CN104931448B - A kind of detection reagent and its detection method of lactate dehydrogenase isoenzyme 1 - Google Patents
A kind of detection reagent and its detection method of lactate dehydrogenase isoenzyme 1 Download PDFInfo
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Abstract
The present invention relates to a kind of detection reagent that lactate dehydrogenase isoenzyme 1 is detected using Chemical Inhibition Method.This reagent includes reagent R1 and reagent R2, it is characterised in that the reagent R1 is included：Concentration of the biological buffer wherein solute in reagent R1 is 50 150mmol/L, the 1.5mmol/L of L lithium lactates 1.0, the 2.5mol/L of sodium perchlorate 1.5, the ml/L of 1-hydroxy ethylidene-1,1-diphosphonic acid 15, the 1g/L of Cetyltrimethylammonium bromide 0.1, the ml/L of polyoxyethylene alkyl ether 0.1 1, the 5ml/L of preservative 0.5；The reagent R2 is included：Concentration of the buffer solution wherein solute in reagent R1 is 50 150mmol/L, NAD+8 12mmol/L, the ml/L of preservative 0.5 5.The detection reagent of this lactate dehydrogenase isoenzyme 1 has the advantages of degree of accuracy is high, cheap.
Description
Technical field
The present invention relates to the detection field of lactate dehydrogenase isoenzyme 1, and in particular to one kind is examined using Chemical Inhibition Method
Survey the detection reagent and detection method of lactate dehydrogenase isoenzyme 1.
Background technology
Lactic acid dehydrogenase has 5 kinds of isodynamic enzyme forms, i.e. LDH1, LDH2, LDH3, LDH4, LDH5, can be divided with electrophoresis
From.Human body cardiac muscle, kidney, red blood cell are most using LDH1 and LDH2.Liver and striated muscle are then based on LDH4 and LDH5.Spleen, pancreas, first
LDH3 is more in shape gland, adrenal gland.Lactate dehydrogenase isoenzyme is one of index for observing cardiomyopathies, disease in the liver and gallbladder etc..
Lactate dehydrogenase isoenzyme refers to be catalyzed same reaction (Lactate-Pyruvate) and the different one group of enzyme of structure.Its
Be distributed widely in whole body respectively to organize, and the LDH1 overwhelming majority is distributed in cardiac muscle cell, thus cardiac muscle cells occur lesion or
During damage, LDH1 is drastically raised, therefore measure LDH1 has higher sensitivity to Diagnosing Cardiac disease.In addition, detection LDH1
Vigor be alternatively arranged as treat myocardial infarction, myocarditis, an index of myocardial damage new medicament screen.
Detection method currently used for isodynamic enzyme mainly have Chemical Inhibition Method, immunodepression, heating denaturalization, electrophoresis,
Affinity chromatography.Detection method for lactate dehydrogenase isoenzyme mainly has the continuous detection method of chemistry, workable chemistry suppression
Preparation mainly has perchlorate, hydroxy compounds and guanidine thiocyanate, and this several method has advantage and disadvantage, and wherein electrophoresis accurately may be used
To lean on, the situation of whole LDH isozymograms can be understood, information is comprehensive, and shortcoming is poor sensitivity, and the operating time is grown, and to instrument
The requirement of device and operating personnel is high, and the result of ion exchange chromatography is also very accurate, but operate require also very it is high simultaneously
And the operating time is also very long, and it is simple to operate using Chemical Inhibition Method and immune rule, and usage time is short, is especially suitable for pushing away
Extensively, and relative to immunization, then the price of Chemical Inhibition Method is more cheap, but Chemical Inhibition Method is due to available suppression
Agent is more, can cause the specific poor of detection reagent because the species difference of inhibitor is different with the content of inhibitor, easily
Disturbed by other lactate dehydrogenase isoenzymes, or the situation of extra-inhibitory, the present invention press down according to this problem in chemistry
A kind of detection examination of the Chemical Inhibition Method detection serum lactate dehydrogen ase isoenzyme 1 of high specificity is established on the basis of preparation method
Agent.
The content of the invention
The utilization Chemical Inhibition Method detection serum lactic that problem to be solved by this invention is to provide a kind of high accuracy takes off
The detection reagent of hydrogen enzyme isoenzyme 1.
A kind of detection method of the detection reagent detection lactate dehydrogenase isoenzyme 1 using the present invention is additionally provided simultaneously.
Lactate dehydrogenase catalyzed Pfansteihl has specificabsorption peak with NAD+ reaction generations NADH, NADH at 340nm, and it is given birth to
Into speed it is active directly proportional to the LDH1's in serum.Chemical inhibitor is the specific inhibitor of LDH M subunits, in 340nm
Place measure NADH generating rate, you can measure LDH1 activity.
The particular content of the present invention is as follows：
The invention discloses a kind of detection method for detecting lactate dehydrogenase isoenzyme 1, it is characterised in that includes following step
Suddenly：
(1) reagent preparation R1, comprising：
Concentration of the biological buffer wherein solute in reagent R1 is 50-150mmol/L, and biological buffer buffers for CAPS
Liquid,
Pfansteihl lithium 1.0-1.5mmol/L,
Sodium perchlorate 1.5-2.5mol/L,
1-hydroxy ethylidene-1,1-diphosphonic acid 1-5ml/L,
Cetyltrimethylammonium bromide 0.1-1g/L,
Polyoxyethylene alkyl ether 0.1-1ml/L,
Preservative 0.5-5ml/L；
Also include：Ascorbic acid oxidase 1-5KU/L, and/or bilirubin oxidase 1-3KU/L.
(2) reagent preparation R2, comprising：
Concentration of the buffer solution wherein solute in reagent R2 is 50-150mmol/L, and buffer solution is winestone in the reagent R2
Acid buffer,
NAD+ 8-12mmol/L,
Preservative 0.5-5ml/L；
Also include：Gelatin 1-5g/L；
(3) reagent R1 and reagent R2, respectively 250 μ L and 50 μ L are measured；
(4) the μ L of test serum 5 are mixed with reagent R1, measures the absorbance A 1 when wavelength is 340nm；
(5) reagent R2 is added in the mixed solution of step (4), after 3-8min, measures absorbance when wavelength is 340nm
A2, calculate its difference DELTA A measure；
(6) the Δ A standards and C standards of above step measurement standard product are used；
(7) content of lactate dehydrogenase isoenzyme 1 is calculated as follows：The content of lactate dehydrogenase isoenzyme 1 (U/L)=
(Δ A measure ÷ Δ A standards) × C standards.
Above-mentioned detection method, it is preferred that concentration of the solute in reagent R1 is 100mmol/L, at 25 DEG C, CAPS
The PH of buffer solution is 9.7.
Above-mentioned detection method, it is preferred that concentration of the solute in reagent R2 is 100mmol/L, at 25 DEG C, tartaric acid
The pH of buffer solution is 3.5.
Above-mentioned detection method, it is preferred that the preservative is serial 300 types of Proclin.
Above-mentioned detection method, it is preferred that the reaction time in step (5) is 5min.
The present invention used respectively in reagent R1 and R2 CAPS (3- (Cyclohexylamino) -1- propane sulfonic acid) buffer solutions and
Tartaric acid buffer, the CAPS used in reagent (3- (Cyclohexylamino) -1- propane sulfonic acid) buffer solution is biological buffer,
To that while buffer capacity is ensured, will not be had a negative impact to reaction system；Octadecyl is added in the R1 of reagent
Two kinds of surfactants of trimethylammonium bromide and polyoxyethylene alkyl ether (i.e. EMULGEN-707), both surfactant energy
There is the emulsification for preferably improving reagent, so as to improve the degree of accuracy of reagent and specificity；Preferentially selected in R1 biochemical reagents
Sodium perchlorate has been selected as chemical inhibitor, can preferably improve the specificity of detection reagent；Hydroxyl is added in reagent R1
Ethylene-diphosphonic acid, heavy metal ion can be effectively chelated, and can preferably improve the accuracy of reagent；The present invention also exists
Anti- chemical acid oxidase and bilirubin oxidase are added in reagent, can effectively remove the dry of ascorbic acid and bilirubin
Disturb.
The detection reagent of this lactate dehydrogenase isoenzyme 1 has the advantages of degree of accuracy is high, cheap, can widely promote
In the detection field of lactate dehydrogenase isoenzyme 1.
The detection method of this lactate dehydrogenase isoenzyme 1, its principle are exactly A2 and A1 difference, the use with titer
The ratio between difference of numerical value that same method measures is multiplied by the LDH1 of titer concentration, is exactly the concentration of test serum, has speed
The advantages of degree is fast, and the degree of accuracy is high.
Embodiment
The present invention is further described with reference to specific embodiment：
(1) it is formulated 1：Reagent R1 component is：
TRIS buffer solution 100mmol/L,
Pfansteihl lithium 1.3mmol/L,
Guanidine thiocyanate 2mol/L,
Sodium azide (preservative) 0.5ml/L；
Reagent R2 component is：
Phosphate buffer 1 00mmol/L,
NAD+ 10mmol/L,
Sodium azide (preservative) 0.5ml/L；
(2) 2 are formulated：Reagent R1 component is：
CAPS (3- (Cyclohexylamino) -1- propane sulfonic acid) buffer solution 50mmol/L,
Pfansteihl lithium 1.3mmol/L,
Sodium perchlorate 2mol/L,
1-hydroxy ethylidene-1,1-diphosphonic acid 1ml/L
Cetyltrimethylammonium bromide 0.1g/L,
EMULGEN-707 0.1ml/L,
Ascorbic acid oxidase 1KU/L,
Bilirubin oxidase 1KU/L,
PC-300 (preservative) 0.5ml/L；
Reagent R2 component is：
Tartaric acid buffer 100mmol/L,
NAD+ 10mmol/L,
Gelatin 1g/L,
PC-300 (preservative) 0.5ml/L；
(3) 3 are formulated：Reagent R1 component is：
CAPS (3- (Cyclohexylamino) -1- propane sulfonic acid) buffer solution 100mmol/L,
Pfansteihl lithium 1.3mmol/L,
Sodium perchlorate 2mol/L,
1-hydroxy ethylidene-1,1-diphosphonic acid 5ml/L
Cetyltrimethylammonium bromide 1g/L,
EMULGEN-707 1ml/L,
Ascorbic acid oxidase 5KU/L,
Bilirubin oxidase 3KU/L,
PC-300 (preservative) 3ml/L；
Reagent R2 component is：
Tartaric acid buffer 100mmol/L,
NAD+ 10mmol/L,
Gelatin 5g/L,
PC-300 (preservative) 0.5ml/L；
(4) detection method：
It is complete using Hitachi 7180 using the automatic clinical chemistry analyzer with double reagent function, this detection experiment when in use
Automatic analyzer, be measured using end-point method, detection dominant wavelength is 340nm, is measured using performance rate method, respectively by R1 and
R2 is according to 5:1 ratio is placed on corresponding reagent position, and distilled water, standard items and sample are placed in the correspondence position of sample disc
This, operating procedure such as table 1：
Table 1
Computational methods：The content of lactate dehydrogenase isoenzyme 1 (U/L)=(Δ A measure ÷ Δ A standards) × C standards.
(5) interference is tested：
Fresh mix serum is taken, is divided into 2 equal portions, then 5 equal portions will be separated into per equal portions, and add different interfering materials,
Its concentration in serum is reached the requirement of table 2, then use the reagents of formula 2 and formula 3, while comparative determination serum respectively
The content of middle lactate dehydrogenase isoenzyme 1, the measurement result of each group is shown in Table 2 after adding disturbance material, relative deviation (%)
The measure average of=(the measure average of the sample of measure average-noiseless material of interference sample)/noiseless material ×
100%.
As can be seen from Table 2, the test of ascorbic acid, bilirubin, hemoglobin and triglycerides to formula 2 and formula 3
As a result do not significantly interfere with, illustrate that this detection reagent has stronger antijamming capability.
Table 2
(6) correlation is tested：
20 clinical serum samples are taken, catalogue number(Cat.No.) is respectively 1,2,3 --- 20, each sample is well mixed, and is divided into 4
Part, respectively by being formulated 1, formula 2, the performance rate method for being formulated 3 and electrophoresis totally 4 kinds of methods, detect the same work of serum lactic dehydrogenase (SLDH)
The content of enzyme 1, testing result are as shown in table 3.Shown by testing result, by being formulated 2 reagents and being formulated the detection knot of 3 reagents
The coefficient correlation of the result of fruit and electrophoresis detection serum lactate dehydrogen ase isoenzyme 1 is respectively 0.9993 and 0.9977, explanation
The detection reagent of this lactate dehydrogenase isoenzyme 1 has high accuracy.
Table 3
Claims (5)
1. a kind of detection method for detecting lactate dehydrogenase isoenzyme 1, it is characterised in that comprise the following steps：
（1）Reagent preparation R1, comprising：
Concentration of the biological buffer wherein solute in reagent R1 is 50-150mmol/L, and biological buffer is CAPS buffer solutions,
Pfansteihl lithium 1.0-1.5mmol/L,
Sodium perchlorate 1.5-2.5mol/L,
1-hydroxy ethylidene-1,1-diphosphonic acid 1-5 ml/L,
Cetyltrimethylammonium bromide 0.1-1g/L,
Polyoxyethylene alkyl ether 0.1-1 ml/L,
Preservative 0.5-5ml/L；
Also include：Ascorbic acid oxidase 1-5KU/L, and/or bilirubin oxidase 1-3KU/L。
（2）Reagent preparation R2, comprising：
Concentration of the buffer solution wherein solute in reagent R2 is 50-150mmol/L, and buffer solution is tartaric acid in the reagent R2
Buffer solution,
NAD+ 8-12mmol/L,
Preservative 0.5-5 ml/L；
Also include：Gelatin 1-5 g/L；
（3）Measure reagent R1 and reagent R2, respectively 250 μ L and 50 μ L；
（4）The μ L of test serum 5 are mixed with reagent R1, measure the absorbance A 1 when wavelength is 340nm；
（5）Reagent R2 is added into step（4）Mixed solution in, after 3-8min, measure the absorbance A 2 when wavelength is 340nm,
Calculate its difference A measure；
（6）Using the A standards of above step measurement standard product；
（7）The content of lactate dehydrogenase isoenzyme 1 is calculated as follows：The content of lactate dehydrogenase isoenzyme 1（U/L）=（A is surveyed
Determine ÷ A standards）× C standards.
2. detection method according to claim 1, it is characterised in that concentration of the solute in reagent R1 is 100mmol/L,
At 25 DEG C, the PH of CAPS buffer solutions is 9.7.
3. detection method according to claim 2, it is characterised in that concentration of the solute in reagent R2 is 100mmol/L,
At 25 DEG C, the pH of tartaric acid buffer is 3.5.
4. detection method according to claim 3, it is characterised in that the preservative is serial 300 types of Proclin.
5. detection method according to claim 1, it is characterised in that step（5）In reaction time be 5min.
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| CN105385751B (en) * | 2015-12-24 | 2019-10-15 | 山东博科生物产业有限公司 | A kind of accuracy is high, strong antijamming capability total cholesterol detection reagent |
| CN105651763A (en) * | 2016-03-08 | 2016-06-08 | 山东博科生物产业有限公司 | Alpha-hydroxybutyrate dehydrogenase detection reagent with good stability and high accuracy |
| CN108982851A (en) * | 2018-07-27 | 2018-12-11 | 金华市强盛生物科技有限公司 | A kind of lactate dehydrogenase isozyme Ⅰ detection kit continuously monitored |
| CN110346311A (en) * | 2019-07-15 | 2019-10-18 | 三诺生物传感股份有限公司 | A kind of lactic dehydrogenase detection reagent |
| CN114381494B (en) * | 2021-12-01 | 2023-12-22 | 天津中成佳益生物科技有限公司 | Detection reagent and detection method for lactic dehydrogenase isozyme 1 |
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| US4157279A (en) * | 1974-09-12 | 1979-06-05 | Kommanditgesellschaft Schwarzhaupt | Process for the determination of at least one of the isoenzymes of lactatedehydrogenase |
| CN1202259C (en) * | 2003-01-09 | 2005-05-18 | 中国人民解放军第二军医大学 | Kit for detecting lactate dehydrogenase isoenzyme |
| CN100342028C (en) * | 2005-01-26 | 2007-10-10 | 中国科学院上海微系统与信息技术研究所 | Detection of activity of isoenzyme by micro-fluid chip electrophoretic separation |
| CN102766677A (en) * | 2012-07-31 | 2012-11-07 | 武汉生之源生物科技有限公司 | Lactic dehydrogenase detection kit and preparation method thereof |
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Effective date of registration: 20210111 Address after: No.2717, Gongye 1st Road, Mingshui Economic Development Zone, Zhangqiu District, Jinan City, Shandong Province, 250200 Patentee after: Shandong Zhongan biosafety Testing Co.,Ltd. Address before: 250200 no.1920 Huiquan Road, Mingshui sub district office, Zhangqiu City, Jinan City, Shandong Province Patentee before: Chen Xiliang |
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