I know that if the affinity is too low, assays such as ELISA would not be appropriate, because the off rate of the ligand would be so fast that it would not hold on for the 2-3 hours that it takes to run the ELISA. Does anyone know at what Kd ELISA stops being an appropriate assay?
I have been running ELISAs to determine Kd for an interaction. For some interactions, the Kd, as calculated from the curve by Graphpad Prism, is 300 nM. Is that Kd so high that an ELISA theoretically should not work?
This paper explores this question.
Article Detection of High- and Low-Affinity Antibodies Against a Hum...
It would be interesting to know how you measured the Kd of an antibody using an ELISA. Depending in the type of ELISA, you may have two antibodies involved which both would have different affinities. I don't know how you read the Kd from an ELISA curve, but I hope you didn't make a common mistake of confusing the similarity of the shape of an ELISA curve with a ligand binding assay curve both of which are sigmoidal but the only one giving you a Kd would be the the ligand binding curve. It's an equilibrium curve that is constant over time and only depends on the concentration of the ligand (Kd being the concentration where 50% of the binding sites occupied). However, the ELISA curve is different in that is only similar in shape but not in its biochemical nature. If you stopped the reaction earlier you would get a more linear curve that indicates the time- and concentration-dependent turnover reaction of a substrate,so no equilibrium, hence no dissociation constant Kd could be calculated from that.
I agree with Hüseyin Besir that ELISA is not an appropriate method for measuring the Kd (equilibrium dissociation constant) of an interaction because it is not an equilibrium method. That is because of the washing steps required to remove unbound components.
Thanks very much for your answers. It's not that I'm confused by the shape of the curve being similar. There are people publishing the use of ELISA for this purpose.
Eble JA. Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction. J Vis Exp. 2018 Feb 15;(132):57334. doi: 10.3791/57334. PMID: 29553569; PMCID: PMC5912426.
Syedbasha M, Linnik J, Santer D, O'Shea D, Barakat K, Joyce M, Khanna N, Tyrrell DL, Houghton M, Egli A. An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions. J Vis Exp. 2016 Mar 14;(109):53575. doi: 10.3791/53575. PMID: 27023275; PMCID: PMC4828978.
Maybe I need to reevaluate whether my particular binding interaction is at equilibrium in the assay I've designed and also whether there is some other molecule (for example, in the blocking buffer) interfering with this experiment.
The method of Eble seems to use ELISA after binding and fixation of the ligand with a crosslinker to the receptor (in your case this would be the antibody) and then performs an ELISA afterwards. You need to consider for this approach that the equlibrium conditions for an antibody with a Kd in nanomolar range requires the ligand concentrations in the dilution series to be in that range and lower. This could be a problem after the binding reaction when you wash the wells and crosslink the ligand to the antibody for an extended period of time which could be long enough for the ligand to dissociate before crosslinking. Also, crosslinking could modify the binding site of the second antibody leading to even lower number of bound antibodies. Therefore, I would be careful with the results of such an experiment.
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