CN106565809B - A kind of enzyme donor conjugate of beta galactosidase and its purposes in glycocholic acid detection - Google Patents
A kind of enzyme donor conjugate of beta galactosidase and its purposes in glycocholic acid detection Download PDFInfo
- Publication number
- CN106565809B CN106565809B CN201610946486.7A CN201610946486A CN106565809B CN 106565809 B CN106565809 B CN 106565809B CN 201610946486 A CN201610946486 A CN 201610946486A CN 106565809 B CN106565809 B CN 106565809B
- Authority
- CN
- China
- Prior art keywords
- glycocholic acid
- synthesis
- reagent
- acid detection
- leu
- 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.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
- C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
- C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
- C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
- C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
-
- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- 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
Abstract
A kind of enzyme donor conjugate this application involves beta galactosidase and its purposes in glycocholic acid detection.Specifically, this application provides a kind of liver and gall acid detection kit, it includes the first reagent, the second reagent and optional glycocholic acid calibration object or quality-control product.First reagent includes the enzyme acceptor of beta galactosidase and the specific antibody of glycocholic acid, and the second reagent includes glycocholic acid derivative and enzyme donor conjugate.In this application, glycocholic acid derivative is directed in the particular amino acid residue for being coupled to enzyme donor.The competence exertion enzymatic activity when enzyme acceptor is combined with enzyme donor, and such a combination in by sample content of glycocholic acid controlled.Compared with radioimmunoassay method, detection reagent high sensitivity that the application provides, high specificity, testing result is accurate and is not involved in the problems, such as isotopic contamination.
Description
Technical field
This application involves clinical examination, field of biology.Content of glycocholic acid in serum is detected more particularly, to a kind of
Method, and further relate to a kind of enzyme donor conjugate of beta galactosidase.
Background technology
Serum CG (Cholyglycine, CG) is one of mating type cholic acid that cholic acid is combined into glycine.
In liver cell, cholesterol passes through and its enzymatic reaction of complexity, is transformed into primary bile acid, wherein having cholic acid (CA) and goose deoxidation
Cholic acid (CDCA).Cholic acid is combined with glycine forms glycocholic acid.
CG eubolisms approach circulates for intestines liver, and after CG is synthesized by liver cell, gall-bladder is discharged into through bile capillaries, bile duct, with
Enter duodenum with bile, help food digestion.95% cholic acid returns liver again in terminal ileum reabsorption, trans-portal vein, by
Liver cell intake recycles.Mainly exist in serum with protein-bound form, the total amount for overflowing into body circulation is less than 1%.
Under normal circumstances, cholic acid content is little in peripheral blood, normal adult either empty stomach or postprandial, its change of serum C G
Concentration stabilization is in low-level.When liver cell is damaged, liver cell intake CG abilities decline, and cause CG contents in blood to increase；Bile
During stasis, obstacle occurs for hepatic excretion cholic acid, and sanguimotor CG contents of backflowing increase, and also increase blood CG contents.Measure
Serum CG (SCG) is to evaluate one of hepatocyte function and its sensitive indicator of liver and gall system material circulatory function.
Cloned enzyme donor immune detection (Enzyme Donor Immunoassay, CEDIA) is a kind of homogeneous immune inspection
Survey technology, combines the specific binding characteristics of beta galactosidase α complementarity principles and antigen-antibody.By small molecule antigens (Ag)
Be coupled with enzyme donor (ED), form ED-Ag conjugates, this conjugate can with enzyme acceptor (EA) is spontaneous combines to form active β
Galactosidase；But if in the reaction system add for the small molecule antigens antibody (Ab), antigen-antibody it is special
Property combination can form ED-Ag-Ab compounds, at this time due to the influence of antibody steric hindrance, ED-Ag-Ab compounds cannot again with
EA combines to form active beta galactosidase.If contain small molecule antigens to be detected in serum, at this time reaction system
In the antigen containing two kinds of forms, a kind of is the free antigen in serum, it is a kind of be in kit with ED coupling antigen, i.e.,
ED-Ag conjugates, the antigen of both forms all have the ability combined with antibody specificity.Obviously, both forms is anti-
Original understands the emulative antibody binding with reaction system, and free antigen is more in serum, and the antibody that it is combined is more, so that
Cause to have more residue ED-Ag conjugates (not with the part of antibody binding) can with EA is spontaneous combines to form active β
Galactosidase, and the height of enzymatic activity is directly proportional to the content of free antigen in serum.
The conventional method of clinical assays Serum CG includes chemiluminescence (CLIA) and enzyme linked immunological (ELISA).The former
High sensitivity, but need expensive chemiluminescence instrument；The latter is cumbersome, and detection time length, the degree of automation is low, repeats
Property is poor.
Recently a kind of glycocholic acid detection method (CN103760348A) based on EMIT occurred, is a kind of homogeneous immune inspection
Survey method, can detect in the high throughput of the clear content of glycocholic acid of the enterprising promoting circulation of blood of full automatic biochemical apparatus, but specificity and reagent heat
Stability has much room for improvement.
The content of the invention
In order to provide more valuable diagnostic message to clinical, according to some embodiments of the disclosure, there is provided a kind of
Glycocholic acid derivative, it is as shown in logical formula (I)：
Wherein n is the arbitrary integer between 0 to 20, and such as, but not limited to n is 1,2,3,4,5,6,7,8,9 or 10, more excellent
It is 2,3,4,5 or 6 to select n；
X is the group that can be reacted with sulfydryl, such as, but not limited to maleimide, acetyl bromide, vinyl sulfone, nitrogen
Third pyridine.In a specific embodiment, X is maleimide.
In some specific embodiments, there is provided glycocholic acid derivative, it is such as logical formula (II) to any one of (V) institute
Show：
Can be with enzyme donor (hereinafter referred to as ED) molecule of beta galactosidase according to 1 according to the glycocholic acid derivative of the application：
The orientation coupling of 1 molar ratio.
In this application, the enzyme donor of any of or following beta galactosidase is suitable for implementing the application's
Technical solution, as long as can have beta galactosidase activity after being combined with enzyme acceptor, (enzymatic activity refers to：It can urge
Change substrate colour developing).For example, the enzyme of the enzyme donor of wild-type beta galactosidase or engineered beta galactosidase supplies
Body (such as the enzyme donor announced in CN105524898A).
According to some embodiments, there is provided a kind of conjugate, its by the application glycocholic acid derivative and ED molecule coupling labeleds
Form.In the context of this application, " ED- glycocholic acid conjugate " can also be known as.This conjugate can be by the spy of glycocholic acid
Heterogenetic antibody (monoclonal antibody is how anti-) identification, can also form active holoenzyme with EA complementary elements.
The substrate of beta galactosidase includes but not limited to：P- aminobenzene-β-D- synthesis, 2 '-N- (16
Alcohol)-N- (amino -2 '-nitrobenzene)-β-D- synthesis, 4-methyl umbelliferone acyl-β-D- synthesis, naphthyl -
AS-B1- β-D- synthesis, 1- naphthyl-β-D- synthesis, 2- naphthyl-β-D- synthesis, o- nitros
Benzene-β-D- synthesis, m- nitrobenzene-β-D- synthesis, p- nitrobenzene-β-D- synthesis, phenyl-
The chloro- 3- indoles-β-D- synthesis of β-D- synthesis, the bromo- 4- of 5-, (the different phenoxazine ketone of 9- hydroxyls -3-)-β-D-
Synthesis, 7- hydroxyls -4- trifluoromethyls-cumarin, ω-nitrostyrolene base-β-D- synthesis, fluorescence
Element-D- synthesis.In a specific embodiment, substrate is o- nitrobenzene-β-D- synthesis (abbreviation ONPG).
According to some embodiments, there is provided a kind of glycocholic acid antibody, it can specifically be identified and cholylglycine molecule.
The glycocholic acid antibody is monoclonal antibody or resists more.The analogue cross reaction of the glycocholic acid antibody and glycocholic acid molecule is very
It is small even without the analogue of wherein glycocholic acid molecule includes but not limited to：Cholic acid, deoxycholic acid, chenodesoxycholic acid, ox
Jaundice acid, glycodesoxycholic acid, glycochenodeoxycholate, sweet ammonia cholyltaurine.
Compound is formed after the specific antibody of glycocholic acid is combined with ED- glycocholic acid conjugates, then EA molecules no longer can
Combined with above-mentioned compound, so that the activity of holoenzyme cannot be produced.
According to other embodiments, there is provided a kind of glycocholic acid detection kit, it includes the first reagent, the second reagent,
And optionally calibration object and/or quality-control product.
In some embodiments, the sweet courages of ED- according to the application are included according to the glycocholic acid detection kit of the application
Sour conjugate.
In some embodiments, the first reagent includes the enzyme acceptor and glycocholic acid antibody of beta galactosidase.
In some embodiments, the second reagent includes the ED- glycocholic acid conjugates according to the application.
In the context of this application, the enzyme acceptor of any of or following beta galactosidase can be used for reality
The technical solution of the application is applied, as long as it produces beta galactosidase activity i.e. after being combined with the enzyme donor of beta galactosidase
Can.In a specific embodiment, the enzyme acceptor of beta galactosidase is public in Chinese patent application 201610841474.8
The enzyme acceptor opened, the amino acid sequence of the enzyme acceptor is shown in SEQ ID No.2；Or the enzyme acceptor is by SEQ ID
Coded by the polynucleotides of No.1.Compared with wild type, the enzyme acceptor shown in SEQ ID No.2 lacks the 13rd to 33 amino acids
Residue.Inventor it was unexpectedly found that, by this engineered beta galactosidase acceptor have it is low-down detection this
Bottom, while be conducive to expression and the raising of complementary activity.The full content of Chinese patent application 201610841474.8 is by drawing
With being incorporated herein by.
In one embodiment, there is provided a kind of detection kit for detecting content of glycocholic acid in sample, it includes the
One reagent and the second reagent, wherein
First reagent, comprising：
Second reagent, comprising：
In some embodiments, the concentration of buffer solution is 50mM to 100mM.
In some embodiments, stabilizer concentration is 0.1g/L to 0.5g/L.
In some embodiments, surfactant concentration is 0.1g/L to 0.5g/L.
In some embodiments, concentration of preservatives is 0.1g/L to 0.5g/L.
In some embodiments, MgCl2Concentration is 2mM to 5mM.
In some embodiments, the enzyme acceptor concentration of beta galactosidase is 0.01mg/ml to 0.05mg/ml.
In some embodiments, glycocholic acid antibody concentration is 10mg/L to 50mg/L.
In some embodiments, the concentration of the application conjugate is 5mg/L to 50mg/L.
In some embodiments, the concentration of substrate is 0.5g/L to 3.0g/L.
In some embodiments, buffer solution is selected from：Phosphate buffer, glycine buffer, Tris buffer solutions, boric acid delay
Fliud flushing and MOPS buffer solutions；In a specific embodiment, buffer solution is phosphate buffer；
In some embodiments, the pH of buffer solution be 6.0 to 8.0, preferably 7.0 to 7.5, including 7.1,7.2,7.3,
7.4 or 7.5.
In some embodiments, stabilizer is selected from：Bovine serum albumin(BSA), trehalose, sucrose, mannitol, glycerine, sweet ammonia
Acid, Macrogol 6000 and combinations thereof.In a specific embodiment, stabilizer is bovine serum albumin(BSA).
In some embodiments, surfactant is selected from：Triton X-100、TritonX-405、Tween 80、
Tween 20, Brij 35, Brij 23 and combinations thereof.In a specific embodiment, surfactant is Tween 20.
In some embodiments, preservative is selected from：Azido compound, MIT and biological preservative PC.Specific
In embodiment, azido compound is Sodium azide or nitrine lithium；In specific embodiments, the biological preservative PC is
PC-300。
In some embodiments, substrate is selected from：P- aminobenzene-β-D- synthesis, 2 '-N- (hexadecanol)-N-
(amino -2 '-nitrobenzene)-β-D- synthesis, 4-methyl umbelliferone acyl-β-D- synthesis, naphthyl-AS-B1-
β-D- synthesis, 1- naphthyl-β-D- synthesis, 2- naphthyl-β-D- synthesis, o- nitrobenzenes-β-
D- synthesis, m- nitrobenzene-β-D- synthesis, p- nitrobenzene-β-D- synthesis, phenyl-β-D-
The chloro- 3- indoles-β-D- synthesis of the bromo- 4- of synthesis, 5-, (the different phenoxazine ketone of 9- hydroxyls -3-)-β-D- galas
Pyranoside, 7- hydroxyls -4- trifluoromethyls-cumarin, ω-nitrostyrolene base-β-D- synthesis, fluorescein-D-
Synthesis and combinations thereof, preferably o- nitrobenzene-β-D- synthesis.
In specific embodiments, there is provided a kind of detection kit for detecting content of glycocholic acid in sample, it includes
First reagent and the second reagent, wherein
First reagent, comprising：
Second reagent, comprising：
Optionally, calibration object
PB buffer solutions 10mM
Glycocholic acid 0 is to 40mg/L；
Optionally, quality-control product
PB buffer solutions 10mM
Glycocholic acid 4.0 is to 15.0mg/L.
According to some embodiments, there is provided the ED- glycocholic acid conjugate of the application is in glycocholic acid detection device is prepared
Purposes.In specific embodiments, the detection device is kit, especially CEDIA kits.
According to some embodiments, there is provided the β galas shown in the ED- glycocholic acid conjugate and SEQ ID No.2 of the application
Purposes of the combination of both enzyme acceptors of glycosidase in glycocholic acid detection device is prepared.In specific embodiments, it is described
Detection device is kit, especially CEDIA kits.
Brief description of the drawings
Fig. 1：The synthetic route of glycocholic acid derivative.
Fig. 2：Calibration curve.
Fig. 3：Correlation in kit (CEDIA methods) and HPLC methods the detection serum of the application between content of glycocholic acid
Property.
Fig. 4：37 DEG C of thermal accelerations are destroyed, the calibration absorbance reactive residual of different number of days.■ is compareed；◆ the reagent of the application
Box.
Embodiment
Embodiment
Embodiment 1：The synthesis of glycocholic acid derivative
Specific synthesis step is following (Fig. 1)：
Into the 25mL two-mouth bottles of dried and clean add glycocholic acid (1.0eq), dimaleoyl imino ethamine (1.0g,
1.0eq), triethylamine (3.0eq), adds dimethylformamide (5mL) and stirs to complete molten, addition dichloroethanes (1.25eq).
2h is stirred at 25 DEG C.HPLC is monitored, until reaction finishes.Above-mentioned reaction mixture is added in water (25mL), is added
Enter ethyl acetate 20mL × 3 to be extracted.Merge organic phase, anhydrous Na2SO4It is dry, it is concentrated under reduced pressure, gained grease column layer
Analysis method purifies to obtain 1.04g cream powder solids, yield 45%.M+：589.4.
Embodiment 2：The coupling of glycocholic acid derivative and ED molecules
According to the ED- glycocholic acid conjugates of the application, there can be a variety of coupling modes：On glycocholic acid derivative molecular
Amino on carboxyl and ED molecules, sulfydryl reactive group (such as maleimide base group) and ED on glycocholic acid derivative molecular
Sulfydryl on molecule.
1. solution is prepared：
Glycocholic acid derivative solution：Glycocholic acid derivative 10mg/ml prepared by embodiment 1 is dissolved in DMF；
ED solution (see US7135325B2)：1.5mg/mL ED, PB 100mmol, NaCl 100mmol, pH=7.0；
Mops solution：Mops 100mmol, NaCl 1%, pH=7.3.
Desalting soln：PB 100mM, 0.1%NaN3, 1%NaCl, pH=7.0.
2. COUPLING PROCEDURE：6.0ml ED solution, 22.5ml Mops solution and 4.5ml glycocholic acid derivative solutions, in room temperature
(20 to 25 DEG C) reaction 4h.
3. after above-mentioned reaction system shaken at room temperature is reacted 4h, eluted, received using desalting column with above-mentioned desalting soln
Collect protein peak, products therefrom, that is, ED- glycocholic acid conjugates.
Embodiment 3. prepares the enzyme acceptor of beta galactosidase
Enzyme acceptor is prepared according to the method disclosed in Chinese patent application 201610841474.8.
Embodiment 4：The preparation of the glycocholic acid reagent of cloned enzyme donor immunologic detection method
1. the preparation of the first reagent R1：
A) Na of 28.65g is accurately weighed into beaker A2HPO4.12H2O, the NaH of 3.12g2PO4.2H2O, 1.02g
MgCl2.6H2O, 1.0g/L Tween 20,1.0g BSA, 1.0g NaN3, 900ml deionized water, 30min is stirred at room temperature,
Solution 1 is made；
B) the glycocholic acid antibody that 25ml concentration is 2mg/ml is added in above-mentioned beaker A, and (laboratory is made by oneself, can also be used
Any commercially available antibody), the volume ratio of antibody and solution 1 is 1:10 to 1:1000, preferable 1:40,5min is stirred at room temperature；
C) enzyme acceptor (any enzyme acceptor, such as wild type that 2ml concentration is 10mg/ml are added in above-mentioned beaker A
EA.Preferably, when using embodiment 3 prepare EA when, complementary activity can be further improved and reduce Background), enzyme by
The volume ratio of body and solution 1 is 1:10 to 1:2000, preferable 1:500,5min is stirred at room temperature；
D) pH value of solution is adjusted to 1000ml is settled to after 7.3 in above-mentioned beaker A, is the first reagent.
2. the preparation of the second reagent R2：
A) 28.65 Na is accurately weighed into beaker B2HPO4.12H2O, the NaH of 3.12g2PO4.12H2O, the ONPG of 3g,
Tween 20, the NaN of 1.0g of 1.0g/L3, 1.0g BSA, 950ml deionized water, 30min is stirred at room temperature, solution is made
2；
B) enzyme donor-glycocholic acid conjugate that 20ml concentration is 0.5mg/ml, enzyme donor-sweet are added in above-mentioned beaker B
The volume ratio of cholic acid conjugate and solution 2 is 1:10 to 1:1000, specific ratio is 1 in the present embodiment:50, it is stirred at room temperature
5min；
C) solution is settled to 1000ml after adjusting pH to 7.3 in above-mentioned beaker B, is the second reagent.
By the first reagent and the second reagent, and optional calibration object and/or quality-control product, kit is assembled into.
Test case
The Activity determination of 1. enzyme acceptor of test case
The enzyme acceptor of the E. coli-galactosidase of the gained of embodiment 3 is subjected to Activity determination.
E. coli-galactosidase EA can be complementary with ED, forms beta galactosidase.Beta galactosidase can incite somebody to action
ONPG is hydrolyzed, and generates galactolipin and the o-nitrophenol (ONP) of yellow.The amount of measure ONP can determine the enzyme of galactosidase
It is living, so that it is determined that the complementary activity of EA.
(1) EA PB buffer solutions (100mM PBS, 150mM NaCl, 0.1%NaN3, pH 7.3) be diluted to 0.2mg/ml,
ED is diluted to 1 μ g/ml with PB buffer solutions.Complementary activity is measured with Siemens's Biochemical Analyzer 1800：
100 μ l EA solution are as R1；
100 μ l ONPG substrate solutions (5mg/ml ONPG, 100mM PBS, 150mM NaCl, 10mM MgCl2, 0.1%
NaN3, pH7.3) and it is used as R2；
10 μ l enzyme donors solution are as sample S, for measuring the complementary activity of EA.
10 μ l PB buffer solutions are as blank control sample, for measuring the background activity of EA；
EA-ED programs are run, determine reaction rate,
(2) using M15 as control, the parallel progress aforesaid operations (paper that Langley KE and Zabin I are delivered
Biochemistry.1976, Vol 2；15:A kind of beta galactosidase EA mentioned in 4866-75, is compared with wild type EA, is lacked
The amino acid residue of the 11st to 41 is lost, is referred to as M15).
(3) result：
The EA complementary activities of the application are than M15 high 11.2%, and background activity low 21%.
1. complementary activity of table
| EA | OD571 | % |
| M15 | 0.21874 | 100 |
| The EA of the application | 0.24332 | 111.2 |
2. background activity of table
| EA | OD571 | % |
| M15 | 0.00167 | 100 |
| The EA of the application | 0.00132 | 79 |
Test case 2：Cross reaction of the specific glycocholic acid antibody to various cholic acid
1. chaff interferent includes：
Cholic acid ＜ 5.0%, deoxycholic acid ＜ 1.0%, chenodesoxycholic acid ＜ 1.0%, taurocholate ＜ 2.0%, sweet ammonia take off
Oxycholic acid ＜ 3.0%, glycochenodeoxycholate ＜ 1.0%, sweet ammonia taurocholate ＜ 2.0%.
2. contrast agents：
The testing principle of contrast agents：Dissociate glycocholic acid and G6PD-glycocholic acid conjugate in sample
The anti-glycocholic acid specific antibody of competitive binding.The glycocholic acid to dissociate in sample is more, and the antibody of competition binding is more, and not
It is more with enzyme-glycocholic acid conjugate of antibody binding.Free enzyme-glycocholic acid conjugate catalysis two core of β-nicotinamide adenine
Thuja acid oxidized form (NAD+) be converted into β-nicotinamide adenine dinucleotide reduced form (NADH), the glycocholic acid concentration in sample with
The growing amount of NADH is directly proportional, and the content of glycocholic acid in sample is can obtain by the change of 340nm absorbances.
Contrast agents concentration of component：
First reagent R1,50mM buffer solution, 0.1mM glucose hexaphosphates, 0.1mM β-nicotinamide adenine dinucleotide oxygen
Change type ,≤1.0% anti-glycocholic acid antibody, 0.05% preservative；
Second reagent R2,100mM buffer solution ,≤1.0% G6PD-glycocholic acid conjugate, 0.05%
Preservative.
3. the detection method of cross reaction：
Using buffer solution glycocholic acid sterling, various concentrations glycocholic acid calibration object is prepared.Prepared using same buffer
Concentration is 10mg/ml cholic acid, deoxycholic acid, chenodesoxycholic acid, taurocholate, glycodesoxycholic acid, sweet ammonia volume deoxycholic acid, sweet
Ammonia taurocholate sample.
The reagent of the application is calibrated using glycocholic acid calibration object, measures above-mentioned sample 3 times, the ratio of measured value and theoretical value
As cross reacting rate.The results are shown in Table 3.
The various cholic acid of the specific glycocholic acid antibody determination of table 3.
| Chemical name | The application reagent | Contrast agents |
| Glycocholic acid | 100% | 100% |
| Cholic acid | 4.6% | 18.2% |
| Deoxycholic acid | 0.2% | 0.1% |
| Chenodesoxycholic acid | 0.1% | 0.1% |
| Taurocholate | 1.4% | 3.5% |
| Glycodesoxycholic acid | 2.3% | 5.1% |
| Glycochenodeoxycholate | 0.1% | 0.2% |
| Sweet ammonia taurocholate | 1.7% | 3.6% |
Test case 3：The clinical detection of the homogeneous immune detection of glycocholic acid (clone enzyme recipient immune detection method)
1. reaction principle：Glycocholic acid in sample to be checked and the sweet courage of ED- glycocholic acid conjugate competition bindings in reagent R1
Sour antibody；The ED- glycocholic acid conjugate with glycocholic acid antibody binding can not form holoenzyme with the EA complementary elements in R2 reagents；
And the ED- glycocholic acid conjugates being selectively bound by the antibody cannot be with EA complementation stroke holoenzymes due to steric hindrance；The β of formation
Galactosidase holoenzyme has enzymatic activity, can be catalyzed substrate colour developing.
2. sample type：Sample to be checked is various physiology samples, and such as serum, blood plasma, urine, sample preferably to be checked is blood
Clearly.
3. testing result：
4. set factors of table
5. accuracy of table, precision, the rate of recovery (Fig. 2)
| Blood serum sample | 1 | 2 | 3 |
| Sample concentration (mg/L) | 1.50 | 5.00 | 20.00 |
| 1 | 1.47 | 5.05 | 21.05 |
| 2 | 1.52 | 5.12 | 20.55 |
| 3 | 1.63 | 5.07 | 20.73 |
| 4 | 1.52 | 4.88 | 19.95 |
| 5 | 1.49 | 4.95 | 19.64 |
| 6 | 1.45 | 4.94 | 20.40 |
| 7 | 1.62 | 5.06 | 21.21 |
| 8 | 1.50 | 5.02 | 20.72 |
| 9 | 1.55 | 4.93 | 19.77 |
| 10 | 1.44 | 4.89 | 20.39 |
| Accuracy (preceding to measure three times) | 2.67% | 1.60% | 3.88% |
| Average | 1.52 | 4.99 | 20.44 |
| SD | 0.065 | 0.083 | 0.524 |
| CV | 4.3% | 1.7% | 2.6% |
| The rate of recovery | 101.3% | 99.8% | 102.2% |
Table 6. is linear
Test case 4：Correlation analysis
1. test method：
80, fresh serum sample is taken, every sample is divided into two parts, and every part of volume is no less than 500 μ l, uses the application
Reagent measure a copy of it sample twice on 7180 instrument of Hitachi, measure another sample using Shimadzu HPLC.Two kinds of sides
Method measured value carries out correlation analysis using scatter diagram.
2. result of the test：
Gained function is y=1.0057x-0.0247, coefficient R2=0.9962.
The result shows that glycocholic acid concentration value in the reagent measure sample of the application, measures with HPLC methods (can be considered goldstandard)
Glycocholic acid concentration value has good correlation in sample.
Correlation analysis of the reagent (CEDIA methods) of 7. the application of table between HPLC methods
(unit：Mg/L) (Fig. 3)
Test case 5：Thermal stability analysis
Heat stability testing the results show is in table 8 and table 9.
8. 37 DEG C of thermal accelerations of table destroy different number of days calibration absorbance activity surplus
| 37 DEG C of thermal accelerations destroy number of days | The application reagent | Contrast agents |
| 0 day | 100% | 100% |
| 1 day | 91% | 25% |
| 4 days | 70% | 15% |
| 7 days | 55% | 10% |
Linear determination (Fig. 4) after 9. 37 DEG C of thermal accelerations of table are destroyed 7 days
Cloned enzyme donor immunologic detection method is used for the detection of glycocholic acid by the application first.The application, which uses, passes through gene
Engineered enzyme acceptor, background activity is extremely low, the sensitivity of the detection effectively improved.The application is a kind of homogeneous immune detection
Method, easy to operate suitable for a variety of automatic clinical chemistry analyzers, accuracy is high, high specificity.
Sequence table
<110>Beijing Strong Biotechnologies, Inc.
<120>A kind of enzyme donor conjugate of beta galactosidase and its purposes in glycocholic acid detection
<130> 360243CG-360165
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 3006
<212> DNA
<213>Artificial sequence
<220>
<223>Beta galactosidase receptor coding sequence
<400> 1
accatgatta cggattcact ggccgtcgtt ttacaagcca gctggcgtaa tagcgaagag 60
gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgctttgcc 120
tggtttccgg caccagaagc ggtgccggaa agctggctgg agtgcgatct tcctgaggcc 180
gatactgtcg tcgtcccctc aaactggcag atgcacggtt acgatgcgcc catctacacc 240
aacgtgacct atcccattac ggtcaatccg ccgtttgttc ccacggagaa tccgacgggt 300
tgttactcgc tcacatttaa tgttgatgaa agctggctac aggaaggcca gacgcgaatt 360
atttttgatg gcgttaactc ggcgtttcat ctgtggtgca acgggcgctg ggtcggttac 420
ggccaggaca gtcgtttgcc gtctgaattt gacctgagcg catttttacg cgccggagaa 480
aaccgcctcg cggtgatggt gctgcgctgg agtgacggca gttatctgga agatcaggat 540
atgtggcgga tgagcggcat tttccgtgac gtctcgttgc tgcataaacc gactacacaa 600
atcagcgatt tccatgttgc cactcgcttt aatgatgatt tcagccgcgc tgtactggag 660
gctgaagttc agatgtgcgg cgagttgcgt gactacctac gggtaacagt ttctttatgg 720
cagggtgaaa cgcaggtcgc cagcggcacc gcgcctttcg gcggtgaaat tatcgatgag 780
cgtggtggtt atgccgatcg cgtcacacta cgtctgaacg tcgaaaaccc gaaactgtgg 840
agcgccgaaa tcccgaatct ctatcgtgcg gtggttgaac tgcacaccgc cgacggcacg 900
ctgattgaag cagaagcctg cgatgtcggt ttccgcgagg tgcggattga aaatggtctg 960
ctgctgctga acggcaagcc gttgctgatt cgaggcgtta accgtcacga gcatcatcct 1020
ctgcatggtc aggtcatgga tgagcagacg atggtgcagg atatcctgct gatgaagcag 1080
aacaacttta acgccgtgcg ctgttcgcat tatccgaacc atccgctgtg gtacacgctg 1140
tgcgaccgct acggcctgta tgtggtggat gaagccaata ttgaaaccca cggcatggtg 1200
ccaatgaatc gtctgaccga tgatccgcgc tggctaccgg cgatgagcga acgcgtaacg 1260
cgaatggtgc agcgcgatcg taatcacccg agtgtgatca tctggtcgct ggggaatgaa 1320
tcaggccacg gcgctaatca cgacgcgctg tatcgctgga tcaaatctgt cgatccttcc 1380
cgcccggtgc agtatgaagg cggcggagcc gacaccacgg ccaccgatat tatttgcccg 1440
atgtacgcgc gcgtggatga agaccagccc ttcccggctg tgccgaaatg gtccatcaaa 1500
aaatggcttt cgctacctgg agagacgcgc ccgctgatcc tttgcgaata cgcccacgcg 1560
atgggtaaca gtcttggcgg tttcgctaaa tactggcagg cgtttcgtca gtatccccgt 1620
ttacagggcg gcttcgtctg ggactgggtg gatcagtcgc tgattaaata tgatgaaaac 1680
ggcaacccgt ggtcggctta cggcggtgat tttggcgata cgccgaacga tcgccagttc 1740
tgtatgaacg gtctggtctt tgccgaccgc acgccgcatc cagcgctgac ggaagcaaaa 1800
caccagcagc agtttttcca gttccgttta tccgggcaaa ccatcgaagt gaccagcgaa 1860
tacctgttcc gtcatagcga taacgagctc ctgcactgga tggtggcgct ggatggtaag 1920
ccgctggcaa gcggtgaagt gcctctggat gtcgctccac aaggtaaaca gttgattgaa 1980
ctgcctgaac taccgcagcc ggagagcgcc gggcaactct ggctcacagt acgcgtagtg 2040
caaccgaacg cgaccgcatg gtcagaagcc ggacacatca gcgcctggca gcagtggcgt 2100
ctggctgaaa acctcagcgt gacactcccc gccgcgtccc acgccatccc gcatctgacc 2160
accagcgaaa tggatttttg catcgagctg ggtaataagc gttggcaatt taaccgccag 2220
tcaggctttc tttcacagat gtggattggc gataaaaaac aactgctgac gccgctgcgc 2280
gatcagttca cccgtgcacc gctggataac gacattggcg taagtgaagc gacccgcatt 2340
gaccctaacg cctgggtcga acgctggaag gcggcgggcc attaccaggc cgaagcagcg 2400
ttgttgcagt gcacggcaga tacacttgct gatgcggtgc tgattacgac cgctcacgcg 2460
tggcagcatc aggggaaaac cttatttatc agccggaaaa cctaccggat tgatggtagt 2520
ggtcaaatgg cgattaccgt tgatgttgaa gtggcgagcg atacaccgca tccggcgcgg 2580
attggcctga actgccagct ggcgcaggta gcagagcggg taaactggct cggattaggg 2640
ccgcaagaaa actatcccga ccgccttact gccgcctgtt ttgaccgctg ggatctgcca 2700
ttgtcagaca tgtatacccc gtacgtcttc ccgagcgaaa acggtctgcg ctgcgggacg 2760
cgcgaattga attatggccc acaccagtgg cgcggcgact tccagttcaa catcagccgc 2820
tacagtcaac agcaactgat ggaaaccagc catcgccatc tgctgcacgc ggaagaaggc 2880
acatggctga atatcgacgg tttccatatg gggattggtg gcgacgactc ctggagcccg 2940
tcagtatcgg cggaattcca gctgagcgcc ggtcgctacc attaccagtt ggtctggtgt 3000
caaaaa 3006
<210> 2
<211> 1002
<212> PRT
<213>Artificial sequence
<220>
<223>Beta galactosidase acceptor
<400> 2
Thr Met Ile Thr Asp Ser Leu Ala Val Val Leu Gln Ala Ser Trp Arg
1 5 10 15
Asn Ser Glu Glu Ala Arg Thr Asp Arg Pro Ser Gln Gln Leu Arg Ser
20 25 30
Leu Asn Gly Glu Trp Arg Phe Ala Trp Phe Pro Ala Pro Glu Ala Val
35 40 45
Pro Glu Ser Trp Leu Glu Cys Asp Leu Pro Glu Ala Asp Thr Val Val
50 55 60
Val Pro Ser Asn Trp Gln Met His Gly Tyr Asp Ala Pro Ile Tyr Thr
65 70 75 80
Asn Val Thr Tyr Pro Ile Thr Val Asn Pro Pro Phe Val Pro Thr Glu
85 90 95
Asn Pro Thr Gly Cys Tyr Ser Leu Thr Phe Asn Val Asp Glu Ser Trp
100 105 110
Leu Gln Glu Gly Gln Thr Arg Ile Ile Phe Asp Gly Val Asn Ser Ala
115 120 125
Phe His Leu Trp Cys Asn Gly Arg Trp Val Gly Tyr Gly Gln Asp Ser
130 135 140
Arg Leu Pro Ser Glu Phe Asp Leu Ser Ala Phe Leu Arg Ala Gly Glu
145 150 155 160
Asn Arg Leu Ala Val Met Val Leu Arg Trp Ser Asp Gly Ser Tyr Leu
165 170 175
Glu Asp Gln Asp Met Trp Arg Met Ser Gly Ile Phe Arg Asp Val Ser
180 185 190
Leu Leu His Lys Pro Thr Thr Gln Ile Ser Asp Phe His Val Ala Thr
195 200 205
Arg Phe Asn Asp Asp Phe Ser Arg Ala Val Leu Glu Ala Glu Val Gln
210 215 220
Met Cys Gly Glu Leu Arg Asp Tyr Leu Arg Val Thr Val Ser Leu Trp
225 230 235 240
Gln Gly Glu Thr Gln Val Ala Ser Gly Thr Ala Pro Phe Gly Gly Glu
245 250 255
Ile Ile Asp Glu Arg Gly Gly Tyr Ala Asp Arg Val Thr Leu Arg Leu
260 265 270
Asn Val Glu Asn Pro Lys Leu Trp Ser Ala Glu Ile Pro Asn Leu Tyr
275 280 285
Arg Ala Val Val Glu Leu His Thr Ala Asp Gly Thr Leu Ile Glu Ala
290 295 300
Glu Ala Cys Asp Val Gly Phe Arg Glu Val Arg Ile Glu Asn Gly Leu
305 310 315 320
Leu Leu Leu Asn Gly Lys Pro Leu Leu Ile Arg Gly Val Asn Arg His
325 330 335
Glu His His Pro Leu His Gly Gln Val Met Asp Glu Gln Thr Met Val
340 345 350
Gln Asp Ile Leu Leu Met Lys Gln Asn Asn Phe Asn Ala Val Arg Cys
355 360 365
Ser His Tyr Pro Asn His Pro Leu Trp Tyr Thr Leu Cys Asp Arg Tyr
370 375 380
Gly Leu Tyr Val Val Asp Glu Ala Asn Ile Glu Thr His Gly Met Val
385 390 395 400
Pro Met Asn Arg Leu Thr Asp Asp Pro Arg Trp Leu Pro Ala Met Ser
405 410 415
Glu Arg Val Thr Arg Met Val Gln Arg Asp Arg Asn His Pro Ser Val
420 425 430
Ile Ile Trp Ser Leu Gly Asn Glu Ser Gly His Gly Ala Asn His Asp
435 440 445
Ala Leu Tyr Arg Trp Ile Lys Ser Val Asp Pro Ser Arg Pro Val Gln
450 455 460
Tyr Glu Gly Gly Gly Ala Asp Thr Thr Ala Thr Asp Ile Ile Cys Pro
465 470 475 480
Met Tyr Ala Arg Val Asp Glu Asp Gln Pro Phe Pro Ala Val Pro Lys
485 490 495
Trp Ser Ile Lys Lys Trp Leu Ser Leu Pro Gly Glu Thr Arg Pro Leu
500 505 510
Ile Leu Cys Glu Tyr Ala His Ala Met Gly Asn Ser Leu Gly Gly Phe
515 520 525
Ala Lys Tyr Trp Gln Ala Phe Arg Gln Tyr Pro Arg Leu Gln Gly Gly
530 535 540
Phe Val Trp Asp Trp Val Asp Gln Ser Leu Ile Lys Tyr Asp Glu Asn
545 550 555 560
Gly Asn Pro Trp Ser Ala Tyr Gly Gly Asp Phe Gly Asp Thr Pro Asn
565 570 575
Asp Arg Gln Phe Cys Met Asn Gly Leu Val Phe Ala Asp Arg Thr Pro
580 585 590
His Pro Ala Leu Thr Glu Ala Lys His Gln Gln Gln Phe Phe Gln Phe
595 600 605
Arg Leu Ser Gly Gln Thr Ile Glu Val Thr Ser Glu Tyr Leu Phe Arg
610 615 620
His Ser Asp Asn Glu Leu Leu His Trp Met Val Ala Leu Asp Gly Lys
625 630 635 640
Pro Leu Ala Ser Gly Glu Val Pro Leu Asp Val Ala Pro Gln Gly Lys
645 650 655
Gln Leu Ile Glu Leu Pro Glu Leu Pro Gln Pro Glu Ser Ala Gly Gln
660 665 670
Leu Trp Leu Thr Val Arg Val Val Gln Pro Asn Ala Thr Ala Trp Ser
675 680 685
Glu Ala Gly His Ile Ser Ala Trp Gln Gln Trp Arg Leu Ala Glu Asn
690 695 700
Leu Ser Val Thr Leu Pro Ala Ala Ser His Ala Ile Pro His Leu Thr
705 710 715 720
Thr Ser Glu Met Asp Phe Cys Ile Glu Leu Gly Asn Lys Arg Trp Gln
725 730 735
Phe Asn Arg Gln Ser Gly Phe Leu Ser Gln Met Trp Ile Gly Asp Lys
740 745 750
Lys Gln Leu Leu Thr Pro Leu Arg Asp Gln Phe Thr Arg Ala Pro Leu
755 760 765
Asp Asn Asp Ile Gly Val Ser Glu Ala Thr Arg Ile Asp Pro Asn Ala
770 775 780
Trp Val Glu Arg Trp Lys Ala Ala Gly His Tyr Gln Ala Glu Ala Ala
785 790 795 800
Leu Leu Gln Cys Thr Ala Asp Thr Leu Ala Asp Ala Val Leu Ile Thr
805 810 815
Thr Ala His Ala Trp Gln His Gln Gly Lys Thr Leu Phe Ile Ser Arg
820 825 830
Lys Thr Tyr Arg Ile Asp Gly Ser Gly Gln Met Ala Ile Thr Val Asp
835 840 845
Val Glu Val Ala Ser Asp Thr Pro His Pro Ala Arg Ile Gly Leu Asn
850 855 860
Cys Gln Leu Ala Gln Val Ala Glu Arg Val Asn Trp Leu Gly Leu Gly
865 870 875 880
Pro Gln Glu Asn Tyr Pro Asp Arg Leu Thr Ala Ala Cys Phe Asp Arg
885 890 895
Trp Asp Leu Pro Leu Ser Asp Met Tyr Thr Pro Tyr Val Phe Pro Ser
900 905 910
Glu Asn Gly Leu Arg Cys Gly Thr Arg Glu Leu Asn Tyr Gly Pro His
915 920 925
Gln Trp Arg Gly Asp Phe Gln Phe Asn Ile Ser Arg Tyr Ser Gln Gln
930 935 940
Gln Leu Met Glu Thr Ser His Arg His Leu Leu His Ala Glu Glu Gly
945 950 955 960
Thr Trp Leu Asn Ile Asp Gly Phe His Met Gly Ile Gly Gly Asp Asp
965 970 975
Ser Trp Ser Pro Ser Val Ser Ala Glu Phe Gln Leu Ser Ala Gly Arg
980 985 990
Tyr His Tyr Gln Leu Val Trp Cys Gln Lys
995 1000
Claims (20)
- A kind of 1. compound shown in logical formula (I)：Wherein, n is the arbitrary integer between 0 to 20；X represents maleimide.
- 2. compound according to claim 1, wherein n are 1,2,3,4,5,6,7,8,9 or 10.
- 3. compound according to claim 2, wherein n are 2,3,4,5 or 6.
- 4. a kind of conjugate, it is formed as the enzyme donor covalent coupling of the compound described in claim 1 and beta galactosidase.
- 5. conjugate according to claim 4, the enzyme donor of compound and beta galactosidase described in claim 1 Molar ratio is 1:1.
- 6. a kind of glycocholic acid detection kit, it includes the first reagent and the second reagent,Wherein, first reagent includes enzyme acceptor, the glycocholic acid antibody of beta galactosidase；Second reagent includes the conjugate described in claim 4.
- 7. glycocholic acid detection kit according to claim 6, it also includes calibration object and/or quality-control product.
- 8. glycocholic acid detection kit according to claim 6, it includes：First reagent, it includes：10mM to 500mM buffer solutions,The enzyme acceptor of beta galactosidase,Glycocholic acid antibody,2mM to 10mM MgCl2、0.1g/L to 5g/L stabilizers,0.1g/L to 5g/L surfactants,0.1g/L is to 5g/L preservatives；Second reagent, it includes：10mM to 500mM buffer solutions,Conjugate described in claim 4,0.5g/L to 10g/L substrates,0.1g/L to 5g/L stabilizers,0.1g/L to 5g/L surfactants,0.1g/L is to 5g/L preservatives；The stabilizer is selected from：Bovine serum albumin(BSA), trehalose, sucrose, mannitol, glycerine, glycine, Macrogol 6000 and It is combined；The surfactant is selected from：Triton X-100、TritonX-405、Tween 80、Tween 20、Brij 35、 Brij 23 and combinations thereof；The preservative is selected from：Azido compound, MIT and biological preservative PC；The substrate is selected from：P- aminobenzene-β-D- synthesis, 2 '-N- (hexadecanol)-N- (amino -2 '-nitrobenzene) - β-D- synthesis, 4-methyl umbelliferone acyl-β-D- synthesis, naphthyl-AS-B1- β-D- synthesis, 1- naphthyl-β-D- synthesis, 2- naphthyl-β-D- synthesis, o- nitrobenzene-β-D- synthesis, m- Nitrobenzene-β-D- synthesis, p- nitrobenzene-β-D- synthesis, phenyl-β-D- synthesis, 5- are bromo- The chloro- 3- indoles-β-D- synthesis of 4-, (the different phenoxazine ketone of 9- hydroxyls -3-)-β-D- synthesis, 7- hydroxyls -4- Trifluoromethyl-cumarin, ω-nitrostyrolene base-β-D- synthesis, fluorescein-D- synthesis and its group Close.
- 9. glycocholic acid detection kit according to claim 8, the buffer solution are phosphate buffers.
- 10. glycocholic acid detection kit according to claim 8, the stabilizer are bovine serum albumin(BSA)s.
- 11. glycocholic acid detection kit according to claim 8, the surfactant is Tween 20.
- 12. glycocholic acid detection kit according to claim 8, the azido compound is Sodium azide or nitrine lithium.
- 13. glycocholic acid detection kit according to claim 8, the biological preservative PC is PC-300.
- 14. glycocholic acid detection kit according to claim 8, the substrate is o- nitrobenzene-β-D- gala pyranoses Glycosides.
- 15. glycocholic acid detection kit according to claim 6, the enzyme acceptor of the beta galactosidase is SEQ ID Shown in No.2.
- 16. glycocholic acid detection kit according to claim 7, the calibration object or quality-control product include 10mM to 500mM Buffer solution and 0mg/L are to 40mg/L glycocholic acid.
- 17. purposes of the conjugate in glycocholic acid detection device is prepared described in claim 4.
- 18. purposes according to claim 17, the detection device is kit.
- 19. the enzyme acceptor combination of the beta galactosidase shown in conjugate and SEQ ID No.2 described in claim 4 is used for Prepare the purposes in glycocholic acid detection device.
- 20. purposes according to claim 19, the detection device is kit.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610536792 | 2016-07-08 | ||
| CN2016105367923 | 2016-07-08 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106565809A CN106565809A (en) | 2017-04-19 |
| CN106565809B true CN106565809B (en) | 2018-05-01 |
Family
ID=58535083
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610946486.7A Active CN106565809B (en) | 2016-07-08 | 2016-10-26 | A kind of enzyme donor conjugate of beta galactosidase and its purposes in glycocholic acid detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106565809B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107656064B (en) * | 2017-08-11 | 2020-03-24 | 中山市创艺生化工程有限公司 | Composite stabilizer for immunoglobulin M determination kit and application thereof |
| CN107501378B (en) * | 2017-08-31 | 2019-05-14 | 湖南华腾制药有限公司 | A kind of glycocholic acid polyethyleneglycol derivative and preparation method thereof |
| CN108761068A (en) * | 2018-04-02 | 2018-11-06 | 深圳上泰生物工程有限公司 | Cloned enzyme donor immunologic function test reagent, preparation method, detecting system, detection method and kit |
| CN110174363A (en) * | 2019-01-09 | 2019-08-27 | 北京九强生物技术股份有限公司 | Glucose-6-phosphate dehydrogenase mutant and its purposes in preparation detection reagent |
| CN111999500B (en) * | 2019-04-19 | 2023-04-18 | 北京九强生物技术股份有限公司 | Carbamazepine detection kit |
| CN115825455A (en) * | 2019-07-04 | 2023-03-21 | 北京九强生物技术股份有限公司 | Application of valproic acid derivative in immunoassay |
| CN112285345A (en) * | 2020-08-31 | 2021-01-29 | 北京九强生物技术股份有限公司 | Glycocholic acid detection kit |
| CN112114127A (en) * | 2020-09-09 | 2020-12-22 | 武汉生之源生物科技股份有限公司 | Glycocholic acid homogeneous enzyme immunoassay kit and preparation method and application thereof |
| CN113567662A (en) * | 2021-07-08 | 2021-10-29 | 重庆中元汇吉生物技术有限公司 | Kit for determining glycocholic acid and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1632578A (en) * | 2004-12-24 | 2005-06-29 | 上海复星长征医学科学有限公司 | Bile acid determination reagent |
| CN102955033A (en) * | 2012-10-22 | 2013-03-06 | 金华市强盛生物科技有限公司 | Kit for determining glycocholic acid in human blood |
| CN103940816A (en) * | 2014-04-18 | 2014-07-23 | 安徽大千生物工程有限公司 | Kit for determining glycocholic acid content in human body and preparation method |
| CN103760348B (en) * | 2014-02-11 | 2015-03-11 | 苏州博源医疗科技有限公司 | Glycocholic acid immunodetection reagent and preparing method and detecting method thereof |
| CN105524898A (en) * | 2016-01-22 | 2016-04-27 | 宁波美康生物科技股份有限公司 | Cloned enzyme donor fragment and preparation method thereof |
-
2016
- 2016-10-26 CN CN201610946486.7A patent/CN106565809B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1632578A (en) * | 2004-12-24 | 2005-06-29 | 上海复星长征医学科学有限公司 | Bile acid determination reagent |
| CN102955033A (en) * | 2012-10-22 | 2013-03-06 | 金华市强盛生物科技有限公司 | Kit for determining glycocholic acid in human blood |
| CN103760348B (en) * | 2014-02-11 | 2015-03-11 | 苏州博源医疗科技有限公司 | Glycocholic acid immunodetection reagent and preparing method and detecting method thereof |
| CN103940816A (en) * | 2014-04-18 | 2014-07-23 | 安徽大千生物工程有限公司 | Kit for determining glycocholic acid content in human body and preparation method |
| CN105524898A (en) * | 2016-01-22 | 2016-04-27 | 宁波美康生物科技股份有限公司 | Cloned enzyme donor fragment and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106565809A (en) | 2017-04-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106565809B (en) | A kind of enzyme donor conjugate of beta galactosidase and its purposes in glycocholic acid detection | |
| US9964537B2 (en) | Method and device for immunoassay using nucleotide conjugates | |
| JP2870704B2 (en) | Homocysteine assay | |
| CN102014929A (en) | Enzymatic substrates for multiple detection systems | |
| CN113549673B (en) | Methods involving testing for lysosomal storage disorders | |
| JP6466907B2 (en) | Compounds and methods for testing for lysosomal disease | |
| Rani et al. | Measurement of bile acid in serum and bile with arylamine-glass-bound 3α-hydroxysteroid dehydrogenase and diaphorase | |
| Holmes | Coupled enzymatic assay for the determination of sucrose | |
| JP2016522677A5 (en) | ||
| CN110078711A (en) | A kind of carbamazepine derivative and its purposes in immune detection | |
| JP2000275247A (en) | Measuring method for d-cysteine | |
| Bier et al. | A redox-label immunosensor on basis of a bi-enzyme electrode | |
| Schölmerich et al. | [19] Immobilized enzymes for assaying bile acids | |
| JPH04253991A (en) | Novel galactopyranoside, preparation thereof and process and reagent for detecting analytical object in test solution using beta- galactosedase substrate comprising the same and cedia-system | |
| JPS5913199B2 (en) | Amylase activity measurement method | |
| JP3760250B2 (en) | Method for quantifying sulfate-conjugated bile acids and kits thereof | |
| JP4544598B2 (en) | Liquid reagent and storage method | |
| JPH01295165A (en) | Immunoassay | |
| US6818415B2 (en) | Sodium activation of amylase | |
| WO1990011526A1 (en) | Cancer diagnosis | |
| Dhar et al. | Determination of dehydroepiandrosterone sulfate in plasma by a one-step enzyme immunoassay with a microtitre plate. | |
| JP2005512601A5 (en) | ||
| AU625242B2 (en) | Method for the quantitative determination of gangliosides of the gangliotetraose series by cholera toxin conjugated with an enzyme | |
| JPH08168377A (en) | Method for stabilizing aqueous alkaline phosphatase solution | |
| Dörner | Quantitative determination of lactose, maltose, and sucrose in urine |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |