CN103760348A - Glycocholic acid immunodetection reagent and preparing method and detecting method thereof - Google Patents
Glycocholic acid immunodetection reagent and preparing method and detecting method thereof Download PDFInfo
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Abstract
The invention relates to a glycocholic acid detecting reagent and a preparing method and a detecting method thereof, in particular to a glycocholic acid immunodetection reagent and a preparing method and a detecting method thereof. The glycocholic acid immunodetection reagent comprises a glycocholic acid specificity resisting antibody and an indicating reagent, wherein the indicating reagent is used for detecting the glycocholic acid specificity resisting antibody and a glycocholic acid composite; the glycocholic acid specificity resisting antibody is obtained from glycocholic acid immunogen immune animals. The glycocholic acid immunodetection reagent has the benefits that the glycocholic acid immunogen specificity is strong, the immunogenicity is high, and the prepared glycocholic acid specificity resisting antibody has strong specificity and high valence and does not have any cross reaction with 45 common drugs; a homogeneous enzyme immunodetection reagent containing the glycocholic acid specificity resisting antibody can conveniently, rapidly and accurately determine the content of glycocholic acid in a sample and can simultaneously test multiple samples on a fully-automatic biochemical analysis instrument; the high-throughout rapid measurement of the glycocholic acid is realized, the accuracy is high, the specificity is strong, and both the precision and the detection efficiency are greatly improved.
Description
Technical field
The present invention relates to a kind of glycocholic acid and detect reagent and preparation and determination methods method thereof, be specifically related to a kind of glycocholic acid homogeneous enzyme immunoassay and detect reagent and preparation and determination methods method thereof.
Background technology
Glycocholic acid (Cholylglycine, CG) structural formula is as shown in (III):
Formula (III)
Glycocholic acid is one of mating type cholic acid of being combined into of cholic acid and glycocoll, by liver cell, synthesized, with bile, enter enteron aisle, through portal vein, return again liver, when liver cell is impaired, liver cell picked-up glycocholic acid ability declines, and causes content in blood to increase, therefore, glycocholic acid is that evaluation hepatocyte function and liver and gall thereof are one of sensitive indicator of material recycle function.Studies show that, the hepatopaths' such as cirrhosis, liver cancer, oxyhepatitis and chronic active hepatitis glycocholic acid content is apparently higher than normal person, glycocholic acid is also the important indicator that detects cholestasia and early stage alcoholic hepatic injury, and the detection of glycocholic acid simultaneously has important clinical meaning to the diagnosis of pregnant women's intrahepatic cholestasis (ICP).
At present, external quantitative measurement glycocholic acid is mainly used radio immunoassay (RIA), chemiluminescence immunoassay (CLIA), enzyme linked immunosorbent assay (ELISA) etc.Radioimmunology need to have specialty to put to exempt from facility, and common lab is difficult to carry out, and puts that to exempt from method accuracy low, and radioactive ray also can produce harm greatly to operating personnel's health, seldom uses in the world.Chemoluminescence method sensitivity is higher, but finding speed is slower, and accuracy and the reagent stability of test result are poor, and needs expensive special chemiluminescence checkout equipment, is unfavorable for that Routine Test Lab carries out, and clinical practice limitation is obvious.Euzymelinked immunosorbent assay (ELISA) is generally used for semiquantitative determination, and complex operation, and detection time is long, and automaticity is low, and repeatability is poor, is unfavorable for being widely used in clinical examination.
Lack in the market glycocholic acid highly sensitive, high specificity and detect the measured Automated inspection reagent of reagent, especially matter.
Homogeneous enzyme immunoassay detection method,, high specificity fast, simple to operate, highly sensitive with its detection speed and can realize the advantage of the rapid detection of high flux to small-molecule substance on automatic clinical chemistry analyzer, starts more and more to be paid close attention to.
Summary of the invention
For solving the deficiencies in the prior art, the glycocholic acid homogeneous enzyme immunoassay that the object of the present invention is to provide a kind of not only safety but also can quick, efficient, sensitive, accurately detect glycocholic acid content in sample to be tested detects reagent and preparation method thereof, and can be with various types of automatic biochemistry analyzer couplings, to the less demanding glycocholic acid homogeneous enzyme immunoassay of testing staff detection method.
In order to realize above-mentioned target, the technical solution used in the present invention is:
A kind of glycocholic acid immunologic function test reagent, is characterized in that, comprising: anti-glycocholic acid specific antibody, for detection of the indicator of anti-glycocholic acid specific antibody-glycocholic acid compound; Above-mentioned anti-glycocholic acid specific antibody is obtained by glycocholic acid immunogen immune animal, the immunogenic structural formula of glycocholic acid as shown in the formula (I):
Formula (I)
In formula, R is linking group-(CH
2)
n-O-CH
2-COO-, n is the arbitrary integer between 1 to 20, and carrier has immunogenic protein, and carrier is haemocyanin, hemocyanin or thyroglobulin; Above-mentioned indicator is selected from enzyme reagent, radioactive isotope reagent, fluorescent reagent or chemical illuminating reagent.
Aforesaid glycocholic acid immunologic function test reagent, R is-(CH
2)
2-O-CH
2-COO-.
Aforesaid glycocholic acid immunologic function test reagent, above-mentioned indicator is selected from enzyme reagent, comprising: the substrate of enzyme mark conjugate and enzyme; Above-mentioned enzyme mark conjugate comprises glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate; The substrate of above-mentioned enzyme is G-6-P.
Aforesaid glycocholic acid immunologic function test reagent, above-mentioned glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate is formed by glucose-6-phosphate dehydrogenase (G6PD) and the coupling of glycocholic acid derivant, the structural formula of above-mentioned glycocholic acid derivant as shown in the formula (II):
Formula (II)
Above-mentioned R is-(CH
2)
n-O-CH
2-COO-, n is the integer between 1 to 20.
Aforesaid glycocholic acid immunologic function test reagent, above-mentioned R is-(CH
2)
2-O-CH
2-COO-.
A preparation method for glycocholic acid immunologic function test reagent, is characterized in that, comprises the steps:
(1) the synthetic and purifying of glycocholic acid derivant, and carry out Structural Identification;
(2) glycocholic acid is immunogenic synthetic: make glycocholic acid derivant-(CH
2)
n-O-CH
2-COO-group with there is immunogenic protein carrier and be connected, n is the integer between 1 to 20;
(3) with glycocholic acid immunogen immune animal, preparation the anti-glycocholic acid specific antibody of purifying;
(4) preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing: prepare glucose-6-phosphate dehydrogenase (G6PD) solution, activate glycocholic acid derivant, G6PDH is connected with glycocholic acid derivant, purifying connects product;
(5) glycocholic acid homogeneous enzyme immunoassay detects the preparation of reagent:
The preparation of reagent A: mixed by anti-glycocholic acid specific antibody and homogeneous phase zymolyte;
The preparation of reagent B: mixed by glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing and Tris damping fluid.
The preparation method of aforesaid a kind of glycocholic acid immunologic function test reagent, in described step (2), protein carrier is BSA, n=2, concrete synthesis step is as follows:
1) 20mg BSA is dissolved in the PBS of 5ml0.2M, pH8.5, above-mentioned solution is placed in beaker A;
2) following chemicals is joined to stirring and dissolving in beaker B: the kaliumphosphate buffer of 20mg glycocholic acid derivant, 0.35ml dimethylformamide DMF, 0.35ml ethanol, 0.7ml10mM pH5.0,40mg1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, 5mg N-hydroxy thiosuccinimide, stirring and dissolving under room temperature, reacts 30 minutes;
3) solution in beaker B is dropped in beaker A, obtain mixed solution, at 2～8 ℃, stir and spend the night; Mixed solution after above-mentioned stirring, through neutral phosphor phthalate buffer dialysis purifying, is obtained to BSA-glycocholic acid immunogene, be stored in-20 ℃.
The preparation method of aforesaid a kind of glycocholic acid immunologic function test reagent, described step (4) detailed process is:
1) preparation of glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) solution:
A. take the G6PDH that 15mg specification is 100KU, room temperature is dissolved in 12mL and contains 72.6mg(0.05M) Tris, 8mg MgCl
2(3.3mM) and in the solution of 100mg NaCl, these pH value of solution=9.0;
B. add the nicotinamide adenine dinucleotide NADH of 225mg reduction-state, 135mg G-6-P G-6-P and 0.75mL carbitol;
C. dropwise add 2mL dimethyl sulfoxide (DMSO);
2) activation of glycocholic acid derivant
A) under anhydrous state, take 10mg glycocholic acid derivant, be dissolved in 600 μ L DMF;
B) make above-mentioned solution temperature drop to-2～-8 ℃;
C) add 3 μ L tri-n-butylamines;
D) add 1.5 μ L isobutyl chlorocarbonates;
E)-2～-8 ℃ are stirred 30 minutes;
2) G6PDH and glycocholic acid derivant is connected
A) the glycocholic acid derivative solution of above-mentioned activation is dropwise joined in the G6PDH solution of above-mentioned dissolving;
B) 2-8 ℃ of stirring spent the night;
4) purified product
By G-25 gel chromatography column purifying, connect product, the final product of acquisition is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, at 2-8 ℃, stores.
The preparation method of aforesaid a kind of glycocholic acid immunologic function test reagent, the detailed process of step (5) is as follows:
The preparation of reagent A: by 4.036g(11.25mM) nicotinamide adenine dinucleotide NAD, the 1.711g(11.25mM of oxidation state) the Tris damping fluid of G-6-P 1L55mM, pH=8.0 for G6P dissolves and makes homogeneous phase zymolyte; The anti-glycocholic acid specific antibody of preparation is added in above-mentioned homogeneous phase zymolyte, and the volume ratio of antibody and homogeneous phase zymolyte is 1:100～1:10000;
The preparation of reagent B: glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing of preparation is added in the Tris damping fluid of 120mM, pH=8.2, and the volume ratio of above-mentioned conjugate and Tris damping fluid is 1:100～1:10000.
The detection method of utilizing glycocholic acid immunologic function test reagent, is characterized in that, comprises the following steps:
1) sample to be tested is contacted with anti-glycocholic acid specific antibody;
2) according to the combination situation of glycocholic acid in sample to be tested and anti-glycocholic acid specific antibody,
Utilize the content of glycocholic acid in indicator judgement sample;
Described sample to be tested is serum, blood plasma, saliva or urine.
Usefulness of the present invention is: glycocholic acid immunogene high specificity of the present invention, immunogenicity are high, the anti-glycocholic acid specific antibody high specificity of preparing, the height of tiring, and with 45 kinds of common medicines without any cross reaction; The homogeneous enzyme immunoassay that contains above-mentioned anti-glycocholic acid specific antibody detects reagent can determine the glycocholic acid content in sample easily and fast, exactly, and can on automatic clinical chemistry analyzer, measure multiple samples simultaneously, realize the rapid mensuration of high flux of glycocholic acid, accuracy is high, high specificity, degree of accuracy is all enhanced before comparing with detection efficiency, has realized the full-automation of testing process simultaneously, less demanding to testing staff, is easy to realize and promote the use of.
Accompanying drawing explanation
Fig. 1 is glycocholic acid homogeneous enzyme immunoassay reaction normal curve map;
Fig. 2 is glycocholic acid homogeneous enzyme immunoassay correlation analysis figure.
Embodiment
The technical solution used in the present invention is:
Glycocholic acid immunogene, its structural formula as shown in the formula (I):
Formula (I)
In formula, R is linking group, can be-(CH
2)
n-O-CH
2-COO-, n is the integer between 1 to 20, special, R is-(CH
2)
2-O-CH
2-COO-; Carrier has immunogenicity, and preferred, carrier is for having immunogenic protein.Although other enough the large immunogenic material that possesses also can be used as carrier, select under normal circumstances protein as carrier.The most frequently used immunogenic carrier comprises haemocyanin, hemocyanin (KLH) and thyroglobulin.Carrier in the present invention is preferably haemocyanin.
A kind of anti-glycocholic acid specific antibody, is obtained by the glycocholic acid immunogen immune animal shown in formula (I).
In the present invention, " antibody " of indication not only refers to complete antibody molecule, also comprises the antibody fragment or the derivant that retain complete antibody specific binding capacity.Antibody of the present invention can be that polyclonal antibody can be also monoclonal antibody, is preferably polyclonal antibody.
The method that obtains polyclonal antibody is the glycocholic acid immunogene shown in use formula (I), adding or do not add after adjuvant, at one or more position of animal, carries out immunity, and host animal comprises: rabbit, goat, mouse, sheep, cavy or horse.Lasting immunity is carried out always, until that antibody titer reaches is the highest.Animal timing blood sampling obtains appropriate specific corrosioning anteserum, and antiserum can purifying.
Monoclonal antibody can be made by somatocyte hybriding technology.
A kind of glycocholic acid homogeneous enzyme immunoassay detects reagent, comprising: above-mentioned anti-glycocholic acid specific antibody, for detection of the indicator of anti-glycocholic acid specific antibody-glycocholic acid compound.Indicator is selected from enzyme reagent, radioactive isotope reagent, fluorescent reagent and chemical illuminating reagent.Preferably, indicator is enzyme reagent, comprising: the substrate of enzyme mark conjugate and enzyme.Wherein, enzyme mark conjugate comprises glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate, and it can obtain by chemical synthesis process.
Above-mentioned glycocholic acid homogeneous enzyme immunoassay detects the using method of reagent, comprises the following steps:
1) sample to be tested is contacted with above-mentioned anti-glycocholic acid specific antibody;
2), according to the combination situation of glycocholic acid in sample to be tested and above-mentioned anti-glycocholic acid specific antibody, utilize the content of glycocholic acid in indicator judgement sample.
Sample to be tested is various physiology samples, such as serum, blood plasma, urine, saliva etc.Preferably, sample to be tested is serum or blood plasma.
Below in conjunction with specific embodiment, further illustrate the present invention.
Embodiment mono-: the synthetic and structural confirmation of glycocholic acid derivant
The glycocholic acid derivatives chemical structure of using in following examples is suc as formula shown in (IV):
Formula (IV)
The synthetic route of this glycocholic acid derivant is as follows:
Glycocholic acid derivant
Concrete synthesis step is as follows:
Synthesizing of compound 3
1) take 10.0g(24.5mmol) compound 1 cholic acid, compound 1 is dissolved in 100mL anhydrous dimethyl formamide (DMF), under room temperature, add 7.04g(36.7mmol) carbodiimide hydrochloride (EDCI), 4.96g(36.7mmol) hydroxy benzo triazole (HOBT) and 10.7mL(61.2mmol) diisopropylethylamine (DIEA), add again 3.07g(24.5mmol) compound 2 glycocoll, stir 4 hours, obtain resulting solution;
2) will after above-mentioned resulting solution dilute with water, use again ethyl acetate (EtOAc) extraction, make water and bittern rinse organic layer, add Na
2sO
4dry, by decompression method, make solvent evaporation, finally obtain 9.38g white solid compound 3, i.e. glycocholic acid, productive rate 80%.
Synthesizing of compound 4
1) take 8.27g(17.2mmol) compound 3 is dissolved in the pyridine of 90mL, adds 2.37g(20.7mmol at 0 ℃) methylsufonyl chloride (MsCl), this solution is at room temperature stirred 1 hour, obtain resulting solution.
2) will after above-mentioned resulting solution dilute with water, use again ethyl acetate (EtOAc) extraction, make water and bittern rinse organic layer, add Na
2sO
4dry, by decompression method, make solvent evaporation, finally obtain 6.67g white solid compound 4, productive rate 81%.
Synthesizing of compound 5
1) taking 10.0g(18.0mmol) compound 4 is dissolved in the pyridine of 100mL, adds the ethylene glycol of 20mL under room temperature, and this reaction mixture is heated to 110 ℃ and spends the night.
2) above-mentioned reaction mixture is removed to desolventizing, after residue dilute with water, use again ethyl acetate (EtOAc) extraction, use bittern to rinse organic layer, add Na
2sO
4dry, filter, concentrate and pass through FCC(DCM/MeOH=30/1) method purifying obtains 1.33g white solid compound 5 crude products, productive rate 11%.
Synthesizing of compound 7
1) take 100mg(0.191mmol) compound 5 and 64mg(0.21mmol) cesium carbonate (Cs
2cO
3), be jointly suspended in the anhydrous dimethyl formamide (DMF) of 5mL, under condition of ice bath, add 41mg(0.21mmol) compound 6(bromo-acetic acid tert-butyl), under room temperature, stirring reaction spends the night.
2) will after above-mentioned reactant liquor dilute with water, use again ethyl acetate (EtOAc) extraction, make water and bittern rinse organic layer, add Na
2sO
4dry, filter, concentrate and pass through FCC(DCM/MeOH=35/1) method purifying obtains 15mg yellow solid compound 7, productive rate 12%.
Synthesizing of glycocholic acid derivant
1) taking 80mg(0.13mmol) compound 7 is dissolved in the methylene chloride (DCM) of 5mL, adds 1mL trifluoroacetic acid (CF under condition of ice bath
3cOOH), stirring reaction 2 hours under room temperature.
2) NaOH (NaOH) the adjusting pH value to 9.0 except use 1N after desolventizing by above-mentioned reactant liquor, then use ethyl acetate (EtOAc) extraction.Water is regulated to pH value to 6.0 with the hydrochloric acid (HCl) of 1N, then use ethyl acetate (EtOAc) extraction.
3) use bittern to rinse organic layer, add Na
2sO
4dry, the concentrated 65mg colorless solid product, i.e. glycocholic acid derivant shown in formula (IV), productive rate 89%, purity >95% of obtaining.
Above-mentioned colorless solid purified product is carried out to Structural Identification
1, utilize Bruker Avance III plus400MHz and VARIAN MERCURY plus300M to carry out NMR (Nuclear Magnetic Resonance) spectrum scanning to above-mentioned colorless solid compounds, adopt TMS as interior mark.Result is as follows:
1h NMR (400MHz, CD
3oD): δ 4.61 (s, 2H), 4.27 (t, J=5.2Hz, 2H), 3.95 (s, 1H), 3.90 (s, 2H), 3.79 (s, 1H), 3.71 (s, 3H), 3.60-3.61 (m, 3H), 2.14-2.43 (m, 4H), 1.73-1.92 (m, 6H), 1.55-1.65 (m, 7H), 1.28-1.45 (m, 7H), 1.03 (d, J=6.4Hz, 3H), 0.92 (s, 3H), 0.71 (s, 3H).Be characterized by the glycocholic acid derivant shown in formula (IV).
2, utilize Chromatography/Mass Spectrometry technology (LCMS) to carry out Analysis and Identification to the derivant obtaining, adopt the series connection level Four bar mass spectrometer LC/MSD1200 series of Agilent company, ion gun adopts positive ion or negative ionization pattern.Chromatographic column specification is: and Symmetry C18 (50 × 4.6mm, m), column temperature is 30 ℃ to 5 μ, and flow velocity is 1.5mL/min, and detection wavelength is 214nm, and mobile phase is 5-60% acetonitrile-0.02%NH
4oAc.LCMS result shows: purity >95%; Retention time 3.637min.
Comprehensive the above results, can determine that this colorless solid compounds is the glycocholic acid derivant shown in formula (IV).
Embodiment bis-: BSA-glycocholic acid is immunogenic synthetic
BSA-glycocholic acid immunogene by the glycocholic acid derivant shown in bovine serum albumin(BSA) BSA and formula (IV)-(CH
2)
n-O-CH
2-COO-group is formed by connecting, and in the present embodiment, describes this immunogenic synthetic method as an example of n=2 example in detail, and concrete steps are as follows:
1) 20mg BSA is dissolved in to 5ml0.2M, in the phosphate buffer (Phosphate buffer solution, PBS) of pH8.5, above-mentioned solution is placed in beaker A;
2) following chemicals is joined to stirring and dissolving in beaker B: 20mg glycocholic acid derivant, 0.35ml dimethylformamide (dimethylformamide, DMF), 0.35ml ethanol, the kaliumphosphate buffer of 0.7ml10mM pH5.0.40mg1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, 5mg N-hydroxy thiosuccinimide (N-hydroxysulfosuccinimide, Sulfo-NHS), stirring and dissolving under room temperature, reacts 30 minutes;
3) solution in beaker B is dropped in beaker A, obtain mixed solution, at 2～8 ℃, stir and spend the night; Mixed solution after above-mentioned stirring, through neutral phosphor phthalate buffer dialysis (4 × 4L) purifying, is obtained to BSA-glycocholic acid immunogene, be stored in-20 ℃.
Similarly, when n gets other integers in 1～20 scope, use the same method and can prepare glycocholic acid immunogene as shown in the formula (I).Certainly, carrier, for having immunogenic protein, can be still haemocyanin, hemocyanin (KLH) and thyroglobulin.Preferably, carrier is bovine serum albumin(BSA).
The present invention only discloses linking group R
2)
n-O-CH
2-COO-, and the synthetic example of the glycocholic acid derivant of n=2 has also been carried out relevant subsequent experiment, because linking group mainly plays the connection function of little molecule derivant and carrier, immunogenicity is strong and weak relevant with glycocholic acid derivative molecular structure and the selected kind of carrier of synthesized, when therefore n gets the arbitrary integer between 1 to 20 in theory, experimental result there is no significant difference, the glycocholic acid immunogene of using the glycocholic acid derivant of different n values to prepare all possesses strong immunogenicity, and the specific antibody of corresponding preparation all has excellent properties.
Embodiment tri-: the preparation of anti-glycocholic acid specific antibody
The above-mentioned BSA-glycocholic acid immunogene making is adopted to conventional method inoculation experiments animal rabbit, get antiserum after booster immunization, concrete steps are as follows:
Above-mentioned synthetic BSA-glycocholic acid immunogene is diluted to 1.0mg/ml with PBS, obtains antigenic solution, then with 1.0ml antigenic solution, mix with Freund's complete adjuvant, animal used as test rabbit is injected.
After 2～3 weeks, then with the identical antigenic solution of 1.0ml and incomplete Freund's adjuvant, above-mentioned animal used as test rabbit is injected once, every surrounding, inject once afterwards, inject altogether 4 times.
Above-mentioned animal used as test rabbit is got to blood, and separation and purification obtains anti-glycocholic acid specific antibody, after measured, and the tiring as 1:30000 of this anti-glycocholic acid specific antibody.
Embodiment tetra-: the preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing
1) preparation of glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) solution:
1) accurately take the G6PDH that 15mg specification is 100KU, room temperature is dissolved in 12mL and contains 72.6mg(0.05M) Tris, 8mg MgCl
2(3.3mM) and in the solution of 100mg NaCl, these pH value of solution=9.0, this step is carried out in beaker C.
2) in above-mentioned beaker C, add nicotinamide adenine dinucleotide NADH, 135mg G-6-P G-6-P and the 0.75mL carbitol (Carbitol) of 225mg reduction-state.
3) in above-mentioned beaker C, dropwise add again 2mL dimethyl sulfoxide (DMSO) (dimethy sulfoxide, DMSO).
1) activation of glycocholic acid derivant
1) under anhydrous state, take the above-mentioned glycocholic acid derivant of 10mg, be dissolved in 600 μ LDMF.
2) make above-mentioned solution temperature drop to-2～-8 ℃.
3) add 3 μ L tri-n-butylamines (tributylamine).
4) add 1.5 μ L isobutyl chlorocarbonates (isobutylchloroformate).
5)-2～-8 ℃ are stirred 30 minutes.
2) G6PDH and glycocholic acid derivant is connected
1) the glycocholic acid derivative solution of above-mentioned activation is dropwise joined in the G6PDH solution of above-mentioned dissolving.
2) 2-8 ℃ of stirring spent the night.
3) purified product
By G-25 gel chromatography column purification step 3) in solution, the final product of acquisition is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, at 2-8 ℃ store.
Embodiment five: glycocholic acid homogeneous enzyme immunoassay detects the preparation of reagent
Glycocholic acid homogeneous enzyme immunoassay detects reagent, comprising: above-mentioned anti-glycocholic acid specific antibody, and for detection of the indicator of anti-glycocholic acid specific antibody-glycocholic acid compound.Indicator is selected from enzyme reagent, radioactive isotope reagent, fluorescent reagent and chemical illuminating reagent.Preferably, indicator is enzyme reagent, comprising: the substrate of enzyme mark conjugate and enzyme.Wherein, enzyme mark conjugate comprises glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate, and it obtains by above-mentioned chemical synthesis process.
Glycocholic acid homogeneous enzyme immunoassay detected reagent before using, for fear of the enzyme mark conjugate in indicator and the substrate of enzyme, react, the substrate of enzyme mark conjugate and enzyme is separated, does not mix, so the substrate of enzyme and above-mentioned anti-glycocholic acid specific antibody are mixed.That is to say, glycocholic acid homogeneous enzyme immunoassay detects reagent and comprises two kinds of points of reagent that are arranged, specific as follows:
1. the preparation of reagent A: by 4.036g(11.25mM) nicotinamide adenine dinucleotide NAD, the 1.711g(11.25mM of oxidation state) G-6-P G6P is placed in beaker D, and with the Tris damping fluid of 1L55mM, pH=8.0, dissolve and make homogeneous phase zymolyte; The anti-glycocholic acid specific antibody of above-mentioned preparation is added in above-mentioned homogeneous phase zymolyte, and the volume ratio of antibody and homogeneous phase zymolyte can be 1:100～1:10000, and concrete ratio is 1:400 in the present embodiment.
2. the preparation of reagent B: glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing of above-mentioned preparation is added in the Tris damping fluid of 120mM, pH=8.2, the volume ratio of above-mentioned conjugate and Tris damping fluid can be 1:100～1:10000, and concrete ratio is 1:1500 in the present embodiment.
Above-mentioned glycocholic acid homogeneous enzyme immunoassay detects the using method of reagent, comprises the following steps:
1) sample to be tested is contacted with above-mentioned anti-glycocholic acid specific antibody;
2), according to the combination situation of glycocholic acid in sample to be tested and above-mentioned anti-glycocholic acid specific antibody, utilize indicator to judge the content of glycocholic acid in sample to be tested.
Concrete, during detection, sample to be tested being added in reagent A, the anti-glycocholic acid specific antibody generation specific binding in the glycocholic acid in sample to be tested and reagent A, generates anti-glycocholic acid specific antibody-glycocholic acid compound; Add again reagent B, now glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation the thing in reagent B mixes, contacts with the substrate of the enzyme in reagent A, there is enzymatic reaction, form the indicator that detects anti-glycocholic acid specific antibody-glycocholic acid compound, indicator judges the content of glycocholic acid in sample to be tested according to the combination situation of glycocholic acid in sample to be tested and above-mentioned anti-glycocholic acid specific antibody.
Due to the anti-glycocholic acid specific antibody of the glycocholic acid competitive binding in glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing and sample to be tested, so, in sample to be tested, the amount of glycocholic acid is more, in homogeneous phase enzyme solutions, the amount of free glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing is more, enzymatic reaction is faster, causes OD
340rise.
Above-mentioned sample to be tested is physiology sample, such as serum, blood plasma, urine, saliva etc.
As the preferred scheme of one, above-mentioned sample to be tested is serum or blood plasma.
Embodiment six: glycocholic acid homogeneous enzyme immunoassay check
1, obtain typical curve: arrange and step auspicious BS200 automatic clinical chemistry analyzer response parameter (in Table 1), operating process is: first reagent adding A, then add standard items, finally add reagent B.Add after reagent B, measure the OD of different time points
340light absorption value, the reaction rate while calculating various criterion product concentration, needs constantly to adjust the volume ratio of reagent A and reagent B in actual mechanical process, adjust photometry point simultaneously, finally draws comparatively ideal reaction normal curve map, as shown in Figure 1.
Table 1 steps auspicious BS200 automatic clinical chemistry analyzer response parameter
By homogeneous enzyme immunoassay of the present invention, detect the typical curve that reagent obtains, the basic, normal, high concentration Quality Control of replication sample 10 times, above-mentioned Quality Control sample is: glycocholic acid standard items are dissolved in human serum, are respectively 1.20 to concentration, 5.00,30.00 μ g/ml.Detect data and data analysis in Table 2.
Table 2 sample determination and precision and recovery assessment
| Blood sample | Low | In | High |
| Sample concentration (μ g/ml) | 1.20 | 5.00 | 30.00 |
| 1 | 1.23 | 5.25 | 31.90 |
| 2 | 1.19 | 5.14 | 30.45 |
| 3 | 1.25 | 4.97 | 29.16 |
| 4 | 1.15 | 5.23 | 32.39 |
| 5 | 1.18 | 5.29 | 31.07 |
| 6 | 1.27 | 5.12 | 30.52 |
| 7 | 1.25 | 5.05 | 30.25 |
| 8 | 1.17 | 5.15 | 28.98 |
| 9 | 1.20 | 5.09 | 31.03 |
| 10 | 1.26 | 4.94 | 30.41 |
| Mean value (μ g/ml) | 1.22 | 5.12 | 30.62 |
| Standard deviation (SD) | 0.0422 | 0.1154 | 1.0637 |
| Precision (CV%) | 3.46 | 2.25 | 3.47 |
| Recovery % | 101.7 | 102.4 | 102.1 |
Testing result: the accuracy that homogeneous enzyme immunoassay detection reagent of the present invention is measured is high, and the recovery reaches 95%-105%, and precision is high, and CV is all lower than 4%.
Embodiment seven: interfering effects of drug test
Choose 45 kinds of Common drugs, adjust concentration to 10.0 μ g/ml, carry out interference test mensuration.Common 45 kinds of medicines and measurement result are specifically referring to table 3.
Table 3 common interference medicine
Measurement result: the concentration that above-mentioned 45 kinds of Common drugs are equivalent to glycocholic acid is all less than 0.1 μ g/ml.Visible, antibody of the present invention is the specific antibody of anti-glycocholic acid.
Embodiment eight: correlation analysis
To 105 routine clinical samples including 81 routine positive samples and 24 routine ' negative ' specimens, use respectively the radioimmunology of Abbott Laboratories and homogeneous enzyme immunoassay method of the present invention to carry out correlation analysis, the data of mensuration are referring to table 4.
Table 4 authentic specimen measured value
To above-mentioned data mapping, referring to Fig. 2, the linear equation obtaining is: y=0.995x+0.1105, coefficient R
2=0.9993, show that the glycocholic acid clinical samples accuracy of detection reagent mensuration of the present invention is high.
Because testing process of the present invention is to be completed by instrument full-automation, so less demanding to testing staff is easy to realize and promote the use of.
It should be noted that; the foregoing is only embodiments of the invention; not thereby limit the scope of the claims of the present invention; every equivalent structure or conversion of equivalent flow process that utilizes instructions of the present invention and accompanying drawing content to do; or be directly or indirectly used in other correlative technology fields, be all in like manner included in scope of patent protection of the present invention.
Claims (10)
1. a glycocholic acid immunologic function test reagent, is characterized in that, comprising: anti-glycocholic acid specific antibody, for detection of the indicator of anti-glycocholic acid specific antibody-glycocholic acid compound;
Above-mentioned anti-glycocholic acid specific antibody is obtained by glycocholic acid immunogen immune animal, the immunogenic structural formula of glycocholic acid as shown in the formula (I):
Formula (I)
In formula, R is linking group-(CH
2)
n-O-CH
2-COO-, n is the arbitrary integer between 1 to 20, and carrier is for having immunogenic protein, and carrier is haemocyanin, hemocyanin or thyroglobulin; Above-mentioned indicator is selected from enzyme reagent, radioactive isotope reagent, fluorescent reagent or chemical illuminating reagent.
2. glycocholic acid immunologic function test reagent according to claim 1, is characterized in that, R is-(CH
2)
2-O-CH
2-COO-.
3. glycocholic acid immunologic function test reagent according to claim 1, is characterized in that, above-mentioned indicator is selected from enzyme reagent, comprising: the substrate of enzyme mark conjugate and enzyme; Above-mentioned enzyme mark conjugate comprises glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate; The substrate of above-mentioned enzyme is G-6-P.
4. glycocholic acid immunologic function test reagent according to claim 3, it is characterized in that, above-mentioned glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate is formed by glucose-6-phosphate dehydrogenase (G6PD) and the coupling of glycocholic acid derivant, the structural formula of above-mentioned glycocholic acid derivant as shown in the formula (II):
Formula (II)
Above-mentioned R is-(CH
2)
n-O-CH
2-COO-, n is the integer between 1 to 20.
5. glycocholic acid immunologic function test reagent according to claim 4, is characterized in that, above-mentioned R is-(CH
2)
2-O-CH
2-COO-.
6. a preparation method for glycocholic acid immunologic function test reagent, is characterized in that, comprises the steps:
(1) the synthetic and purifying of glycocholic acid derivant claimed in claim 4, and carry out Structural Identification;
(2) glycocholic acid is immunogenic synthetic: make glycocholic acid derivant-(CH
2)
n-O-CH
2-COO-group with there is immunogenic protein carrier and be connected, n is the integer between 1 to 20;
(3) with glycocholic acid immunogen immune animal, preparation the anti-glycocholic acid specific antibody of purifying;
(4) preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing: prepare glucose-6-phosphate dehydrogenase (G6PD) solution, activate glycocholic acid derivant, G6PDH is connected with glycocholic acid derivant, purifying connects product;
(5) glycocholic acid homogeneous enzyme immunoassay detects the preparation of reagent:
The preparation of reagent A: mixed by anti-glycocholic acid specific antibody and homogeneous phase zymolyte;
The preparation of reagent B: mixed by glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing and Tris damping fluid.
7. the preparation method of a kind of glycocholic acid immunologic function test reagent according to claim 6, is characterized in that, in described step (2), protein carrier is BSA, n=2, and concrete synthesis step is as follows:
1) 20mg BSA is dissolved in the PBS of 5ml0.2M, pH8.5, above-mentioned solution is placed in beaker A;
2) following chemicals is joined to stirring and dissolving in beaker B: the kaliumphosphate buffer of 20mg glycocholic acid derivant, 0.35ml dimethylformamide DMF, 0.35ml ethanol, 0.7ml10mM pH5.0,40mg1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, 5mg N-hydroxy thiosuccinimide, stirring and dissolving under room temperature, reacts 30 minutes;
3) solution in beaker B is dropped in beaker A, obtain mixed solution, at 2～8 ℃, stir and spend the night; Mixed solution after above-mentioned stirring, through neutral phosphor phthalate buffer dialysis purifying, is obtained to BSA-glycocholic acid immunogene, be stored in-20 ℃.
8. the preparation method of a kind of glycocholic acid immunologic function test reagent according to claim 6, is characterized in that, described step (4) detailed process is:
1) preparation of glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) solution:
A. take the G6PDH that 15mg specification is 100KU, room temperature is dissolved in 12mL and contains 72.6mg(0.05M) Tris, 8mg MgCl
2(3.3mM) and in the solution of 100mg NaCl, these pH value of solution=9.0;
B. add the nicotinamide adenine dinucleotide NADH of 225mg reduction-state, 135mg G-6-P G-6-P and 0.75mL carbitol;
C. dropwise add 2mL dimethyl sulfoxide (DMSO);
2) activation of glycocholic acid derivant
A) under anhydrous state, take 10mg glycocholic acid derivant, be dissolved in 600 μ L DMF;
B) make above-mentioned solution temperature drop to-2～-8 ℃;
C) add 3 μ L tri-n-butylamines;
D) add 1.5 μ L isobutyl chlorocarbonates;
E)-2～-8 ℃ are stirred 30 minutes;
3) G6PDH and glycocholic acid derivant is connected
A) the glycocholic acid derivative solution of above-mentioned activation is dropwise joined in the G6PDH solution of above-mentioned dissolving;
B) 2-8 ℃ of stirring spent the night;
4) purified product
By G-25 gel chromatography column purifying, connect product, the final product of acquisition is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, at 2-8 ℃, stores.
9. the preparation method of a kind of glycocholic acid immunologic function test reagent according to claim 6, is characterized in that, the detailed process of step (5) is as follows:
The preparation of reagent A: by 4.036g(11.25mM) nicotinamide adenine dinucleotide NAD, the 1.711g(11.25mM of oxidation state) the Tris damping fluid of G-6-P 1L55mM, pH=8.0 for G6P dissolves and makes homogeneous phase zymolyte; The anti-glycocholic acid specific antibody of preparation is added in above-mentioned homogeneous phase zymolyte, and the volume ratio of antibody and homogeneous phase zymolyte is 1:100～1:10000;
The preparation of reagent B: glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing of preparation is added in the Tris damping fluid of 120mM, pH=8.2, and the volume ratio of above-mentioned conjugate and Tris damping fluid is 1:100～1:10000.
10. the detection method of utilizing the glycocholic acid immunologic function test reagent described in claim 1 to 5 any one, is characterized in that, comprises the following steps:
1) sample to be tested is contacted with anti-glycocholic acid specific antibody;
2) according to the combination situation of glycocholic acid in sample to be tested and anti-glycocholic acid specific antibody,
Utilize the content of glycocholic acid in indicator judgement sample;
Described sample to be tested is serum, blood plasma, saliva or urine.
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