Radioligand Binding Assays
The first radio-receptor assay (RRA) was done in 1970 by Lefkowitz et al., using a radiolabeled hormone to determine the binding affinity for its receptor.
A radio-receptor assay requires the separation of the bound from the free ligand. This is done by filtration, centrifugation or dialysis.
A method that does not require separation is the scintillation proximity assay that relies on the fact that β-rays from 3H travel extremely short distances. The receptors are bound to beads coated with a polyhydroxy scintillator. Only the bound ligands to be detected.
Today, the fluorescence method is preferred to radioactive materials due to a much lower cost, lower hazard, and the possibility of multiplexing the reactions in a high-throughput manner. One problem is that fluorescent-labeled ligands have to bear a bulky fluorophore that may cause it to hinder the ligand binding. Therefore, the fluorophore used, the length of the linker, and its position must be carefully selected.
An example is by using FRET, where the ligand's fluorophore transfers its energy to the fluorophore of an antibody raised against the receptor.
Other detection methods such as surface plasmon resonance do not even require fluorophores.
Read more about this topic: Schild Regression
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