Solution phase homogeneous assays
View Patent ↗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.
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.