Name: titration elisa as a method to determine the dissociation constant of receptor ligand interaction
Uploaded: 2018-02-15T12:00:00
Description: Watch this Scientific Journal Video about Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction at JoVE.com

The protocol and algorithm were developed within a project financed by the Deutsche Forschungsgemeinschaft (DFG grant SFB1009 A09 and EB177/13-1). The author thanks Barbara Schedding and Felix Schmalbein for technical support and Dr. Niland for critically reading the manuscript.

1. Stock Solutions
<ol>
	<li>To prepare 100 mL of 10x&#160;TBS pH 7.4 solution, dissolve 6.06 g of TRIS and 8.77 g of NaCl in 90 mL of deionized water, adjust the pH to 7.4 with 37% HCl solution, fill the volume to 100 mL with deionized water, and filter the solution.</li>
	<li>To prepare 100 mL of 1 M HEPES/NaOH, pH 7.4 solution, dissolve 23.83 g of HEPES in 90 mL of deionized water, adjust the pH to 7.4 with 1.5 M NaOH, fill the volume to 100 mL with deionized water, and filter the solution.</li>
	<li>To prepare 100 ...

<ol>
	<li>Klotz, I. 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+application+of+the+law+of+mass+action+to+binding+by+proteins;+interactions+with+calcium.">The application of the law of mass action to binding by proteins; interactions with calcium.</a> Arch Biochem. 9, 109-117 (1946).</li><li>Klotz, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-receptor+complexes:+origin+and+development+of+the+concept.">Ligand-receptor complexes: origin and development of the concept.</a> J Biol Chem. 279 (1), 1-12 (2004).</li><li>Bisswanger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+1:+Multiple+Equilibria,+Principles+and+Derivations.">Ch. 1: Multiple Equilibria, Principles and Derivations.</a> Enzyme Kinetics: Principles and Methods. , 1-26 (2017).</li><li>Shimura, K., Kasai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+gel+titration:+quantitative+analysis+of+the+binding+equilibrium+between+immobilized+protein+and+free+ligand+by+a+continuous+titration+procedure.">Affinity gel titration: quantitative analysis of the binding equilibrium between immobilized protein and free ligand by a continuous titration procedure.</a> Anal Biochem. 149 (2), 369-378 (1985).</li><li>Gong, M., Nikcevic, I., Wehmeyer, K. R., Limbach, P. A., Heineman, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-aptamer+binding+studies+using+microchip+affinity+capillary+electrophoresis.">Protein-aptamer binding studies using microchip affinity capillary electrophoresis.</a> Electrophoresis. 29 (7), 1415-1422 (2008).</li><li>Hanes, M. S., Ratcliff, K., Marqusee, S., Handel, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-protein+binding+affinities+by+pulse+proteolysis:+application+to+TEM-1/BLIP+protein+complexes.">Protein-protein binding affinities by pulse proteolysis: application to TEM-1/BLIP protein complexes.</a> Protein Sci. 19 (10), 1996-2000 (2010).</li><li>Latour, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Langmuir+isotherm:+a+commonly+applied+but+misleading+approach+for+the+analysis+of+protein+adsorption+behavior.">The Langmuir isotherm: a commonly applied but misleading approach for the analysis of protein adsorption behavior.</a> J Biomed Mater Res A. 103 (3), 949-958 (2015).</li><li>Stockell, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+binding+of+diphosphopyridine+nucleotide+by+yeast+glyceraldehyde-3-phosphate+dehydrogenase.">The binding of diphosphopyridine nucleotide by yeast glyceraldehyde-3-phosphate dehydrogenase.</a> J Biol Chem. 234 (5), 1286-1292 (1959).</li><li>Heyn, M. P., Weischet, W. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+dichroism+and+fluorescence+studies+on+the+binding+of+ligands+to+the+&#945;+subunit+of+tryptophan+synthase.">Circular dichroism and fluorescence studies on the binding of ligands to the &#945; subunit of tryptophan synthase.</a> Biochem. 14 (13), 2962-2968 (1975).</li><li>Campbell, I. D., Humphries, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+structure,+activation,+and+interactions.">Integrin structure, activation, and interactions.</a> Cold Spring Harb Perspect Biol. 3 (3), (2011).</li><li>Zeltz, C., Gullberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+integrin-collagen+connection--a+glue+for+tissue+repair?.">The integrin-collagen connection--a glue for tissue repair?.</a> J Cell Sci. 129 (4), 653-664 (2016).</li><li>Kanchanawong, P., 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=Nanoscale+architecture+of+integrin-based+cell+adhesions.">Nanoscale architecture of integrin-based cell adhesions.</a> Nature. 468 (7323), 580-584 (2010).</li><li>Luo, B. H., Carman, C. V., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+integrin+regulation+and+signaling.">Structural basis of integrin regulation and signaling.</a> Annu Rev Immunol. 25, 619-647 (2007).</li><li>Luo, B. H., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+structures+and+conformational+signaling.">Integrin structures and conformational signaling.</a> Curr Opin Cell Biol. 18 (5), 579-586 (2006).</li><li>Ricard-Blum, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+collagen+family.">The collagen family.</a> Cold Spring Harb Perspect Biol. 3 (1), a004978 (2011).</li><li>Eble, J. A., Tuckwell, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+&#945;2&#946;1+integrin+inhibitor+rhodocetin+binds+to+the+A-domain+of+the+integrin+&#945;2+subunit+proximal+to+the+collagen-binding+site.">The &#945;2&#946;1 integrin inhibitor rhodocetin binds to the A-domain of the integrin &#945;2 subunit proximal to the collagen-binding site.</a> Biochem J. 376 (Pt 1), 77-85 (2003).</li><li>Eble, J. 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=The+&#945;2&#946;1+integrin-specific+antagonist+rhodocetin+is+a+cruciform,+heterotetrameric+molecule.">The &#945;2&#946;1 integrin-specific antagonist rhodocetin is a cruciform, heterotetrameric molecule.</a> FASEB J. 23 (9), 2917-2927 (2009).</li><li>Eble, J. 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=Dramatic+and+concerted+conformational+changes+enable+rhodocetin+to+block+&#945;2&#946;1+integrin+selectively.">Dramatic and concerted conformational changes enable rhodocetin to block &#945;2&#946;1 integrin selectively.</a> PLoS Biol. 15 (7), e2001492 (2017).</li><li>Bracht, T., Figueiredo de Rezende, F., Stetefeld, J., Sorokin, L. M., Eble, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+reveal+the+alteration+of+the+rhodocetin+structure+upon+&#945;2&#946;1+integrin+binding.">Monoclonal antibodies reveal the alteration of the rhodocetin structure upon &#945;2&#946;1 integrin binding.</a> Biochem J. 440 (1), 1-11 (2011).</li><li>Harlow, E., Lane, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+5:+Immunizations.">Chapter 5: Immunizations.</a> Antibodies, a laboratory manual. 5, 53-138 (1988).</li><li>Lu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+interactions+between+SSB+and+proteins+by+the+use+of+fluorescence+anisotropy.">Analyzing interactions between SSB and proteins by the use of fluorescence anisotropy.</a> Methods Mol Biol. 922, 155-159 (2012).</li><li>Fielding, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+methods+for+the+determination+of+protein-ligand+dissociation+constants.">NMR methods for the determination of protein-ligand dissociation constants.</a> Curr Top Med Chem. 3 (1), 39-53 (2003).</li><li>Leavitt, S., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+protein+binding+energetics+by+isothermal+titration+calorimetry.">Direct measurement of protein binding energetics by isothermal titration calorimetry.</a> Curr Opin Struct Biol. 11 (5), 560-566 (2001).</li><li>McDonnell, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+plasmon+resonance:+towards+an+understanding+of+the+mechanisms+of+biological+molecular+recognition.">Surface plasmon resonance: towards an understanding of the mechanisms of biological molecular recognition.</a> Curr Opin Chem Biol. 5 (5), 572-577 (2001).</li><li>Rich, R. L., Myszka, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+surface+plasmon+resonance+biosensor+analysis.">Advances in surface plasmon resonance biosensor analysis.</a> Curr Opin Biotechnol. 11 (1), 54-61 (2000).</li><li>Pesquero, N. C., 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=Real-time+monitoring+and+kinetic+parameter+estimation+of+the+affinity+interaction+of+jArtinM+and+rArtinM+with+peroxidase+glycoprotein+by+the+electrogravimetric+technique.">Real-time monitoring and kinetic parameter estimation of the affinity interaction of jArtinM and rArtinM with peroxidase glycoprotein by the electrogravimetric technique.</a> Biosens Bioelectron. 26 (1), 36-42 (2010).</li><li>Scheuermann, T. H., Padrick, S. B., Gardner, K. H., Brautigam, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+acquisition+and+analysis+of+microscale+thermophoresis+data.">On the acquisition and analysis of microscale thermophoresis data.</a> Anal Biochem. 496, 79-93 (2016).</li></ol>

The titration ELISA is a versatile test system to determine the dissociation of a receptor-ligand interaction. As the titration ELISA circumvents the necessity to separate free and bound ligands effectively and to analyze their concentrations quantitatively, substantially more studies and publications have employed titration ELISAs instead of recording binding curves. Moreover, titration ELISAs are easy to perform and require reasonably low amounts of receptor and ligand. For accurate analysis of dissociation constants, ...

The dissociation constant K is a key parameter to describe the affinity of a receptor (R) for its ligand (L). Based on the law of mass action, K is defined for the equilibrium, in which the receptor-ligand complex RL dissociates into the receptor R and the ligand L:
<img alt="Equation 1 " src="/files/ftp_upload/57334/57334eq1.jpg" />&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;Equation 1
with the indices f indicating the free/unbound state of receptor and ligand. The concentration of the receptor-ligand complex, RL, is identical to the concentration of the receptor-bound ligand Lb. As the total concentration of receptor Rt is the sum of the free receptor Rf and ligand-bound receptor Rb = Lb, the dissociation constant can also be written as:
<img alt="Equation 2 " src="/files/ftp_upload/57334/57334eq2.jpg" />&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;Equation 2
Therefore, the saturation yield Y, defined as the fraction of bound ligand Lb in relation of the total concentration of receptor Rt,
<img alt="Equation 3 " src="/files/ftp_upload/57334/57334eq3.jpg" />&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;Equation 3
depends on the concentration of free ligand Lf:
<img alt="Equation 4 " src="/files/ftp_upload/57334/57334eq4.jpg" />&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;Equation 4
This hyperbolic relation describes the binding curve of a receptor-ligand interaction and its plot shows the concentration of bound ligand Lb as a function of the concentration of free ligand Lf. From the binding curve, the dissociation constant K can be derived as the concentration of free ligand at half-maximal saturation yield. Moreover, different algorithms to linearize binding curves have been established, such as the double-reciprocal plot by Klotz1,2, or transformations according to Scatchard or Hanes (reviewed by Bisswanger3). However, all algorithms suffer from the problem that the maximum value of the saturation yield, which is asymptotically approached at high concentrations of free ligand in the binding curve, has to be estimated in a graphical pre-evaluation and therefore is error-prone.
In addition, the determination of a binding curve requires the quantification of free and bound ligand during the binding equilibrium. To this end, the free ligand has to be separated from the receptor-bound ligand and quantified. Therefore, the ligand and receptor have to differ in their properties, such as a non-protein ligand as opposed to a protein receptor. If both binding partners are proteins, they have to be distinguishable in their sizes, charges, or other molecular features. Nevertheless, the quantification of ligand concentrations in small-scale binding approaches is a difficult task. Radioactive labeling of the ligand has often been necessary to detect the low concentration of bound ligand, especially if substantial amounts of receptors were not available or affordable. Moreover, the receptor-bound ligand may dissociate during and after isolation in a non-negligible manner. Hence, complex methods, such as equilibrium gel filtration4, capillary electrophoresis5, and pulse proteolysis6, are required to quantify receptor-bound ligand and separate it from free ligand.
In contrast to these binding assays, titration experiments do not require the quantitative separation of bound and free ligand. To this end, a receptor at a constant concentration is titrated with different concentrations of added ligand. By binding to the receptor, the bound ligand has a biophysical property which distinguishes it from the free ligand and is measurable by, e.g., photometry, fluorometry, or antibody detection. Thus, a signal S, which is proportional to the saturation yield Y and consequently also to the concentration of receptor-bound ligand (Lb), is detected as a function of the total concentration of added ligand (Lt). Both parameters, the signal S and the total concentration of added ligand are quantified in a direct and easier manner than the concentrations of bound and free ligand. Especially, the detection of receptor-bound ligand by enzyme-linked immunosorbent assay (ELISA) allowed the reduction of sample volumes to below 100 &#181;L as well as parallel measurements of several ligand concentrations in multi-well microtiter plates. In a titration ELISA, a receptor is physically adsorbed to a microtiter plate at the same concentration and titrated with soluble ligand. The receptor is immobilized to the plastic surface essentially by hydrophobic adsorption. The surface concentration of immobilized receptor correlates with the coating concentration of the receptor in a nonlinear relation, likely according to Langmuir&#180;s adsorption isotherm7. In addition to the overall number of adsorbed receptor molecules, their activity state is another important parameter for titration assays. Only immobilized receptors which have retained ligand binding activity, are relevant for the titration assay and eventually contribute to the total concentration of active receptors Rt of the titration assay, which cannot be determined directly.
Sites on the plastic surface, which are not covered by the immobilized receptor are prone to adsorb other proteins, such as the ligand. Physical adsorption of the ligand to such plastic surface sites would result in a similar signal as the receptor-bound ligand, yet in a nonspecific manner. To reduce this nonspecific signal, the plastic surface sites of the microtiter plates which have not been coated with protein yet will be blocked with bovine serum albumin (BSA). However, for some receptor-ligand titration assays, nonspecific background signals may be observed. Then, other blocking agents, such as a solution of 0.2% gelatin or of 0.04% Tween 20, are recommended.
After binding to the receptor, the free ligand is removed by two washing steps. Bound ligand remains with the receptor, which is immobilized to the plastic surface of the microtiter well, and optionally reinforced by chemical fixation. For the subsequent covalent cross-linkage of bound ligand and immobilized receptor with glutaraldehyde, the buffer substance TRIS is replaced for HEPES, without any change in ligand binding. HEPES, in contrast to TRIS, does not inactivate glutaraldehyde. The covalent cross-linkage with glutaraldehyde fixes the bound ligand with its receptor and prevents its dissociation during subsequent washing and incubation steps. Thus, the receptor-ligand interaction is chemically frozen and warrants a titration curve which is unaffected by subsequent steps of washing and incubation. However, glutaraldehyde fixation may chemically modify the ligand and receptor in such a way that their interaction is reduced or abolished. Moreover, modification of epitopes within the ligand may change the binding affinity of the detecting antibody, especially if a monoclonal antibody is used to quantify bound ligand. Although neither of these adverse effects of glutaraldehyde fixation occurs in this titration ELISA, the sensitivity of the test towards glutaraldehyde must be tested for every receptor-ligand interaction prior to the titration experiment. After fixation, excess glutaraldehyde is removed in three washing steps with TRIS-containing buffer. TRIS inactivates remaining aldehyde groups, which might nonspecifically react with detecting antibodies in the subsequent step.
The amount of bound ligand is quantified with enzyme-linked antibodies, which provide a photometric ELISA signal S. This is plotted versus the total ligand concentration Lt added to each well. Despite its easier acquisition, the titration curve is not a hyperbolic function in contrast to the binding curve. Furthermore, it has been unclear how to calculate the dissociation constant K from a titration curve. Although algorithms to linearize spectroscopically acquired titration curves have been independently reported by Stockell8 and Heyn and Weischet9, they fell short due to their uncertainty of estimating the maximum signal value that the saturation yield approaches at high concentrations of added ligand.
Here, a titration ELISA and a non-linear regression algorithm are described to derive the dissociation constant K for a receptor ligand interaction from a titration curve. This protocol is exemplified for the interaction of the collagen-binding A-domain of the integrin &#945;2&#946;1 with a snake venom-derived inhibitor. Integrins are cell adhesion molecules, which mediate the anchorage of cells to the surrounding extracellular matrix or the underlying basement membrane10,11. Moreover, integrins convey important signals between cells and the extracellular matrix by recruiting additional signaling molecules and forming new cell organelles, adhesomes, upon cell-matrix interaction12,13,14. Collagen, the ligand of &#945;2&#946;1 integrin, is the most abundant protein of the human body and is a crucial scaffolding component of the connective tissue15. The interaction between &#945;2&#946;1 integrin and collagen is mediated by the A-domain of the integrin &#945;2 subunit. The integrin &#945;2A-domain contains a divalent cation, which is required for collagen binding and stabilizes its structure. The wild-type form as well as mutants of the &#945;2A domain, such as the one in which the surface-exposed residue Y216 had been replaced for a glycine, can easily be produced recombinantly in a bacterial expression system and isolated via their oligo-His-tags with a NiNTA superflow column with a subsequent dialysis against TRIS-buffered saline (TBS; 50 mM TRIS/HCl, pH 7.4, 150 mM NaCl) containing 2 mM MgCl216. Their concentrations were determined with the bicinchoninic acid assay (BCA) and their purities are tested by conventional SDS-PAGE and stained with Coomassie-Brilliant Blue R250.
The interaction between &#945;2&#946;1 integrin and collagen is blocked by binding of the snake venom component, rhodocetin, from the Malayan pit viper (Calloselasma rhodostoma)16,17. Used as a soluble ligand in this titration ELISA, rhodocetin was purified from the crude venom as described previously16. It is dissolved in HEPES-buffered saline (HBS; 10 mM HEPES/NaOH, pH 7.4, 150 mM NaCl) and is stored frozen at -20 &#176;C. Its concentration was determined by BCA and its purity was proven by SDS-PAGE. As an antagonist, rhodocetin not only blocks collagen binding to the integrin &#945;2&#946;1 A-domain, but also stabilizes the inactive conformation of the integrin thereby preventing any signaling from collagen into cells or platelets18. It is of great biomedical importance to determine the dissociation constant of rhodocetin with its receptor target and thus unravel its molecular mechanism and pharmaceutical potential e.g., as an antithrombotic agent. To this end, a titration ELISA is described including its evaluation, which is applicable to almost any receptor-ligand interaction with a 1:1 interaction stoichiometry.

<table>
	<tbody>
		<tr>
			<td>TRIS</td>
			<td>neoFrox</td>
			<td>1125KG001</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Applichem</td>
			<td>1,316,591,214</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Merck</td>
			<td>172571</td>
			<td></td>
		</tr>
		<tr>
			<td>integrin a2A, wild-type and mutant, recombinant</td>
			<td></td>
			<td>isolated in author&#39;s lab</td>
			<td></td>
		</tr>
		<tr>
			<td>NiNTA superflow column&#160;</td>
			<td>Qiagen, Germany</td>
			<td>30821</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie-Brilliant Blue R250</td>
			<td>Serva</td>
			<td>35050</td>
			<td></td>
		</tr>
		<tr>
			<td>bicinchoninic acid assay (BCA), protein concentration determination kit</td>
			<td>Fisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumine (BSA), fraction V</td>
			<td>Applichem</td>
			<td>A1391</td>
			<td></td>
		</tr>
		<tr>
			<td>25 % solution of glutaraldehyde</td>
			<td>Merck</td>
			<td>354400</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit immunglobulin-antibodies from goat, conjugated with alkaline phosphatase</td>
			<td>Sigma-Aldrich</td>
			<td>A9919</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Applichem</td>
			<td>A1377</td>
			<td></td>
		</tr>
		<tr>
			<td>Zn(II)-acetate</td>
			<td>Applichem</td>
			<td>A4324</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Applichem</td>
			<td>A1551</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline phosphatase substrate tablet (5 mg)</td>
			<td>Sigma-Aldrich</td>
			<td>S0942</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar half-area microtiter plate</td>
			<td>Thermo Scientific</td>
			<td>Corning 3690</td>
			<td></td>
		</tr>
		<tr>
			<td>micro reaction tubes</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate ELISA reader</td>
			<td>BioTek</td>
			<td>Synergy HT</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

After the ELISA has been developed, the yellow color of the converted alkaline phosphatase substrate, para-nitrophenolate, indicates that the amount of bound rhodocetin ligand decreases with decreasing concentrations of added rhodocetin from columns 1 to 11 (Figure 1). The colorless wells in the rhodocetin-free wells in column 12 show a low background signal.
Photometric quantification at 4...

Watch this Scientific Journal Video about Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction at JoVE.com

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

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 ...

Research

JoVE Journal

Biochemistry

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

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 ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...