Preparation of multi-hapten mutant g6pdh conjugates and their use for detection of multiple analytes

Abstract

The present invention relates to multi-hapten mutant G6PDH conjugates, methods of their preparation and their use for detection of multiple analytes. The compositions of the invention comprise different types of haptens (molecules) require to be immobilized into the G6PDH to make one multi-hapten-G6PDH conjugate. Both thiol and amine functional groups on G6PDH are utilized for immobilization of different haptens.

1 . A composition which comprises at least two distinct specific binding pair (sbp) members conjugated to a glucose-6-phosphate dehydrogenase (G6PDH), wherein at least a first specific binding pair member is conjugated to G6PDH via a thiol group and wherein at least a further specific binding pair member is conjugated to G6PDH via a amino group. 
     
     
         2 . The composition according to  claim 1 , wherein the G6PDH is a mutant G6PDH, the mutant G6PDH being derived from a precursor G6PDH by the insertion of at least one cysteine per subunit, or substitution of at least one amino acid with cysteine per subunit, as compared to the precursor G6PDH. 
     
     
         3 . The composition according to  claim 1 , wherein G6PDH is a bacterial G6PDH derived from the genus of bacterium selected from the group consisting of Leuconostoc. 
     
     
         4 . The composition according to  claim 1 , wherein the two distinct binding pair members are serologically not cross-reactive. 
     
     
         5 . The composition according to  claim 1 , comprising three or more distinct specific binding pair (sbp) members conjugated to G6PDH. 
     
     
         6 . The composition according to  claim 1 , comprising a plurality of distinct further specific binding pair members conjugated to a respective plurality of amino groups. 
     
     
         7 . The composition according to  claim 1 , comprising a plurality of distinct first specific binding pair members conjugated to a respective plurality of thiol groups. 
     
     
         8 . The composition according to  claim 1 , wherein the first and second specific binding pair members are selected from the group consisting of opium, opioid analgesics, amphetamines, cocaine, methadone, alkaloids, catecholamines, methylendioxyamphetamines (MDMA, MDA, and MDEA, etc.), PCP, propoxyphene, methaqualone, barbiturates, benzodiazepines, tricyclic antidepressants, tranquilizers, tetrahydrocannabinol, LSD, ketamine, GHB, and other drugs of abuse, including amino acids, hormones, and steroids, buprenorphine, norbuprenorphine, and analogs, metabolites, and derivatives thereof. 
     
     
         9 . The composition according to  claim 1 , further comprising a linker between G6PDH and either or both of specific binding pair member and further specific binding pair member. 
     
     
         10 . The composition according to  claim 1 , wherein the first binding pair member is MDA or an analog, metabolites, or derivative thereof and the second binding pair member is Amphetamine or an analog, metabolite, or derivative thereof or Methamphetamine or an analog, metabolite, or derivative thereof. 
     
     
         11 . The composition according to  claim 10 , wherein the first binding pair member is MDA or an analog, metabolites, or derivative thereof and the second binding pair member is Amphetamine or an analog, metabolite, or derivative thereof and a third binding pair member is Methamphetamine or an analog, metabolites, or derivative thereof. 
     
     
         12 . A method for producing a composition according to  claim 1 , comprising the steps of:
 conjugating a first specific binding pair member to G6PDH via a thiol group; and then   conjugating at least a further specific binding pair member to G6PDH via a amino group.   
     
     
         13 . The method according to  claim 12 , wherein the step of conjugating includes providing a linker between G6PDH and either or both of specific binding pair member and further specific binding pair member. 
     
     
         14 . A method of detecting a first analyte and a further analyte in a sample, comprising the steps of:
 (i) combining in a liquid medium:
 (a) said sample, 
 (b) a composition comprising G6PDH wherein a first specific binding pair member is conjugated to G6PDH via a thiol group and a further specific binding pair member is conjugated to G6PDH via an amino group, 
 (c) a first antibody capable of binding the first specific binding pair member, 
 (d) a further antibody capable of binding the further specific binding pair member, and 
 (e) a substrate for said G6PDH; and 
   (ii) determining the enzymatic activity of said G6PDH in said medium.