Antibody conjugates

Abstract

Antibody/signal-generating moiety conjugates are disclosed that include an antibody covalently linked to a signal-generating moiety through a heterobifunctional polyalkyleneglycol linker. The disclosed conjugates show exceptional signal-generation in immunohistochemical and in situ hybridization assays on tissue sections and cytology samples. In one embodiment, enzyme-metallographic detection of nucleic acid sequences with hapten-labeled probes can be accomplished using the disclosed conjugates as a primary antibody without amplification.

1 . An antibody-signal-generating moiety conjugate comprising an antibody covalently linked to a signal-generating moiety through a heterobifunctional PEG linker.  
   
   
       2 . The conjugate of  claim 1 , wherein a thiol-reactive group of the linker is covalently linked to the antibody and an amine-reactive group of the heterobifunctional linker is covalently linked to the signal-generating moiety.  
   
   
       3 . The conjugate of  claim 2 , wherein the thiol-reactive group of the linker is covalently linked to a cysteine residue of the antibody.  
   
   
       4 . The conjugate of  claim 2 , wherein the thiol-reactive group of the linker is covalently linked to a thiol group that is introduced to the antibody.  
   
   
       5 . The conjugate of  claim 1 , wherein an aldehyde-reactive group of the linker is covalently linked to the antibody and a thiol-reactive group of the linker is covalently linked to the signal-generating moiety.  
   
   
       6 . The conjugate of  claim 5 , wherein the aldehyde-reactive group of the linker is covalently linked to an aldehyde formed on a glycosylated portion of the antibody.  
   
   
       7 . The conjugate of  claim 1 , wherein the linker has the formula:  
     
       
         
         
             
             
         
       
     
     wherein n 1 to 50; or,  
     
       
         
         
             
             
         
       
     
     wherein m is from 1 to 50.  
   
   
       8 . The conjugate of  claim 7 , wherein the conjugate has the formula:  
     
       
         
         
             
             
         
       
     
     wherein Ab is an antibody, SM is a signal-generating moiety, n=1 to 50 and s=1 to 10.  
   
   
       9 . The conjugate of  claim 8 , wherein the signal-generating moiety comprises an enzyme.  
   
   
       10 . The conjugate of  claim 9 , wherein the enzyme comprises horseradish peroxidase or alkaline phosphatase.  
   
   
       11 . The conjugate of  claim 8 , wherein s=2 to 6.  
   
   
       12 . The conjugate of  claim 8 , wherein n=4 to 12.  
   
   
       13 . The conjugate of  claim 7 , wherein the conjugate has the formula:  
     
       
         
         
             
             
         
       
     
     wherein Ab is an antibody, SM is a signal-generating moiety, m=1 to 50 and t=1 to 10.  
   
   
       14 . The conjugate of  claim 13 , wherein the signal-generating moiety comprises an enzyme.  
   
   
       15 . The conjugate of  claim 14 , wherein the enzyme comprises horseradish peroxidase or alkaline phosphatase.  
   
   
       16 . The conjugate of  claim 13 , wherein t=2 to 6.  
   
   
       17 . The conjugate of  claim 13 , wherein m=4 to 12.  
   
   
       18 . A heterobifunctional linker having the formula:  
     
       
         
         
             
             
         
       
     
     wherein m=1 to 50.  
   
   
       19 . The linker of  claim 18 , wherein m=2 to 30.  
   
   
       20 . The linker of  claim 18 , wherein m=3 to 20.  
   
   
       21 . The linker of  claim 18 , wherein m=4 to 12.  
   
   
       22 . A method for preparing an antibody-signal-generating moiety conjugate, comprising: 
 forming a thiolated antibody from an antibody;    reacting a signal-generating moiety having an amine group with a PEG maleimide/active ester bifunctional linker to form an activated signal-generating moiety; and    reacting the thiolated antibody with the activated signal-generating moiety to form the antibody-signal-generating moiety conjugate.    
   
   
       23 . The method of  claim 22 , wherein forming the thiolated antibody comprises reacting the antibody with a reducing agent to form the thiolated antibody.  
   
   
       24 . The method of  claim 23 , wherein reacting the antibody with a reducing agent to form a thiolated antibody comprises forming an antibody with an average number of thiols per antibody of between about 1 and about 10.  
   
   
       25 . The method of  claim 23 , wherein reacting the antibody with a reducing agent comprises reacting the antibody with a reducing agent selected from the group consisting of 2-mercaptoethanol, 2-mercaptoethylamine, DTT, DTE and TCEP, and combinations thereof.  
   
   
       26 . The method of  claim 25 , wherein reacting the antibody with a reducing agent comprises reacting the antibody with a reducing agent selected from the group consisting of DTT and DTE, and combinations thereof.  
   
   
       27 . The method of  claim 26 , wherein reacting the antibody with a reducing agent comprises reacting the antibody with the reducing agent at a concentration of between about 1 mM and about 40 mM.  
   
   
       28 . The method of  claim 22 , wherein forming the thiolated antibody comprises introducing a thiol group to the antibody.  
   
   
       29 . The method of  claim 28 , wherein introducing the thiol group to the antibody comprises reacting the antibody with a reagent selected from the group consisting of 2-Iminothiolane, SATA, SATP, SPDP, N-Acetylhomocysteinethiolactone, SAMSA, and cystamine, and combinations thereof.  
   
   
       30 . The method of  claim 28 , wherein introducing the thiol group to the antibody comprises reacting the antibody with an oxidant to convert a sugar moiety of the antibody into an aldehyde group and reacting the aldehyde group with cystamine.  
   
   
       31 . The method of  claim 30 , wherein the oxidant comprises periodate ion, I 2 , Br 2 , or a combination thereof.  
   
   
       32 . The method of  claim 22 , wherein reacting the signal-generating moiety with a PEG maleimide/active ester bifunctional linker to form an activated signal-generating moiety comprises reacting the signal-generating moiety with a PEG maleimide/active ester having the formula:  
     
       
         
         
             
             
         
       
     
     wherein n=1 to 50.  
   
   
       33 . The method of  claim 22 , wherein the signal-generating moiety comprises an enzyme.  
   
   
       34 . The method of  claim 33 , wherein the enzyme comprises horseradish peroxidase or alkaline phosphatase.  
   
   
       35 . A method for preparing an antibody-signal-generating moiety conjugate, comprising: 
 reacting an antibody with an oxidant to form an aldehyde-bearing antibody;    reacting the aldehyde-bearing antibody with a PEG maleimide/hydrazide bifunctional linker to form a thiol-reactive antibody; and    reacting the thiol-reactive antibody with a thiolated signal-generating moiety to form the antibody-signal-generating moiety conjugate.    
   
   
       36 . The method of  claim 35 , wherein reacting the antibody with an oxidant to form the aldehyde-bearing antibody comprises oxidizing a glycosylated region of the antibody to form the aldehyde-bearing antibody.  
   
   
       37 . The method of  claim 35 , wherein oxidizing the glycosylated region of the antibody comprises treating the antibody with periodate, I 2 , Br 2 , or a combination thereof.  
   
   
       38 . The method of  claim 35 , further comprising forming the thiolated signal-generating moiety from a signal-generating moiety.  
   
   
       39 . The method of  claim 38 , wherein forming the thiolated signal-generating moiety comprises reacting the signal-generating moiety with a reducing agent to form the thiolated signal-generating moiety.  
   
   
       40 . The method of  claim 39 , wherein the reducing agent is selected from the group consisting of 2-mercaptoethanol, 2-mercaptoethylamine, DTT, DTE and TCEP, and combinations thereof.  
   
   
       41 . The method of  claim 38 , wherein forming the thiolated signal-generating moiety comprises introducing a thiol group to the signal-generating moiety.  
   
   
       42 . The method of  claim 38 , wherein introducing the thiol group to the signal generating moiety comprises reacting the signal-generating moiety with a reagent selected from the group consisting of 2-Iminothiolane, SATA, SATP, SPDP, N-Acetylhomocysteinethiolactone, SAMSA, and cystamine, and combinations thereof.  
   
   
       43 . The method of  claim 57 , wherein reacting the aldehyde-bearing antibody with the PEG maleimide/hydrazide bifunctional linker to form the thiol-reactive antibody comprises reacting the aldehyde-bearing antibody with a linker having the formula:  
     
       
         
         
             
             
         
       
     
     wherein m=1 to 50.  
   
   
       44 . The method of  claim 35 , wherein the thiolated signal-generating moiety comprises an enzyme.  
   
   
       45 . The method of  claim 44 , wherein the enzyme comprises horseradish peroxidase or alkaline phosphatase.  
   
   
       46 . A method for detecting a molecule of interest in a biological sample, comprising: 
 contacting the biological sample with an antibody-signal-generating moiety conjugate comprising an antibody covalently linked to a signal-generating moiety through a heterobifunctional PEG linker; and    detecting a signal generated by the antibody-signal-generating moiety conjugate that indicates the presence of the molecule of interest in the sample.    
   
   
       47 . The method of  claim 46 , wherein the biological sample comprises a tissue section or a cytology sample.  
   
   
       48 . The method of  claim 46 , wherein the conjugate comprises an antibody covalently linked to an enzyme.  
   
   
       49 . The method of  claim 48 , wherein the enzyme comprises horseradish peroxidase or alkaline phosphatase.  
   
   
       50 . The method of  claim 46 , wherein the conjugate has the formula:  
     
       
         
         
             
             
         
       
     
     wherein Ab is an antibody, SM is a signal-generating moiety, n=1 to 50 and s=1 to 10.  
   
   
       51 . The method of  claim 46 , wherein the conjugate has the formula:  
     
       
         
         
             
             
         
       
     
     wherein Ab is an antibody, SM is a signal-generating moiety, m=1 to 50 and t=1 to 10.  
   
   
       52 . The method of  claim 48 , wherein the method further comprises contacting the biological sample with a water-soluble metal ion and a redox-inactive substrate of the enzyme that is converted to a redox-active agent by the enzyme, which redox-active agent reduces the metal ion causing it to precipitate.  
   
   
       53 . The method of  claim 52 , wherein the enzyme comprises alkaline phosphatase.  
   
   
       54 . The method of  claim 48 , wherein the signal-generating moiety comprises an oxido-reductase enzyme and the method further comprises contacting the biological sample with a water soluble metal ion, an oxidizing agent and a reducing agent.  
   
   
       55 . The method of  claim 54 , wherein the oxido-reductase enzyme comprises horseradish peroxidase.  
   
   
       56 . The method of  claim 46 , wherein the antibody comprises an anti-antibody antibody.  
   
   
       57 . The method of  claim 46 , wherein the antibody comprises an anti-hapten antibody, and the method further comprises contacting the sample with a hapten-labeled antibody or a hapten-labeled nucleic acid sequence.  
   
   
       57 . The method of  claim 46 , wherein the signal-generating moiety comprises an enzyme and the method further comprises contacting the biological sample with a chromogenic, fluorogenic and/or luminogenic compound.