In addition to binding to receptors of interest, radioligands also bind to other sites. Binding to the receptor of interest is called specific binding, while binding to the other sites is called nonspecific binding. This means that nonspecific binding can represent several phenomena:
•In most cases, the bulk of nonspecific binding represents some sort of interaction of the ligand with membranes. The molecular details are unclear, but nonspecific binding depends on the charge and hydrophobicity of a ligand – but not its exact structure.
•Nonspecific binding can also be binding to receptors, transporters, or other proteins not of interest to the investigator. For example binding of the adrenoceptor agonist, epinephrine, to serotonin receptors or metabolic enzymes can be considered “nonspecific”.
•Nonspecific binding can also be binding to the filters used to separate bound from free ligand.
Nonspecific binding is usually (but not necessarily) proportional to the concentration of radioligand (within the range it is used). Add twice as much radioligand, and you will see twice as much nonspecific binding.
Nonspecific binding is detected by measuring radioligand binding in the presence of a saturating concentration of an unlabeled drug that binds to the receptors. Under those conditions, virtually all the receptors are occupied by the unlabeled drug so the radioligand can only bind to nonspecific sites. Subtract the nonspecific binding at a particular concentration of radioligand from the total binding at that concentration to calculate the specific radioligand binding to receptors.
Which unlabeled drug should you use for determining nonspecific binding? The obvious answer is to use the same compound as the radioligand, but in its unlabeled form. In many cases this is necessary, as no other drug is known to bind to the receptors. But most investigators avoid using the same compound as the hot and cold ligand and prefer to define nonspecific binding with a drug that is chemically distinct from the radioligand but which binds to the same receptor.
What concentration of unlabeled drug should you use? You want to use enough to block virtually all the specific radioligand binding, but not so much that you cause more general physical changes to the membrane that might alter binding. If you are studying a well-characterized receptor, a useful rule-of-thumb is to use the unlabeled compound at a concentration equal to 100 times its Kd for the receptors, or 100 times the highest concentration of radioligand, whichever is higher.
Ideally, you should get the same results defining nonspecific binding with a range of concentrations of several drugs, and you should test this when possible. In many assay systems, nonspecific binding is only 10-20% of the total radioligand binding. If the nonspecific binding makes up more than half of the total binding, you will find it hard to get quality data. If your system has a lot of nonspecific binding, try different kinds of filters, a larger volume of washing buffer, warmer washing buffer, or a different radioligand.