Chemiluminescent enzyme assay method and apparatus

Abstract

A chemiluminescent enzyme immunoassay method for quantifying antigen or antibody using 1,1′-oxalyldiimidazole (ODI) derivative or 1,1′-oxalyldisodium benzoate (ODB) derivative chemiluminescence (CL) detection was developed. Also, various enzymes were quantified using ODI derivative or DOB derivative CL detection. Fluorescent compound formed from a substrate (non-fluorescent compound) through the enzyme assay methods emitted CL when the fluorescent compound received energy from high-energy intermediate formed in ODI derivative or ODB derivative CL reaction.

What is claimed is: 
     
         1 . A method of quantifying antigen or antibody-labeled enzymes, comprising:
 adding a peroxyoxalate reagent and hydrogen peroxide to a chemiluminescent immunoassay comprising the antigen or antibody-labeled enzyme to be quantified, wherein the peroxyoxalate reagent is selected from the group consisting of 1,1′-oxalyldiimidazole (ODI) derivatives and 1,1′-oxalyldisodium benzoate (ODB) derivatives; and   correlating chemiluminescence emission intensity to antigen or antibody-labeled enzyme quantity.   
     
     
         2 . The method of  claim 1 , wherein the correlating step comprises plotting a calibration curve based on sample concentration on the x-axis and chemiluminescence intensity on the y-axis, wherein concentration of the sample is determined using the calibration curve and chemiluninescence emission measured in the presence of each test sample. 
     
     
         3 . The method of  claim 1 , further comprising adding an enzyme appropriate substrate. 
     
     
         4 . The method of  claim 1 , wherein the ODI derivative is 1,1′-Oxalyldiimidazole; 1,1′-Oxalyldi-2-methyl-imidazole (OD2MI); 1,1′-Oxalyldi-2-ethyl-imidazole (OD2EI); or  1 , 1 ′-Oxalyldi-4-methyl-imidazole (OD4MI). 
     
     
         5 . The method of  claim 1 , wherein the ODB derivative is 1,1′-oxalyldi-2-sodiumbenzoate (OD2B) or 1,1′-oxalyldi-4-sodiumbenzoate (OD4B). 
     
     
         6 . The method of  claim 1 , wherein the substrate comprises Amplex Red, OPDA, FDP, MUP, 9H-(1,3-dichlor-9,9-dimethylacridin-2-one-7-yl)phosphate, diammonium salt phosphate, 6,8-Difluoro-4-methylumbelliferyl phosphate (DiFMUP), Resorufin-7-O-phosphate, diammonium salt (Res-Phos), or 3-O-methyl fluorscein phosphate. 
     
     
         7 . The method of  claim 1 , wherein the reaction of hydrogen peroxide to an ODI derivative or a DOB derivative yields a high energy intermediate which energizes a fluorescent dye. 
     
     
         8 . The method of  claim 7 , wherein the fluorescent dye is Dimethylacridinone (DDA), Resorufin, or Fluorescein. 
     
     
         9 . The method of  claim 1 , wherein the enzyme comprises horseradish peroxidase or alkaline phosphatase. 
     
     
         10 . The method of  claim 1 , wherein the chemiluminescent immunoassay (CLEIA) is a sandwich CLEIA, a competitive CLEIA, or an indirect CLEIA. 
     
     
         11 . An immunoassay kit, comprising:
 an antigen or antibody-linked enzyme;   a substrate; and   a peroxyoxalate chemiluminescence (POCL) reagent.   
     
     
         12 . The kit of  claim 11 , wherein the POCL reagent is a 1,1′-oxalyldiimidazole (ODI) derivative or a 1,1′-oxalyldisodium benzoate (ODB) derivative. 
     
     
         13 . The method of  claim 12 , wherein the ODI derivative is 1,1′-Oxalyldiimidazole; 1,1′-Oxalyldi-2-methyl-imidazole (OD2MI); 1,1′-Oxalyldi-2-ethyl-imidazole (OD2EI); or  1 , 1 ′-Oxalyldi-4-methyl-imidazole (OD4MI). 
     
     
         14 . The method of  claim 12 , wherein the ODB derivative is 1,1′-oxalyldi-2-sodiumbenzoate (OD2B) or 1,1′-oxalyldi-4-sodiumbenzoate (OD4B). 
     
     
         15 . The method of  claim 11 , wherein the substrate comprises Amplex Red, OPDA, FDP, MUP, 9H-(1,3-dichlor-9,9-dimethylacridin-2-one-7-yl)phosphate, diammonium salt phosphate, 6,8-Difluoro-4-methylumbelliferyl phosphate (DiFMUP), Resorufin-7-O-phosphate, diammonium salt (Res-Phos), or 3-O-methyl fluorscein phosphate. 
     
     
         16 . The method of  claim 11 , wherein the enzyme comprises horseradish peroxidase or alkaline phosphatase. 
     
     
         17 . A method of quantifying concentration of one or more enzymes in a human sample, comprising quantifying hydrogen peroxide concentration which is formed when the one or more enzymes reacts with a specific enzyme oxidase, and the hydrogen peroxide concentration is determined using HRP, a substrate, and CL detection. 
     
     
         18 . The method of  claim 17 , wherein the CL detection comprises ODI derivative chemiluminescence or ODB derivative chemiluminescence.