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Method for detecting blood homocysteine and kit

A homocysteine, detection kit technology, applied in the measurement of color/spectral properties, material analysis by observing the effect on chemical indicators, material excitation analysis, etc., can solve the problem of high sensitivity, high price, color development Reaction and other problems, to achieve the effects of good reagent stability, sensitive method and high repeatability

Inactive Publication Date: 2013-01-30
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the operation is more complicated, and it is not suitable for rapid analysis of large samples
(3) Fluorescence polarization immunoassay (FPLA) method, which has high sensitivity and fast detection speed, but is expensive, so it is not easy to popularize in a short period of time
However, the color reaction is susceptible to interference from some blood components

Method used

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  • Method for detecting blood homocysteine and kit
  • Method for detecting blood homocysteine and kit
  • Method for detecting blood homocysteine and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] HCY generates H under the catalysis of recombinant rHCYase 2 S, H 2 S and dimethyl-p-aniline in F 3+ Under oxidation, the reaction produces blue methyl blue. The depth of blue is proportional to the concentration of HCY. The darker the color, the higher the concentration of HCY. Based on this principle, the kit is prepared, such as figure 1Shown is the color chart of HCY detection. Add HCY and cysteine ​​at concentrations of 0, 10, 15, 20, and 25 μM to the well plate, and perform detection according to the color detection method. There is no obvious cysteine ​​in the upper row. Color, the lower row of color, with the increase of HCY concentration, the darker the color, the absorbance is proportional to the concentration of HCY.

[0032] Fluorescence detection kit consists of: a 96-well blackboard, 27.03 μg of HCY standard substance, 3 mg of HCY-specific converting enzyme, 50 ml of diluent, 0.771 μg of reducing agent, 10 ml of chromogenic solution A, and 10 ml of chrom...

Embodiment 2

[0042] The composition of the colorimetric detection kit: a 96-well transparent microplate, HCY standard 27.03μg, HCY-specific converting enzyme 2mg, diluent 50ml, reducing agent 0.771μg, chromogenic solution A10ml, chromogenic solution B10ml.

[0043] Steps:

[0044] 1) Prepare HCY standard substance with diluent to make 1ml solution, the concentration is 200μmol / L, after serial doubling dilution, dilute to 100μmol / L, 50μmol / L, 25μmol / L, 10μmol / L, 5μmol / L L, 2.5μmol / L, 0μmol / / L, the diluent is directly used as the standard concentration of 0μmol / / L, and it is prepared within 15 minutes before use. Dissolve the reducing agent with 5ml of diluent.

[0045] 2) Add 100 μl of HCY standard and sample to a 96-well transparent microtiter plate, and then add 10 μl of reducing agent to reduce for 1 h;

[0046] 3) Dissolve HCY-specific invertase with 5ml diluent, then add 20 μl of HCY-specific invertase solution to the above reaction solution for 5 minutes;

[0047] 4) Add 50 μl of c...

Embodiment 3

[0051] The HCY of the serum sample is measured by the detection method of the present invention, and the result is compared with the measurement result of high performance liquid chromatography (HPLC).

[0052] Select 10 parts of serum samples measured by HPLC method, measure with the method of the present invention, measure the result and compare with HPLC method measure result, the result is as shown in Table 1, and the calculated coincidence rate is 0.9998. It shows that the result of this method is reliable.

[0053] Table 1 comparison of measurement results

[0054]

[0055]

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Abstract

The utility model discloses a method for detecting blood homocysteine and a kit in the field of biological detection technologies. The kit comprises a kit body, a 96-pore plate, HCY standard substance, HCY specific convertase, a reducing agent, a buffer solution, color developing liquid A and color developing liquid B, wherein the 96-pore plate, the HCY standard substance, the HCY specific convertase, the reducing agent, the buffer solution, the color developing liquid A and the color developing liquid B are arranged in the kit body. The kit utilizes HCY in rHCYase decomposed blood to produce H2S, produced H2S is reacted with dimethyl-p-toluidine to produce methyl blue, and the HCY content in the blood is reflected through the methyl blue generating amount. The kit is simple in operation, a reagent is good in stability and high in repeatability, and the method is flexible and suitable for quick clinical HCY detection of laboratories.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a blood homocysteine ​​detection method and a kit. Background technique [0002] Studies in the past ten years have confirmed that a high concentration of homocysteine ​​(Homocysteine, HCY) in the blood is an independent risk factor for coronary artery disease, cerebrovascular and peripheral vascular diseases. HCY is a sulfur-containing amino acid produced by demethylation of methionine. It is metabolized in the body through two pathways: remethylation and transsulfurization: first, under the catalysis of methionine synthase, vitamin B12 is used as the The coenzyme uses N5-methyltetrahydrofolate as the methyl donor, and HCY is methylated to generate methionine. The key enzyme in this process is methylenetetrahydrofolate reductase (MTHFR) with vitamin B2 as a coenzyme. This enzyme reduces N5, N10-methylenetetrahydrofolate to N5-methyltetrahydrofolate, whi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N21/78G01N21/31
Inventor 弓景波钱令嘉
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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