Concentated liquid single component TMB substrate for detection assay of horseradish peroxidase

Abstract

Through special formulations, stable twenty-fold concentrate of single component TMB substrate is produced to minimize the volume and reduce the transportation cost. Reconstitution of the concentrate upon delivery to the distal site can be made with deionized water to yield single component TMB substrate solution for routine immunodiagnostic applications.

Claims

1. A 3,3,5,5-tetramethylbezidine (TMB) 20-fold concentrate solution, said concentrate solution comprising 0.1 ml Glycerol, 0.1 g PEG2000, 0.1 ml DMSO, 0.013 g TMB, 0.02 g -cyclodextrin (BCD), 0.0024 g EDTA, 0.02 g Citric Acid, 0.0022% H.sub.2O.sub.2, 0.05 ml Isopropanol, 0.01 g Fumaric acid, and 0.7 ml deionized water; wherein the final volume of the concentrate solution is 1 ml and the concentrate solution has a pH of about 3.6.

Description

DETAILS DESCRIPTION OF THE INVENTION

(1) Abbreviated names of organic compounds. DMSOdimethyl sulfoxide TMB3,3,5,5 tetramethylbenzidine BCD-cyclodextrin PEGpolyethyle glycol of defined molecular weight (<2000) EDTAethylediamine tetra-acetic acid H.sub.2O.sub.2 hydrogen peroxide HClHydrochloric acid

(2) As noted, TMB has been widely accepted as an excellent peroxidase substrate. The mechanism of action in using TMB substrate during enzyme immunoassay with peroxidase has been well established for decades. Methods of preventing spontaneous oxidation of TMB and subsequent precipitation have been elucidated previously. This will lead to the storage shell life of TMB reagent to as long as 36 months or longer. Therefore, polyhydroxy alcohol has been shown to increase solubility of TMB or encased TMB in mixture of organic solvents and H.sub.2O.

EXAMPLE 1

(3) The Production of A twenty fold concentrated TMB Solution for ELISA Methods is described as follows: The reagents that are required to formulate twenty fold plus TMB concentrate are listed in the preferred weigh or final volume per 100 ml by the following table:

(4) TABLE-US-00001 Name ml (g)/100 ml a. Glycerol 10 ml b. PEG2000 10 g c. DMSO 10 ml d. TMB 1.3 g e. BCD 2 g f. EDTA 0.24 g g. Citric Acid 2 g h. H.sub.2O.sub.2 0.22% i. Isopropanol 5 ml j. Fumaric Acid 1 g k. Distill H.sub.2O 70 ml

(5) Reagent Preparations Prior to Final Production of concentrate of one step TMB. 1. The resistance of deionized water should be greater than 18 megohm. 2. Preparation of BCD solution. 10 ml deionized water, 2 g, stirred until 2 g BCD is dissolved. 3. Additions of other chemicals are given as listed in the Table except for TMB and DMSO (sec 4.) and H.sub.2O.sub.2. 4. Prepare 1.3 g TMB in 40 ml DMSO with constant stirring 5. The final solution should consist of all the chemical reagents listed on the table with PH of the final solution adjusted to pH3.6+ or 0.02 with N HCl. 6. Deionized water was added until the final solution one step TMB concentrate to reach to 100 ml with 5-10 hours of constant stirring at 40-50 C. until all solid materials are fully dissolved.