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Tetramethylbenzidine (TMB) coloration solution and preparation method thereof

A technology of color developing solution and B solution, which is applied in the direction of chemical reaction of materials and material analysis by observing the influence of chemical indicators, can solve the problems of complicated operation, poor stability, low sensitivity, etc. The effect of simplifying operation steps, reducing random errors and improving sensitivity

Active Publication Date: 2013-04-24
杭州联科生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The usual TMB chromogenic reagent is composed of multiple components, which must be prepared before use, and is prone to precipitation, which is relatively inconvenient to use, and it is easy to cause unstable test results
The TMB chromogenic reagents produced by some manufacturers are usually divided into two storage solutions, A and B. Mix A and B in proportion before use, which is cumbersome to operate and prone to mismatch, resulting in experimental failure.
In addition, some manufacturers also produce single-phase TMB chromogenic solution, but there are disadvantages such as low solubility, poor stability, short storage time, low sensitivity, and high color background.
[0005] Such as the Chinese patent, the patent No. is 200610028624X and the name is a method for preparing TMB chromogenic solution disclosed in "Preparation of Chromogenic Agent in ELISA In Vitro Diagnostic Reagent". The reagent finally obtained by this method has low stability, 2~ 8 degrees can only be stored for about 15 months, and the color rendering background is relatively high

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: the preparation of TMB chromogenic solution:

[0020] a. Preparation of liquid A: add 0.5mol of citric acid, 0.5mol of disodium hydrogen phosphate, 0.5mol of hydrogen peroxide, and 1mmol of EDTA into 1L of water, stir well, and fully dissolve;

[0021] b. Prepare liquid B: add TMB1mmol and HCl0.5mol to 1L water, then add polyvinylpyrrolidone with a final mass concentration of 10%; stir evenly and fully dissolve;

[0022] c. Finally, after mixing equal volumes of liquid A and liquid B, adjust the pH=4.0 to obtain the crude TMB chromogenic liquid;

[0023] d. Suction filter the crude TMB chromogenic solution with a 0.22um microporous membrane to obtain the TMB chromogenic solution.

Embodiment 2

[0024] Example 2: The TMB chromogenic solution prepared by the method of Example 1 was compared with the TMB ONE chromogenic solution commercialized by Kem-En-Tec Diagnostics, Denmark.

[0025] Taking the Human IL-17A double-antibody sandwich ELISA as an example, the color was developed for 15 minutes, and the OD value (λ=450nm) was measured as shown in Table 1:

[0026] Human IL-17A pg / ml KEM-OD value Example 1-OD value 500.00 2.680 2.976 200.00 1.308 1.576 80.00 0.606 0.738 32.00 0.292 0.361 12.80 0.166 0.198 5.12 0.116 0.131 2.05 0.094 0.101 0.00 0.076 0.078

[0027] It can be drawn from Table 1 that the signal-to-noise ratio of KEM ONE at a Human IL-17A concentration of 2.05 pg / ml is 0.094 / 0.076=1.24, while the TMB chromogenic solution obtained by the method of Example 1 was obtained at a Human IL-17A concentration When it is 2.05pg / ml, the signal-to-noise ratio is 0.101 / 0.078=1.29, which is obviously better ...

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Abstract

The invention relates to a tetramethylbenzidine (TMB) coloration solution and a preparation method of the TMB coloration solution and belongs to the technical field of biological in-vitro diagnostic reagents. A solution and B solution are mixed in an isopyknic mode to obtain the TMB coloration solution, the A solution comprises, by molarity, citric acid 0.01-0.5 mol / L, sodium hydrogen phosphate 0.01-0.5 mol / L, hydrogen peroxide 0.01-0.5 mol / L and EDTA 0.1-1 mmol / L; the B solution comprises the TMB, hydrochloric acid and polyvinylpyrrolidone, wherein TMB 0.1-1 mmol / L, hydrochloric acid 0.01-0.5 mol / L and by weight percentage, polyvinylpyrrolidone 0.1-10%. The dissolving system of the TMB is adjusted to increase solubility of the TMB, and the polyvinylpyrrolidone enables the one-phase TMB solution to be capable of being stored for two years at the storage temperature of 2-8 DEG C.

Description

technical field [0001] The invention relates to a TMB chromogenic solution and a preparation method thereof, belonging to the technical field of biological in vitro diagnostic reagents. Background technique [0002] Enzyme-linked immunosorbent assay (ELISA) has been widely used in the detection of antigens, antibodies, cytokines, and biochemical substances due to its advantages of rapidity, sensitivity, simplicity, and ease of standardization. There are many enzyme-linked immunosorbent assay (ELISA) detection methods, but each method is inseparable from enzyme conjugates and chromogenic reagents. The enzyme most widely used in ELISA reagents is horseradish peroxidase (HRP). 3,3',5,5'-Tetramethylbenzidine (TMB). [0003] TMB, or 3,3’,5,5’-tetramethylbenzidine, is a common substrate for horseradish peroxidase. During the detection, HRP catalyzes the peroxide to release oxygen, and the colorless TMB is oxidized into a blue product. The blue product can usually measure the ab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/78
Inventor 袁志波
Owner 杭州联科生物技术股份有限公司
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