for the identification of the Qdm¹. Second, a putative Qa-1-binding motif from the analysis of Qdm was used for the identification of the HSP60p216-224 and a TCRBV8.1 epitope^9,18. Third, individual overlapping pe...

Materiały

Name	Company	Catalog Number	Comments
The protein to be analyzed	N/A	N/A	Sequence of the protein can be obtained from NCBI
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	Cat#: D2650 SIGMA	DMSO should be sterile and cell culture tested.
Kb-/-Db-/- mice	The Lackson Laboratory	Stock#: 019995	We used Taconic H2-KbH2-Db doube knockout mice (Cat#: 4215-F and 4215-M) which however are not available anymore.
AIM V Serum Free Medium	ThermoFisher Scientific	Cat#: 12055091
2-mercaptoethanol	ThermoFisher Scientific	Cat#: 21985023
Sodium pyruvate	ThermoFisher Scientific	Cat#: 11360070
Nonessential Amino Acids	ThermoFisher Scientific	Cat#: 11140076
Dynabeads CD8 Positive Isolation Kit	ThermoFisher Scientific	Cat#: 11333D
Bio-Gel P-100	Bio-Rad	Cat#: 150-4171
Phoshate Balanced Solution (PBS)	ThermoFisher Scientific	Cat#: 20012027
Trasfer pipette	Globe Scientific	Mfg#: 137238
Murine M-CSF	PeproTech	Cat#: 315-02
48-well tissue culture plates	USA Scientific	Cat#: CC7682-7548
Corning Costar TC-treated Multiple well Plates, 96-well, V-shaped bottom	Sigma-Aldrich	Cat#: Z372129 Sigma
1ml deep 06-well PP plate, sterile	USA Scientific	Item#: 1896-1110
Recombinant murine IL-2	PeproTech	Cat#: 212-12
Recombinant murine IL-7	PeproTech	Cat#L: 217-17
Capture anti-IFN-γ antibody	BD Biosciences	Cat#: 551881
ELISPOT plate	Sigma-Aldrich	Cat#: S2EM004M99
C1R	ATCC	Cat#: ATCC CRL-1993
C1R.Qa-1b			Custom made (GenBank access#: NM_010398.3)
Qa-1 lentiviral vector	GeneCopoeia	Product#: Mm02955
Detection anti-IFN-γ antibody	BD Biosciences	Cat#: 551881
Tween20	Sigma-Aldrich	Cat#: P9416
Streptavidin-HRP	BD Biosciences	Cat#: BD557630
AEC substrate	BD Biosciences	Cat#: 551951
ImmunoSpot Analyzer	ImmunoSpot		Any immunoSpot analyer should work for this purpose.