Molecular Conjugate

Abstract

A method is disclosed for making a conjugate of two molecules using a hydrazide thiol linker. In a particular working embodiment, an Fc-specific antibody-enzyme conjugate is made using the method and demonstrated to provide exceptional staining sensitivity and specificity in immunohistochemical and in situ hybridization assays.

1 . A conjugate comprising an antibody covalently bonded to a detectable label through a hydrazide thiol linker, wherein the hydrazide thiol linker is one or more of MBH, MBCH, MAMBH, THMBH, BTAL, BTHL, TAGD, THGD, a PEG-based hydrazide-thiol linker, a multifunctional hydrazide thiol linker, a PEG-based multifunctional hydrazide thiol linker, and a polyacrylamide hydrazide thiol linker, wherein a hydrazide group of the linker is covalently bonded to an Fc region of the antibody; and wherein the detectable label is an enzyme having at least one thiol reactive group, wherein a thiol group of the hydrazide thiol linker is coupled to the at least one thiol reactive group of the enzyme. 
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . The conjugate of  claim 1 , wherein the hydrazide thiol linker comprises MBH or MBCH. 
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . The conjugate of  claim 1 , wherein the enzyme is selected from the group consisting of alkaline phosphatase and horseradish peroxidase. 
     
     
         8 . The conjugate of  claim 1 , wherein the enzyme comprises alkaline phosphatase. 
     
     
         9 . The conjugate of  claim 8 , wherein the alkaline phosphatase comprises cross-linked alkaline phosphatase. 
     
     
         10 . The conjugate of  claim 1 , wherein the antibody is selected from the group consisting of an anti-hapten antibody or an anti-antibody antibody. 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . The conjugate of  claim 1 , wherein the at least one thiol reactive group is a maleimide. 
     
     
         14 . The conjugate of  claim 1 , wherein the enzyme comprises at least four thiol reactive groups. 
     
     
         15 . The conjugate of  claim 14 , wherein the at least four thiol reactive groups are maleimides. 
     
     
         16 . The conjugate of  claim 1 , wherein the conjugate has a median molecular weight of about 270 kDa. 
     
     
         17 . The conjugate of  claim 1 , wherein the at least one thiol reactive group is selected from the group consisting of pyridyl disulfide and iodylacetamide. 
     
     
         18 . The conjugate of  claim 1 , wherein the antibody is covalently bonded to more than one hydrazide thiol linker. 
     
     
         19 . A conjugate comprising an antibody covalently bonded to a phosphatase enzyme through a mercaptobutyric acid hydrazide linker, wherein a hydrazide group of the mercaptobutyric acid hydrazide linker is covalently bonded to an Fc region of the antibody; the phosphatase enzyme having at least one maleimide group and wherein a thiol group of the mercaptobutyric acid hydrazide linker is covalently bonded to the at least one maleimide group of the phosphatase enzyme. 
     
     
         20 . The conjugate of  claim 19 , wherein the phosphatase enzyme is an alkaline phosphatase. 
     
     
         21 . The conjugate of  claim 19 , wherein the phosphatase enzyme has at least four maleimide groups. 
     
     
         22 . A conjugate of an antibody and a modified phosphatase enzyme, the modified phosphatase enzyme comprising at least one thiol reactive group, wherein the antibody and the modified phosphatase enzyme are coupled through a mercaptobutyric acid hydrazide linker, and wherein a hydrazide group of the mercaptobutyric acid hydrazide linker is covalently bonded to an Fc region of the antibody 
     
     
         23 . The conjugate of  claim 22 , wherein the at least one thiol reactive group is a maleimide. 
     
     
         24 . The conjugate of  claim 22 , wherein the at least one thiol reactive group is selected from the group consisting of pyridyl disulfide and iodylacetamide. 
     
     
         25 . The conjugate of  claim 22 , wherein the conjugate comprises at least four thiol reactive groups. 
     
     
         26 . The conjugate of  claim 22 , wherein a thiol group of the mercaptobutyric acid hydrazide linker is covalently bonded to the at least one thiol reactive group of the modified phosphatase enzyme.