We thank Prof. J. Stelling (Department of Biosystems Science and Engineering, ETH Zurich and Swiss Institute for Bioinformatics, Basel, Switzerland) for his critical review of the manuscript.

1. Reagent Preparation
<ol>
	<li>To prepare carbonate coating buffer, dissolve 0.36 g Na2CO3 and 0.84 g NaHCO3in 100 ml distilled water; sterile filter the buffer by using a vacuum driven 0.22 &#181;m polyethersulfone (PES) membrane filter and store at RT until usage.</li>
	<li>Prepare washing solution by adding 0.05% v/v Tween 20 in Phosphate buffered saline (PBS).</li>
	<li>Prepare a 5% Bovine Serum Albumin (BSA) (blocking solution) in PBS solution by dissolving 5 g BSA (&#8805;98%) in 100 ml PBS and store at 4 &#176;C.</li>
	<li>Recombinant Receptor, Ligands and Blocking Peptides
	<ol>
		<li>Reconstitutethe recombinant human interleukin receptor alpha subunit (IL28RA) and recombinant His-tagged ligands of human IFN (IFNL1-3) according to the manufacturer&#39;s instructions and store at -80&#176;C. Synthetize blocking peptides and used as previously described 4. Use PBS to prepare different concentrations of ligands and peptides for use in the assays.</li>
	</ol>
	</li>
	<li>To prepare the primary antibody, dilute 6x His Mouse monoclonal antibody in PBS with 0.1% BSA at 1:1,000 dilution. To prepare the secondary antibody, dilute horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (H+L) in PBS with 0.1% BSA at 1:10,000 dilution.</li>
	<li>Prepare TMB solution by mixing the reagents A and B according to the manufacturer&#39;s instructions.</li>
	<li>Prepare stop solution by adding 5 N sulphuric acid (H2SO4) in distilled water and store at RT.</li>
</ol>
2. Enzyme-linked Immunosorbent Assays (ELISAs)
NOTE: The direct ligand-receptor interaction ELISA (direct LRA, Figure 1) can be used to measure the receptor-ligand dissociation constant (KD), as a measure of the receptor-ligand binding affinity. The competition ligand-receptor interaction ELISA (competition LRA, Figure 2) allows screening of peptides (and other blocking compounds), which act to interfere with the interaction between ligand and receptor. The basic protocol that was previously published 5 was further optimized. 
NOTE: In both ELISA methods use multichannel pipette for adding solutions to the wells of 96-well plate in each step. In solution decant or washing steps, throw out the solutions directly into the sink.
<ol>
	<li>Direct Ligand-Receptor-Interaction Assay (direct LRA) 
	NOTE: For an illustration of the workflow (see Figure 1).
	<ol>
		<li>Coating Plate with Recombinant Receptor
		<ol>
			<li>Dilute the recombinant receptor in carbonate buffer to a final concentration of 100 ng/&#181;l. Coat wells of 96-well microtitre plate with fixed receptor concentration (100 ng/&#181;l) by pipetting 100 &#181;l to each well using a multichannel pipette. Exclude outer walls of the plate to avoid well edge artifact. Cover the plate with a lid and incubate the plate at 4 &#176;C O/N.</li>
		</ol>
		</li>
		<li>Blocking and Addition of Ligands
		<ol>
			<li>The next day, remove the coating solution by tilting the plate against the sink and wash the plate 3 times with washing solution (PBS + 0.05% v/v Tween 20).</li>
			<li>Block the free receptor-binding sites in the coated plate using 200 &#181;l of 5% BSA solution to each well using a multichannel pipette and incubate the plate for 2 hr at RT.</li>
			<li>Discard the blocking solution (see step 2.1.2.1.) and wash the plate 3 times with washing solution.</li>
			<li>Prepare the recombinant His-tagged ligands at different concentrations (e.g., 8 &#181;g/ml, 4 &#181;g/ml, 2 &#181;g/ml, 1 &#181;g/ml, 0.5 &#181;g/ml, 0.25 &#181;g/ml, 0.125 &#181;g/ml, 0.063 &#181;g/ml, 0.031 &#181;g/ml, 0.0 &#181;g/ml) in PBS. Add only PBS in the blank wells.</li>
			<li>Add 100 &#181;l of each ligand concentration to the wells in duplicate and incubate the plate for 2 hr at RT allowing receptor-ligand interaction.</li>
		</ol>
		</li>
		<li>Incubation with Antibodies
		<ol>
			<li>Following incubation with the ligands, wash the plate 3 times with washing solution.</li>
			<li>Pipette 100 &#181;l of primary anti-His mouse monoclonal antibody solution (1:1,000) to each well.</li>
			<li>Incubate the plate at RT for 2 hr; after incubation, discard the antibody solution (see step 2.1.2.1.) and wash the plate 3 times with washing solution.</li>
			<li>Add 100 &#181;l of HRP coupled goat anti-mouse IgG secondary antibody solution (1:10,000) to each well. Incubate the plate for 45 min at RT.</li>
			<li>Discard the antibody solution (see step 2.1.2.1.) and wash the plate 3 times with washing solution.</li>
		</ol>
		</li>
		<li>Addition of Substrate and Development
		<ol>
			<li>Bring the TMB substrate solutions to RT and prepare TMB substrate solution A and B at 1:1 ratio. Add 100 &#181;l freshly prepared substrate to each well and keep the plate at RT for 15-30 min. After sufficient color development add 50 &#181;l stop solution.</li>
		</ol>
		</li>
		<li>Reading the Plate and Data Analysis 
		NOTE: The described protocol is based on the assumption that the measured signal rises from specific binding. It might be necessary to estimate the contribution of unspecific binding to the signal but this is out of the scope of this protocol.
		<ol>
			<li>Read the absorbance (optical density, OD) directly at 450 nm.</li>
			<li>Subtract the background signal from the measured OD values and normalize them. Transform all values of the ligand concentration to logarithmic scale (base 10, log10).</li>
			<li>Plot the normalized and background corrected OD values (Y-axis, corresponds to the fraction of occupied receptor binding sites) against the logarithm of the ligand concentration (X-axis, log10 scale).</li>
			<li>To estimate the KD value, fit the data to the following form of the Hill equation: 
			<img alt="Equation 1" src="/files/ftp_upload/53575/53575eq1.jpg" /> 
			NOTE: Here Y denotes the fraction of occupied receptor binding sites and Ymax the maximal binding; [L] denotes the concentration of free ligand and the Hill coefficient. If there is only one binding site for the ligand, the Hill coefficient is n = 1. For systems with more than one ligand binding site, the binding exhibits positive cooperativity if n &#62;1, negative cooperativity if n&#60;1 and no cooperativity if n = 1. The microscopic dissociation constant is termed and corresponds to the half maximal effective concentration EC506. The apparent dissociation constant is Kd = (KD)n. In the simplest case where n = 1, the dissociation constant corresponds to the ligand concentration at which half of the receptor binding sites are occupied and Kd = KD. This model assumes mass action binding under equilibrium conditions, as well as that only a small fraction of the added ligand is bound to the receptor, i.e., [L] &#62;&#62; [RL].</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53575/53575fig1.jpg" /> 
Figure 1.&#160;Direct ligand-receptor-interaction assay (direct LRA). Step-by-step protocol for direct LRA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53575/53575fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Competition Ligand-Receptor-Interaction Assay (competition LRA) 
	NOTE: For an illustration of the workflow see Figure 2. The competition LRA procedure follows the same steps as the direct LRA (coating the plate, antibody incubation, plate development) except for important changes in the ligand and peptides addition step. Proper negative controls are essential for this assay. In a previous screening study 4, the scrambled blocking peptide did not show antagonistic effects.
	<ol>
		<li>Blocking - Addition of Ligands and Blocking Peptides
		<ol>
			<li>The next day, remove the coating solution and wash the plate (see 2.1.2.1).</li>
			<li>Block the coated plate by adding 200 &#181;l of 5% BSA solution to each well and incubate the plate for 2 hr at RT.</li>
			<li>Prepare the recombinant His-tagged ligands (IFNL1-3) at a fixed concentration (2x-20 ng/ml) in PBS.</li>
			<li>Prepare the blocking peptide (cf. Table 3) with different concentrations ranging from 10 nM to 100 &#181;M in PBS to guarantee a dose-response curve. 
			NOTE: This enables subsequent determination of the IC50 value for the blocking peptide. In control wells, add only fixed ligand concentration without peptide to derive the maximum (100%) binding. In the blank, add only PBS without ligand or peptide.</li>
			<li>Add 50 &#181;l of the ligands (IFNL1-3) and 50 &#181;l of each peptide concentration to the wells in duplicates.</li>
			<li>Incubate the plate for 2 hr at RT.</li>
		</ol>
		</li>
		<li>Reading the Plate and data Analysis 
		NOTE: The described protocol is based on the assumption that the measured signal rises from specific binding. It might be necessary to estimate the contribution of unspecific binding to the signal but this is out of the scope of this protocol.
		<ol>
			<li>Read the absorbance (optical density, OD) directly at 450 nm.</li>
			<li>Subtract the background signal from the measured OD values and normalize them. Transform all values of the peptide concentration to logarithmic scale (base 10, log10).</li>
			<li>Plot the normalized and background corrected OD values (Y-axis, corresponds to the fraction of occupied receptor binding sites) against the logarithm of the ligand concentration (X-axis, log10 scale).</li>
			<li>To estimate the IC50 value, fit the data to the following equation: 
			<img alt="Equation 2" src="/files/ftp_upload/53575/53575eq2.jpg" /> 
			NOTE: Here [P] is the peptide concentration and the Hill slope. The Hill slope describes the steepness of the dose-response curve. The IC50 corresponds to the inhibitor concentration at which 50% inhibition of binding between ligand and receptor is observed.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/53575/53575fig2.jpg" /> 
Figure 2.&#160;Competition ligand-receptor-interaction assay (competition LRA). Step-by-step protocol for competition LRA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53575/53575fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol>
	<li>Schneider, P., Willen, L., Smulski, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+and+techniques+to+study+ligand-receptor+interactions+and+receptor+activation+by+TNF+superfamily+members.">Tools and techniques to study ligand-receptor interactions and receptor activation by TNF superfamily members.</a> Methods in enzymology. 545, 103-125 (2014).</li><li>Rossi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensor+analysis+of+anti-citrullinated+protein/peptide+antibody+affinity.">Biosensor analysis of anti-citrullinated protein/peptide antibody affinity.</a> Analytical biochemistry. 465, 96-101 (2014).</li><li>van der Merwe, P. A., Barclay, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell-adhesion+molecule+interactions+using+surface+plasmon+resonance.">Analysis of cell-adhesion molecule interactions using surface plasmon resonance.</a> Curr Opin Immunol. 8, 257-261 (1996).</li><li>Egli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-28B+is+a+key+regulator+of+B-+and+T-cell+vaccine+responses+against+influenza.">IL-28B is a key regulator of B- and T-cell vaccine responses against influenza.</a> PLoS Pathog. 10, e1004556 (2014).</li><li>Rosenbluh, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positively+charged+peptides+can+interact+with+each+other,+as+revealed+by+solid+phase+binding+assays.">Positively charged peptides can interact with each other, as revealed by solid phase binding assays.</a> Analytical biochemistry. 352, 157-168 (2006).</li><li>Goutelle, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hill+equation:+a+review+of+its+capabilities+in+pharmacological+modelling.">The Hill equation: a review of its capabilities in pharmacological modelling.</a> Fundamental &amp; clinical pharmacology. 22, 633-648 (2008).</li><li>Levin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptides+derived+from+HIV-1+integrase+that+bind+Rev+stimulate+viral+genome+integration.">Peptides derived from HIV-1 integrase that bind Rev stimulate viral genome integration.</a> PLoS One. 4, e4155 (2009).</li><li>Egli, A., Santer, M. D., O'Shea, D., Tyrrell, D. L., Houghton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+the+interferon-lambda+family+on+the+innate+and+adaptive+immune+response+to+viral+infections.">The impact of the interferon-lambda family on the innate and adaptive immune response to viral infections.</a> Emerging infectious diseases. , e51 (2014).</li><li>Gad, H. H., Hamming, O. J., Hartmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+human+interferon+lambda+and+what+it+has+taught+us.">The structure of human interferon lambda and what it has taught us.</a> J Interferon Cytokine Res. 30, 565-571 (2010).</li><li>Folch, B., Rooman, M., Dehouck, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermostability+of+salt+bridges+versus+hydrophobic+interactions+in+proteins+probed+by+statistical+potentials.">Thermostability of salt bridges versus hydrophobic interactions in proteins probed by statistical potentials.</a> Journal of chemical information and modeling. 48, 119-127 (2008).</li><li>Yuzlenko, O., Lazaridis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+ionizable+amino+acid+side+chains+at+a+lipid+bilayer-water+interface.">Interactions between ionizable amino acid side chains at a lipid bilayer-water interface.</a> The journal of physical chemistry. B. 115, 13674-13684 (2011).</li><li>Tissot, A. C., Vuilleumier, S., Fersht, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+two+buried+salt+bridges+in+the+stability+and+folding+pathway+of+barnase.">Importance of two buried salt bridges in the stability and folding pathway of barnase.</a> Biochemistry. 35, 6786-6794 (1996).</li></ol>

A.E. was supported by a research grants from the "SNSF Ambizione Score" program (PZ00P3_154709), "Forschungsfond, F&#246;rderung strategischer Projekte" University of Basel, Stiftungsinfektionskrankheiten Basel, and Bangeter Rhyner Stiftung. J.L. acknowledges support by an iPhD fellowship of the SystemsX.ch initiative in systems biology program (9th call).

ELISA is a standard and well-established method for many laboratories. We have further modified and improved a previously published method 5,7. The demonstrated step-by-step protocol shows how it can be used in a simple way to determine the KD values of ligand-receptor interactions. In addition, the IC50 of a blocking peptide that interferes with the ligand-receptor interaction can be determined.
Major advantages are the rapid setup, easy preparation of reagents and familiar handling, as most researchers have used an ELISA protocol before. The direct LRA protocol is highly flexible and can be adapted to measure many protein-protein interactions. Recombinant proteins with His6- or alternative tag should be used as the binding partner to an immobilized partner. The competition LRA can be exploited as a screening tool to (i) determine the inhibitory potential of blocking compounds (peptides, antibodies, or small molecules) and to (ii) determine the critical interaction sites by using blocking peptides designed to mimic the receptor or the ligand.
Negative controls are essential for a proper interpretation of the presented assays. In a previous screening study 4, the scrambled sequence of the used blocking peptide did not show antagonistic effects. However, other peptides showed a blocking capacity also after scrambling, likely due to unspecific electrostatic interactions.
A potential limitation is that this assay reflects an in vitro situation. In particular, heterodimeric receptors often form a more complex structure. It is not possible to distinguish whether the ligand just binds to the receptor or whether the ligand also activates the receptor by triggering a conformational change or a dimerization, which in turn leads to an intracellular signal. In the presented assay, we used a recombinant receptor, which is immobilized to a solid phase. This setup does not work to test the activation or to investigate the interaction of receptors, which require the membrane environment or membrane cholesterol such as G protein coupled receptors (GPCRs). Also the use of recombinant protein raises caveats. For example, the folding and tertiary structure of a recombinant protein may be different compared to an in vivo situation. The binding of ligand and receptor usually occurs at RT, however in humans the optimal temperature would be 37 &#176;C. Finally, the use of commercial recombinant ligand and receptor can prove expensive. Despite these limitations, these two ELISA protocols show potential to rapidly explore the ligand-receptor interaction.
The presented results show that IFNL1 has a slightly higher affinity for IL28RA compared to IFNL2, and the affinity of IFNL3 is three-fold lower than IFNL2. This is remarkable considering the similarity between IFNLs. IFNL1 and IFNL2 differ in 33 amino acids while IFNL2 and IFNL3 differ only in seven amino acids 8. The interaction between IL28RA and IFNLs involves Helix A and the AB-loop of IFNL 9. Alignment of IFNL sequences reveals four significant differences in Helix A and the AB-loop between IFNL1 and IFNL3 (Figure 5A). One affects the salt bridge Arg54-Glu119. The amino acid residues in this section are enumerated according to the UniProt entries Q8IU54 (IFNL1), Q8IZI9 (IFNL3), Q8IZJ0 (IFNL2) and Q8IU57 (IL28RA), which has been found in the crystal structure of the IFNL1-IL28R1 complex (Figure 5B). Structural alignment shows that Arg57 in IFNL1 is replaced by Lys57 in INFL3, which is also able to form a salt bridge with Glu118 (Figure 5C). Consistent with the decreased affinity of IFNL3 and IL28RA, computational 10,11 and mutational 12 analyses show, that Lys-Glu salt bridges are in general less stable than Arg-Glu salt bridges.
However, the differences in Helix A do not satisfactorily explain the lower affinity of IFNL3, since the amino acid sequence of Helix A is identical for IFNL2 and IFNL3. It is thought that the main difference arises from the mutations in the AB-loop, where Arg74 and His76 in IFNL2 are replaced by Lys70 and Arg72 in IFNL3 8.
Moreover, differences in stability and solubility between IFNLs may also affect the outcome of the assay. Since the direct LRA assay uses concentrations from nM to M and the physiological concentration of cytokines in serum lies in the pM to nM range, aggregation of IFNL cannot be excluded. We can assume that the observed positive cooperativity of the IFN binding is caused by a specific or non-specific increase in ligand-receptor binding, e.g., a dimerization of the recombinant IL28RA receptor in solution, or a binding of a second ligand or ligand fragment with higher affinity. However, further studies are required to verify this.
The blocking peptide used in the competition LRA mimics the AB-loop of IFNL3 (Figure 6). As previously described, the AB-loop plays an essential role in the interaction between IFNLs and IL28RA 9, particularly IFNL2 and IFNL3. Homology modeling of IFNL3 with the IL28RA/IFNL1 crystal structure shows that the region, which corresponds to the blocking peptide lies in close spatial proximity to the interaction interface with IL28RA (Figure 6). Supposed that the peptide blocks the interaction of the AB-loop of IFNL and IL28RA. The results of the competition LRA support this: The peptide has an inhibitory effect on the binding of all IFNLs, however the peptide is a more effective inhibitor of the interaction between IL28RA and either IFNL3 or IFNL2 than of the interaction of IFNL1 and IL28A. As expected, the peptide blocks the binding of IFNL3 most effectively since it occupies exactly the same binding pocket as the AB-loop of IFNL3.
As shown in Table 3, the AB-loops of IFNL1 and IFNL2 aligned to the blocking peptide differ. Consistent with the lower IC50 value of the peptide for inhibition of the IFNL1/IL28RA interaction, twelve amino acids differ between IFNL1 and the peptide whereas and only two amino acids differ between IFNL2 and the peptide. This indicates that there are slightly different binding modes for interaction between the AB-loops of the IFNLs and IL28RA and predicts that peptides which mimic IFNL1 would be more effective at blocking the interaction between IFNl1 and IL28RA than the interaction between IFNL3 and IL28RA.
Further, the peptide is overall positively charged (four arginine and two lysine residues but only two aspartate residues) and computational analysis shows that the peptide has no defined secondary motifs. Due to its coiled, flexible structure the peptide might also bind to other regions of the receptor. This could potentially block glutamate and aspartate residues such as D118, which forms a salt bridge to stabilize the ligand-receptor complex (Figure 5B and 5C).
<img alt="Figure 5" src="/files/ftp_upload/53575/53575fig5.jpg" /> 
Figure 5.&#160;Structural comparison of IFNL1 (green) and IFNL3 (blue). Oxygen atoms are shown in red, nitrogen atoms are shown in blue. (A) Superposition of the IL28RA-bound IFNL1 and IFNL3 (IL28RA not shown). The root mean square deviation (RMSD) of all aligned atoms is 0.672 &#197;. Side chains of IFNL3 that differ from IFNL1 are highlighted in light-pink. (B) Salt bridge between Arg54 and D118. (C) IFNL3 aligned to IL28RA-bound IFNL1 (IL28RA is shown in grey). The alignment shows that the Arg-Glu salt bridge is probably replaced by a less stable Lys-Glu, a salt-bridge between Lys57 and D118. PyMol was used for the preparation of figures and for the alignment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53575/53575fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53575/53575fig6.jpg" /> 
Figure 6.&#160;Alignment of IFNL3 and the IFNL1-IL28RA complex (IFNL1 not shown). IFNL3 is shown in blue and the IL28RA in grey. The regions corresponding to the blocking peptide are highlighted in purple. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53575/53575fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 3" src="/files/ftp_upload/53575/53575table3.jpg" /> 
Table 3: Sequence alignment of IFNL1-3 and inhibitory peptide. The peptide mimics the AB-loop of INFL3. Amino acid mismatches are highlighted in red.

A comprehensive understanding of signaling pathways requires detailed knowledge about the ligand-receptor interaction. Most methods for assessing the interaction of a particular ligand with its specific receptor are expensive, time consuming, labor intensive and require specific equipment and expertise 1.
This article describes two fast and reliable point-by-point protocols to investigate the ligand-receptor interaction based on an enzyme linked immunosorbent assay (ELISA): the direct ligand-receptor interaction assay (LRA) and the competition LRA. ELISA is a highly sensitive, specific and readily available technique, routinely used in almost every laboratory. ELISA can be performed and adapted in various fashions. The presented protocols are optimized for the investigation of the interaction between different lambda interferons (INFLs) and their receptor.
The direct LRA allows for a quantification of ligand-receptor binding with respect to ligand concentration and thus yields a binding curve. Using an appropriate model for the ligand-receptor interaction, the data can be further analyzed to estimate the dissociation constant (KD).
In the presented protocol, the commonly used Hill equation is applied to model the ligand-receptor binding. Although other methods such as the surface plasmon resonance technology 2,3 allow the determination of the binding affinities between two proteins, this technology is often labor intensive, expensive, and requires special laboratory equipment.
The competition LRA enables the screening of inhibitory peptides: The ligand-receptor binding is quantified with respect to peptide concentration. This yields a dose-response curve describing the inhibitory effect of the peptide. The data can be further analyzed to estimate the half maximal inhibitory concentration (IC50) of the blocking peptide.
Both ELISA protocols are easy to use and can be adapted to a broad range of research questions. Recombinant proteins of any kind can be used to reliably and fast determine the interaction parts. In addition, the competition LRA can be used to determine critical interaction sites of ligands and receptors by using blocking peptides, which are designed to mimic either the ligand or the receptor. If the blocking peptide shows efficient and specific inhibition, the peptide occupies a critical interaction site of the ligand (if the peptide mimics the receptor) or of the ligand (if the peptide mimics the ligand).
The first protocol describes the KD value determination of different INFLs and the alpha subunit of their receptor, i.e., the interleukin-28 receptor (IL28RA) using the direct LRA. Next, the second protocol shows how to determine the capability of a 20 amino acid long peptide to inhibit the INFL-IL28RA interactions. The peptide is designed to compete with IFNLs at their receptor binding site and thus enables a molecular understanding of the interaction. Furthermore, this peptide can be used to block IL28RA in in vitro experiments to determine the impact on downstream signaling effects4.

<table><tbody><tr><td>Nunc-Immunoplate (F96 Maxi sorp)</td><td>Thermo Scientific</td><td>442404</td><td>ELISA plate</td></tr><tr><td>Sodium carbonate (Na2CO3)</td><td>Merck</td><td>497-19-8</td><td>For ELISA plate coating buffer</td></tr><tr><td>Sodium hydrogen carbomnate(NaHCO3)</td><td>Merck</td><td>144-55-8</td><td>For ELISA plate coating buffer</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A7030-100G</td><td>5% BSA in PBS for Blocking</td></tr><tr><td>rhIL-28R&amp;alpha;/IFN&amp;lambda;R1</td><td>R&amp;amp;D systems</td><td>5260-MR</td><td>Recombinant human interlukin-28 Receptor alpha</td></tr><tr><td>rhIL-29/IFN&amp;lambda;1</td><td>R&amp;amp;D systems</td><td>1598-IL/CF</td><td>Recombinant human interlukin-29/Carrier free/C-terminal 10-His tag</td></tr><tr><td>rhIL-28A/IFN&amp;lambda;2</td><td>R&amp;amp;D systems</td><td>1587-IL/CF</td><td>Recombinant human interlukin-28A/Carrier free/C-terminal 6-His tag</td></tr><tr><td>rhIL-28B/IFN&amp;lambda;3</td><td>R&amp;amp;D systems</td><td>5259-IL/CF</td><td>Recombinant human interlukin-28B/Carrier free/C-terminal 6-His tag</td></tr><tr><td>6x His Monoclonal antibody (Mouse)</td><td>Clontech</td><td>631212</td><td>Primary antiboy to capture His tagged Ligands</td></tr><tr><td>Goat anti-Mouse igG (H+L)</td><td>Jackson Immuno Research</td><td>115-035-166</td><td>Horseradish Peroxidase conjucated secondary antibody</td></tr><tr><td>BDoptEIA TMB reagent set</td><td>BD Biosciences</td><td>555214</td><td>ELISA - TMB substrate solution</td></tr><tr><td>Sulfuric acid (H2SO4)</td><td>Fulka</td><td>84720</td><td>5 N H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt; (Enzyme reaction stop solution)</td></tr><tr><td>Synergy/H1 - Microplate reader</td><td>BioTeK</td><td></td><td>ELISA plate reader</td></tr></tbody></table>

an elisa based binding and competition method to rapidly determine ligand-receptor interactions

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

The dissociation constants between INFL1-3 and their receptor alpha subunit IL28RA were determined using the direct LRA. The results are shown in Figure 3: The fraction of occupied binding sites is plotted against the logarithm of the respective IFN concentration. The Scatchard plot of the data is shown in the bottom right corner. The results illustrate that the direct LRA yields a binding curve, which can be further analyzed to estimate the KD value. The KD value was determined by fitting the data to the Hill equation (Equation 1).
IFNL1 has the highest binding affinity, followed by IFNL2 and IFNL3. The Hill coefficient n &#62;1 suggests increased affinity for additional ligands after the initial ligand-receptor interaction (see Discussion). The estimated dissociation constants and Hill coefficients are summarized in Table 1.
Competitive LRA was used to quantitate the impact of a blocking peptide on the interaction between IFNL1-3 and the IL28RA (Figure 4). The fraction of occupied binding sites for a ligand concentration of 10 ng/ml is plotted against the logarithm of the peptide concentration. To estimate the IC50 values, the data is fitted to Equation 2.
The blocking peptide inhibited the interaction between IFNL3 and IL28RA (IC50 = 0.26 &#956;M) to the greatest extent. The IC50 is twice as high for the IFNL2-IL28RA interaction (IC50 = 0.50 &#956;M) and one order of magnitude higher for the IFNL1-IL28RA interaction, indicative the peptide was less effective at disrupting IFNL1-IL28RA interactions. The determined IC50 values and Hill slopes are summarized in Table 2.
Proper data analysis is essential for understanding the ligand-receptor interaction. The shown results were generated using a scientific graphing software such as GraphPad PRISM. For the KD value determination, the data was fitted to the &#39;One site - specific binding with Hill slope&#39; (corresponds to Equation 1 in Sec. 2.2.1). For the IC50 value determination, the data is fitted to the function &#39;log(inhibitor) vs. normalized response - variable slope&#39; (see Equation 2 in Sec. 2.2.2). However, any software for non-linear regression analysis can be used.
<img alt="Figure 3" src="/files/ftp_upload/53575/53575fig3.jpg" /> 
Figure 3.&#160;Results of the direct ligand-receptor assay (direct LRA). Binding curves for the binding of IFNL1 (green), IFNL2 (red) and IFNL3 (blue) to IL28RA. The respective Scatchard plot in the bottom right corner suggests positive co-operativity of the binding. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53575/53575fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53575/53575fig4.jpg" /> 
Figure 4.&#160;Results of the competitive ligand-receptor assay (competitive LRA). Dose-response curves showing inhibition of the binding of IFNL1 (10 ng/ml, green), IFNL2 (10 ng/ml, red) and IFNL3 (10 ng/ml, blue) to IL28RA by the 20 aa peptide. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53575/53575fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/53575/53575table1.jpg" /> 
Table 1: Estimated dissociation constants (KD) and Hill coefficients of IFNL1-3 binding to IL28RA. The standard error (SE) is given for a sample size of four replicates per data point.
<img alt="Table 2" src="/files/ftp_upload/53575/53575table2.jpg" /> 
Table 2: Estimated half maximal inhibitory concentrations (IC50) of the blocking peptides and the Hill slope of the dose-response curve for the binding of IFNL1-3 to IL28RA. The IFN concentration is 10 ng/ml. The number of replicates is three.

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An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

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Nanyang Technological University

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JoVE Journal

Chemistry

18.9K Views.  University of Basel. The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions. 

Video: An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia coli&#160;ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit &#949;&#160;could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of &#949;&#160;undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of &#949;&#39;s conformations is predominant. The assay measures kinetics of &#949;&#39;s binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound &#949;&#160;to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on &#949;&#39;s conformational changes.

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its &#949; subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with &#949;&#8217;s inhibitory C-terminal domain.

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia ...

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a major pharmacological target for multiple indications, including analgesia, blood pressure regulation, and the treatment of psychiatric disease. Upon ligand binding, GPCRs catalyze the activation of intracellular G-proteins by stimulating the incorporation of guanosine triphosphate (GTP). Activated G-proteins then stimulate signaling pathways that elicit cellular responses. GPCR signaling can be monitored by measuring the incorporation of a radiolabeled and non-hydrolyzable form of GTP, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTP&#947;S binding measures a proximal event in GPCR signaling and, importantly, can distinguish agonists, antagonists, and inverse agonists. The present protocol outlines a sensitive and specific method for studying GPCR signaling using crude membrane preparations of an archetypal GPCR, the &#181;-opioid receptor (MOR1). Although alternative approaches to fractionate cells and tissues exist, many are cost-prohibitive, tedious, and/or require non-standard laboratory equipment. The present method provides a simple procedure that enriches functional crude membranes. After isolating MOR1, various pharmacological properties of its agonist, [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO), and antagonist, naloxone, were determined.

Guanosine triphosphate (GTP) binding is one of the earliest events in G-Protein-Coupled Receptor (GPCR) activation. This protocol describes how to pharmacologically characterize specific GPCR-ligand interactions by monitoring the binding of the radio-labeled GTP analog, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), in response to a ligand of interest.

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a ...

Biochemistry

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of &#945;2&#946;1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

A detailed protocol to perform a titration ELISA is described. Moreover, a novel algorithm is presented to evaluate titration ELISAs and to obtain a dissociation constant of binding of a soluble ligand to a microtiter plate-immobilized receptor.

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial ...

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The linker was created using molecular modeling software as a loop structure. Here, we introduce a protocol forin silico analysis of a complete scFv antibody that interacts with the epidermal growth factor receptor (EGFR). The homology modeling, with Pyrx of protein-protein docking and molecular dynamic simulation of the interacting scFv antibody and EGFR First, the authors used a protein structure modeling program and Python for homology modeling, and the antibody scFv structure was modeled for homology. The investigators downloaded Pyrx software as a platform in the docking study. The Molecular dynamic simulation was run using modeling software. Results show that when the MD simulation was subjected to energy minimization, the protein model had the lowest binding energy (-5.4 kcal/M). In addition, the MD simulation in this study showed that the docked EGFR-scFv antibody was stable for 20-75 ns when the movement of the structure increased sharply to 7.2 &#197;. In conclusion, in silicoanalysiswas performed, and the molecular docking and molecular dynamics simulations of the scFv antibody proved the effectiveness of the designed immune-therapeutic drug scFv as a specific drug therapy for EGFR.

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

This antibody homology modeling prediction protocol is followed by antibody-receptor Pyrx docking and molecular dynamic simulation. These three primary methods are used to visualize the accurate antibody-receptor binding areas and the binding stability of the final structure.

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The ...

Genetics

Optimized Methods for the Surface Immobilization of Collagens and Collagen Binding Assays

Fibrosis occurs in various tissues as a reparative response to injury or damage. If excessive, however, fibrosis can lead to tissue scarring and organ failure, which is associated with high morbidity and mortality. Collagen is a key driver of fibrosis, with type I and type III collagen being the primary types involved in many fibrotic diseases. Unlike conventional protocols used to immobilize other proteins (e.g., elastin, albumin, fibronectin, etc.), comprehensive protocols to reproducibly immobilize different types of collagens in order to produce stable coatings are not readily available. Immobilizing collagen is surprisingly challenging because multiple experimental conditions may affect the efficiency of immobilization, including the type of collagen, the pH, the temperature, and the type of microplate used. Here, a detailed protocol to reproducibly immobilize and quantify type I and III collagens resulting in&#160;stable and reproducible gels/films is provided. Furthermore, this work demonstrates how to perform, analyze, and interpret in vitro time-resolved fluorescence binding studies to investigate the interactions between collagens and candidate collagen-binding compounds (e.g., a peptide conjugated to a metal chelate carrying, for example, europium [Eu(III)]). Such an approach can be universally applied to various biomedical applications, including&#160;the field of molecular imaging to develop targeted imaging probes, drug development, cell toxicity studies, cell proliferation studies, and immunoassays.

This work presents an optimized protocol to reproducibly immobilize and quantify type I and III collagen onto microplates, followed by an improved in vitro binding assay protocol to study collagen-compound interactions using a time-resolved fluorescence method. The subsequent step-by-step data analysis and data interpretation are provided.

Fibrosis occurs in various tissues as a reparative response to injury or damage. If excessive, however, fibrosis can lead to tissue scarring and organ ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

Neuroscience

ELISA-Based Reporter Assay to Study the Receptor-Ligand Interaction in BW Cells

Source: Elias, S. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58685">A BW Reporter System for Studying Receptor-Ligand Interactions.</a> J. Vis. Exp. (2019)
This video demonstrates the reporter assay-based detection of the receptor-ligand interaction. The successful receptor-ligand interaction activates the downstream signaling pathway, causing the generation and release of cytokines as secretory reporter molecules in the solution, which is confirmed using ELISA.

Source: Elias, S. et al., A BW Reporter System for Studying Receptor-Ligand Interactions. J. Vis. Exp. (2019)
This video demonstrates the reporter ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-protein interactions

Ligand Binding Sites

Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the ligand-binding site. The specificity of a protein&#8217;s ligand-binding site is determined by the arrangement of its amino acid chain which gives the area its shape and chemical reactivity. Hence, a ligand-binding site provides a complementary shape to its ligand and keeps the ligand in place via chemical interactions. These chemical interactions are often noncovalent; however, since these interactions are reversible and weak, many of these interactions need to occur simultaneously to hold the protein and the ligand together.
Research that elucidates interaction mechanisms at ligand binding sites generally involves in silico modeling and in vitro approaches. In silico modeling uses computers to compare previously known protein structures and evolutionary data to make predictions to determine the optimal binding shape and energy state of the protein-ligand complex. In vitro approaches compliment in silico predictions by providing evidence for ligand binding through binding and kinetic assays in the laboratory. Ligand binding research is important for understanding the functions of proteins and how they perform specific cellular processes in both healthy, as well as in diseased conditions. For instance, certain genetic conditions and cancers can alter the sequence of a protein, ultimately affecting its ability to bind a ligand. In addition, this research also allows scientists to design drugs with specific interactions and minimal side effects by targeting the ligand-binding site of an implicated protein.

Ligand Binding Sites and Protein-Ligand Interactions

Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their ...

Education

JoVE Core

Molecular Biology

Unit 1

Protein Function

KEY TERMS AND CONCEPTS

The Equilibrium Binding Constant and Binding Strength

The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:
<img alt="Eq1" src="/files/ftp_upload/11513/Equation 1.png" style="text-align: center;" />
where&#160; P and L are the unbound protein and ligand, respectively, and PL is the protein-ligand complex.&#160;
As the amount of bound ligand is also related to the rate of ligand binding, experiments can also determine Kb by examining the rates of protein-ligand association (kon) and dissociation (koff) using the following ratio:
<img alt="Eq1" src="/files/ftp_upload/11513/Equation 2.png" style="text-align: center;" />
Thus, two categories of&#160; binding assays are used to determine the equilibrium binding constant &#8211; those that measure equilibrium concentrations and those that measure a reaction&#8217;s kinetics. In case, the reaction must be at equilibrium at the time of measurement.
The method of determining the equilibrium concentrations depends on the desired sensitivity and ease of signal detection. For these reasons, spectroscopic assays are most widely used. In these experiments, the reaction produces an absorbance change of a reactant or a product at a given wavelength, detectable by a UV-Vis spectrophotometer. Alternatively, the reactant or product can be tagged with a fluorescent probe or may contain an intrinsic fluorophore. Then, the reaction progress can be measured from the change in fluorescence. These assays are performed by varying one reactant&#8217;s concentrations while the rest of the experiment is held constant. The results can then be graphed and analyzed with various curve fitting methods.
Interactions between proteins and ligands are also studied using a variety of biochemical and spectroscopic techniques. Structural analysis, using X-ray crystallography and NMR spectroscopy, aids in predicting protein-ligand interactions through molecular simulations. Theoretical and computational approaches, such as protein-ligand docking studies, are used extensively to characterize the position and interactions of small molecule ligands, including drug candidates. Computer-aided drug design is a fast and low-cost alternative to accelerate the pace of conventional trial and error drug testing.

The Equilibrium Binding Constant and Binding Strength of Proteins

The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at ...

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

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