Azobenzene derivatives as labeling agents and intermediates thereof

Abstract

A compound of the formula I wherein R is H or —N═N-2-carboxyphenyl; A is (CH 2 ) n or —CH═CH—, wherein n is an integer from 0 to 10, or A may also be —CH(COOH)— when R is —N═N-2-carboxyphenyl; and X is a radical selected from the group consisting of: (i) Cl; (ii) COOR 1 , wherein R 1 is p-nitrophenyl or N-succinimidyl; (iii) CONH—NHR 2 , wherein R 2 is H, COO(t-butyl) or COObenzyl; (iv) CONH—[B]—NHR 3 , wherein R 3 is H, COOR 1 , or CO-[B′]-maleimido, wherein R 1 is t-butyl, p-nitrophenyl or N-succinimidyl, and B and B′, the same or different, are (CH 2 ) n wherein n is an integer from 2 to 10; (v) CONH—[B]—COOR 4 , wherein R 4 is H, C 1 -C 8 alkyl, N-succinimidyl; (vi) CONH—[B]—OH; (vii) CONH—[B]—CONH—NHR 2 , wherein R 2 is H, COO(t-butyl) or COObenzyl; and (viii) NHR 2 , wherein R 2 is H, COO(t-butyl) or COObenzyl, when A is —CH(COOH)— and R is —N═N-2-carboxyphenyl. The 4′-hydroxyazobenzene-2-carboxylic acid (HABA) compounds are novel reagents for labeling, isolation and detection of biological molecules.

What is claimed is:  
     
         1 . Anti-HABA antibodies.  
     
     
         2 . Anti-HABA antibodies according to  claim 1 , which are polyclonal antibodies.  
     
     
         3 . Polyclonal anti-HABA antibodies according to  claim 2  which recognize the entire HABA molecule.  
     
     
         4 . Polyclonal anti-HABA antibodies according to  claim 2  which are specific for the azophenyl-2-carboxylic acid moiety of the HABA core.  
     
     
         5 . Anti-HABA antibodies according to  claim 1 , which are monoclonal antibodies.  
     
     
         6 . A method for qualitative or quantitative detection of a molecule I in a sample which comprises the steps of: 
 a. contacting the sample with a HABAylated molecule II that recognizes molecule I;    b. reacting with labeled anti-HABA antibodies or labeled avidin;    c. visualizing and quantifying the HABA/avidin or HABA/anti-HABA complex formed.    
     
     
         7 . The method according to  claim 6  for the detection of a DNA molecule transferred to a specific membrane after digestion of a DNA sample with specific restriction enzymes and separation by agarose electrophoresis, which comprises hybridizing the DNA molecule with the HABAylated DNA probe.  
     
     
         8 . The method according to  claim 6  for detecting an RNA molecule transferred to a specific membrane after separation of an RNA sample by agarose electrophoresis, which comprises hybridizing said RNA molecule with the hybridized DNA or RNA probe.  
     
     
         9 . The method according to  claim 6  for the detection of a protein molecule transferred to a specific membrane after separation of a protein mixture by SDS-PAGE, which comprises reacting said protein molecule with the corresponding HABAylated antibody.