Separation-free assay method
A separation-free assay including a binding partner, a binding partner labeled with a directly luminescent label, and a nonspecifically binding label able to affect the signal of the unbound labeled binding partner, in which a signal of the binding partner label or the signal of the nonspecific binding label is measured in a binding event, and where at least one of the binding partners is a mobile binding partner.
1. A separation-free assay method to determine a concentration of a substance in a sample, the method comprising
adding a binding partner to said sample,
allowing the binding partner to bind specifically to said substance,
adding a labeled ligand having a directly luminescent label adsorbed and/or covalently coupled thereto to said sample,
allowing the labeled ligand to specifically bind with said binding partner to form a complex with the binding partner,
adding a nonspecifically binding label to the sample,
allowing the nonspecifically binding label to affect a signal of unbound, labeled ligand which has not formed a complex with said binding partner,
measuring directly without separation of said complex or said unbound, labeled ligand from the sample
a signal of the labeled ligand of said complex, or a signal of said unbound, labeled ligand,
wherein at least one of said binding partner and said labeled ligand is mobile, and
determining the concentration of said substance by measuring said signal of the labeled ligand of said complex or measuring said signal of said unbound, labeled ligand.
2. The method according to claim 1 wherein the method is a non-competitive assay.
3. The method according to claim 1 wherein the method is a competitive assay, the method further comprising
adding a competitive binding partner, and
allowing the competitive binding partner to bind to the binding partner that is mobile.
4. The method according to claim 1 , wherein the label of the binding partner that is mobile is an acceptor of a donor-acceptor label pair or a donor of a donor-acceptor label pair.
5. The method according to claim 1 , wherein the nonspecifically binding label is a quencher, an enhancer, an acceptor of a donor-acceptor label pair, or a donor of a donor-acceptor label pair.
6. The method according to claim 1 , the method further comprising
adding a separate enhancer or quencher to said sample, the separate enhancer or quencher being configured to enhance or quench the signal of the labeled ligand upon binding to the binding partner that is mobile.
7. The method according to claim 1 , the method further comprising
adding an enhancer to said sample, the enhancer being configured to enhance the signal of the labeled ligand upon binding to or interacting with the binding partner that is mobile.
8. The method according to claim 1 , wherein the label of the labeled ligand is protected from the nonspecifically binding label upon binding to a binding partner that is a receptor.
9. The method according to claim 4 , wherein the label pair is a resonance energy transfer label pair.
10. The method according to claim 4 , wherein the label of the labeled ligand comprises a lanthanide.
11. The method of claim 1 , wherein the signal of said labeled ligand is increased or decreased upon binding of said labeled ligand to said binding partner.