Compositions and methods for in vitro diagnostic tests including zwitterionic solubilization reagent
View Patent ↗The disclosure is directed to compositions, kits, and methods for performing colorimetric analysis. A composition for a colorimetric assay can be prepared that includes an aromatic amine chromogenic substrate and a zwitterionic solubilization reagent that is N,N,N-trimethylglycine (betaine). The zwitterionic solubilization reagent is highly useful as a non-encapsulating solubilization reagent and provided faster kinetics and increased dye content of an aromatic amine chromogenic substrate in an enzymatic reaction. In turn, this can result in improvements in detection of an analyte in a sample and also can increase the sample throughput. The aromatic amine chromogenic substrate and N,N,N-trimethylglycine solubilization reagent can be used in assays such as ELISAs in order to provide a more accurate and faster detection of analytes in a biological sample.
1. An aqueous composition for colorimetric analysis comprising a benzidine chromogenic substrate and N,N,N-trimethylglycine, wherein the benzidine chromogenic substrate is 3,3′,5,5′ tetramethylbenzidine (3,3′,5,5′ TMB) wherein the N,N,N-trimethylglycine is present in the composition in the range of 0.25 M to 1 M, wherein the composition has a pH in the range of 3 to 4, wherein the N,N,N-trimethylglycine and 3,3′,5,5′ TMB are present in the composition in a molar ratio in the range of 20:1 to 1,000:1, and wherein the N,N,N-trimethylglycine improves the solubility of the 3,3′,5,5′ TMB in the aqueous composition.
2. The composition of claim 1 , wherein the N,N,N-trimethylglycine is present in the composition in the range of 0.5 M to 1 M.
3. The composition of claim 1 further comprising a peroxidase, an oxidase, or a conjugate of a peroxidase or an oxidase with an analyte-specific binding moiety.
4. The composition of claim 3 wherein the peroxidase and/or oxidase is selected from the group consisting of horseradish peroxidase, soybean peroxidase, glucose oxidase, galactose oxidase, xanthine oxidase, lactoperoxidase, microperoxidase, NADH peroxidase NADPH peroxidase, fatty-acid peroxidase, and catalase.
5. The composition of claim 4 wherein the peroxidase is a derivative of horseradish peroxidase (HRP).
6. A kit for colorimetric analysis comprising the composition of claim 1 , and one or more of the following: (a) an analysis plate upon which a colorimetric analysis can be performed; (b) an analyte specific binding member; (c) an analyte positive control; or (d) an analyte negative control.