# Patent

## Title
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.

## Background
This application claims the benefit of U.S. Provisional Application No. 62/307,549 filed on Mar. 13, 2016, entitled “Hao Zhang”


BACKGROUND OF INVENTION
The present invention relates to the concentrated forms of TMB substrate in the field of immunoassays by using horseradish peroxidase (HRP) for the enzyme labeling of detection antibodies. Preferably, single component TMB substrate is used for the signal detection during various forms of immunoassays. The stability and concentrated forms of such TMB substrate become desirable during the large scale practical applications in immunodiagnostics to facilitate the transportation to distal or remote sites.
SUMMARY OF INVENTION
The present invention relates to the formulations of single component TMB substrate in concentrated form to facilitate the distant transport of liquid substrate component in its concentrated form. The key production protocols required the formulations of TMB substrate in high concentration (20 fold concentrate or higher) with good solubility and stability. To achieve this goal, a protocol is disclosed have to ensure TMB solubility as stability under such concentration at room temperature for 24-36 months.
In the process of the present invention, the addition of polyhydroxy alcohol such as glycerol, and low molecular weight PEG would enhance the solubility of TMB in high concentrations. EDTA at specific PH provides protection of TMB oxidation by heavy transition metals. Notably, encasing of TMB with β-cyclodextrin in essential for the stability of TMB during the long term storage. This encasing process of TMB has been widely disclosed in several previous inventions. Thus in brief summary, the current invention involves the inclusion of special polyhydroxyl organic solvents to enhance the solubility of the encasing TMB in the final concentration forms for long term stability during storage and transport at room temperature.


DETAILS DESCRIPTION OF THE INVENTION
Abbreviated names of organic compounds.

    DMSO—dimethyl sulfoxide
    TMB—3,3′,5,5′ tetramethylbenzidine
    BCD—β-cyclodextrin
    PEG—polyethyle glycol of defined molecular weight (<2000)
    EDTA—ethylediamine tetra-acetic acid
    H2O2— hydrogen peroxide
    HCl—Hydrochloric acid


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 H2O.
EXAMPLE 1
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:













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. H2O2
0.22%











i. Isopropanol
5
ml



j. Fumaric Acid
1
g



k. Distill H2O
70
ml









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 H2O2.
        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.

## 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 β-cyclodextrin (BCD), 0.0024 g EDTA, 0.02 g Citric Acid, 0.0022% H2O2, 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.