CN106434847A - Kit for detecting activity of MTHFR (5,10-methylene tetrahydrofolate reductase) - Google Patents
Kit for detecting activity of MTHFR (5,10-methylene tetrahydrofolate reductase) Download PDFInfo
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Abstract
The invention discloses a kit for detecting activity of MTHFR (5,10-methylene tetrahydrofolate reductase), a method and an application of the kit. The kit comprises a reagent 1, a reagent 2, a reagent 3, a reagent 4 and a reagent 5 which are packaged independently, wherein the reagent 1 is a buffer system formed by a buffer solution 1 and an enzyme activity stabilizer 1, and the pH value of the reagent 1 ranges from 5 to 8; the reagent 2 is methylene tetrahydrofolate; the reagent 3 is NADPH; the reagent 4 is a stop buffer formed by a stop reagent and an enzyme activity stabilizer 2; the reagent 5 is a diluent formed by a buffer solution 2 and the enzyme activity stabilizer 2, and the pH value of the reagent 5 ranges from 5 to 8. The method can be applied to clinics and physical examination screening, meets the requirements for physical examination screening, diagnosis and treatment of MTHFR deficiency, folic acid utilization obstacle and folic acid deficiency, and is easy to popularize and use.
Description
Technical field
The present invention relates to a kind of test kit of detection enzymatic activity is and in particular to a kind of detect Methylene tetrahydrofolate reductase
The test kit of enzymatic activity, method and its application, belong to biological medicine detection field.
Background technology
Folic Acid is the methyl donor in many important substance such as synthesis of protein, nucleic acid, hemoglobin etc. in vivo, Folic Acid
Shortage or the shortage of folic acid metabolism enzyme, can lead to cell cycle abnormal, DNA, protein methylation abnormal reaction, DNA alkali
It is abnormal, thus leading to many major diseases that base such as cannot normally synthesize at multiple biochemical reactions.
The metabolism of Folic Acid relies on a series of enzyme catalysiss to realize, and Folic Acid will be through transforming into activated 5- methyl
Tetrahydrofolic acid form just can complete normal function in vivo, and the key enzyme realizing the conversion of this active substance is exactly that 5,10- is sub-
Methyl tetrahydrofolate reductase（5,10-methylenetetrahydrofolatereductase, abbreviation MTHFR）.Its function
It is that catalysis 5,10-CH2-THFA is converted into 5-methyltetrahydrofolate, and with 5-methyltetrahydrofolate together with other enzymes
As homocysteine（Homocysteine, writes a Chinese character in simplified form Hcy）Cosubstrate by homocysteine methylate generation first sulfur
Propylhomoserin.Under the different degrees of enzymatic activity of MTHFR, general who has surrendered leads to folate metabolism disorder, and then leads to a series of diseases, is such as born and lacks
Fall into, the common birth defect of China has neural tube defects, congenital heart disease, harelip, Down's syndrome etc., wherein neurocele
Deformity accounts for the 1/3 about of all birth defects.The folate metabolism disorder that MTHFR enzymatic activity defect leads to is to cause high homotype
The principal element of cysteinaemia, hyperhomocysteinemiainjury causes cardiovascular and cerebrovascular disease further, including coronary artery
Atherosclerotic heart disease, peripheral vascular disease, cerebrovascular and venothrombotic formation, clinically can behave as apoplexy, the heart
Flesh infarction, pulmonary infarction, Renal vascular and peripheral vascular disease etc..Additionally, folate metabolism disorder also causes other series of problems, such as
Neonatal development is slow, realize and take action obstacle, epilepsy, neurologic impairment, the recurrent abortion of Women of childbearing age, gestation
The problems such as hypertension, mankind spermatozoon dyspoiesis or low sperm activity；Also the dementia of Ahl tribulus sea silent sickness and other forms,
Autism, diabetes, colon cancer are closely related with many diseases such as acute leukemia.
Therefore MTHFR enzymatic activity assessment for prevention congenital disorders, sterility and infertility, serious cardiovascular and cerebrovascular disease etc. is many
There is very important realistic meaning in field.
At present, there is no MTHFR enzyme activity detectable or the test kit being applied to Clinical detection both at home and abroad.It is worth proposition
It is that, under normal physiological conditions, MTHFR enzyme is that irreversible catalysis 5,10-CH2-THFA is converted into 5- methyl tetrahydrochysene
Folic Acid, we are in this, as the reaction in physiology direction（Forward reaction）, under in vitro conditions, the MTHFR extracting has three kinds
Enzyme activity, is physiology forward reaction, physiology back reaction, NAD (P) H- menadione oxygen also enzymatic activity respectively.
Existing scientific research MTHFR enzyme activity detection method is all to set up about eighties of last century eighties, mainly includes two
Kind, it is a kind of that to be change based on light absorbs be built upon NAD (P) H in 340nm carrying out enzyme activity detection, optical absorption method detection
Under light absorbs change to assess the process of reaction, and the method detection is physiology back reaction.This method should be only suitable
For the MTHFR enzyme after extraction purification.Reaction system for complex biological sample（As Clinical detection）, this method is uncomfortable
?.
Another kind is to be detected using labelled with radioisotope method, and its principle is that the physiology of detection MTHFR enzyme is reversely anti-
Should.Because methylene tetrahydrofolate is unstable, so the enzyme of MTHFR is assessed using the oxidability method that opposite direction detects this enzyme
Vigor, this method is also used for studying mankind's MTHFR deficiency disease.Because isotope is a kind of relatively unstable radioactive material
Matter, easily causes environmental pollution, and the method is not suitable for clinic yet.
Now in the urgent need to a kind of method of the detection normal physiological reaction direction MTHFR enzyme activity being applied to clinical demand
And reagent.
There is document report to adopt HPLC method to detect the normal physiological reaction of MTHFR, but this method is used for detecting Hepar Sus domestica
The extract of tissue and the fibroblast of culture, and first have to synthesize basic bottom by formaldehyde and tetrahydrofolic acid in reaction system
Thing 5,10-CH2-THFA, is further continued for follow-up reaction, and this point makes must accurately determine the kinetic parameter of natural substrate
Highly difficult.
Content of the invention
An object of the present invention is to provide a kind of detection Methylene tetrahydrofolate reductase（5,10-
Methylenetetrahydrofolatereductase, abbreviation MTHFR, EC 1.5.1.20）The test kit of enzymatic activity, this
When bright test kit is detected, with natural substrate 5,10-CH2-THFA as substrate, therefore detection is normal in vivo for people
The enzyme activity in physiological reaction direction, the test kit of the present invention has higher detection sensitivity, can be directly used for Enzyme assay；
The test kit of the present invention can use on the fluorescence detection device of chromatograph of liquid manually or automatically, and easy to use.
The invention provides a kind of test kit of detection Methylene tetrahydrofolate reductase enzymatic activity, mentioned reagent box includes
The reagent 1 of independent packaging, reagent 2, reagent 3, reagent 4 and reagent 5；
Mentioned reagent 1 is the buffer system being made up of buffer 1 and enzyme activity stabilizer 1, and the pH value of mentioned reagent 1 is 5 ~ 8；
Mentioned reagent 2 is 5,10- methylene tetrahydrofolate；
Mentioned reagent 3 is NADPH；
Mentioned reagent 4 is the terminate liquid being made up of termination reagent and enzyme activity stabilizer 2；
Mentioned reagent 5 is the diluent being made up of buffer 2 and enzyme activity stabilizer 2, and the pH value of mentioned reagent 5 is 5 ~ 8.
Optionally, in mentioned reagent 1, the molar concentration of above-mentioned buffer 1 is 50 ~ 500mmol/L, above-mentioned enzyme activity stabilizer
1 molar concentration is 5 ~ 100mmol/L；
In mentioned reagent 2, the molar concentration of above-mentioned 5,10-CH2-THFA is 0.05 ~ 0.5mmol/L；
In mentioned reagent 3, the molar concentration of above-mentioned NADPH can be 0.05 ~ 2mmol/L；
In mentioned reagent 4, the concentration of volume percent of above-mentioned terminate liquid can be 20 ~ 80ml/L, above-mentioned enzyme activity stabilizer 2 mole
Concentration can be 10 ~ 100mmol/L；
In mentioned reagent 5, the molar concentration of above-mentioned buffer 2 can be 20 ~ 200mmol/L, above-mentioned enzyme activity stabilizer 2 mole dense
Degree can be 5 ~ 50mmol/L.
Optionally, in mentioned reagent 1, above-mentioned buffer 1 is kaliumphosphate buffer, and molar concentration is 80 ~ 150mmol/L,
Above-mentioned enzyme activity stabilizer 1 is dithiothreitol, DTT, and molar concentration is 5mmol/L；
In mentioned reagent 2, the molar concentration of above-mentioned 5,10-CH2-THFA is 0.15 ~ 0.5mmol/L；
In mentioned reagent 3, the molar concentration of above-mentioned NADPH can be 1 ~ 2mmol/L；
In mentioned reagent 4, above-mentioned terminate liquid is high chloro acid solution, and concentration of volume percent is 60 ~ 80ml/L, and above-mentioned enzyme activity is steady
Determining agent 2 is aqueous ascorbic acid, and molar concentration is 60mmol/L；
In mentioned reagent 5, above-mentioned buffer 2 is kaliumphosphate buffer, and molar concentration is 60mmol/L, above-mentioned enzyme activity stabilizer 2
For aqueous ascorbic acid, molar concentration is 30mmol/L.
Optionally, in mentioned reagent 1, above-mentioned buffer 1 is kaliumphosphate buffer, and molar concentration is 80mmol/L, pH value
For 6.8, above-mentioned enzyme activity stabilizer 1 is dithiothreitol, DTT, and molar concentration is 5mmol/L；
In mentioned reagent 2, the molar concentration of above-mentioned 5,10-CH2-THFA is 0.5mmol/L；
In mentioned reagent 3, the molar concentration of above-mentioned NADPH can be 2mmol/L；
In mentioned reagent 4, above-mentioned terminate liquid is high chloro acid solution, and concentration of volume percent is 60ml/L, and above-mentioned enzyme activity is stable
Agent 2 is aqueous ascorbic acid, and molar concentration is 60mmol/L；
In mentioned reagent 5, above-mentioned buffer 2 is kaliumphosphate buffer, and molar concentration is 60mmol/L, above-mentioned enzyme activity stabilizer 2
For aqueous ascorbic acid, molar concentration is 30mmol/L.
Optionally, in mentioned reagent 1, above-mentioned buffer 1 is kaliumphosphate buffer, and molar concentration is 100mmol/L, pH value
For 7.0, above-mentioned enzyme activity stabilizer 1 is dithiothreitol, DTT, and molar concentration is 5mmol/L；
In mentioned reagent 2, the molar concentration of above-mentioned 5,10-CH2-THFA is 0.4mmol/L；
In mentioned reagent 3, the molar concentration of above-mentioned NADPH can be 1.5mmol/L；
In mentioned reagent 4, above-mentioned terminate liquid is high chloro acid solution, and concentration of volume percent is 60ml/L, and above-mentioned enzyme activity is stable
Agent 2 is aqueous ascorbic acid, and molar concentration is 60mmol/L；
In mentioned reagent 5, above-mentioned buffer 2 is kaliumphosphate buffer, and molar concentration is 60mmol/L, above-mentioned enzyme activity stabilizer 2
For aqueous ascorbic acid, molar concentration is 30mmol/L.
Optionally, in mentioned reagent 1, above-mentioned buffer 1 is kaliumphosphate buffer, and molar concentration is 150mmol/L, pH value
For 7.2, above-mentioned enzyme activity stabilizer 1 is dithiothreitol, DTT, and molar concentration is 5mmol/L；
In mentioned reagent 2, the molar concentration of above-mentioned 5,10-CH2-THFA is 0.15mmol/L；
In mentioned reagent 3, the molar concentration of above-mentioned NADPH can be 1mmol/L；
In mentioned reagent 4, above-mentioned terminate liquid is high chloro acid solution, and concentration of volume percent is 80ml/L, and above-mentioned enzyme activity is stable
Agent 2 is aqueous ascorbic acid, and molar concentration is 60mmol/L；
In mentioned reagent 5, above-mentioned buffer 2 is kaliumphosphate buffer, and molar concentration is 60mmol/L, above-mentioned enzyme activity stabilizer 2
For aqueous ascorbic acid, molar concentration is 30mmol/L.
Optionally, above-mentioned enzyme activity stabilizer is at least one of beta -mercaptoethanol, dithiothreitol, DTT or ascorbic acid.
The invention provides a kind of method that employing mentioned reagent box detects Methylene tetrahydrofolate reductase enzymatic activity：
Mentioned reagent 1, mentioned reagent 2 are mixed with sample to be tested, above-mentioned sample to be tested and mentioned reagent 2 and mentioned reagent 1
Volume ratio is 1：（2~4）：（6~3）, form mentioned reagent 1, mentioned reagent 2 and the mixed system of sample to be tested, add above-mentioned
Reagent 3, mentioned reagent 3 is 1 with the ratio of above-mentioned mixed system:9, after mixing, reaction system is placed in 37 DEG C of lucifuge reactions
20min, reaction adds mentioned reagent 4 terminating reaction after terminating and above-mentioned sample to be tested is placed in 30min on ice, mentioned reagent 4
Volume ratio with reaction system is 1：10, after termination, above-mentioned sample to be tested pipe 18000g is centrifuged 5min, takes supernatant, adds above-mentioned
The ratio of reagent 5, wherein supernatant and mentioned reagent 5 is 1：5, the system after dilution is filtered with the filter of 0.45um,
And be positioned over low temperature, treat that HPLC detects under the conditions of lucifuge.
Optionally, mentioned reagent 1, mentioned reagent 2 are mixed with sample to be tested, above-mentioned sample to be tested and mentioned reagent 2 and
The volume ratio of mentioned reagent 1 is 1：2：6, form mentioned reagent 1, mentioned reagent 2 and the mixed system of sample to be tested, add
State reagent 3, mentioned reagent 3 is 1 with the ratio of above-mentioned mixed system:9, after mixing, reaction system is placed in 37 DEG C of lucifuge reactions
20min, reaction adds mentioned reagent 4 terminating reaction after terminating and above-mentioned sample to be tested is placed in 30min on ice, mentioned reagent 4
Volume ratio with reaction system is 1：10, after termination, above-mentioned sample to be tested pipe 18000g is centrifuged 5min, takes supernatant, adds above-mentioned
The ratio of reagent 5, wherein supernatant and mentioned reagent 5 is 1：5, the system after dilution is filtered with the filter of 0.45um,
And be positioned over low temperature, treat that HPLC detects under the conditions of lucifuge.
The invention provides one kind by mentioned reagent box in auxiliary diagnosis methylenetetrahydrofolate reductase deficiency and leaf
Application in acid-utilising obstacle, the health check-up examination of folic acid deficiency.
The test kit that the present invention provides can be also used for analyzing and detect all kinds of specimen from human body or animal（Including each
Plant body fluid, cell or tissue）In Methylene tetrahydrofolate reductase enzyme activity, such as can be used for blood, hemocyte, cultured cells
And liver organization etc., applicable specimen scope is wide.
Our researchs to MTHFR method for detecting enzymatic activity in human peripheral leucocytes, are groped by substantial amounts of experiment, I
Overcome many technical barriers, finally establish the enzyme activity detection side in a set of stable, sensitive MTHFR physiological reaction direction
Method, further combined with gene sequencing checking etc. means, We conducted clinical sample checking it was confirmed the accuracy of the method and
Specificity.
When being detected using the detection kit that the present invention provides, it is possible to obtain the methylene in normal physiological reaction direction
The quantitative result of tetrahydrofolate reductase activity, therefore can be used for analysis, detection and diagnosis Methylene tetrahydrofolate reductase
The various diseases that extremely lead to of enzyme activity and the various disorder in screening that lead to of Folic Acid Use barriers, folic acid deficiency.
Brief description
Fig. 1 is the standard curve between the 5-methyltetrahydrofolate concentration and peak area making in embodiment 1.
Fig. 2 is the 5-methyltetrahydrofolate HPLC detection peak figure in embodiment 5（Wherein 5-MTHF refers to 5- methyl tetrahydrochysene leaf
Acid）.
Fig. 3 is the 5-methyltetrahydrofolate HPLC detection peak figure in embodiment 6（Wherein 5-MTHF refers to 5- methyl tetrahydrochysene leaf
Acid）.
Fig. 4 is the 5-methyltetrahydrofolate HPLC detection peak figure in embodiment 7（Wherein 5-MTHF refers to 5- methyl tetrahydrochysene leaf
Acid）.
Specific embodiment
Experimental technique used in following embodiments if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, all commercially obtain.
Embodiment 1, the detection kit of Methylene tetrahydrofolate reductase enzymatic activity, including following component：
Reagent 1：
Kaliumphosphate buffer（pH6.8） 80mmol/L
Dithiothreitol, DTT 5mmol/L
Reagent 2：
5,10- methylene tetrahydrofolate aqueous solution 0.5mmol/L
Reagent 3：
NADPH aqueous solution 2mmol/L
Reagent 4：
High chloro acid solution 60ml/L
Aqueous ascorbic acid 60mmol/L
Reagent 5：
Kaliumphosphate buffer（pH6.8） 60mmol/L
Aqueous ascorbic acid 30mmol/L
Embodiment 2, the detection kit of Methylene tetrahydrofolate reductase enzymatic activity, including following component：
Reagent 1：
Kaliumphosphate buffer（pH7.0） 100mmol/L
Dithiothreitol, DTT 5mmol/L
Reagent 2：
5,10- methylene tetrahydrofolate aqueous solution 0.4mmol/L
Reagent 3：
NADPH aqueous solution 1.5mmol/L
Reagent 4：
High chloro acid solution 60ml/L
Aqueous ascorbic acid 60mmol/L
Reagent 5：
Kaliumphosphate buffer（pH6.8） 60mmol/L
Aqueous ascorbic acid 30mmol/L
Embodiment 3, the detection kit of Methylene tetrahydrofolate reductase enzymatic activity, including following component：
Reagent 1：
Kaliumphosphate buffer（pH7.2） 150mmol/L
Dithiothreitol, DTT 5mmol/L
Reagent 2：
5,10- methylene tetrahydrofolate aqueous solution 0.15mmol/L
Reagent 3：
NADPH aqueous solution 1mmol/L
Reagent 4：
High chloro acid solution 80ml/L
Aqueous ascorbic acid 60mmol/L
Reagent 5：
Kaliumphosphate buffer（pH6.8） 60mmol/L
Aqueous ascorbic acid 30mmol/L
Embodiment 4, the preparation of sample
（1）Leukocyte separates
The sample of Methylene tetrahydrofolate reductase is the peripheral vein whole blood of healthy volunteer, separates from peripheral vein whole blood
Leukocyte, particular by the preparation of following method：With containing EDTA.K2The vacuum test tube of anticoagulant collects the outer of health adult
All venous whole 4ml, add 15ml lysate（0.1mM EDTA）, process 15min with broken red blood cell, 3000rpm is centrifuged
10min, abandons supernatant, and collecting precipitation is leukocyte, and with brine leukocyte twice, 3500rpm is centrifuged 5min, abandons
Clearly, collect leukocyte cell pellet, leukocyte cell pellet can frozen preserve under the conditions of -80 DEG C.
（2）Prepared by Methylene tetrahydrofolate reductase crude enzyme liquid
Add 1ml reagent 1 suspension cell, then ultrasonic, ultrasonic 10s in leukocyte cell pellet, interval 10s, at common ultrasonic 10 times
Reason, is then 1ug/ul with the legal protein concentration of BCA, and this suspension is the thick enzyme containing Methylene tetrahydrofolate reductase
Liquid, is put in stand-by in 4 DEG C of environment after reserving concentration.
Embodiment 5, the detection of Methylene tetrahydrofolate reductase enzymatic activity
（1）The mensure of sample enzymatic activity
Using kit measurement sample enzymatic activity described in embodiment 1, reagent 1, reagent 2 are mixed with 40ug sample, sample and examination
The volume ratio of agent 2 and reagent 1 is 1：2：6, add reagent 3, reagent 3 is 1 with the ratio of aforementioned mixed system:9, will after mixing
Reaction system is placed in 37 DEG C of lucifuge reaction 20min, and reaction adds reagent 4 terminating reaction after terminating and is placed on ice sample
30min, reagent 4 is 1 with the volume ratio of reaction system：10.After termination, sample cell 18000g centrifugation 5min, takes supernatant, adds
The ratio of reagent 5, wherein supernatant and reagent 5 is 1：5；
System after dilution is filtered with the filter of 0.45um, and is positioned over low temperature, to be detected under the conditions of lucifuge.
（2）The mensure of blank
Reagent 1, reagent 2 are mixed with 40ug sample, sample is 1 with the volume ratio of reagent 2 and reagent 1：2：6, will be anti-after mixing
System is answered to be placed in 37 DEG C of lucifuge reaction 20min, reaction adds reagent 3 after terminating, and reagent 3 with the ratio of aforementioned mixed system is
1:9, and add reagent 4 terminating reaction immediately and sample is placed in 30min on ice, reagent 4 is 1 with the volume ratio of reaction system：
10.After termination, sample cell 18000g centrifugation 5min, takes supernatant, adds reagent 5, the wherein ratio of supernatant and reagent 5 is 1：
5；
System after dilution is filtered with the filter of 0.45um, and is positioned over low temperature, to be detected under the conditions of lucifuge.
（3）The preparation of standard substance
Take variable concentrations 5-methyltetrahydrofolate standard substance, its concentration is respectively 0,20,40,80,150,300 nmol/L, respectively
Carry out described termination diluting reaction with described reagent 4 and reagent 5, and detect described reacted system in excitation wavelength
Peak area under 290nm and launch wavelength 350nm；With the concentration of 5-methyltetrahydrofolate as abscissa, sat with peak area for vertical
Mark, makes standard curve as shown in Figure 1.
（4）HPLC detects
Chromatographic column used is C18 post, 250*4.6mm, internal diameter 5um, and mobile phase is 50mmol/L potassium dihydrogen phosphate（pH 2.8）Contain
10% acetonitrile, flow velocity is 1ml/min, and column temperature keeps 25 DEG C, and sample size is 50ul, and excitation wavelength is set to 290nm and launch wavelength
It is set to 350nm.A length of 20min during detection.
Average peak area value according to made standard curve and sample and the average peak area value of blank
Difference, calculates health adult's blood methylene tetrahydrofolate reductase enzymatic activity, wherein, the unit definition of enzymatic activity
For：The nanomole number of the 5-methyltetrahydrofolate being generated with the described methylene tetrahydrofolate of every milligram of sample reduction per minute, table
It is shown as nmol/mg/min.
Testing result as shown in Fig. 2 as can be seen from the figure 5-MTHF can efficiently separate through HPLC, peak figure is clearly differentiated
Preferably, therefore described in embodiment 1, test kit can be with effective detection Methylene tetrahydrofolate reductase enzymatic activity.
The detection of embodiment 6 Methylene tetrahydrofolate reductase enzymatic activity
（1）The mensure of sample enzymatic activity
Using kit measurement sample enzymatic activity described in embodiment 2, reagent 1, reagent 2 are mixed with 40ug sample, sample and examination
The volume ratio of agent 2 and reagent 1 is 1：3：5, add reagent 3, reagent 3 is 1 with the ratio of aforementioned mixed system:9, will after mixing
Reaction system is placed in 37 DEG C of lucifuge reaction 20min, and reaction adds reagent 4 terminating reaction after terminating and is placed on ice sample
30min, reagent 4 is 1 with the volume ratio of reaction system：10.After termination, sample cell 18000g centrifugation 5min, takes supernatant, adds
The ratio of reagent 5, wherein supernatant and reagent 5 is 1：5；
System after dilution is filtered with the filter of 0.45um, and is positioned over low temperature, to be detected under the conditions of lucifuge.
（2）The mensure of blank
Reagent 1, reagent 2 are mixed with 40ug sample, sample is 1 with the volume ratio of reagent 2 and reagent 1：3：5, will be anti-after mixing
System is answered to be placed in 37 DEG C of lucifuge reaction 20min, reaction adds reagent 3 after terminating, and reagent 3 with the ratio of aforementioned mixed system is
1:9, and add reagent 4 terminating reaction immediately and sample is placed in 30min on ice, reagent 4 is 1 with the volume ratio of reaction system：
10.After termination, sample cell 18000g centrifugation 5min, takes supernatant, adds reagent 5, the wherein ratio of supernatant and reagent 5 is 1：
5；
System after dilution is filtered with the filter of 0.45um, and is positioned over low temperature, to be detected under the conditions of lucifuge；
Product adopts in detection method such as embodiment 5 after dilute filtration（4）HPLC detection is described.
Testing result as shown in figure 3, as can be seen from the figure 5-MTHF can efficiently separate through HPLC, peak figure is clearly differentiated
Preferably, therefore described in embodiment 2, test kit can be with effective detection Methylene tetrahydrofolate reductase enzymatic activity.
The detection of embodiment 7 Methylene tetrahydrofolate reductase enzymatic activity
（1）The mensure of sample enzymatic activity
Using kit measurement sample enzymatic activity described in embodiment 3, reagent 1, reagent 2 are mixed with 40ug sample, sample and examination
The volume ratio of agent 2 and reagent 1 is 1：4：3, add reagent 3, reagent 3 is 1 with the ratio of aforementioned mixed system:8, will after mixing
Reaction system is placed in 37 DEG C of lucifuge reaction 20min, and reaction adds reagent 4 terminating reaction after terminating and is placed on ice sample
30min, reagent 4 is 1 with the volume ratio of reaction system：9.After termination, sample cell 18000g centrifugation 5min, takes supernatant, adds examination
The ratio of agent 5, wherein supernatant and reagent 5 is 1：5；
System after dilution is filtered with the filter of 0.45um, and is positioned over low temperature, to be detected under the conditions of lucifuge.
（2）The mensure of blank
Reagent 1, reagent 2 are mixed with 40ug sample, sample is 1 with the volume ratio of reagent 2 and reagent 1：4：3, will be anti-after mixing
System is answered to be placed in 37 DEG C of lucifuge reaction 20min, reaction adds reagent 3 after terminating, and reagent 3 with the ratio of aforementioned mixed system is
1:8, and add reagent 4 terminating reaction immediately and sample is placed in 30min on ice, reagent 4 is 1 with the volume ratio of reaction system：
9.After termination, sample cell 18000g centrifugation 5min, takes supernatant, adds reagent 5, the wherein ratio of supernatant and reagent 5 is 1：5；
System after dilution is filtered with the filter of 0.45um, and is positioned over low temperature, to be detected under the conditions of lucifuge；
Product adopts in detection method such as embodiment 5 after dilute filtration（4）HPLC detection is described.
Testing result as shown in figure 4, as can be seen from the figure 5-MTHF can efficiently separate through HPLC, peak figure is clearly differentiated
Preferably, therefore described in embodiment 3, test kit can be with effective detection Methylene tetrahydrofolate reductase enzymatic activity.
The foregoing is only the alternative embodiment of the present invention, be not limited to the present invention, for those skilled in the art
For member, the present invention can have various modifications and variations.All any modifications within the spirit and principles in the present invention, made,
Equivalent, improvement etc., should be included within the scope of the present invention.
Claims (10)
1. a kind of detection Methylene tetrahydrofolate reductase enzymatic activity test kit it is characterised in that：Described test kit includes solely
The reagent 1 of vertical packaging, reagent 2, reagent 3, reagent 4 and reagent 5；
Described reagent 1 is the buffer system being made up of buffer 1 and enzyme activity stabilizer 1, and the pH value of described reagent 1 is 5 ~ 8；
Described reagent 2 is 5,10- methylene tetrahydrofolate；
Described reagent 3 is NADPH；
Described reagent 4 is the terminate liquid being made up of termination reagent and enzyme activity stabilizer 2；
Described reagent 5 is the diluent being made up of buffer 2 and enzyme activity stabilizer 2, and the pH value of described reagent 5 is 5 ~ 8.
2. test kit according to claim 1 it is characterised in that：In described reagent 1, the molar concentration of described buffer 1
For 50 ~ 500mmol/L, the molar concentration of described enzyme activity stabilizer 1 is 5 ~ 100mmol/L；
In described reagent 2, the molar concentration of described 5,10-CH2-THFA is 0.05 ~ 0.5mmol/L；
In described reagent 3, the molar concentration of described NADPH can be 0.05 ~ 2mmol/L；
In described reagent 4, the concentration of volume percent of described terminate liquid can be 20 ~ 80ml/L, described enzyme activity stabilizer 2 mole
Concentration can be 10 ~ 100mmol/L；
In described reagent 5, the molar concentration of described buffer 2 can be 20 ~ 200mmol/L, described enzyme activity stabilizer 2 mole dense
Degree can be 5 ~ 50mmol/L.
3. test kit according to claim 2 it is characterised in that：In described reagent 1, described buffer 1 delays for potassium phosphate
Rush liquid, molar concentration is 80 ~ 150mmol/L, described enzyme activity stabilizer 1 is dithiothreitol, DTT, molar concentration is 5mmol/L；
In described reagent 2, the molar concentration of described 5,10-CH2-THFA is 0.15 ~ 0.5mmol/L；
In described reagent 3, the molar concentration of described NADPH can be 1 ~ 2mmol/L；
In described reagent 4, described terminate liquid is high chloro acid solution, and concentration of volume percent is 60 ~ 80ml/L, and described enzyme activity is steady
Determining agent 2 is aqueous ascorbic acid, and molar concentration is 60mmol/L；
In described reagent 5, described buffer 2 is kaliumphosphate buffer, and molar concentration is 60mmol/L, described enzyme activity stabilizer 2
For aqueous ascorbic acid, molar concentration is 30mmol/L.
4. test kit according to claim 3 it is characterised in that：In described reagent 1, described buffer 1 delays for potassium phosphate
Rush liquid, molar concentration is 80mmol/L, pH value is 6.8, described enzyme activity stabilizer is dithiothreitol, DTT, molar concentration is 5mmol/
L；
In described reagent 2, the molar concentration of described 5,10-CH2-THFA is 0.5mmol/L；
In described reagent 3, the molar concentration of described NADPH can be 2mmol/L；
In described reagent 4, described terminate liquid is high chloro acid solution, and concentration of volume percent is 60ml/L, and described enzyme activity is stable
Agent 2 is aqueous ascorbic acid, and molar concentration is 60mmol/L；
In described reagent 5, described buffer 2 is kaliumphosphate buffer, and molar concentration is 60mmol/L, described enzyme activity stabilizer 2
For aqueous ascorbic acid, molar concentration is 30mmol/L.
5. test kit according to claim 3 it is characterised in that：In described reagent 1, described buffer 1 delays for potassium phosphate
Rush liquid, molar concentration is 100mmol/L, pH value is 7.0, described enzyme activity stabilizer is dithiothreitol, DTT, molar concentration is
5mmol/L；
In described reagent 2, the molar concentration of described 5,10-CH2-THFA is 0.4mmol/L；
In described reagent 3, the molar concentration of described NADPH can be 1.5mmol/L；
In described reagent 4, described terminate liquid is high chloro acid solution, and concentration of volume percent is 60ml/L, and described enzyme activity is stable
Agent 2 is aqueous ascorbic acid, and molar concentration is 60mmol/L；
In described reagent 5, described buffer 2 is kaliumphosphate buffer, and molar concentration is 60mmol/L, described enzyme activity stabilizer 2
For aqueous ascorbic acid, molar concentration is 30mmol/L.
6. test kit according to claim 3 it is characterised in that：In described reagent 1, described buffer 1 delays for potassium phosphate
Rush liquid, molar concentration is 150mmol/L, pH value is 7.2, described enzyme activity stabilizer is dithiothreitol, DTT, molar concentration is
5mmol/L；
In described reagent 2, the molar concentration of described 5,10-CH2-THFA is 0.15mmol/L；
In described reagent 3, the molar concentration of described NADPH can be 1mmol/L；
In described reagent 4, described terminate liquid is high chloro acid solution, and concentration of volume percent is 80ml/L, and described enzyme activity is stable
Agent 2 is aqueous ascorbic acid, and molar concentration is 60mmol/L；
In described reagent 5, described buffer 2 is kaliumphosphate buffer, and molar concentration is 60mmol/L, described enzyme activity stabilizer 2
For aqueous ascorbic acid, molar concentration is 30mmol/L.
7. according to the arbitrary described test kit of claim 1 or 2 it is characterised in that：Described enzyme activity stabilizer be beta -mercaptoethanol,
At least one of dithiothreitol, DTT or ascorbic acid.
8. the method that a kind of arbitrary described test kit of employing claim 1 to 7 detects Methylene tetrahydrofolate reductase enzymatic activity,
It is characterized in that：
Described reagent 1, described reagent 2 are mixed with sample to be tested, described sample to be tested and described reagent 2 and described reagent 1
Volume ratio is 1：（2~4）：（6~3）, form described reagent 1, described reagent 2 and the mixed system of sample to be tested, add described
Reagent 3, described reagent 3 is 1 with the ratio of described mixed system:9, after mixing, reaction system is placed in 37 DEG C of lucifuge reactions
20min, reaction adds described reagent 4 terminating reaction after terminating and described sample to be tested is placed in 30min on ice, described reagent 4
Volume ratio with reaction system is 1：10, after termination, described sample to be tested pipe 18000g is centrifuged 5min, takes supernatant, adds described
Reagent 5, the wherein ratio of supernatant and described reagent 5 are 1：5, the system after dilution is filtered with the filter of 0.45um,
And be positioned over low temperature, treat that HPLC detects under the conditions of lucifuge.
9. method according to claim 8 it is characterised in that：Described reagent 1, described reagent 2 are mixed with sample to be tested,
Described sample to be tested is 1 with the volume ratio of described reagent 2 and described reagent 1：2：6, form described reagent 1, described reagent 2 and treat
This mixed system of test sample, adds described reagent 3, and described reagent 3 is 1 with the ratio of described mixed system:9, will after mixing
Reaction system is placed in 37 DEG C of lucifuges reaction 20min, and reaction adds described reagent 4 terminating reaction and by described sample to be tested after terminating
It is placed in 30min on ice, described reagent 4 is 1 with the volume ratio of reaction system：10, after termination described sample to be tested pipe 18000g from
Heart 5min, takes supernatant, adds described reagent 5, the wherein ratio of supernatant and described reagent 5 is 1：5, the system after dilution is used
The filter of 0.45um is filtered, and is positioned over low temperature, treats that HPLC detects under the conditions of lucifuge.
10. in claim 1 to 7 arbitrary described test kit in auxiliary diagnosis methylenetetrahydrofolate reductase deficiency and Folic Acid
Application in Use barriers, the health check-up examination of folic acid deficiency.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610239025 | 2016-04-18 | ||
| CN2016102390256 | 2016-04-18 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115436540A (en) * | 2022-09-26 | 2022-12-06 | 汤臣倍健股份有限公司 | Method and kit for simultaneously determining contents of folic acid and homocysteine in blood |
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| CN1333375A (en) * | 2001-04-02 | 2002-01-30 | 四川宇康生物技术有限公司 | Pregestational gene warning diagnostic kit |
| WO2004013284A2 (en) * | 2002-08-02 | 2004-02-12 | Tufts University | A method to assess genomic dna methylation using high-performance liquid chromatography-electospray ionization mass spectrometry |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1333375A (en) * | 2001-04-02 | 2002-01-30 | 四川宇康生物技术有限公司 | Pregestational gene warning diagnostic kit |
| WO2004013284A2 (en) * | 2002-08-02 | 2004-02-12 | Tufts University | A method to assess genomic dna methylation using high-performance liquid chromatography-electospray ionization mass spectrometry |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115436540A (en) * | 2022-09-26 | 2022-12-06 | 汤臣倍健股份有限公司 | Method and kit for simultaneously determining contents of folic acid and homocysteine in blood |
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