CN104931448A - Lactic dehydrogenase isozyme 1 detection reagent and method - Google Patents
Lactic dehydrogenase isozyme 1 detection reagent and method Download PDFInfo
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- CN104931448A CN104931448A CN201510337664.1A CN201510337664A CN104931448A CN 104931448 A CN104931448 A CN 104931448A CN 201510337664 A CN201510337664 A CN 201510337664A CN 104931448 A CN104931448 A CN 104931448A
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Abstract
The invention relates to a detection reagent for detecting lactic dehydrogenase isozyme 1 through a chemical inhibition method. The detection reagent comprises a reagent R1 and a reagent R2. The detection reagent is characterized in that the reagent R1 comprises a biological buffer solution in which a solute in the reagent R1 ranges from 50 mmol/L to 150 mmol/L in concentration, 1.0 mmol/L to 1.5 mmol/L of L-lithium lactate, 1.5 mmol/L to 2.5 mmol/L of sodium perchlorate, 1ml/L to 5ml/L of hydroxy ethidene diphosphonic acid, 0.1 g/L to 1g/L of octadecyl trimethyl ammonium bromide, 0.1 ml/L to 1 ml/L of polyoxyethylene alkyl ether, and 0.5 ml/L to 5 ml/L of preservative. The reagent R2 comprises a buffer solution in which a solute in the reagent R2 ranges from 50 mmol/L to 150 mmol/L in concentration, 8-12 mmol/L NAD+ and 0.5-5 ml/L of preservative. The lactic dehydrogenase isozyme 1 detection reagent has the advantages of being high in accuracy and cheap in price.
Description
Technical field
The present invention relates to the detection field of lactate dehydrogenase isoenzyme 1, be specifically related to a kind of Chemical Inhibition Method that utilizes to detect detection reagent and the 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, and available electrophoresis is separated.Human body cardiac muscle, kidney, red blood cell are maximum with LDH1 and LDH2.Liver and striated muscle are then based on LDH4 and LDH5.In spleen, pancreas, thyroid gland, adrenal gland, LDH3 is more.Lactate dehydrogenase isoenzyme is one of index of observing cardiomyopathies, disease in the liver and gallbladder etc.
Lactate dehydrogenase isoenzyme refers to the same reaction of catalysis (Lactate-Pyruvate) and the different one group of enzyme of structure.It is distributed widely in whole body and respectively organizes, and the LDH1 overwhelming majority is distributed in cardiac muscle cell, so when cardiac muscle cells generation pathology or damage, LDH1 sharply raises, therefore measures LDH1 and has higher sensitivity to Diagnosing Cardiac disease.In addition, the vigor detecting LDH1 also can be used as an index for the treatment of myocardial infarction, myocarditis, myocardial damage new medicament screen.
Detection method at present for isodynamic enzyme mainly contains Chemical Inhibition Method, immunodepression, heating denaturalization, electrophoresis, affinity chromatography.Detection method for lactate dehydrogenase isoenzyme mainly contains chemical continuous detecting method, spendable chemical inhibitor mainly contains perchlorate, hydroxyl compound and guanidine thiocyanate, this several method has relative merits, wherein electrophoresis has something accurately and reliably, the situation of whole LDH isozymogram can be understood, information is comprehensive, shortcoming is poor sensitivity, running time is long, and it is high to the requirement of instrument and operating personnel, the result of ion exchange chromatography also very accurate, but operation requirements is also very high and the running time is also very long, and use Chemical Inhibition Method and immune rule simple to operate, service time is short, be applicable to very much promoting, and relative to immunization, then the price of Chemical Inhibition Method is more cheap, but Chemical Inhibition Method due to available inhibitor more, different can the cause specificity that detect reagent different from the content of inhibitor of kind due to inhibitor is poor, easily be subject to the interference of other lactate dehydrogenase isoenzymes, or the situation of extra-inhibitory, the present invention is according to this problem, the Chemical Inhibition Method that the basis of chemical inhibitor method is set up a kind of high specificity detects the detection reagent of serum lactate dehydrogen ase isoenzyme 1.
Summary of the invention
The Chemical Inhibition Method that utilizes that problem to be solved by this invention is to provide a kind of pin-point accuracy detects the detection reagent of serum lactate dehydrogen ase isoenzyme 1.
Additionally provide a kind of detection method utilizing detection reagent of the present invention to detect lactate dehydrogenase isoenzyme 1 simultaneously.
Lactate dehydrogenase catalyzed Pfansteihl and NAD+ react and generate NADH, NADH and have specificabsorption peak at 340nm place, and its speed generated is directly proportional to the activity of the LDH1 in serum.Chemical inhibitor is the specific inhibitor of the M subunit of LDH, measures the generating rate of NADH, can measure LDH1 activity at 340nm place.
Particular content of the present invention is as follows:
A detection reagent for lactate dehydrogenase isoenzyme 1, comprises reagent R1 and reagent R2, it is characterized in that, described reagent R1 comprises: the biological buffer wherein concentration of solute in reagent R1 is 50-150mmol/L,
Pfansteihl lithium 1.0-1.5mmol/L,
Sodium perchlorate 1.5-2.5mol/L,
HEDP 1-5ml/L,
Cetyltrimethylammonium bromide 0.1-1g/L,
Polyoxyethylene alkyl ether 0.1-1ml/L,
Antiseptic 0.5-5ml/L;
Described reagent R2 comprises: the damping fluid wherein concentration of solute in reagent R1 is 50-150mmol/L,
NAD+ 8-12mmol/L，
Antiseptic 0.5-5ml/L.
More optimize, also comprise in described reagent R1: ascorbic acid oxidase 1-5KU/L, and/or bilirubin oxidase 1-3KU/L.
More optimize, also comprise in described reagent R2: gelatin 1-5g/L.
Preferably, in described reagent R1, biological buffer is CAPS damping fluid, and the concentration of its solute in reagent R1 is 100mmol/L, and 25 DEG C time, the PH of CAPS damping fluid is 9.7.
Preferably, in described reagent R2, damping fluid is tartaric acid buffer, and the concentration of its solute in reagent R1 is 100mmol/L, and 25 DEG C time, the pH of tartaric acid buffer is 3.5.
Preferably, described antiseptic is Proclin series 300 types.
Preferably, the volume ratio of described reagent R1 and reagent R2 is 5:1.
Above-mentioned detection reagent, for detecting a detection method for lactate dehydrogenase isoenzyme 1, is characterized in that, comprises following steps:
(1) reagent R1 and reagent R2 is measured;
(2) test serum is mixed with reagent R1, record the absorbance A 1 when wavelength is 340nm;
(3) reagent R2 is added in the mixed solution of step (2), after 3-8min, record the absorbance A 2 when wavelength is 340nm.
Above-mentioned detection method, is changed to more again, and the reaction time in step (3) is 5min.
The preparation method of reagent R1 and reagent R2, for be measured by each raw material, joins in container, mixes.
Proclin series 300 types are referred to as PC-300.
The present invention employs CAPS (3-(Cyclohexylamino)-1-propane sulfonic acid) damping fluid and tartaric acid buffer respectively in reagent R1 and R2, CAPS (3-(the Cyclohexylamino)-1-propane sulfonic acid) damping fluid used in reagent is biological buffer, to while guarantee surge capability, can not have a negative impact to reaction system; Cetyltrimethylammonium bromide and polyoxyethylene alkyl ether (i.e. EMULGEN-707) two kinds of surfactants are added in the R1 of reagent, these two kinds of surfactants can have the emulsification improving reagent preferably, thus improve accuracy and the specificity of reagent; In R1 biochemical reagents, prioritizing selection sodium perchlorate is as chemical inhibitor, to cross the specificity improving preferably and detect reagent; HEDP is added in reagent R1, can effective sequester heavy metal ions, and the accuracy of reagent can be improved preferably; The present invention also adds chemical resistance acid oxidase and bilirubin oxidase in reagent, effectively can remove ascorbic acid and bilirubinic interference.
The detection reagent of this lactate dehydrogenase isoenzyme 1 has high, the cheap advantage of accuracy, can be extended to the detection field of lactate dehydrogenase isoenzyme 1 widely.
The detection method of this lactate dehydrogenase isoenzyme 1, its principle is exactly, the difference of A2 and A1, the ratio of the difference of the numerical value recorded with the use same method of titer is multiplied by the concentration of the LDH1 of titer, be exactly the concentration of test serum, there is speed fast, the advantage that accuracy is high.
Accompanying drawing explanation
Fig. 1 is formula 2 and electrophoresis correlation curve figure
Fig. 2 is formula 3 and electrophoresis correlation curve figure
Embodiment
Below in conjunction with specific embodiment, the present invention is further described:
(1) component of formula 1: reagent R1 is:
TRIS damping fluid 100mmol/L,
Pfansteihl lithium 1.3mmol/L,
Guanidine thiocyanate 2mol/L,
Sodium azide (antiseptic) 0.5ml/L;
The component of reagent R2 is:
Phosphate buffer 1 00mmol/L,
NAD+ 10mmol/L，
Sodium azide (antiseptic) 0.5ml/L;
(2) component of formula 2: reagent R1 is:
CAPS (3-(Cyclohexylamino)-1-propane sulfonic acid) damping fluid 50mmol/L,
Pfansteihl lithium 1.3mmol/L,
Sodium perchlorate 2mol/L,
HEDP 1ml/L
Cetyltrimethylammonium bromide 0.1g/L,
EMULGEN-707 0.1ml/L，
Ascorbic acid oxidase 1KU/L,
Bilirubin oxidase 1KU/L,
PC-300 (antiseptic) 0.5ml/L;
The component of reagent R2 is:
Tartaric acid buffer 100mmol/L,
NAD+ 10mmol/L，
Gelatin 1g/L,
PC-300 (antiseptic) 0.5ml/L;
(3) component of formula 3: reagent R1 is:
CAPS (3-(Cyclohexylamino)-1-propane sulfonic acid) damping fluid 100mmol/L,
Pfansteihl lithium 1.3mmol/L,
Sodium perchlorate 2mol/L,
HEDP 5ml/L
Cetyltrimethylammonium bromide 1g/L,
EMULGEN-707 1ml/L，
Ascorbic acid oxidase 5KU/L,
Bilirubin oxidase 3KU/L,
PC-300 (antiseptic) 3ml/L;
The component of reagent R2 is:
Tartaric acid buffer 100mmol/L,
NAD+ 10mmol/L，
Gelatin 5g/L,
PC-300 (antiseptic) 0.5ml/L;
(4) detection method:
Adopt the automatic clinical chemistry analyzer with double reagent function in use, this detection experiment adopts Hitachi 7180 fully-automatic analyzer, end-point method is utilized to measure, detection predominant wavelength is 340nm, rate method is utilized to measure, be placed on corresponding reagent position according to the ratio of 5:1 by R1 and R2 respectively, place distilled water, standard items and sample at the correspondence position of sample disc, operation steps is as table 1:
Table 1
Computing method: lactate dehydrogenase isoenzyme 1 content (U/L)=(Δ A measures ÷ Δ A standard) × C standard.
(5) interference test:
Get fresh mix serum, be divided into 2 equal portions, then every equal portions are divided into 5 equal portions again, add different interfering materials, its concentration in serum is made to reach the requirement of table 2, then respectively with the reagent of formula 2 with formula 3, the simultaneously content of lactate dehydrogenase isoenzyme 1 in comparative determination serum, after adding disturbance material, the measurement result of each group is in table 2, mensuration average × 100% of relative deviation (%)=(the mensuration average of the sample of the mensuration average-noiseless material of interference sample)/noiseless material.
As can be seen from Table 2, ascorbic acid, cholerythrin, haemoglobin and the triglyceride test result to formula 2 and formula 3 is not obviously disturbed, and illustrates that this detection reagent has stronger antijamming capability.
Table 2
(6) correlativity experiment:
Get 20 clinical serum samples, catalogue number(Cat.No.) is respectively 1,2,3---and 20, each sample mixes, be divided into 4 parts, respectively by rate method and electrophoresis totally 4 kinds of methods of formula 1, formula 2, formula 3, detect the content of serum lactate dehydrogen ase isoenzyme 1, testing result is as shown in table 3.And obtain with formula 2 and formula 3 two kinds of reagent respectively with the correlation curve (as depicted in figs. 1 and 2) of electrophoresis testing result, shown by testing result, the related coefficient being detected the result of serum lactate dehydrogen ase isoenzyme 1 by fill a prescription 2 reagent and the formula testing result of 3 reagent and electrophoresis is respectively 0.9993 and 0.9977, illustrates that the detection reagent of this lactate dehydrogenase isoenzyme 1 has high accuracy.
Table 3
R
2。
Claims (9)
1. a detection reagent for lactate dehydrogenase isoenzyme 1, comprises reagent R1 and reagent R2, it is characterized in that, described reagent R1 comprises: the biological buffer wherein concentration of solute in reagent R1 is 50-150mmol/L,
Pfansteihl lithium 1.0-1.5mmol/L,
Sodium perchlorate 1.5-2.5mol/L,
HEDP 1-5 ml/L,
Cetyltrimethylammonium bromide 0.1-1g/L,
Polyoxyethylene alkyl ether 0.1-1 ml/L,
Antiseptic 0.5-5ml/L;
Described reagent R2 comprises: the damping fluid wherein concentration of solute in reagent R1 is 50-150mmol/L,
NAD+
8-12mmol/L，
Antiseptic 0.5-5 ml/L.
2. the detection reagent of lactate dehydrogenase isoenzyme 1 according to claim 1, is characterized in that, also comprises: ascorbic acid oxidase 1-5KU/L in described reagent R1,
And/or bilirubin oxidase
1-3KU/L.
3. the detection reagent of lactate dehydrogenase isoenzyme 1 according to claim 2, is characterized in that, also comprises: gelatin in described reagent R2
1-5 g/L.
4. according to the detection reagent of the arbitrary described lactate dehydrogenase isoenzyme 1 of claim 1-3, it is characterized in that, in described reagent R1, biological buffer is CAPS damping fluid, and the concentration of its solute in reagent R1 is 100mmol/L's, 25 DEG C time, the PH of CAPS damping fluid is 9.7.
5. the detection reagent of lactate dehydrogenase isoenzyme 1 according to claim 4, is characterized in that, in described reagent R2, damping fluid is tartaric acid buffer, and the concentration of its solute in reagent R1 is 100mmol/L, and 25 DEG C time, the pH of tartaric acid buffer is 3.5.
6. the detection reagent of lactate dehydrogenase isoenzyme 1 according to claim 5, is characterized in that, described antiseptic is Proclin series 300 types.
7. the detection reagent of lactate dehydrogenase isoenzyme 1 according to claim 6, is characterized in that, the volume ratio of described reagent R1 and reagent R2 is 5:1.
8. the detection reagent of lactate dehydrogenase isoenzyme 1 according to claim 7 is for detecting a detection method for lactate dehydrogenase isoenzyme 1, it is characterized in that, comprises following steps:
(1) reagent R1 and reagent R2 is measured;
(2) test serum is mixed with reagent R1, record the absorbance A 1 when wavelength is 340nm;
(3) reagent R2 is added in the mixed solution of step (2), after 3-8min, record the absorbance A 2 when wavelength is 340nm.
9. detection method according to claim 8, is characterized in that, the reaction time in step (3) is 5min.
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Cited By (5)
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| CN105385751A (en) * | 2015-12-24 | 2016-03-09 | 山东博科生物产业有限公司 | Total cholesterol detection reagent with high accuracy and disturbance resisting capacity |
| 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 |
| CN114381494A (en) * | 2021-12-01 | 2022-04-22 | 天津中成佳益生物科技有限公司 | Detection reagent and detection method for lactate dehydrogenase isozyme 1 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105385751A (en) * | 2015-12-24 | 2016-03-09 | 山东博科生物产业有限公司 | Total cholesterol detection reagent with high accuracy and disturbance resisting capacity |
| 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 |
| CN114381494A (en) * | 2021-12-01 | 2022-04-22 | 天津中成佳益生物科技有限公司 | Detection reagent and detection method for lactate dehydrogenase isozyme 1 |
| 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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