Homogeneous enzyme immunoassay for keratinized structures

Abstract

This disclosure relates to methods and kits for determining the presence and/or amount of one or more analytes in a keratinized structure (e.g., hair) sample.

1 . A method for determining the presence or absence of an analyte of interest in a keratinized structure sample comprising:
 (a) combining in an aqueous medium:
 i. a conjugate comprising an enzyme and the analyte of interest; 
 ii. an antibody specific for the analyte of interest; and 
 iii. the keratinized structure sample; and 
   (b) determining if the analyte of interest is present or absent in the keratinized structure sample.   
     
     
         2 . The method of  claim 1 , wherein the enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase (G6PDH), alcohol dehydrogenase, glutamic dehydrogenase, malic dehydrogenase, isocitric dehydrogenase, α-glycerol phosphate dehydrogenase, lactic dehydrogenase, glyceraldehydes-3-phosphate dehydrogenase, gluthathione reductase, quinine reductase, nitrate reductase, and glutamic dehydrogenase. 
     
     
         3 . The method of  claim 1 , wherein the enzyme is glucose-6-phosphate dehydrogenase (G6PDH). 
     
     
         4 . The method of  claim 3 , wherein the G6PDH is covalently linked to the analyte of interest. 
     
     
         5 . The method of  claim 1 , wherein the antibody specific for the analyte of interest is specific for an analyte selected from the group consisting of cocaine, benzoylecgonine, cocaethylene, norcocaine, PCP, amphetamine, methamphetamine, cannabinoids, THC, carboxy-THC, heroin, benzodiazepines, methadone, codeine, morphine, 6-monoacetylmorphine (MAM), oxycodone, 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxymethamphetamine (MDMA). 
     
     
         6 . The method of  claim 1 , wherein the antibody is detectably labeled. 
     
     
         7 . The method of  claim 3 , wherein the G6PDH is obtained from a natural source. 
     
     
         8 . The method of  claim 3 , wherein the G6PDH is a recombinant enzyme. 
     
     
         9 . The method of  claim 3 , wherein the aqueous medium further comprises:
 (a) an enzyme substrate for G6PDH; and   (b) a coenzyme for G6PDH.   
     
     
         10 . The method of  claim 9 , further comprising:
 detecting a change in the enzymatic activity of the enzyme-analyte conjugate, wherein the change in enzymatic activity is related to the amount of the analyte of interest present in the sample.   
     
     
         11 . The method of  claim 1 , wherein the antibody is present at a concentration from about 0.4 μg/mL to about 1.0 μg/mL. 
     
     
         12 . The method of  claim 1 , wherein the conjugate comprising an enzyme and the analyte of interest is present at a concentration from about 0.4 μg/mL to about 0.5 μg/mL. 
     
     
         13 . The method of  claim 1 , wherein the determining the presence or absence of the analyte of interest is determined at a temperature of about 4° C. to about 45° C. 
     
     
         14 . The method of  claim 1 , wherein the determining the presence or absence of the analyte of interest is determined at a temperature of about 30° C. to about 40° C. 
     
     
         15 . The method of  claim 1 , wherein the determining the presence or absence of the analyte of interest is determined at a temperature of about 37° C. 
     
     
         16 . The method of  claim 1 , wherein the aqueous medium has a pH range of about 4.0 to about 11.0. 
     
     
         17 . The method of  claim 1 , wherein the aqueous medium has a pH of about 7.0. 
     
     
         18 . The method of  claim 1 , wherein the aqueous medium contains less than about 20% of a polar solvent. 
     
     
         19 . The method of  claim 1 , wherein the aqueous medium contains less than about 20% of N,N-Dimethylformamide (DMF), acetonitrile, dimethylsulfoxide (DMSO), methanol, or a combination thereof. 
     
     
         20 . The method of  claim 1 , wherein the analyte of interest is selected from the group consisting of a drug of abuse, a toxic chemical, an environmental chemical, a petroleum product, a natural product, an organic compound, a nutrient, a prescription and over-the-counter medication, or a metabolite, derivative, or breakdown product of any of the foregoing. 
     
     
         21 . The method of  claim 20 , wherein the analyte of interest is a drug of abuse or metabolite thereof. 
     
     
         22 . The method of  claim 21 , wherein the drug of abuse or metabolite thereof is selected from the group consisting of cocaine, benzoylecgonine, cocaethylene, norocaine, PCP, amphetamine, methamphetamines, cannabinoids, THC, carboxy-THC, nicotine, cotinine, benzodiazepines, heroin, codeine, morphine, 6-monoacetylmorphine (MAM), oxycodone, 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxymethamphetamine (MDMA). 
     
     
         23 . The method of  claim 1 , wherein the analyte of interest is selected from the group consisting of opioids, amphetamines, NSAIDS, steroids, cannabinoids, benzodiazepines, barbiturates, tricyclics, nicotine, and ephedrines, or metabolites, derivatives, or breakdown products of any of the foregoing. 
     
     
         24 . A method for determining the presence or absence of an analyte of interest in a keratinized structure sample comprising:
 (a) combining in an aqueous medium:
 i. an enzyme-analyte conjugate comprising glucose-6-phosphate dehydrogenase (G6PDH) covalently linked to the analyte of interest; 
 ii. an antibody specific for the analyte of interest; 
 iii. a keratinized structure sample; 
 iv. an enzyme substrate for G6PDH; and 
 v. a coenzyme for G6PDH; and 
   (b) determining if the analyte of interest is present or absent in the keratinized structure sample.   
     
     
         25 . A method for determining the presence or absence of an analyte of interest in a keratinized structure sample comprising:
 (a) combining in an aqueous medium:
 i. an enzyme-analyte conjugate comprising glucose-6-phosphate dehydrogenase (G6PDH) covalently linked to the analyte of interest; 
 ii. an antibody specific for the analyte of interest; 
 iii. a keratinized structure sample; 
 iv. an enzyme substrate for G6PDH; and 
 v. a coenzyme for G6PDH; and 
   (b) determining if the analyte of interest is present or absent in the keratinized structure sample, wherein the analyte of interest is a drug of abuse or metabolite thereof.   
     
     
         26 . The method of  claim 1 , wherein the keratinized structure sample is a hair sample. 
     
     
         27 . The method of  claim 1 , wherein the keratinized structure sample is a nail sample. 
     
     
         28 . The method of  claim 1 , further comprising extracting the analyte of interest from the keratinized structure using a buffer. 
     
     
         29 . The method of  claim 28 , wherein the buffer is a phosphate buffer. 
     
     
         30 . The method of  claim 25 , further comprising extracting the analyte of interest from the keratinized structure using a buffer.