CN107655870A - The detection method and detection kit of acidic hydrolysis enzymatic activity in a kind of lysosome - Google Patents
The detection method and detection kit of acidic hydrolysis enzymatic activity in a kind of lysosome Download PDFInfo
- Publication number
- CN107655870A CN107655870A CN201710781253.0A CN201710781253A CN107655870A CN 107655870 A CN107655870 A CN 107655870A CN 201710781253 A CN201710781253 A CN 201710781253A CN 107655870 A CN107655870 A CN 107655870A
- Authority
- CN
- China
- Prior art keywords
- substrate
- concentration
- enzyme
- enzymatic activity
- sulfatase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
- G01N2333/938—Hydrolases (3) acting on glycosyl compounds (3.2) acting on beta-galactose-glycoside bonds, e.g. beta-galactosidase
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Optics & Photonics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to biochemical analysis and detection technical field, the detection method and detection kit of acidic hydrolysis enzymatic activity specially in lysosome.Present invention design first includes the detecting system of mucopolysaccharide acid hydrolase in 5 kinds of lysosomes, and this five kinds of enzymes are respectively：α L idose glycosides enzyme, iduronate sulfatase, the sulfatase of β N acetylgalactosamines 6, beta galactosidase and aryl sulfatase B enzyme.The present invention has the characteristic of fluorescence using 4 methyl umbelliferones, the 4 methylumbelliferyl ketone groups obtained using chemical synthesis new substrate with reference to caused by with zymolyte, reacted using the acidic buffer solution containing new substrate and mucopolysaccharide hydrolase, 4 free methyl umbelliferone fluorescent materials are discharged after hydrolases substrate.The activity of hydrolase is higher in sample, and the amount of 4 methyl umbelliferones of hydrolysis substrate release is more, also higher by fluorescence intensity caused by fluoroscopic examination, so as to realize the quantitative detection to hydrolytic enzyme activities.Detection sensitivity of the present invention is high, selectivity is good, strong antijamming capability, reproducibility are good, easy to operate.
Description
Technical field
The invention belongs to biochemical analysis and detection technical field, and in particular to the detection side of acidic hydrolysis enzymatic activity in lysosome
Method and detection kit.
Background technology
Hydrolase is the general name of the class of enzymes of catalytic hydrolysis reaction, is distributed widely in human body, to human metabolism and
The vital movements such as bioelectric detecting play an important role.Lysosome is that the blister rich in a variety of high-concentration acidic wastewater hydrolases is thin in cell
Born of the same parents' device, it is responsible for intracellular various substance decompositions and the important organelle of digestion.And caused by lysosomal enzyme activities lack
A series of inherited metabolic disorders be collectively referred to as lysosomal disease, and the lysosomal disease clinically often said at present mainly refers in particular to elder generation
Nature lysosomal disease (Congenital Lysosomal Disease).Lysosomal disease be due to it is a kind of in lysosome or
The defects of a variety of enzymes, its metabolin is caused to be stored up in histoorgan, therefore the lysosomal storage disease (Lysosomal that is otherwise known as
Storage Disorders).Therefore the detection of lysosomal hydrolysis enzymatic activity is in the sieving and diagnosis of lysosomal disease and prognosis etc.
All have very important significance.
Wherein mucopolysaccharidosis (Mucopolysaccharidoses, MPS) is that the most common one group of progressive in China glues
More metabolism obstacles of blood glucose hereditary diseases, accounting are up to 40%.Mucopolysaccharidosis is divided into 7 types according to the difference of defect enzyme：Respectively
It is I, II, III, IV, VI, VII and IX type, wherein III type is divided into tetra- hypotypes of D of III A, III B, III C, III, IV type is divided into again
Two hypotypes of IV A and IV B, although various Disease-causing gene and clinical manifestation are variant, because the substrate stored up all is mucopolysaccharide
Therefore it is referred to as mucopolysaccharidosis.Wherein mucopolysaccharidosis II types belong to x linked recessive hereditary disease, and other types are
Autosomal recessive inheritance.Mucopolysaccharidosis is to be proposed by Brante by tissue chemical analysis earliest.But in fact, early in
1917 have just delivered the description of related clinical manifestation to Hunter in 1919 to Hurler, but due to its cause of disease always not
It is bright, until just confirming that it belongs to lysosomal storage disease the beginning of the seventies.MPS II types therein are only second to the disease of Gaucher disease for distribution
Disease, it is the second single lysosomal storage disease occurred frequently in China（20%）.In addition, MPS I types and MPS IV types are also Chinese population
In relatively conventional mucopolysaccharidosis type.The most common performance of this group of disease be mainly face is coarse and crude, skeletal abnormality, companion or
Without mental retardation etc., it is China's understanding and studies one group of slightly more diseases.
Because enzyme has a highly single-minded catalytic activity, thus can by determine its corresponding substrate or production concentration change into
And realize the detection to enzymatic activity.Detection for hydrolytic enzyme activities at present both at home and abroad more uses XRF and colorimetric analysis
Method, colorimetric method is more to be received as chromogenic substrate with p-nitrophenyl di(2-ethylhexyl)phosphate, because substrate hydrolyzes under the catalytic action of alkaline phosphatase
The end productses of yellow are generated, and have maximum absorption band at 405nm, so as to realize the quantitative ratio to alkaline phosphatase activities
Colour analysis, but the shortcomings of sensitivity to be present low for this method, and detection range is narrow and antijamming capability is weak, and the stability of substrate
Difference, easily crystallization, easily being decomposed under illumination condition causes " false positive " result.And fluorogenic substrate stability is strong, fluoroscopic examination has
The features such as sensitivity is strong, specificity is high, strong antijamming capability.
The content of the invention
It is an object of the invention to provide a kind of cost is low, efficiency high, stability are high, it is strong antijamming capability can be quick
Detect the method and kit of acidic hydrolysis enzymatic activity in lysosome.
The detection method of acidic hydrolysis enzymatic activity, comprises the following steps in lysosome proposed by the present invention：4 methyl will be contained
The substrate solution of umbelliferone fluorophor is incubated with hydrolysis enzyme solutions to be measured, and hydrolases substrate discharges fluorophor；It is right
In sulfatase, hydrolyzed organo-sulfate substrate and be allowed to dissociate and one is formed after inorganic sulfuric acid new be free of sulfate group
Substrate compounds, the substrate can by another hydrolases, by adding a certain amount of hydrolase, what hydrolysis was newly formed
Substrate, discharge fluorophor；The measure reaction emitted luminescence intensity that 460nm goes out under exciting light at 355nm；According to fluorescence intensity with
The standard curve relation of fluorescent material concentration, is calculated fluorescent material concentration, by between fluorescent material concentration and enzymatic activity
Standard curve relation, be calculated it is to be measured hydrolysis enzyme solutions reclaimed water solution enzyme activity.
Specifically, the method proposed by the present invention for detecting acidic hydrolysis enzymatic activity in lysosome, is comprised the following steps that：
（1）By the substrate solution containing 4 methyl umbelliferone fluorophors（PH is 3.5-5.5）With hydrolysis enzyme solutions to be measured at 37 DEG C
1-17 hours are incubated under constant temperature；Substrate discharges fluorophor after hydrolases and produces fluorescence, and measure reaction exists
Emitted luminescence intensity at 355nm under exciting light at 460nm；
（2）According to fluorescence intensity and the curved line relation of fluorescent material concentration, fluorescent material concentration, fluorescence standard curve is calculated
After concentration gradient dilution being carried out using the methyl umbelliferone of standard fluorescence material 4（0-10nmol/ul, preferably 1-10nmol/ul）Enter
Row fluoroscopic examination, linear analysis is carried out by standard substance concentration gradient and fluorescence intensity and calculates acquisition calibration curve equation；
（3）According to the curved line relation between fluorescent material concentration and enzymatic activity, hydrolysis enzyme solutions reclaimed water solution enzyme to be measured is calculated
Activity, sample to be tested obtains fluorescence intensity through fluoroscopic examination, and florescent intensity value passes through fluorescence intensity and fluorescent material concentration
Calibration curve equation calculate fluorescent material concentration, can be calculated by the molar concentration of fluorescent material and be hydrolyzed substrate
Molar concentration, the amount of hydrolases substrate represents enzymatic activity numerical value in unit concentration albumen in the unit of account time；
The method for detecting acid sulfate enzymatic activity in lysosome, is comprised the following steps that：
（1）By the substrate solution containing 4 methyl umbelliferone fluorophors（PH3.5-5.5）With hydrolysis enzyme solutions to be measured in 37 DEG C of perseverances
4-17 hours are incubated under the conditions of temperature, it hydrolyzes organo-sulfate substrate and is allowed to dissociate after inorganic sulfuric acid, formed one it is new not
The substrate compounds of sulfur-bearing acid groups；5-10ul people's recombinant alpha-L- idose glycosides enzyme solutions are supplemented in reaction system（Concentration is
500-1000ng/ul）Or beta galactosidase solution（Concentration is 0.01-0.1U/ul）Continue to be incubated 2- under 37 DEG C of constant temperatures
24 hours, hydrolyze the substrate compounds after sulfatase enzyme hydrolysis and discharge fluorophor generation fluorescence；Measure reaction exists
Emitted luminescence intensity at 355nm under exciting light at 460nm；
（2）According to fluorescence intensity and the curved line relation of fluorescent material concentration, fluorescent material concentration, fluorescence standard curve is calculated
After concentration gradient dilution being carried out using the methyl umbelliferone of standard fluorescence material 4（0-10nmol/ul, preferably 1-10nmol/ul）Enter
Row fluoroscopic examination, linear analysis is carried out by standard substance concentration gradient and fluorescence intensity and calculates acquisition calibration curve equation；
（3）According to the curved line relation between fluorescent material concentration and enzymatic activity, hydrolysis enzyme solutions reclaimed water solution enzyme to be measured is calculated
Activity, sample to be tested obtains fluorescence intensity through fluoroscopic examination, and florescent intensity value passes through fluorescence intensity and fluorescent material concentration
Calibration curve equation calculate fluorescent material concentration, can be calculated by the molar concentration of fluorescent material and be hydrolyzed substrate
Molar concentration, the amount of hydrolases substrate represents enzymatic activity numerical value in unit concentration albumen in calculating reacting time.
In the present invention, detect the specific of lysosomal enzyme activities and be calculated as follows：
The sample to be tested fluorescent value that detection is read is F samples, blank control in experiment（Sample is replaced with purified water）Fluorescent value
Empty for F, the amount of reaction generation fluorescent material is C, and detection volume is examined for V, reaction volume V, albumen quality M.Assuming that fluorescence
The linear equation of standard curve is Y=a*X+b, and wherein Y is the amount of fluorescent material, and X is fluorescence intensity reading, and a and b are constant.
Formula 1：；
Formula 2：。
Explanation：Fluorescent material and concentration of substrate, which calculate, herein uses molar concentration, therefore sulfatase or hydrolase institute
The molar concentration for hydrolyzing fluorescent material of the molar concentration of substrate with generating is consistent.
In the present invention, described hydrolase is α-L- idose glycosides enzyme, beta galactosidase and aryl sulfatase B enzyme；
Sulfatase is iduronate sulfatase, β-N-acetylgalactosamine-6-sulfatase.Wherein：
Described hydrolase is α-L- idose glycosides enzymes, and substrate is 4-methyl umbelliferone-α-L- idose glycosides.
Described sulfatase is iduronate sulfatase, substrate be 4-methyl umbelliferone-α-L- iduronic acids-
2- sulfuric ester disodium salts.
Described sulfuric acid is β-N-acetylgalactosamine-6-sulfatase, and substrate is 4-methyl umbelliferone-β-D- galactolipins
Glycosides -6- sulfuric ester sodium salts.
Described hydrolase is beta galactosidase, and substrate is 4-methyl umbelliferone-β-D- galactopyranosides.
Described hydrolase is aryl sulfatase B enzyme, and substrate is 4-methyl umbelliferone base sulfuric ester sylvite.
Corresponding to above-mentioned detection method, the present invention provides the detection kit of acidic hydrolysis enzymatic activity in lysosome, and it is wrapped
Include：
4-methyl umbelliferone-α-L- idoses glycosides, 4-methyl umbelliferone-α-L- iduronic acid -2- sulfuric esters disodium salt, 4- first
Base umbelliferone-β-D- galactoside -6- sulfuric esters sodium salt, 4-methyl umbelliferone-β-D- galactopyranoside 4-methyl umbelliferones
This 5 kinds of substrate solutions of base sulfuric ester sylvite, solution concentration 0.68mM-10mM；
D-Glucose diacid-Isosorbide-5-Nitrae lactone solution, concentration 3-7Mm；
People's recombinant alpha-L- idose glycosides enzyme aqueous solutions（LEBT）, concentration 400-600ng/ul, containing 0.2% bovine serum albumin(BSA)
（BSA）；
The beta galactosidase aqueous solution（β-Gal）, concentration 0.01-0.05U/ul, containing 0.2%BSA；
Pi/Ci buffer solutions：0.2M Na2HPO4With 0.1M citric acid mixed liquors, Ph=4.5-5.0；
Pi buffer solutions：0.9M Na2HPO4With 0.9MNaH2PO4Mixed liquor, Ph=4.0-4.5；
And terminate liquid：0.17M glycine-sodium carbonate buffer, Ph=10-11.
The dissolution system of substrate and other reagents is purified water, 0.1M sodium formate buffers, PC buffer solutions in kit
（0.2M phosphate-citrate buffers）, one or more in 0.2M sodium citrate buffer solutions or 0.1M sodium-acetate buffers,
Ph values are 3.5-5.0.
Contain a small amount of preservative in kit reagent（For the 0.02%-0.2% of total system）With a small amount of surfactant（To be total
The 0.025%-0.25% of system）.
Described preservative is received for the misery first, benzoic acid of sorb, one kind in nitrite natrium, proclin series preservatives
It is or several.
Described surfactant is Nonidet P40（NP40）, Tween series, one in Triton X series
Kind is several.
Enzymatic activity fluorescence detection utilizes the decomposition digestion of enzyme, under specific reaction condition and time, user
The fluorescence 4-methyl umbelliferone substrate of work chemical synthesis is detected, and this substrate in itself and does not have fluorescence, but when passing through
4-methyl umbelliferone will be discharged after the digestion decomposition of enzyme, this material has hyperfluorescence property.In normal person,
Enzyme contained by lysosome under certain condition can will can digest Substrate hydrolysis, and discharge fluorescent material（4- methylumbelliferyls
Ketone）, produce fluorescence signal（Other catabolites are without fluorescence）；If due to its lysosome in lysosomal storage disease patient
Interior enzyme defect, during the course of the reaction enzyme do not have or only deposit seldom digestion decomposition function, had no or only with this few
Substrate is digested decomposition, and 4-methyl umbelliferone is still to exist in the form of substrate compounds, will not now produce fluorescence signal.
By multi-function microplate reader device, mesh can be effectively detected under the conditions of excitation wavelength 365nm, wavelength of transmitted light 450nm
Sample in fluorescence intensity, and judge with this activity of enzyme.
The inventive method detection sensitivity is high, selectivity is good, strong antijamming capability, reproducibility are good, easy to operate, can use
The Activity determination of mucopolysaccharide acid hydrolase in lysosomal enzyme.
Brief description of the drawings
Fig. 1 is the canonical plotting of detection fluorescence standard material, concentration and fluorescence intensity.Configure 10nmol/ul fluorescence marks
Quasi- product solution, and gradient dilution fluorescence standard, 10nmol/ul, 5nmol/ul, 2.5nmol/ul, 1.25nmol/ are detected respectively
Ul, 0.625nmol/ul and 0nmol/ul fluorescence intensity, using concentration of specimens as the longitudinal axis, fluorescence intensity is that transverse axis does standard song
Line, obtain the calibration curve equation of fluorescent material concentration and fluorescence intensity.
Fig. 2 is the correlation of concentration of specimens and fluorescence intensity.Wherein, when detecting five kinds of enzyme enzymatic activitys, various concentrations condition
Under, the correlation of sample to be tested concentration and fluorescence intensity is proportional.Various concentrations sample to be tested 100 is chosen, detection respectively is treated
Five kinds of enzyme enzymatic activitys of test sample sheet, it is found that fluorescence intensity raises with the rise of sample protein concentration.
Fig. 3 is the correlation of concentration of specimens and enzymatic activity.Wherein, when detecting five kinds of enzyme enzymatic activitys, different protein concentration bars
Under part, unit enzymatic activity is with sample to be tested concentration without obvious correlation.Various concentrations sample to be tested 100 is taken, is detected respectively
Five kinds of enzyme enzymatic activitys of sample to be tested, because enzymatic activity represents enzyme under unit-protein concentration and decomposes the activity of substrate, therefore detect knot
Fruit finds that enzymatic activity will not raise with the rise of sample to be tested protein concentration, meets expection.
Fig. 4 is that α-L- iduronidase activities detect influence of the auxiliary reagent to fluorescence intensity.α-L- idose glycosides enzyme activity
Property detection when, respectively detect test sample this two（S1 and S2）And blank control.When reaction system prepares, D-Glucose is separately added into
Diacid-Isosorbide-5-Nitrae lactone solution and it is added without D-Glucose diacid-two kinds of Isosorbide-5-Nitrae lactone solution system, observing should under two kinds of systems
The change of intensity.As illustrated, being added without D-Glucose diacid-Isosorbide-5-Nitrae lactone solution, fluorescence intensity substantially weakens, D-Glucose
The lactone solution of diacid -1,4 is zymosthenic auxiliary reagent.
Fig. 5 is influence of the iduronate sulfatase Enzyme assay auxiliary reagent to fluorescence intensity.Iduronic acid
During sulfatase Enzyme assay, test sample this two is detected respectively（S1 and S2）And blank control.When reaction system prepares, respectively
Pi/Ci buffer solutions and LEBT solution are added simultaneously, only add Pi/Ci buffer solutions and only add LEBT three kinds of systems of solution, observation
Under three kinds of systems should intensity change, as illustrated, only add Pi/Ci buffer solutions or only add LEBT solution fluorescence intensity
Slightly it is weaker than while Pi/Ci buffer solutions and LEBT solution, prompting the amount of contained α-L- idoses glycosides enzyme in sample can hydrolyze substantially
New substrate caused by iduronate sulfatase hydrolysis substrate, the extra α-L- idose glycosides enzymes for adding excess may insure to examine
Iduronate sulfatase enzymatic activity can truly be reflected by surveying result, while need to add Pi/Ci buffer solution regulation system Ph values
To increase the hydrolysing activity of α-L- idose glycosides enzymes.
Fig. 6 is β-influence of the N-acetylgalactosamine-6-sulfatase Enzyme assay auxiliary reagent to fluorescence intensity.
During β-N-acetylgalactosamine-6-sulfatase Enzyme assay, test sample this two is detected respectively（S1 and S2）And blank control.
When reaction system prepares, add Pi buffer solutions and β-gal solution simultaneously respectively, only add Pi buffer solutions and only add β-gal and be molten
Three kinds of systems of liquid, observe three kinds of systems under should intensity change.As illustrated, only add Pi buffer solutions or only add β-gal
The fluorescence intensity of solution is markedly less than while Pi buffer solutions and β-gal solution, prompts contained beta galactosidase in sample
Amount is not enough to complete hydrolysis β-new substrate caused by N-acetylgalactosamine-6-sulfatase hydrolysis substrate, adds Pi buffer solutions
Regulation system Ph values can increase the hydrolysing activity of beta galactosidase.
Embodiment
With reference to embodiment, the invention will be further described.
One, leucocyte sample to be measured for taking extraction to complete, add 200ul purified waters and carry out ultrasonic cell disintegration（50W, ice
Bath amounts to ultrasonic 1min）, using BCA kit measurement protein concentrations, sample to be tested concentration is 1259ng/ul.
Described hydrolase is α-L- idose glycosides enzymes, is separately added into the μ l of substrate 10 into EP pipes, D-Glucose diacid -1,
The μ l of 4 lactone solution 10, and the μ l of cell homogenates liquid 10 of the sample to be tested of sample to be tested, are mixed after 37 DEG C of water-bath 1h.Constant temperature
Under the conditions of water-bath 1h terminate after add terminate liquid 200ul terminating reactions, using multi-function microplate reader determine fluorescence intensity, measure glimmering
Luminous intensity is 691519, and fluorescence standard curve equation is Y=0.0000027X, and sample to be tested concentration is 1259ng/ul, during reaction
Between be 1 hour, reactant 230ul, detection volume be 200ul, substitution formula calculating sample to be tested α-L- idose glycosides
Enzyme enzymatic activity is 167.85nmol/mg/h.
Described hydrolase is iduronate sulfatase, and μ l of substrate solution 20 and to be measured are separately added into EP pipes
The μ l of cell homogenates liquid 10 of sample, are mixed after water-bath 4h under 37 DEG C of constant temperatures.Water-bath 4h continues after terminating under constant temperature
The μ l of 20 μ l and LEBT solution of Pi/Ci buffer solutions 10 are separately added into EP pipes, are placed in after mixing under 37 DEG C of secondary constant temperatures
Water-bath 24h.The μ l terminating reactions of terminate liquid 200 are added after water-bath 24h terminates under constant temperature, are determined using multi-function microplate reader glimmering
Luminous intensity is 945952, and fluorescence standard curve equation is Y=0.0000027X, and sample to be tested concentration is 1259ng/ul, permanent first
The water-bath time is 4 hours, reactant 260ul under the conditions of temperature, and detection volume is 200ul, and this of substitution formula calculating is treated
This iduronate sulfatase of test sample enzymatic activity is 262.52nmol/mg/4h.
Described hydrolase is β-N-acetylgalactosamine-6-sulfatase, and the μ of substrate solution 20 is separately added into EP pipes
L and sample to be tested the μ l of cell homogenates liquid 10, are mixed after water-bath 17h under 37 DEG C of constant temperatures.Water-bath 17h under constant temperature
Continue to be separately added into the μ l of 5 μ l and β-gal solution of Pi buffer solutions 10 into EP pipes after end, 37 DEG C of secondary constant temperature are placed in after mixing
Under the conditions of water-bath 2h.The μ l terminating reactions of terminate liquid 200 are added after water-bath 2h terminates under constant temperature, are surveyed using multi-function microplate reader
It is 2658680 to determine fluorescence intensity, and fluorescence standard curve equation is Y=0.0000027X, and sample to be tested concentration is 1259ng/ul, first
The water-bath time is 17 hours, reactant 245ul under secondary constant temperature, and detection volume is 200ul, substitutes into what formula calculated
The sample to be tested iduronate sulfatase enzymatic activity is 693.52nmol/mg/17h.
Described hydrolase is beta galactosidase, and the μ l of substrate solution 20 and sample to be tested are separately added into EP pipes
The μ l of cell homogenates liquid 10, are mixed after water-bath 1h under 37 DEG C of constant temperatures.Water-bath 1h adds terminate liquid after terminating under constant temperature
200 μ l terminating reactions, use multi-function microplate reader determine fluorescence intensity for 2396587, fluorescence standard curve equation for Y=
0.0000027X, sample to be tested concentration are 1259ng/ul, and the water-bath time is 1 hour under constant temperature, and reactant is
230ul, detection volume are 200ul, substitute into the sample to be tested iduronate sulfatase enzymatic activity that formula calculates and are
589.98nmol/mg/h。
Described hydrolase is aryl sulfatase B enzyme, and the μ l of substrate solution 20 are separately added into EP pipes and treat test sample
This μ l of cell homogenates liquid 10, are mixed after water-bath 1h under 37 DEG C of constant temperatures.Added eventually after water-bath 1h terminates under constant temperature
The only μ l terminating reactions of liquid 200, use multi-function microplate reader determine fluorescence intensity for 628476, fluorescence standard curve equation for Y=
0.0000027X, sample to be tested concentration are 1259ng/ul, and the water-bath time is 1 hour under constant temperature first, and reactant is
230ul, detection volume are 200ul, substitute into the sample to be tested iduronate sulfatase enzymatic activity that formula calculates and are
117.30nmol/mg/h。
The fluorescence intensity testing conditions are exciting light：320-360nm, launch light：450-480nm.
Claims (9)
1. the detection method of acidic hydrolysis enzymatic activity in a kind of lysosome, it is characterised in that comprise the following steps：4 first will be contained
The substrate solution of base umbelliferone fluorophor is incubated with hydrolysis enzyme solutions to be measured, and hydrolases substrate discharges fluorophor；
For sulfatase, hydrolyzed organo-sulfate substrate and be allowed to dissociate and one is formed after inorganic sulfuric acid new be free of sulfate
The substrate compounds of group, another hydrolases of the substrate, by adding a certain amount of hydrolase, hydrolyze the bottom newly formed
Thing, discharge fluorophor；The measure reaction emitted luminescence intensity that 460nm goes out under exciting light at 355nm；According to fluorescence intensity with it is glimmering
The standard curve relation of stimulative substance concentration, is calculated fluorescent material concentration, by between fluorescent material concentration and enzymatic activity
Standard curve relation, the activity of hydrolysis enzyme solutions reclaimed water solution enzyme to be measured is calculated.
2. the detection method of acidic hydrolysis enzymatic activity in lysosome according to claim 1, it is characterised in that concrete operations
Step is as follows：
（1）Substrate solution containing 4 methyl umbelliferone fluorophors and hydrolysis enzyme solutions to be measured are incubated under 37 DEG C of constant temperatures
Educate 1-17 hours, the PH of substrate solution is 3.5-5.5；Substrate discharges fluorophor after hydrolases and produces fluorescence, surveys
The emitted luminescence intensity under exciting light at 355nm at 460nm is reacted calmly；
For sulfatase, by the substrate solution containing 4 methyl umbelliferone fluorophors with hydrolysis enzyme solutions to be measured in 37 DEG C of perseverances
4-17 hours are incubated under the conditions of temperature, the PH of substrate solution is 3.5-5.5；Its hydrolyze organo-sulfate substrate be allowed to dissociate it is inorganic
After sulfuric acid, the new substrate compounds without sulfate group are formed；It is 500- that 5-10ul concentration is supplemented in reaction system
The beta galactosidase solution that 1000ng/ul people's recombinant alpha-L- idose glycosides enzyme solutions or concentration are 0.01-0.1U/ul continues
2-24 hours are incubated under 37 DEG C of constant temperatures, the substrate compounds after sulfatase enzyme hydrolysis is hydrolyzed and discharges fluorophor production
Raw fluorescence；Emitted luminescence intensity of the measure reaction under exciting light at 355nm at 460nm；
（2）According to fluorescence intensity and the curved line relation of fluorescent material concentration, fluorescent material concentration, fluorescence standard curve is calculated
Concentration gradient is carried out using the methyl umbelliferone of standard fluorescence material 4 and is diluted to 0-10nmol/ul, is carried out fluoroscopic examination, is passed through mark
Quasi- material concentration gradient carries out linear analysis with fluorescence intensity and calculates acquisition calibration curve equation；
（3）According to the curved line relation between fluorescent material concentration and enzymatic activity, hydrolysis enzyme solutions reclaimed water solution enzyme to be measured is calculated
Activity, sample to be tested obtains fluorescence intensity through fluoroscopic examination, and florescent intensity value passes through fluorescence intensity and fluorescent material concentration
Calibration curve equation calculate fluorescent material concentration, can be calculated by the molar concentration of fluorescent material and be hydrolyzed substrate
Molar concentration, the amount of hydrolases substrate represents enzymatic activity numerical value in unit concentration albumen in the unit of account time.
3. the detection method of acidic hydrolysis enzymatic activity in lysosome according to claim 2, it is characterised in that detection lyase
The specific of body enzymatic activity is calculated as follows：
The sample to be tested fluorescent value that detection is read is F samples, and blank control fluorescent value is F empty in experiment, reacts generation fluorescent material
Amount be C, detection volume be V inspection, reaction volume V, albumen quality M；Assuming that the linear equation of fluorescence standard curve be Y=
A*X+b, wherein Y are the amount of fluorescent material, and X is fluorescence intensity reading, and a and b are constant；
Formula 1：；
Formula 2：。
4. the detection method of acidic hydrolysis enzymatic activity in lysosome according to claim 2, it is characterised in that the hydrolysis
Enzyme is α-L- idose glycosides enzyme, beta galactosidase or aryl sulfatase B enzyme；Described sulfatase is iduronic acid sulfuric acid
Esterase, β-N-acetylgalactosamine-6-sulfatase.
5. the detection method of acidic hydrolysis enzymatic activity in lysosome according to claim 4, it is characterised in that described water
Solution enzyme is α-L- idose glycosides enzymes, and substrate is 4-methyl umbelliferone-α-L- idose glycosides；
Described sulfatase is iduronate sulfatase, and substrate is 4-methyl umbelliferone-α-L- iduronic acid -2- sulphur
Acid esters disodium salt；
Described sulfuric acid is β-N-acetylgalactosamine-6-sulfatase, substrate be 4-methyl umbelliferone-β-D- galactosides-
6- sulfuric ester sodium salts；
Described hydrolase is beta galactosidase, and substrate is 4-methyl umbelliferone-β-D- galactopyranosides；
Described hydrolase is aryl sulfatase B enzyme, and substrate is 4-methyl umbelliferone base sulfuric ester sylvite.
A kind of 6. detection kit of acidic hydrolysis enzymatic activity in lysosome, it is characterised in that including：
4-methyl umbelliferone-α-L- idoses glycosides, 4-methyl umbelliferone-α-L- iduronic acid -2- sulfuric esters disodium salt, 4- first
Base umbelliferone-β-D- galactoside -6- sulfuric esters sodium salt, 4-methyl umbelliferone-β-D- galactopyranoside 4-methyl umbelliferones
This 5 kinds of substrate solutions of base sulfuric ester sylvite, solution concentration 0.68mM-10mM；
D-Glucose diacid-Isosorbide-5-Nitrae lactone solution, concentration 5-10Mm；
People's recombinant alpha-L- idose glycosides enzyme aqueous solutions, concentration 500-1000ng/ul, containing 0.2-0.5%BSA；
The beta galactosidase aqueous solution, concentration 0.01-0.1U/ul, containing 0.2-0.5%BSA；
Pi/Ci buffer solutions：0.2M Na2HPO4With 0.1M citric acid mixed liquors, Ph=4-5；
Pi buffer solutions：0.9M Na2HPO4With 0.9MNaH2PO4Mixed liquor, Ph=4-5；
And terminate liquid：0.17M glycine-sodium carbonate buffer, Ph=10-11.
7. the detection kit of acidic hydrolysis enzymatic activity in lysosome according to claim 6, it is characterised in that kit
The dissolution system of middle substrate and other reagents is purified water, 0.1M sodium formate buffers, PC buffer solutions, 0.2M buffered sodium citrates
One or more in liquid or 0.1M sodium-acetate buffers, Ph values are 3.5-5.0.
8. the detection kit of acidic hydrolysis enzymatic activity in lysosome according to claim 6, it is characterised in that containing anti-
Rotten agent and surfactant.
9. the detection kit of acidic hydrolysis enzymatic activity in lysosome according to claim 8, it is characterised in that described
Preservative receive for the misery first, benzoic acid of sorb, the one or more in nitrite natrium, proclin series preservatives；Described
Surfactant is the one or more in Nonidet P40, Tween series, Triton X series.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710781253.0A CN107655870A (en) | 2017-09-01 | 2017-09-01 | The detection method and detection kit of acidic hydrolysis enzymatic activity in a kind of lysosome |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710781253.0A CN107655870A (en) | 2017-09-01 | 2017-09-01 | The detection method and detection kit of acidic hydrolysis enzymatic activity in a kind of lysosome |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107655870A true CN107655870A (en) | 2018-02-02 |
Family
ID=61129094
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710781253.0A Withdrawn CN107655870A (en) | 2017-09-01 | 2017-09-01 | The detection method and detection kit of acidic hydrolysis enzymatic activity in a kind of lysosome |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107655870A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110632052A (en) * | 2019-10-25 | 2019-12-31 | 山西师范大学 | Fluorescence spectrum detection method for activity of soil arylsulfatase |
| CN111893161A (en) * | 2020-08-07 | 2020-11-06 | 北京中科医学检验实验室有限公司 | Detection kit for activity of alpha-L-iduronidase |
| CN113267555A (en) * | 2021-05-19 | 2021-08-17 | 中国科学技术大学 | Method for classifying lysosomes by using metabolites of lysosomes as markers |
-
2017
- 2017-09-01 CN CN201710781253.0A patent/CN107655870A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110632052A (en) * | 2019-10-25 | 2019-12-31 | 山西师范大学 | Fluorescence spectrum detection method for activity of soil arylsulfatase |
| CN111893161A (en) * | 2020-08-07 | 2020-11-06 | 北京中科医学检验实验室有限公司 | Detection kit for activity of alpha-L-iduronidase |
| CN113267555A (en) * | 2021-05-19 | 2021-08-17 | 中国科学技术大学 | Method for classifying lysosomes by using metabolites of lysosomes as markers |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102721684B (en) | Two-step enzyme measuring method and measuring reagent for creatinine in blood serum | |
| Lorentz et al. | Arylesterase in serum: elaboration and clinical application of a fixed-incubation method. | |
| CN107655870A (en) | The detection method and detection kit of acidic hydrolysis enzymatic activity in a kind of lysosome | |
| CN100595282C (en) | Glutamate-pyruvate transaminase determination reagent kit | |
| CN107796941A (en) | The measure kit and its detection method of a kind of platelet-activating factor acetylhydro-lase | |
| Park et al. | A sensitive dual colorimetric and fluorescence system for assaying the activity of alkaline phosphatase that relies on pyrophosphate inhibition of the peroxidase activity of copper ions | |
| CN109438326A (en) | A kind of fluorescence probe for detecting carboxy-lesterase and preparation method thereof and dedicated test kit | |
| Campbell et al. | Spectrofluorimetric determination of acid phosphatase activity | |
| CN100999758A (en) | Alpha-L-fucosidosidase active tested process and diagnostic reagent of alpha-L-fucosidosidase | |
| Schrenkhammer et al. | Time-resolved fluorescence-based assay for the determination of alkaline phosphatase activity and application to the screening of its inhibitors | |
| CN107238598B (en) | Based on chitosan-platinum simulation oxidizing ferment Assay of acid phosphatase content method | |
| Reljic et al. | New chromogen for assay of glucose in serum | |
| US7323317B2 (en) | Analytical method for detecting alkaline sphingomyelinase and kit for use in such method | |
| Fossati | Phosphate determination by enzymatic colorimetric assay | |
| Makise et al. | Kinetic rate assay of urinary N-acetyl-beta-D-glucosaminidase with 2-chloro-4-nitrophenyl-N-acetyl-beta-D-glucosaminide as substrate. | |
| Fdil et al. | Implementation of an affordable method for MPS diagnosis from urine screening to enzymatic confirmation: results of a pilot study in Morocco | |
| JP2009195239A6 (en) | Analytical method for detecting alkaline sphingomyelinase and kit for use in such method | |
| Rietz et al. | Fluorometric method for measuring serum lipase activity | |
| US5766872A (en) | Method for eliminating hemolysis interference in an amylase analysis | |
| CN102140495A (en) | Method for testing dimethyl arginine dimethylamine hydrolytic enzyme and diagnostic reagent thereof | |
| US4983512A (en) | Reagent for determination of acid phosphatase | |
| Kovar et al. | Determination of cholesterol in sera | |
| Kumar et al. | Determination of serum alkaline phosphatase by amperometric measurement of rate of oxygen depletion in a polyphenol oxidase coupled reaction | |
| CN107576799A (en) | A kind of razaxaban reagent box for detecting content and its method for detecting razaxaban | |
| CN103290098B (en) | A kind of method detecting lithium ion and test kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20180202 |
|
| WW01 | Invention patent application withdrawn after publication |