Rapid and sensitive method for detection of biological targets
The present invention relates to a method of biological labeling that occurs via a free radical chain reaction. The labeling occurs due to deposition of a detectable reporter molecule from a media comprising a substance comprising at least two moieties of a peroxidase enzyme substrate (termed herein ‘cross-linker’) in a target site comprising peroxidase activity and a biological marker. The labeling reaction described herein may generally be used to detect targets in a host of experimental schemes for detecting and visualizing a biological or chemical target, including immunohistochemistry (IHC), in situ hybridization (ISH), antibody-based staining methods such as ELISA, Southern, Northern, and Western blotting, and others.
1. A method of detecting of a biological marker in a biological sample in vitro, comprising the following steps:
a) incubating a biological sample presumably comprising a biological marker with one or more probes capable of specifically binding to the biological marker, wherein at least one of said one or more probes comprises at least one moiety of horse radish peroxidase (HRP) and a member of a specific binding pair or a conjugate comprising a member of a specific binding pair, wherein said member of a specific binding pair is capable of specifically binding to the biological marker, thereby forming a complex of the biological marker with the at least one probe;
b) incubating the sample comprising the complex of (a) in a water solution, the water solution comprising 3,3′-diaminobenzidine, a peroxide compound and a conjugate molecule comprising a combination of different detectable labels, wherein the combination comprises a hapten and a substrate of horse radish peroxidase, thereby depositing said conjugate molecule in a site of the sample where said complex is present; and
c) detecting the deposited conjugate molecules of (b) and thereby detecting the biological marker.
2. The method according to claim 1 , wherein the biological marker is a biological molecule selected from the group consisting of proteins, nucleic acids, lipids, carbohydrates, a molecular complex and a cellular structure.
3. The method according to claim 1 , wherein the 3,3′-diaminobenzidine is present in an amount from about 0.3 mM to about 3 mM.
4. The method according to claim 1 , wherein the method comprises an additional step between step (a) and step (b), wherein the sample is incubated in a water solution comprising 3,3′-diaminobenzidine in an amount from 0.3 mM to 3 mM and a peroxide compound.
5. The method according to claim 1 , wherein step (c) comprises incubating the sample with a substance which is capable of specifically binding to the deposited conjugate.
6. The method according to claim 5 , wherein the substance is detectably labeled.
7. The method according to claim 6 , wherein the substance is an antibody or a nucleic acid.
8. The method according to claim 7 , wherein the method is performed in a format of an immunohistochemistry or in situ hybridization assay.
9. The method according to claim 1 , wherein the combination of different detectable labels comprises ferulic acid and/or tyrosine.
10. The method according to claim 1 , wherein the combination of different detectable labels comprises ferulic acid.
11. The method according to claim 1 , wherein the combination of different detectable labels comprises fluorescein.
12. The method according to claim 1 , wherein the member of a specific binding pair is an antibody or a nucleic acid.