Assays and probes with enzyme labels

Abstract

Probes comprise S1 and P1 nuclease (as an enzyme label) linked to a specific binding member such as a nucleotide sequence or an antibody. Such probes are useful for sandwich assays. As compared with known probes using alkaline phosphatase as a label, advantages include relative insensitivity to phosphate and elevated temperature and reduced risk of nonspecific binding.

1 . A probe for use in an assay comprising a nuclease selected from P1 and S1 nucleases coupled to a specific binding member (“sbm”).  
     
     
         2 . A probe according to  claim 1  wherein the sbm is an antibody or a functional fragment thereof.  
     
     
         3 . A probe according to  claim 1  wherein the sbm is an IgG antibody or a functional fragment thereof.  
     
     
         4 . A probe according to  claim 1  wherein the sbm is a single-stranded nucleic acid.  
     
     
         5 . A probe according to any of  claims 1  to  4  wherein the nuclease is covalently attached, directly or indirectly, to the sbm.  
     
     
         6 . A method of producing a probe according to any preceding claim which comprises coupling a nuclease to an sbm.  
     
     
         7 . A method according to  claim 6  wherein the coupling is between a nucleic acid with a derivatised 5′-end and a nuclease which has been rendered susceptible to disulphide exchange.  
     
     
         8 . An assay employing a probe according to any of  claims 1  to  5  comprising a nuclease and a first sbm, wherein 
 i) a sample believed to contain an analyte is brought into contact with a carrier having a second sbm immobilised to it so that analyte in the sample binds to the second sbm and is thus bound to the carrier, said first and second sbm's being selected such that they bind to different sites on the analyte;  
 ii) the bound analyte is contacted with the probe so that the probe binds to the analyte via the first sbm; and  
 iii) the bound probe is contacted with a signal system such that the activity of the nuclease of the bound probe leads to a detectable signal.  
 
     
     
         9 . An assay according to  claim 8  in which step (iii) is carried out in the presence of phosphate.  
     
     
         10 . An assay according to  claim 9  wherein said phosphate comprises inorganic phosphate.  
     
     
         11 . An assay according to  claim 9  or  claim 10  wherein said phosphate comprises a phosphate monoester.  
     
     
         12 . An assay according to any of  claims 8  to  11  wherein the probe comprises antihuman IgG antibody (as the first sbm) the second sbm is measles antigen, and the analyte is human serum IgG antibodies to measles.  
     
     
         13 . An assay according to any of  claims 8  to  11  wherein the probe comprises an oligonucleotide and the analyte is single-stranded dna.  
     
     
         14 . An assay according to any of  claims 8  to  13  wherein said signal system comprises an amplification system.  
     
     
         15 . An assay according to  claim 14  wherein said amplification system comprises an apoenzyme which is convertable into a holoenzyme by interaction with an accessory subunit; and a masked form of said subunit which is convertible into its active unmasked form by the action of the nuclease of the probe.  
     
     
         16 . An assay according to  claim 15  wherein said subunit is FAD and said masked form is 3′-FADP.  
     
     
         17 . An assay according to  claim 15  or  16  wherein said apoenzyme is apo-glucose oxidase or apo-D-amino oxidase.