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Single-component TMB (Tetramethylbenzidine) developing solution and preparation method thereof

A chromogenic liquid and one-component technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of short storage time, increase the cost of packaging materials, and inconvenient use, and achieve strong sensitivity and stability, and stable solution Sexual problems, the effect of reducing packaging costs

Active Publication Date: 2016-09-28
广州捷倍斯生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Most of the TMB chromogenic solutions used in the existing commercially available enzyme-linked immunosorbent kits are two-component, divided into A solution and B solution, which need to be mixed before use, so there are the following disadvantages: the use is not convenient enough, and the packaging material has been improved. cost, waste of resources, and the mixing process in use is likely to cause uneven quality between batches
Commercially available enzyme-linked immunosorbent assay kits also use a single-component TMB chromogenic solution, but the existing single-component TMB chromogenic solution has problems such as poor stability, short storage time, high color background, and low sensitivity.
Moreover, after the interaction of TMB and HRP, only a blue solution can be produced but no precipitation can be produced. Therefore, the traditional TMB chromogenic solution can only be used for ELISA reactions, but not for Western, immunohistochemical (IHC) or in situ hybridization experiments.

Method used

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  • Single-component TMB (Tetramethylbenzidine) developing solution and preparation method thereof
  • Single-component TMB (Tetramethylbenzidine) developing solution and preparation method thereof
  • Single-component TMB (Tetramethylbenzidine) developing solution and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The one-component TMB chromogenic solution of the present embodiment is prepared by the following method:

[0033] Weigh 25g of citric acid (0.13mol) and 29g of trisodium citrate dihydrate (0.099mol), dissolve them in 800ml of deionized water, adjust the pH to 4.0 with concentrated hydrochloric acid, add 100ml of glycerin and then adjust the volume to 1000ml to obtain a mixture Solution A.

[0034] Weigh 350 mg of TMB powder, dissolve it in 5 ml of dimethyl sulfoxide, and add it to the above mixed solution A after completely dissolving to obtain a new mixed solution B.

[0035] Add 3g of DEAE dextran hydrochloride and 350mg of carbamide peroxide to the above mixed solution B, and mix thoroughly to obtain the one-component TMB color developing solution, which is stored in the dark at 4°C.

Embodiment 2

[0037] The one-component TMB chromogenic solution of the present embodiment is prepared by the following method:

[0038] Weigh 120g of citric acid (0.62mol) and 150g of sodium acetate (1.83mol), dissolve in 800ml of deionized water, adjust the pH to 6.0 with concentrated hydrochloric acid, add 100ml of glycerin and then adjust the volume to 1000ml to obtain the mixed solution A.

[0039] Weigh 300 mg of TMB powder, dissolve it in 5 ml of dimethyl sulfoxide, and add it into the above mixed solution A after completely dissolving to obtain a new mixed solution B.

[0040] Add 3.5 g of dextran and 350 mg of carbamide peroxide to the above mixed solution B, and mix thoroughly to obtain the single-component TMB color developing solution, which is stored in the dark at 4°C.

Embodiment 3

[0041] The sensitivity comparison of embodiment 3TMB chromogenic solution and DAB chromogenic solution

[0042] After gradient dilution with the HRP-labeled goat anti-mouse secondary antibody (product number ab6789) from abcam company, take 1ul and directly mark it on the copy paper. In the chromogenic solution and the DAB chromogenic solution (product of Guangzhou Jabis Biotechnology Co., Ltd., product number G3433), soak for 30 minutes and then rinse with tap water to terminate the color development. Color results as figure 1 As shown, A is TMB chromogenic solution, B is DAB chromogenic solution.

[0043] from figure 1 It can be seen that the single-component TMB chromogenic solution of the present invention can detect the HRP-labeled goat anti-mouse secondary antibody at a dilution of 1:4500, while the DAB chromogenic solution can detect the HRP-labeled goat anti-mouse secondary antibody at a dilution of 1:2000. Antibody also can't be detected, shows that the one-compone...

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Abstract

The invention relates to a single-component TMB (Tetramethylbenzidine) developing solution and a preparation method thereof. The single-component TMB developing solution is prepared from the following components: TMB, dimethyl sulfoxide, glycerol, an oxidant, a sugar type compound or a derivative thereof and a buffering solution; the pH value of the single-component TMB developing solution is 3.0-7.0; the buffering solution is selected from a citric acid-sodium citrate buffering solution, a citric acid-EDTA (Ethylene Diamine Tetraacetic Acid) buffering solution, a citric acid-sodium acetate buffering solution or a citric acid-disodium hydrogen phosphate; and the sugar type compound is selected from at least one of glucoside or a derivative thereof, dextran or a derivative thereof and ficoll or a derivative thereof. The single-component TMB developing solution can be used for experiments including Western, IHC or in-situ hybridization and the like and is high in sensitivity, good in stability and long in preservation time.

Description

technical field [0001] The invention relates to the technical field of biological in vitro diagnostic reagents, in particular to a single-component TMB chromogenic solution and a preparation method thereof. Background technique [0002] In the field of molecular biology and pathology, when performing Western, immunohistochemical (IHC) or in situ hybridization tests, traditional methods generally use DAB (3,3'-diaminobenzidine) as horseradish peroxidase A chromogenic substrate for (HRP), producing a brown precipitate in the chromogenic reaction. DAB has a certain carcinogenicity, and as a substrate of HRP, DAB has poor stability and low sensitivity after being formulated into a solution. TMB (3,3",5,5"-Tetramethylbenzidine) is a non-carcinogenic reagent and one of the chromogenic substrates of HRP. It has high sensitivity and is therefore widely used in ELISA reactions (ELISA). [0003] Most of the TMB chromogenic solutions used in the existing commercially available enzym...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/531
CPCG01N33/531G01N33/54393
Inventor 卢秀劲王小芳
Owner 广州捷倍斯生物科技有限公司
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