How to: Competition-based Binding Assay

A competition-based binding assay enables us to evaluate the binding affinity of a peptide to an HLA type. It allows us to identify high-affinity peptides and, subsequently, select optimal candidates e.g. for a therapeutic HPV vaccine.

Briefly, this method is based on the prevention of a fluorescent consensus peptide binding to its designated HLA binding groove. Therefore we need cells expressing the HLA class I molecule of interest on their surface, fluorescently labeled reference peptides, and unlabeled test peptides. Cells will be stripped from their natural HLA-bound peptides and then incubated with a mix of labeled reference peptide and titrating concentrations of test peptide. Our final read will be the fluorescence intensity measured by flow cytometry.

This assay is adapted from Kessler, Jan H. et al., Current Protocols, 2004.

Reagents

• Peptides (resuspended in DMSO, 10 mg/ml)
• B-LCL cells (with respective HLA class I type)
• B-LCL media (RPMI-1640, 15% FBS, 1% L-Glutamine, 1% 100 mM sodium pyruvate)
• Elution buffer (50% 0.263 M citric acid, 50% 0.123 M Na2HPO4, pH 2.9 or 3.1, adjusted with HCl, in Millipore water)
• PBS
• DMSO
• β-2 microglobulin
• FACS buffer (PBS+ 1% FBS)
• PFA (1%, in PBS)
• 96-well plates (2x, V- or U-bottom)

Protocol

Assay preparation

1. Dissolve lyophilized peptides in sterile DMSO to a final concentration of 10 mg/ml. Ensure proper solution and store aliquots at -80°C. Avoid freeze-thawing and handle peptides under sterile conditions when in use. To ensure that all test peptides experience the same freeze-thaw cycles, pipette the volume of peptide needed for 100 ul 1 mM dilution into PCR tubes. For the fluorescent peptide, prepare working aliquots of 1 ul in a light-protected environment.
   Gather the following peptides:

• Fluorescent peptide (with good binding for the particular HLA molecule being investigated, refer to Kessler et al. for sequences)
• Positive control peptide (the same as the fluorescent peptide, but unlabelled)
• Negative control peptide (known non-binder)
• Test peptides

1. Shortly before the experiment, dilute the test peptides further to 1 mM (100 ul) with PBS.
2. Prepare the titration plate: In the first 96-well plate we prepare a serial dilution of our test peptides ranging from 600 to 4.7 uM using eight 2-fold dilution steps. Fill the wells as indicated in the example plate map and then pipette 75 ul from top to bottom with a multichannel pipette.
3. Prepare the incubation plate: In our second 96-well plate we prepare the fluorescent peptide. Dilute the 1 ul fluorescent peptide (FL) aliquot with 9 ul PBS (1:10). Use this stock to then prepare 2.5 ml FL solution (900 nM). Add 25 ul of this FL solution to the wells of the test peptides and the positive- and negative-control peptides. Add 25 ul plain PBS to the according wells to test background fluorescence. Again, minimum fluorescence is achieved when we only have the fluorescent peptide bound to the HLA molecule and no competition peptide is present. Minimum fluorescence is achieved with no (fluorescence) peptide at all. If a test peptide is a strong binder, it will prevent the FL from binding, causing a decrease in fluorescence.
4. Combine titration and Incubation plate: Transfer 25 ul from each well of the titration plate to the parallel wells of the incubation plate. Use fresh pipette tips for each well and make sure to mix the test peptides before transfer. Store the plate at 4°C in the dark until use. Depending on peptide sequence, this plate can be stored for up to 3 days before adding cells.
5. Prepare cells: We will need 4x10⁴ cells per well. Since some cells are lost during the washing, stripping, etc. procedures, prepare at least 1.5-fold of the total number of cells (6x10⁴ cells per well). For approx. 100 wells this means we need 6x10⁶ cells. Harvest cells, count them, and centrifuge for 5 minutes at 400 xg. Discard the old media and wash cells twice with PBS, again at 400 xg for 5 minutes. Discard the PBS and carefully loosen the cell pellet (e.g. by dragging it along the cell culture hood grid). Cool cells for at least 5 minutes on ice. Resuspend the cell pellet in 2 ml ice-cold elution buffer for PRECISELY 90 seconds. Stop the stripping by adding 12 ml ice-bold B-LCL media. This step removes naturally bound peptides from the HLA molecules, but also the β-2 microglobulin subunit of the HLA itself. IMMEDIATELY centrifuge cells for 3 minutes at 400 xg. Discard the supernatant and quickly resuspend the pellet in media. This will restore a neutral pH. Centrifuge again. Discard media and resuspend pellet in 10 ml B-LCL media containing 2 ug/ml β-2 microglobulin (6x10⁵ cells/ ml).
6. Combine cells and peptides: Transfer the stripped cells to a reservoir and transfer 100 ul of cell suspension to each well of the incubation plate. Carefully pipette up and down when adding the cells. Avoid air bubbles. Use fresh pipette tips for each well.
7. Incubate cells with peptides for 22–26 hours at 4°C in the dark.

Example titration plate.

Example incubation plate.

Cell fixation and data acquisition

1. Resuspend the cells and centrifuge the plate for 3 minutes at 300 xg. Discard the supernatant by quickly flipping the plate in the sink. Once you flip the plate, DO NOT turn it upside-down again. This will remove your cells. Carefully vortex the plate or scratch the bottom to loosen the pellets. Wash the cells with 100 ul cold FACS buffer. Centrifuge again for 3 minutes at 300 xg and discard the supernatant.
2. Resuspend the cells in 100 ul PBS containing 1% PFA for 15 minutes at 44°C in the dark. This will fixate the cells. Add 100 ul FACS buffer to each well and centrifuge again. Wash with FACS buffer as described before.
3. Resuspend the cells in a final volume of 60 ul FACS buffer.
4. Set up your flow cytometer. We will only use the FSC, SSC, and one fluorescence channel (e.g. FITC for a green fluorescently labeled peptide). Check if there are cells, and set the gates based on our cells- and FL-only-control. Acquire 45 ul of each well in these three channels.
5. Export your raw FACS data as .fcs files and analyze them in your preferred analysis software.

FACS analysis

1. When using FlowJo, drag all your .fcs files into the workspace. Open your first sample in FSC-A vs. SSC-A. Find your cell population and gate on it. Apply this gate to all samples. Next, determine the cell count and the geometric mean of FITC in the gate. Drag both quantifications into the table editor and export it as an Excel file.
2. Calculate the mean of all cells-only and all FL-only conditions.
3. Calculate the binding of each positive-, negative-, and test-peptide as the percentage inhibition of FL peptide binding relative to the minimal mean fluorescence (cells only, MFImin) and the maximal mean fluorescence (FL only, MFImax) using the equation from below.
4. Flip the table horizontally, starting with the lowest peptide concentration on top and the highest concentrations at the bottom.
5. Open Sigma-Plot and enter the % inhibition data. Start an analysis and generate a dose-response curve using nonlinear regression (sigmoidal, logistic 4 parameters). The analysis will generate a curve fit and a results sheet, which will include an R2 value (indicates how well the curve fits the data points) and all other metrics: a, b, c, d.
6. Calculate the concentration of test peptide (x) needed for 50% inhibition (y). This value is our IC50. For reference, a peptide with an IC50 < 5 uM is considered a good binder, 5–15 uM an intermediate binder, 15–100 uM a bad binder, and > 100 uM a non-binder.

Left: Calculation % inhibition. Right: IC50 calculation.

Caution: All samples should be processed in an appropriate BSL cell culture hood.

Tips

• Before preparing the peptide plates, make sure you have enough cells. You don’t want to spend 2 hours preparing everything, just to find out there are no cells.
• The elution buffer should be prepared in advance and stored at 4°C. For HLA-A1/A2/A11/A24/B7 we need a pH 3.1 and for HLA-A3/B15 we need pH 2.9.
• When using the fluorescent peptide, make sure to switch off the lights.
• When running the FACS machine, the cells can settle over time. It can make sense to resuspend them after some time to ensure enough cells are being acquired.
• One plate fits 9 test peptides, or 10 if you want to leave out the negative control peptide. When you gain more experience, you can test more 12 peptides by preparing another plate. For this, double all amounts (cells, FL-peptide mix, …).
• B lymphoblastic cell line B-LCL is a safety level 2 cell line, use a bucket cap when centrifugating. They like to be cultured at densities of 0.5–1x10⁶/ml and form aggregates. Resuspend cells regularly disrupt aggregates. When using flasks, store them in an upright position.
• There exist various types of B-LCL, expressing different HLA class 1.
  B-LCL BSM: HLA-A2/B15
  B-LCL EA: HLA-A3
  B-LCL FH8: HLA-A11
  B-LCL E481324: HLA-A1