We produced scFv of an antibody from bacteria. Now we want to establish dissociation Kd of antibody using quantitative elisa.
How can i know the expected Kd value (range in nM, or mM)?
Answers to earlier post:
https://www.researchgate.net/post/How-can-I-determine-antibodys-Kd-value-using-ELISA
Article Titration ELISA as a Method to Determine the Dissociation Co...
Article Evaluation of dissociation constants of antigen-antibody com...
https://www.journalijar.com/article/8983/a-new-method-for-the-determination-of-dissociation-constant-(kd)-on-the-binding-of-ca19-9-to-its-antibody-in-type-2diabetic-patients-by-enzyme-linked-immunosorbent-assay-(elisa)-with-some-modifications/
Hi Saleha,
ELISA is primarily a binding assay, not a kinetic measurement method, which means it measures equilibrium binding rather than the rates of association and dissociation. While ELISA does not directly measure Kd, it can be used to estimate the apparent affinity through metrics like EC50 (the concentration of antibody at which 50% of the maximum binding is observed). However, EC50 is not the same as Kd, though they are related in certain contexts. EC50 is the concentration of the scFv that gives half-maximal binding in an ELISA. It is often used as an indirect measure of affinity. Kd is a more precise measure of the dissociation constant and is best determined using kinetic methods like Biolayer Interferometry (BLI) or Surface Plasmon Resonance (SPR).
Steps to Determine EC50 Using ELISA:
1. Prepare serial dilutions of the scFv in PBS or another appropriate buffer.
2. Coat the ELISA plate with a fixed concentration of the antigen (e.g., 100 ng/well).
3. Block the plate to prevent non-specific binding (commonly with BSA or milk proteins).
4. Incubate the scFv dilutions with the coated antigen.
5. Wash the plate to remove unbound scFvs thoroughly.
6. Detect the bound scFv using an appropriate secondary antibody conjugated with an enzyme (e.g., HRP).
7. Develop the signal with a suitable substrate (e.g., TMB) and measure the absorbance.
8. Plot the binding curve by plotting the concentration of scFv (x-axis) against the absorbance (y-axis).
9. Fit the data to a binding model (e.g., a four-parameter logistic fit) to determine the EC50.
Cheers
Stöpa
Dear Stephen,
Thank you for detailed answer. Problem is how do I know that serial dilutions has to be made in nM range or pM or uM range?
If you do not have prior information on the affinity of your scFv, it's reasonable to start with a broad range that covers common antibody affinities. Based on typical affinities, a starting range of 0.1 nM to 1 µM should be adequate.
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