The goals of this ELISA-based method are first to determine the binding affinity of a cytokine to its receptor and second, to measure the blocking effects of inhibitory peptides of the ligand-receptor interaction. This method can help answer key questions in the field of protein-protein interactions, such as cytokine-receptor binding. Binding affinities and blockade of these may increase the general understanding of interaction dynamics.
The main advantage of this technique is, it is easy to perform, it generates label receptors, and it is relatively cheap, due to the easy access of readily available such as ELISA reader. Though this method can provide insight into to cytokine receptor interaction, it can also be applied to other systems such as general protein-protein interactions and the designing of blocking compounds to inhibit the specific interactions. Prepare the solutions as described in the text protocol.
Make sure to filter the carbonate coating buffer with a 0.22 micron PES membrane. Use a vacuum. Also, make 10 different concentrations of each ligand or peptide.
There are two assays described in this video, the first is the direct ligand receptor interaction, ELISA. It can be used to measure the receptor-ligand dissociation constant, which represents receptor-ligand binding affinity. The second assay is a recently optimized competition ligand-receptor interaction ELISA.
This allows for the screening of peptides and other blocking compounds, which act to interfere with the receptor-ligand interaction. For efficiency, prepare to use multichannel pipettes for 96-well plates, and when decanting, just dump out their contents. Begin this procedure by coating the plate with a recombinant receptor.
First, dilute the receptor in carbonate buffer to 100 nanograms per microliter. Then, load the plate wells with 100 microliters of receptor solution. Exclude the outer walls to avoid dealing with the plate edge artifact.
Cover the plate and incubate it at 4 degrees Celsius overnight. Perform all the plate incubations with gentle swirling. The next day, remove the coating solution and wash the plate three times with the wash solution, PBS with a touch of Tween 20.
Next, block the free receptor binding sites by adding 200 microliters of 5%PSA. Let the plate incubate for two hours at room temperature to complete the block. Later, empty the wells and wash the plate as before.
Now, load the wells with 100 microliters of the various dilutions of recombinant his-tag ligand. Load each concentration in duplicate. Load the blank wells with PBS alone.
Then, incubate the plate for two hours at room temperature. Following the incubation with the ligands, wash the plate three times with washing solution. Then, pipette 100 microliter aliquots of primary anti-his mouse monoclonal antibody into each well and incubate the plate at room temperature for two hours.
After washing out the antibody, to each well, add 100 microliters of HRP coupled goat anti-mouse IGG secondary antibody solution. Wrap the plate with aluminum foil to avoid the light. Then, incubate the plate for 45 minutes at room temperature.
Use the standard washing technique to remove the secondary antibody. Now, to each well, add 100 microliters of freshly-prepared TMB made from equal amounts of stock substrate solutions A and B.Then, keep the plate at room temperature for 15 to 30 minutes. Then, after sufficient color development, add 50 microliters of stock solution to each well.
The protocol is based on the assumption that the messaged signal raises from specific binding. It may be necessary to estimate the contribution of non-specific binding to the signal. Then, read the plate absorbance values at 450 nanometers and proceed with calculating the KD value.
The competition LRA procedure follows the same steps as the direct LRA procedure, except for a few important changes. After washing the excess coating ligands off the plate, block the plate wells. Add 200 microliters of 5%BSA solution to each well and incubate the plate for two hours at room temperature.
During the incubation, prepare the recombinant his-tag ligands at a fixed concentration of 10 nanograms per milliliter in PBS. Then, prepare the blocking peptide at different concentrations in PBS, ranging from 10 nanomolar to 100 micromolar to guarantee a dose-response curve. Thus, the IC50 value for the blocking peptide can be found.
In the control wells, load the fixed ligand concentration without peptide to derive the maximum binding. In the blank wells, add only PBS without ligand or peptide. In the other wells, add 50 microliters of the ligands and 50 microliters of each peptide concentration.
Then, incubate the plate for two hours at room temperature and proceed as usual. The dissociation constants between three different interferon ligand peptides and the receptor alpha subunit IL28R-a were determined using the direct LRA, the fraction of occupied binding sites is plotted against the logarithm of the respective interferon concentration. These results illustrate a binding curve which can be analyzed to estimate the KD value by fitting the data to the Hill equation.
A Scatchard plot illustrates cooperativity in ligand binding, ultimately interferon ligand 1 has the highest binding affinity, followed by interferon ligand 2 and interferon ligand 3. The Hill coefficient value of more than 1 suggests increased affinity for additional ligands after the initial ligand receptor interaction. Next, competitive LRA was used to quantitate the impact of a blocking peptide on the interaction between the interferon ligand peptides and the receptor subunit.
The fraction of occupied binding sites for an interferon ligand peptide at 10 nanograms per milliliter is plotted against the log of the interferon peptide's concentration. From these data, the IC50 values were estimated. According to the calculated values, the blocking peptide inhibited the interaction between interferon ligand 3 and IL28R-a to the greatest extent.
Once mastered, this technique, it can be done in eight hours. If it is performed properly. While attempting this procedure, it is important to remember that each saturation concentration of ligand receptor binding, following this procedure, the other methods like can be performed to obtain a more detailed KD values.
After its development, this technique paved the way for researchers in the field of ligand-receptor interactions to explore inhibitory substances to block these interactions.
