Solution phase homogeneous assays

Abstract

Methods, reagents, kits and systems are disclosed for determining an analyte in a sample suspected of containing the analyte where all reagents are soluble in aqueous solution. One assay method includes treating a sample suspected of containing the analyte under conditions such that if the analyte is present, an activator is brought into reactive configuration with a chemiluminescent compound to activates it. The sample is also treated with an agent to reduce signal not related to analyte. Finally, the sample is treated with a trigger solution thereby producing light from the activated chemiluminescent compound. No reagents are associated with a surface or other solid phase.

The claimed invention is: 
     
       1. A kit for detecting an analyte in a sample comprising:
 a chemiluminescent-labeled specific binding partner comprising a first specific binding partner for the analyte, wherein the first specific binding partner is an antibody, and a chemiluminescent compound conjugated to the first specific binding partner;   an activator-labeled specific binding partner comprising a second specific binding partner for the analyte, wherein the second specific binding partner is an antibody, and an activator compound conjugated to the second specific binding partner, wherein the activator compound is a peroxidase enzyme;   a selective signal inhibiting agent; and   a trigger solution,   wherein all components are soluble in aqueous solution,   wherein the chemiluminescent compound is an acridan ketenedithioacetal compound according to the formula   
       
         
           
           
               
               
           
         
         wherein   designates the point of attachment of the chemiluminescent label to the first specific binding partner, 
         wherein R 1  and R 2  are selected from substituted or unsubstituted lower alkyl, wherein when R 1  or R 2  is a substituted group, it can be substituted with 1-3 groups selected from the group consisting of carbonyl groups, carboxyl groups, a SO 3   −  group, a OSO 3   −2  group, glycosyl groups, a PO 3   −  group, a OPO 3   −2  group, halogen atoms, a hydroxyl group, a thiol group, amino groups, quaternary ammonium groups, and quaternary phosphonium groups; and 
         R 3  is a benzyl group; and 
       
       wherein the selective signal inhibiting agent is selected from the group consisting of ascorbic acid, ascorbate sodium salt, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, phenoxazine, 3-aminotyrosine, and 2-aminophenol. 
     
     
       2. The kit of  claim 1 , wherein the selective signal inhibiting agent is ascorbic acid. 
     
     
       3. The kit of  claim 1 , wherein the chemiluminescent compound is 
       
         
           
           
               
               
           
         
       
       
        
       
     
     
       4. The kit of  claim 1 , wherein the peroxidase enzyme is selected from the group consisting of lactoperoxidase, microperoxidase, myeloperoxidase, haloperoxidase, vanadium bromoperoxidase, horseradish peroxidase (HRP), fungal peroxidases, lignin peroxidase, peroxidase from Arthromyces ramosus, Mn-dependent peroxidase produced in white rot fungi, and soybean peroxidase. 
     
     
       5. The kit of  claim 4 , wherein the peroxidase enzyme is horseradish peroxidase (HRP). 
     
     
       6. The kit of  claim 1 , wherein the trigger solution comprises a peroxide selected from the group consisting of hydrogen peroxide, urea peroxide, and perborate salts. 
     
     
       7. The kit of  claim 1 , wherein the trigger solution comprises an enhancer selected from the group consisting of phenol compounds, aromatic amines, benzoxazoles, hydroxybenzothiazoles, aryl boronic acids and mixtures thereof. 
     
     
       8. The kit of  claim 7 , wherein the enhancer is a phenol compound selected from the group consisting of p-phenylphenol, p-iodophenol, p-bromophenol, p-hydroxycinnamic acid, p-imidazolylphenol, acetaminophen, 2,4-dichlorophenol, 2-naphthol and 6-bromo-2-naphthol. 
     
     
       9. The kit of  claim 1 , wherein at least one of the chemiluminescent-labeled specific binding partner and activator-labeled specific binding partner comprises an auxiliary substance selected from the group consisting of soluble proteins, streptavidin, avidin, neutravidin, biotin, cationized BSA, fos, jun, soluble synthetic dendrimers, soluble synthetic polymers, soluble natural polymers, polysaccharides, dextran, and oligonucleotides. 
     
     
       10. A method for detecting an analyte selected from the group consisting of cells, viruses, bacteria, fungi, and combinations thereof in a sample, the method comprising:
 a) forming an aqueous reaction mixture comprising the following components:   the sample;   a chemiluminescent-labeled specific binding partner comprising a first specific binding partner for the analyte, wherein the first specific binding partner is an antibody, and a chemiluminescent compound conjugated to the first specific binding partner, wherein the chemiluminescent compound is a compound of the formula:   
       
         
           
           
               
               
           
         
       
       wherein   designates the point of attachment of the chemiluminescent label to the first specific binding partner 
       wherein R 1  and R 2  are independently selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted aralkyl groups of 1-20 carbon atoms, wherein when R 1  or R 2  is a substituted group, it can be substituted with 1-3 groups selected from the group consisting of carbonyl groups, carboxyl groups and quaternary ammonium groups, wherein R 3  is selected from the group consisting of alkyl, substituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl groups of 1-20 carbon atoms, phenyl, substituted or unsubstituted benzyl groups, alkoxyalkyl, carboxyalkyl and alkylsulfonic acid groups, wherein when R 3  is a substituted group, it can be substituted with 1-3 groups selected from the group consisting of carbonyl groups, carbox groups, tri(C 1 -C 8  alkyl)silyl groups, a SO 3   −  group, a OSO 3   −2  group, glycosyl groups, a PO 3   −  group, a OPO 3   −2  group, halogen atoms, a hydroxyl group, a thiol group, amino groups, quaternary ammonium groups, and quaternary phosphonium groups;
 an activator-labeled specific binding partner comprising a second specific binding partner for the analyte, wherein the second specific binding partner is an antibody, and an activator compound conjugated to the second specific binding partner, wherein the activator compound is a peroxidase enzyme; 
 a selective signal inhibiting agent selected from the group consisting of L-ascorbic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, 2-aminophenol, 3-amino-L-tyrosine, 4-chlorocatechol, phenoxazine, 2-bromobenzene-1,4-diol, 5,6-isopropylidene ascorbic acid, and ascorbic acid 6-palmitate; 
 
       by adding the components, in any order or concurrently, to an aqueous solution, wherein all of the components are soluble in the aqueous solution and none of the components are immobilized to a solid support, and wherein the chemiluminescent-labeled specific binding partner and the activator-labeled specific binding partner bind to the analyte present in the sample to form a binding complex in the aqueous solution; and
 b) adding to the aqueous reaction mixture a trigger solution comprising a peroxide compound, wherein the trigger solution releases a detectable chemiluminescent signal in the presence of the selective signal inhibiting agent that is correlated to the amount of the analyte-bound chemiluminescent-labeled specific binding partner and the analyte-bound activator-labeled specific binding partner in the aqueous reaction mixture, and wherein the selective signal inhibiting agent causes the ratio of signal produced by reaction between the chemiluminescent label compound and the activator label compound in the complex with the analyte to exceed the signal from reaction between the chemiluminescent label compound and the activator label compound when not in such a complex. 
 
     
     
       11. The method of claim 10, wherein the selective signal inhibiting agent is L-ascorbic acid. 
     
     
       12. The method of claim 10, wherein the chemiluminescent compound is 
       
         
           
           
               
               
           
         
        
       
     
     
       13. The method of claim 10, wherein the peroxidase enzyme is selected from the group consisting of lactoperoxidase, microperoxidase, myeloperoxidase, haloperoxidase, vanadium bromoperoxidase, horseradish peroxidase (HRP), fungal peroxidases, lignin peroxidase, peroxidase from Arthromyces ramosus, Mn-dependent peroxidase produced in white rot fungi, and soybean peroxidase. 
     
     
       14. The method of claim 13, wherein the peroxidase enzyme is horseradish peroxidase (HRP). 
     
     
       15. The method of claim 10, wherein the trigger solution comprises a peroxide selected from the group consisting of hydrogen peroxide, urea peroxide, and perborate salts. 
     
     
       16. The method of claim 10, wherein the trigger solution comprises an enhancer selected from the group consisting of phenol compounds, aromatic amines, benzoxazoles, hydroxybenzothiazoles, aryl boronic acids and mixtures thereof. 
     
     
       17. The method of claim 16, wherein the enhancer is a phenol compound selected from the group consisting of p-phenylphenol, p-iodophenol, p-bromophenol, p-hydroxycinnamic acid, p-imidazolylphenol, acetaminophen, 2,4-dichlorophenol, 2-naphthol and 6-bromo-2-naphthol. 
     
     
       18. The method of claim 10, wherein at least one of the chemiluminescent-labeled specific binding partner and activator-labeled specific binding partner comprises an auxiliary substance selected from the group consisting of soluble proteins, streptavidin avidin, neutravidin, biotin, cationized BSA, fos, jun, soluble synthetic dendrimers, soluble synthetic polymers, soluble natural polymers, polysaccharides, dextran, and oligonucleotides.