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Method for measuring malic acid concentration and malic acid diagnose reagent kit

A technology of a diagnostic kit and a determination method is applied in the field of malic acid diagnostic kit, double amplification method, detection of malic acid concentration, enzyme colorimetric method and enzyme-linked method for determination of malic acid concentration, and can solve the complex sample processing. , affect the process and results of the analysis, etc., to achieve accurate test results, low test costs, and easy operation.

Inactive Publication Date: 2007-12-05
SUZHOU ANJ BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The traditional method of separation and determination of malic acid is gas chromatography, but derivatization must be carried out before analyzing malic acid, which will affect the analysis process and results
Ion chromatography (IC) and high performance liquid chromatography (HPLC) are methods that have been used to measure malic acid in recent years, but the sample processing is more complicated; the HPLC content determination of malic acid adopts Zorbax-sbC18 chromatographic column, and HPLC analyzes malic acid The linear range is 0.280~9.020ug

Method used

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  • Method for measuring malic acid concentration and malic acid diagnose reagent kit
  • Method for measuring malic acid concentration and malic acid diagnose reagent kit

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Experimental program
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Effect test

Embodiment 1

[0045] Embodiment one (single dose)

[0046] Prepare the malic acid diagnostic kit according to the following ingredients and dosage:

[0047] Tris(carboxymethyl)aminomethane-hydrochloric acid buffer 100mmol / L,

[0048] Ethylene glycol 500mmol / L,

[0049] thio-NAD + 3mmol / L,

[0050] Coenzyme A 6mmol / L,

[0051] Malate dehydrogenase 10000U / L,

[0052] Pyruvate dehydrogenase 12000U / L;

[0053] After the reagents are all dissolved and prepared, they are divided into bottles and freeze-dried to make dry powder reagents; before use, add purified water and reconstitute for use.

[0054] Set on the automatic biochemical analyzer: temperature 37°C, reaction time 10 minutes, initial absorbance ≤ 0.1, test main wavelength 340nm, test secondary wavelength 405nm, volume ratio of malic acid sample to reagent is 1:25, The reaction direction is positive reaction (rising reaction), and the detection time is about 5 minutes.

[0055] After adding the samples and ...

Embodiment 2

[0056] Embodiment two (two doses)

[0057] Prepare the malic acid diagnostic kit according to the following ingredients and dosage:

[0058] Reagent I——

[0059] Tris(carboxymethyl)aminomethane-hydrochloric acid buffer 100mmol / L,

[0060] Propylene glycol 50mmol / L,

[0061] NAD + 3mmol / L,

[0062] Coenzyme A 6mmol / L;

[0063] Reagent II——

[0064] Tris(carboxymethyl)aminomethane-hydrochloric acid buffer 100mmol / L,

[0065] Glycerin 500mmol / L,

[0066] Malate dehydrogenase 10000U / L,

[0067] Pyruvate dehydrogenase 12000U / L.

[0068] After the reagents are all dissolved and prepared, they are divided into bottles to make liquid double reagents, which can be used directly.

[0069] Set on the automatic biochemical analyzer: temperature 37°C, reaction time 10 minutes, initial absorbance ≤ 0.1, test main wavelength 340nm, test secondary wavelength 405nm, the volume ratio of the tested malic acid sample to reagent I and reagent II is 2:20:5, t...

Embodiment 3

[0071] Embodiment three (three doses)

[0072] Prepare the malic acid diagnostic kit according to the following ingredients and dosage:

[0073] Reagent I——

[0074] Tris(carboxymethyl)aminomethane-hydrochloric acid buffer 100mmol / L,

[0075] Ammonium sulfate 50mmol / L,

[0076] NADP + 3mmol / L,

[0077] Coenzyme A 6mmol / L;

[0078] Reagent II——

[0079] Tris(carboxymethyl)aminomethane-hydrochloric acid buffer 100mmol / L,

[0080] Glycerin 500mmol / L,

[0081] Pyruvate dehydrogenase 12000U / L;

[0082] Reagent III——

[0083] Tris(carboxymethyl)aminomethane-hydrochloric acid buffer 100mmol / L,

[0084] Propylene glycol 500mmol / L,

[0085] Malate dehydrogenase 10000U / L;

[0086] After all the reagents are dissolved and prepared, they are divided into bottles to make liquid three reagents, which can be used directly.

[0087] When measuring malic acid concentration, set on the automatic biochemical analyzer: temperature 37°C, reaction time 10 mi...

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Abstract

The invention relates to a kind of double enlarge method, enzyme colorimetric method and enzyme linked method and a diagnosis reagent box applying the malic acid dehydrogenase coupling pyruvate dehydrogenase enzymatic reaction ratio colorimetric method / end-point method. The reaction of the malic acid dehydrogenase enzymolysis malic acid generates pyruvate and at the same time makes the coenzyme reducing the reduction coenzyme. The pyruvate generated by acid dehydrogenase enzymolysis malic acid makes the coenzyme reducing the reduction coenzyme again by the coupling reaction of pyruvate dehydrogenase. Because of making the coenzyme reducing the reduction coenzyme two times the reaction sensitivity is enhanced double. So we can assay the degree / speed of the ascent absorbance at the place of 340nm. It can measure and calculate the density of citric acid by measuring the degree / speed of the fall-way absorbance at the place of 340nm. The method has high specificity and it is not polluted by the endogenous and exogenous object and the test result is precise and accurate. The invention can get the array result by the ultraviolet / visible light analytic instrument so it is convenience to extend and apply.

Description

technical field [0001] The present invention relates to the detection of malic acid concentration in the fields of medicine, food, environment, etc., in particular to the method for measuring malic acid concentration by double amplification method, enzyme colorimetric method and enzyme-linked method, and the malic acid diagnostic method prepared therefrom. The kit belongs to the technical field of malic acid concentration analysis and detection. Background technique [0002] The traditional method for separating and measuring malic acid is gas chromatography, but derivatization must be carried out before analyzing malic acid, which will affect the analysis process and results. Ion chromatography (IC) and high performance liquid chromatography (HPLC) are methods that have been used to measure malic acid in recent years, but the sample processing is more complicated; the HPLC content determination of malic acid adopts Zorbax-sbC18 chromatographic column, and HPLC analyzes mali...

Claims

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Application Information

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IPC IPC(8): G01N21/31G01N33/50C12Q1/26C12Q1/00
Inventor 王尔中
Owner SUZHOU ANJ BIOTECHNOLOGY CO LTD
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