Method for chromogenic detection of two or more target molecules in a single sample

Abstract

The present invention provides a method and kit for detection of two or more target molecules in a single tissue sample, such as for gene and protein dual detection in a single tissue sample. Methods comprise treating a tissue sample with a first binding moiety that specifically binds a first target molecule. Methods further comprise treating the tissue sample with a solution containing a soluble electron-rich aromatic compound prior to or concomitantly with contacting the tissue sample with a hapten-labeled binding moiety and detecting a second target molecule. In one example, the first target molecule is a protein and the second is a nucleic acid sequence, the first target molecule being detected by immunohistochemistry and the second by in situ hybridization. The disclosed method reduces background due to non-specific binding of the hapten-labeled specific binding moiety to an insoluble electron rich compound deposited near the first target molecule.

We claim: 
     
         1 . An automated nucleic acid and protein detection method, comprising:
 automatically dispensing a primary antibody onto a tissue sample under conditions sufficient for the primary antibody to specifically bind a first target molecule within the tissue sample;   detecting the first target molecule in the tissue sample with the primary antibody by IHC;   automatically dispensing a hapten-labeled nucleic acid probe onto the tissue sample under conditions sufficient for the hapten-labeled nucleic acid probe to specifically bind a second target molecule, where the hapten-labeled nucleic acid probe comprises an electron-deficient aromatic compound;   treating the tissue sample with a solution containing an electron-rich aromatic compound prior to or concomitantly with automatically dispensing the second, hapten-labeled nucleic acid probe onto the tissue sample; and   detecting the second target molecule by in situ hybridization (ISH), thereby allowing dual nucleic acid and protein detection in the same tissue sample in a single automated run, where the electron-rich aromatic compound has a formula   
       
         
           
           
               
               
           
         
       
       where at least one of R 1 , R 2 , R 3  are electron donating groups, independently selected from —OR 4 , —NR 6 R 7 , where R 6  and R 7  independently are H or lower alkyl or two of R 1 , R 2  and R 3  together form a fused aromatic ring, optionally substituted with one, two or three electron donating substituents. 
     
     
         2 . The method of  claim 1 , where R 2  and R 3  together form a fused aromatic ring, the electron rich aromatic compound having a formula 
       
         
           
           
               
               
           
         
       
       where R 8 , R 9  and R 10  independently are selected from H, —OR 11 , —NR 12 R 13 , or lower alkyl; and R 11 , R 12  and R 13  independently are selected from H and lower alkyl. 
     
     
         3 . The method of  claim 2 , wherein the electron-rich aromatic compound comprises naphthol. 
     
     
         4 . The method of  claim 3 , where the naphthol concentration is effective to allow dual nucleic acid and protein detection in a single sample and ranges from 1 milligrams per milliliter to 30 milligrams per milliliter. 
     
     
         5 . The method of  claim 3 , where the naphthol concentration is effective to allow dual nucleic acid and protein detection in a single sample and ranges from 1 milligrams per milliliter to 7 milligrams per milliliter. 
     
     
         6 . The method of  claim 3 , where the naphthol concentration ranges from about 0.3 milligrams per milliliter to about 1 milligram per milliliter. 
     
     
         7 . The method of  claim 1 , where the hapten of the hapten-labeled nucleic acid probe is a nitroaryl compound. 
     
     
         8 . The method of  claim 7 , where the nitroaryl compound is dinitrophenol. 
     
     
         9 . The method of  claim 8 , where the concentration of the dinitrophenol nucleic acid-labeled probe is at least 5 μg/ml. 
     
     
         10 . The method of  claim 9 , where the concentration of the dinitrophenol nucleic acid-labeled probe ranges from 10 μg/ml to 15 μg/ml. 
     
     
         11 . The method of  claim 1 , where automatically dispensing a hapten-labeled nucleic acid probe onto the tissue sample under conditions sufficient for the hapten-labeled nucleic acid probe to specifically bind a second target molecule, occurs after treating the tissue sample with an electron-rich aromatic compound. 
     
     
         12 . The method of  claim 1 , where automatically dispensing a hapten-labeled nucleic acid probe onto the tissue sample under conditions sufficient for the hapten-labeled nucleic acid probe to specifically bind a second target molecule, occurs simultaneously with treating the tissue sample with an electron-rich aromatic compound. 
     
     
         13 . The method of  claim 1 , where IHC is performed prior to ISH. 
     
     
         14 . The method of  claim 1 , where ISH is performed prior to IHC. 
     
     
         15 . The method of  claim 1 , where ISH comprises detecting the targeted nucleic acid by horseradish peroxidase-silver staining ISH or alkaline phosphatase-red silver staining. 
     
     
         16 . The method of  claim 1 , where IHC comprises detecting the targeted protein by an alkaline phosphatase-red chromogen or a horseradish peroxidase-DAB chromogen. 
     
     
         17 . A kit for chromogenic detection of two or more target molecules in a single tissue sample, comprising a solution containing a first specific binding moiety that specifically binds to a first target molecule; a solution containing a second, hapten-labeled specific binding moiety that specifically binds a second target molecule; a solution containing a soluble, electron-rich aromatic compound. 
     
     
         18 . The kit of  claim 17 , where the soluble, electron-rich aromatic compound is naphthol and the second, hapten-labeled specific binding moiety is a DNP-labeled nucleic acid probe. 
     
     
         19 . The kit of  claim 17 , where the solution containing the soluble, electron-rich aromatic compound further comprises the hapten-labeled nucleic acid probe.