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Method for enzymatically determining creatinine in serum by two steps

A determination method and technology for creatinine, which are applied in the field of medical testing and determination, can solve problems such as affecting the determination results, and achieve the effects of increasing burden, high accuracy and increasing reagent cost.

Inactive Publication Date: 2011-04-20
李立和
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The existing problem is: in the current method, whether it is one-step enzymatic method or two-step enzymatic method to measure creatinine, the creatine produced after creatinine hydrolysis and endogenous creatine are converted into quinoneimine at the same time during the measurement process, and the creatinine measurement results include creatinine and creatine
Zhejiang Dongou Company uses creatinine amidohydrolase, peroxidase and 4-AAP as the second reagent, and adds catalase to reagent I to eliminate the H produced by endogenous creatine. 2 o 2 is eliminated, but the question is whether catalase decomposes the H generated in the second step reaction 2 o 2 , which affects the measurement results

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  • Method for enzymatically determining creatinine in serum by two steps
  • Method for enzymatically determining creatinine in serum by two steps
  • Method for enzymatically determining creatinine in serum by two steps

Examples

Experimental program
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Embodiment 1

[0037] Composition of reagents:

[0038] Phosphate buffer solution configuration: Each liter of phosphate buffer solution (pH7.7) contains 136.10g of potassium dihydrogen phosphate in phosphate buffer solution, add water to dissolve it into 5000ml, take 250ml, add 212.0ml of 0.2mol sodium hydroxide solution Add water to dilute to 1000ml.

[0039] a. Reagent I:

[0040] Each liter of phosphate buffer (pH7.7) contains HDAOS1.0mmol, 4-aminoantipyrine 3.0mmol, ascorbic acid oxidase 20KU, POD 50KU, sarcosine oxidase 40KU, creatine amidinohydrolase 40KU, Proclin-300 200 μl.

[0041] b. Reagent II:

[0042] Each liter of phosphate buffer (pH7.7) contains 500KU of creatinine amidohydrolase and 200μl of Proclin-300.

[0043] c. Standard solution: 265 μmol / L creatinine standard serum.

[0044] Among them, Proclin-300 is a liquid high-efficiency preservative.

Embodiment 2

[0046] In reagent I, change the chromogen HDAOS to DAOS. Content unchanged. All other ingredients remain unchanged, and reagent II remains unchanged.

Embodiment 3

[0048] Measurement procedure

[0049] Two-reagent method: On the Japanese OLYMPUS AU2700 fully automated biochemical analyzer, the instrument automatically adds 2 μl of sample to 150 μl of reagent I and mixes, incubates at 37°C for 3 minutes, adds 50 μl of reagent II and mixes, and incubates at 37°C for 5.1 minutes, fully automatic The analyzer detects at a wavelength of 600nm. The instrument automatically calculates the creatinine result. See Table 1 for details

[0050] Table 1. Automatic biochemical analyzer test conditions of the present invention

[0051]

[0052] response OD Cr Calculated value = OD 2 -OD 1 ×[(SV+R 1 V 1 ) / (SV+R 1 V 1 +R 2 V 2 )]

[0053] Creatinine concentration = F × OD Cr

[0054] where OD Cr is the absorbance produced by creatinine. OD 1 is the absorbance measured after adding reagent I to the sample, OD 2 is the absorbance measured after adding reagent II to the sample, SV is the volume of the serum sample, R 1 V 1 is the volu...

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Abstract

The invention discloses a method for enzymatically determining creatinine in serum by two steps, which belongs to the method for testing materials by testing the color change of reaction results with visible light. The technical scheme is that: a reagent is divided into two parts, wherein the reagent II only contains a creatinine amidohydrolase active ingredient, and the reagent I contains double-chromogen substances and other enzymatically active ingredients; and the determination method comprises the following steps of: performing a warm bath on the serum and the reagent I at 37 DEG C for 3 to 5 minutes, reacting the creatinine in the serum with the reagent I to generate quinoneimine, adding the reagent II to perform the warm bath at 37 DEG C for 4 to 7 minutes, hydrolyzing the creatinine to generate creatine and performing reaction to generate the quinoneimine; and performing detection at the wavelengths of 600 to 610 nm by using an apparatus and calculating creatinine content with the quinoneimine produced by the reaction of the reagent II by taking the quinoneimine produced by the reaction with the reagent I as a blank. In the method, required determination results can be determined completely by biochemical analyzer; and the method is not influenced by endogenous creatine in detection, and convenient to popularize and use, has a using method and application range which are the same as those of the conventional two-step enzymatic method, ensures high sensitivity and accuracy, avoids the pollutions of endogenous and allogenic substances, and is a quick, convenient and high-accuracy creatinine detection method.

Description

technical field [0001] The invention relates to a method for measuring creatinine content, which belongs to the technical field of medical examination and measurement. It belongs to an assay method involving enzymes; or a method for testing materials by using visible light to produce color changes as a result of a test reaction, in particular to a two-step enzyme assay method for quickly and accurately detecting creatinine in serum with a biochemical analyzer . Background technique [0002] Creatinine (Cr) is a polar organic nitrogen-containing compound with a relatively small molecular mass (Mr=113.1188) and a melting point of 255°C. Creatinine, also known as methylguanidinoacetic acid lactam, has the molecular formula C 4 h 7 N 3 O, creatinine is the end product of creatine and phosphocreatine metabolism in muscle. It is an internal dehydrate formed by phosphocreatine through dephosphorylation and closing into a ring. Creatinine in the human body consists of two part...

Claims

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Application Information

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IPC IPC(8): G01N21/78
Inventor 李立和
Owner 李立和
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