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Single-component TMB color developing solution, preparation method thereof and kit

A chromogenic liquid, single-component technology, applied in biochemical equipment and methods, biological testing, instruments, etc., can solve problems such as short storage time, increase test steps, affect ELISA detection results, etc., and achieve enhanced color intensity , the effect of improving the sensitivity

Active Publication Date: 2021-01-29
北京森康维特生物技术研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method is stable, it increases the test steps, and it is easy to produce batch-to-batch differences when the two components are mixed, thus affecting the ELISA detection results.
However, the single-component TMB chromogenic substrate solution currently on the market has poor stability, short storage time, high color background, and poor uniformity.

Method used

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  • Single-component TMB color developing solution, preparation method thereof and kit
  • Single-component TMB color developing solution, preparation method thereof and kit
  • Single-component TMB color developing solution, preparation method thereof and kit

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preparation example Construction

[0048] The embodiment of the present invention also relates to the preparation method of the above-mentioned chromogenic solution, which at least includes the following steps:

[0049] S1. Measure the pH adjuster in proportion and add it to water to obtain a pH adjuster aqueous solution; weigh TMB in proportion and dissolve it in dimethyl sulfoxide to obtain a TMB solution; preferably, the pH of the pH adjuster aqueous solution is 1.69;

[0050] S2, adding polyvinylpyrrolidone K30 in proportion to the pH regulator aqueous solution, and then adding TMB solution;

[0051] S3, adding 1,2,6-hexanetriol and buffer components in proportion, adjusting the pH value, adding hydrogen peroxide, constant volume, and filtering to obtain the color developing solution;

[0052] Preferably, add sodium thiosulfate, 1,2,6-hexanetriol, citric acid, glycerin, trehalose and disodium edetate in proportion, adjust the pH value with sodium acetate solution, and then add hydrogen peroxide .

[0053]...

Embodiment 1

[0057] Embodiment 1: TMB substrate chromogenic solution preparation

[0058] The specific method is:

[0059] 1. Measure 4.32mL of concentrated hydrochloric acid with a molar concentration of 12mol / L, and slowly add it into 200mL of water while stirring to obtain a pH regulator aqueous solution with a pH value of 1.69; add 14g of polyvinylpyrrolidone K30 to fully dissolve it;

[0060] 2. Dissolve 0.5g TMB in 10mL dimethyl sulfoxide, mix thoroughly and add to hydrochloric acid solution; then add 3mg sodium thiosulfate, 5g 1,2,6-hexanetriol, 1g citric acid, 5mL glycerin , trehalose 9.46g, disodium edetate 0.24g, stirred to fully dissolve;

[0061] 3. Use sodium acetate with a concentration of 8.2g / L to adjust the pH value to 3.5-3.8, add 500μL hydrogen peroxide, dilute to 1L, filter with a 0.45μm filter, and store in a brown bottle protected from light.

[0062] During the configuration process, pay attention to avoid strong light exposure. The above-mentioned water is deioni...

Embodiment 3

[0065] Embodiment 3: preparation of TMB substrate chromogenic solution

[0066] The specific method is:

[0067] 1. Measure 4.32mL of concentrated hydrochloric acid with a molar concentration of 12mol / L, and slowly add it into 200mL of water while stirring to obtain a pH regulator aqueous solution with a pH value of 1.69; add 12g of polyvinylpyrrolidone K30 to fully dissolve it;

[0068] 2. Dissolve 0.4g TMB in 10mL dimethyl sulfoxide, mix thoroughly and add to hydrochloric acid solution; then add 2.5mg sodium thiosulfate, 4.5g 1,2,6-hexanetriol, 1g citric acid, Glycerin 4.5mL, trehalose 9g, edetate disodium 0.2g, stir to fully dissolve;

[0069] 3. Use sodium acetate with a concentration of 8.2g / L to adjust the pH value to 3.5-3.8, add 500μL hydrogen peroxide, dilute to 1L, filter with a 0.45μm filter, and store in a brown bottle protected from light.

[0070] During the configuration process, pay attention to avoid strong light exposure. The above-mentioned water is deion...

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Abstract

The invention relates to the field of in-vitro detection, in particular to a single-component TMB color developing solution, a preparation method thereof and a kit. The color developing solution contains TMB, hydrogen peroxide, a buffer solution, a pH regulator, polyvinylpyrrolidone K30, dimethyl sulfoxide and 1,2,6-hexanediol. The single-component TMB color developing solution can be stably placed for 2 years at 2-8 DEG C, the color developing effect can last for 1 hour, the color developing strength is obviously enhanced, meanwhile, a background is not increased, the detection sensitivity isimproved, the purposes of stability and high efficiency are achieved, and various requirements of an enzyme linked immunosorbent assay kit on a color developing substrate solution are met.

Description

technical field [0001] The invention relates to the field of in vitro detection, in particular to a single-component TMB chromogenic solution and a kit for its preparation method. Background technique [0002] TMB (3,3',5,5'-tetramethylbenzidine) is a new type of safe chromogen reagent, as a new substrate of peroxidase, obtained in enzyme-linked immunosorbent assay (ELISA) widely used. HRP catalyzes the oxidation reaction of peroxide, the most representative peroxide is H 2 o 2 , and its reaction formula is as follows: DH 2 +H 2 o 2 →D+H 2 O, in the above formula, DH 2 as oxygen donor, H 2 o 2 for the hydrogen acceptor. In ELISA, DH 2 It is generally a colorless compound, which becomes a colored product after the action of an enzyme for colorimetric determination. The co-products of TMB after the action of HRP appear blue, which is a clear visual contrast. The principle of the test is: when the enzyme labeled on the antibody or antigen reacts with TMB, hydrogen ...

Claims

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Application Information

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IPC IPC(8): C12Q1/28G01N33/58G01N33/543
CPCC12Q1/28G01N33/581G01N33/543C12Q2326/12
Inventor 黄思斯武子函季志华张雨刘伯华杨鹏
Owner 北京森康维特生物技术研究所
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