CN106226512B - A kind of detection method of kit, the preparation method of kit and the peripheral blood glycocholic acid realized using the kit - Google Patents
A kind of detection method of kit, the preparation method of kit and the peripheral blood glycocholic acid realized using the kit Download PDFInfo
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- CN106226512B CN106226512B CN201610620346.0A CN201610620346A CN106226512B CN 106226512 B CN106226512 B CN 106226512B CN 201610620346 A CN201610620346 A CN 201610620346A CN 106226512 B CN106226512 B CN 106226512B
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- glycocholic acid
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- 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
- G01N33/531—Production of immunochemical test materials
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- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The invention discloses a kind of kit, including reagent R1, reagent R2, the reagent R1 contain：Trishydroxymethylaminomethane (Tris HCL) buffer solution, anti-taurine antibody, anti-glycocholic acid antibody, 6 phosphoric acid of glucose (G6P)；The reagent R2 contains：Trishydroxymethylaminomethane (Tris HCL) buffer solution, 6 PD conjugates of glycocholic acid G, NADP, BSA.The present invention discloses the preparation method of kit, especially anti-taurine antibody, anti-glycocholic acid antibody preparation method.Meanwhile invention also discloses the detection methods for the peripheral blood glycocholic acid realized using the kit.The present invention can close the interference of taurocholate, and detection sensitivity is up to 0.1ug/mL, and high specificity detects the advantages that repeatability is high and stability is good.
Description
Technical field
The present invention relates to technical field of immunological detection, specially a kind of kit, the preparation method of kit, Yi Jili
With the detection method for the peripheral blood glycocholic acid that the kit is realized.
Background technology
Serum glycocholic acid (Cholyglycine, CG) is metabolite of the cholesterol in liver cell, is in cholic acid
One of key component.The physiological intestines of the cholic acid of participation-liver cycle.In liver cell, cholesterol passes through a series of enzymatic reactions,
It is transformed into primary cholic acid, gall-bladder is discharged into through bile capillaries and bile duct, enters duodenum in company with bile, help food digestion；
95% cholic acid returns liver in terminal ileum reabsorption, trans-portal vein, is absorbed and is recycled by liver cell；Not by re-absorbed courage
Juice acid forms cholane acid derivative in the case where intestines bacterium acts on, and is discharged by excrement, and the total amount for overflowing into body circulation is less than 1%.In positive reason
Under condition, cholic acid content is little in peripheral blood；No matter on an empty stomach or postprandial normal adult is, and Bile Acid in Serum concentration is stablized in low-level.
When liver cell is damaged, liver cell absorbs cholic acid ability and declines, and cholic acid content in blood is caused to increase；When cholestegnosis, liver row
It lets out cholic acid and obstacle occurs, and sanguimotor cholic acid content of backflowing increases, and also increases blood cholic acid content.Therefore, serum is measured
Cholic acid is to evaluate hepatocyte function and its one of the sensitive indicator of liver and gall system substance circulatory function.However in serum cholic acid composition at
Divide complexity, heterogeneous apparent, total cholic acid measurement tends not to sensitivity reflection hepatocellular injury or cholestasis.Glycocholic acid is
One of key component in cholic acid, and property and stable content, can sensitive reflection hepatocellular injury or cholestasis.
Glycocholic acid is one of the mating type cholic acid that cholic acid is combined into glycine.Cholic acid complex component is multiple in serum
It is miscellaneous.Include conjucated bile acids (glycocholic acid, liver goose glycocholeic acid) and free cholic acid by textural classification；Free cholic acid is by sources
Classification includes primary cholic acid (cholic acid, goose glycocholeic acid) and secondary bile acid (deoxycholic acid, lithocholic acid, ursodesoxycholic acid).
The molecular formula C26H43NO6 of CG, molecular weight 465.5；Belong to small haptens substance.
It is reflection intrahepatic cholestasis of pregnancy (Intrahepatic cholestasis of that glycocholic acid, which increases,
Pregnancy, ICP) sensitive indicator.When normal pregnancy, since progesterone level increases in blood, of smooth muscle is reduced
Power causes gestation gallbladder tension to reduce and empty and inhibits, makes liver that obstacle occur to the intake of bile and excretion, cause courage
The different degrees of siltation of juice.Therefore, part gravid woman serum glycocholic acid value may occur in which that physiological increases, and there is no apparent
Intrahepatic cholestasis of pregnancy symptom.When in pregnant woman's lobuli hepatis, central area and capillary with the presence of cholestasis and courage bolt,
Bile excretion obstacle leads to accumulation of the cholic acid in peripheral circulation, causes glycocholic acid content in blood to increase, and generate skin scabies
The symptoms such as itch, then amniotic fluid-pollution rate, early yield, fetal distress in uterus rate and cesarean delivery rate is made to increase.However to ICP pregnant woman when
Terminal pregnancy is still that many doctors are of interest and stubborn problem.Premature end gestation then can increase peri-natal infant because of premature labor
Illness rate and case fatality rate then have the danger of fetal distress in uterus or even Intrauterine Fetal Death again but if not timely terminal pregnancy.
Clear serum Ievel of total bile acids change is the most important Laboratory evidences of ICP, Serum And Bile sour water in ICP practice guidelines in 2015
Flat measurement includes mainly total bile acid and glycocholic acid.Previously (ICP practice guidelines in 2011) are by total bile acid and sweet ammonia courage
Acid is classified as of equal importance.
Accurately and rapidly detection serum glycocholic acid level is a critical issue.The measurement blood of domestic and foreign literature report
Clear glycocholic acid horizontal process has：It is radiommunoassay, efficient liquid phase (ultraviolet/fluorescence) measurement, gas phase and liquid phase-mass spectrum, homogeneous
Enzyme exempts from method etc..Radiommunoassay, which needs to put, exempts from facility, and has radioactive pollution；(ultraviolet) measurement of efficient liquid phase, gas phase/liquid
Phase-mass spectrum is required to equipment costly, is to measure the confirmation method of serum glycocholic acid level, but be not suitable for clinical labororatory
Conventional detection.Although useful immunoturbidimetry measures the glycocholic acid method in sample, i.e., with anti-glycocholic acid antibody latex
, there is agglutinating reaction in grain and the glycocholic acid antigen-reactive in sample, in the variation of its absorbance of 600nm wavelength detectings, become
Change degree is directly proportional to the glycocholic acid content in sample, energy automatic detection, but its specificity needs further to be confirmed.Homogeneously
Immunoenzyme techniques (Homogeneous Enzyme Immunoassay, HEIA) are mainly used for the measurement of small-molecule substance, are one
Competitive reaction of the kind based on liquid phase homogeneous system, and had both the spy of " antigen and antibody " and " enzyme and substrate " two kinds of systems
Point has many advantages, such as that detection speed is fast, it is accurate to quantify, precision is high, high specificity；It can be applied to various types of biochemistry simultaneously
Analyzer realizes the high throughput and automatic assay of sample, but its detection specificity depends on the specificity of antibody used.
Under normal circumstances, glycocholic acid and taurocholate are existed simultaneously in serum.Glycocholic acid and taurocholate structure
Extremely similar, the corresponding antibodies induced with immune animal can have the cross reaction of specificity, i.e., anti-glycocholic acid antibody can be with
The combination of specificity occurs for taurocholate.When using the immunologic detection method based on antibody, cross reaction easily occurs, causes sweet
The inaccuracy that ammonia cholic acid measures.
Invention content
Technical problem solved by the invention is to provide a kind of kit, the preparation method of kit, and using should
The detection method for the peripheral blood glycocholic acid that kit is realized, to solve the problems in above-mentioned background technology.
Technical problem solved by the invention is realized using following technical scheme：
A kind of kit, including reagent R1, reagent R2,
The reagent R1 contains：Tris-HCL (trishydroxymethylaminomethane) buffer solution, anti-taurine antibody resist sweet ammonia courage
Sour antibody, G6P (G-6-P)；
The reagent R2 contains：Tris-HCL (trishydroxymethylaminomethane) buffer solution, glycocholic acid-G-6-PD conjugates
(Hangzhou Bopu Medical Technology Co., Ltd.), NADP (codehydrogenase Ⅱ), BSA (bovine serum albumin(BSA)).
In the present invention, a kind of kit further includes titer,
The titer is：A concentration of 80 μ g/mL glycocholic acid is taken, with including 5g/L BSA, 50mM trihydroxy methyl amino
Methane (Tris-HCL) buffer solution makees doubling dilution into series concentration solution.
In the present invention, a kind of kit further includes quality-control product,
The quality-control product is：Glycocholic acid sodium salt (purity 97%) 1.000mg is taken to be dissolved in 6.25mL trihydroxy methyl amino
Methane (Tris-HCL) buffer solution 50mM, pH6.8, includes 5g/L BSA；A concentration of 160 μ g/mL of glycocholic acid, then again with strong
Health people's fresh serum makees 1:8 and 1:32 dilutions, i.e., glycocholic acid concentration is respectively 20 μ g/mL and 5 μ g/mL.
Further, the reagent R1 contains：The anti-taurine IgG antibodies of 1-10 μ g/mL, 10-100 μ g/mL resist sweet ammonia
Cholic acid taurine antibody, 50-500 μ g/mL G6P, 0.01-0.5%Tween-20,0.3-10mM MgCl2, 0.01-0.1%
NaN3, solvent is the Tris-HCL buffer solutions of pH6.5-8.0；
The reagent R2 contains：0.2-3U/mL glycocholic acid-G6PDH conjugates, 50-1000ug/mLNADP, 0.1-1%
BSA, solvent are the Tris-HCL buffer solutions of pH6.5-8.0.
The titer process for preparation is as follows：Accurately weigh Sigma Co., USA's glycocholic acid sodium salt (purity 97%)
1.000mg is dissolved in 12.5mL trishydroxymethylaminomethanes (Tris-HCL) buffer solution 50mM, pH6.8, includes 5g/L BSA；It is sweet
Ammonia Bile acid concentrations are 80 μ g/mL, and then again with 5g/L BSA, 50mM is included, trishydroxymethylaminomethane (Tris-HCL) buffers
Liquid make doubling dilution be 40 μ g/mL, 20 μ g/mL, 10 μ g/mL, 5 μ g/mL, 2.5 μ g/mL, 1.25 μ g/mL, 0.625 μ g/mL,
0.3125 μ g/mL, 0.16 μ g/mL and 0.08 μ g/mL etc..
The quality-control product is formulated as follows：Accurately weigh Sigma Co., USA glycocholic acid sodium salt (purity 97%) 1.000mg
It is dissolved in 6.25mL trishydroxymethylaminomethanes (Tris-HCL) buffer solution 50mM, pH6.8, includes 5g/L BSA；Glycocholic acid
Then a concentration of 160 μ g/mL use Healthy People fresh serum to make 1 again:8 and 1:32 dilutions, i.e., glycocholic acid concentration is respectively 20 μ
G/mL and 5 μ g/mL.
Kit as described above, wherein the anti-taurine antibody includes anti-taurine monoclonal IgG antibody or anti-ox
The polyclonal IgG antibody of sulfonic acid.
The anti-taurine monoclonal IgG antibody includes one kind in IgG1, IgG2a, IgG2b, IgG3 or IgG4；It is described
The anti-polyclonal IgG antibody of taurine includes ' 2 F (ab) specific Ab fragments.
The anti-glycocholic acid antibody includes anti-glycocholic acid monoclonal IgG antibody or the polyclonal IgG of anti-glycocholic acid anti-
Body.
The anti-glycocholic acid monoclonal IgG antibody is one kind in IgG1, IgG2a, IgG2b, IgG3 or IgG4；It is described
The anti-polyclonal IgG antibody of glycocholic acid specific Ab fragments containing ' 2 F (ab).
The preparation of kit, key are the preparation of anti-taurine antibody and anti-glycocholic acid antibody.
The preparation method of anti-taurine antibody is as follows：Utilize-the NH on taurine2With-the COOH on protein molecule into
Row coupling；It recycles these conjugates that object is immunized and carries out immune mouse or rabbit as immunogene, finally obtain the anti-of specificity
Taurine antibody.
Wherein, anti-taurine monoclonal IgG antibody is mouse monoclonal IgG type antibody, and the anti-polyclonal IgG of taurine is anti-
Body is the polyclonal IgG antibody of rabbit source property.
In above-mentioned steps, the molar ratio of taurine and protein or poly-D-lysine is 1:20-200, with 1:100 be excellent
Choosing.
The preparation method of anti-glycocholic acid antibody is as follows：Utilize-the COOH and protein or more of glycocholic acid branch terminals
- NH2 on polylysine molecule is coupled；It recycles these conjugates that object is immunized and carries out immune mouse or family as immunogene
Rabbit finally obtains the anti-glycocholic acid antibody of specificity.
Wherein, anti-glycocholic acid monoclonal IgG antibody is mouse monoclonal IgG type antibody, and anti-glycocholic acid is polyclonal
IgG antibody is the polyclonal IgG antibody of rabbit source property.
In above-mentioned steps, the molar ratio of glycocholic acid and protein or poly-D-lysine is 1:20-200, with 1:100 be excellent
Choosing.
When preparing anti-glycocholic acid monoclonal IgG antibody, the sweet ammonia deoxidation of the 10 μ g/mL of analog of glycocholic acid is used respectively
Cholic acid, deoxycholic acid, cholic acid, chenocholic acid, chenodesoxycholic acid, lithocholic acid, taurocholate, tauroursodeoxycholic acid carry out blocking detection
Screening retains cross reacting rate<10% antibody cloning hole.
Trishydroxymethylaminomethane (Tris-HCL) buffer solution, G6P, Tween-20, MgCl are prepared again2, NaN3To get to
Reagent R1.
Simultaneously prepare trishydroxymethylaminomethane (Tris-HCL) buffer solution, NADP, glycocholic acid-G6PDH conjugates,
BSA obtains reagent R2, and preparation method does not repeat.
The detection method for the peripheral blood glycocholic acid realized using the kit is exempted from using immunoassay with homogeneous enzyme
Epidemic disease measuring method is preferential, the interference of taurocholate that may be present in sample is first closed using anti-taurine antibody, then sweet with resisting
Ammonia cholic acid antibody carries out the detection of glycocholic acid.The working concentration of anti-taurine antibody is 1-10 μ g/mL, is excellent with 5 μ g/mL
Choosing.
Detection method includes the following steps：
Reagent R1 is mixed, 25-37 DEG C of reaction 3-5min with sample to be tested, adds reagent R2,25-37 DEG C of reaction 5-
10min detects light absorption value at 340nm, and glycocholic acid concentration in sample to be tested is obtained according to standard curve；
In above-mentioned detection method, the drafting of standard curve is as follows：By glycocholic acid 0.25% cow's serum containing volumetric concentration
The buffer solution of albumin is diluted to the dilution of 0 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 40 μ g/mL, 80 μ g/mL, with
Buffer solution containing the bovine serum albumin(BSA) is blank control, by the glycocholic acid dilution of the various concentration and reagent R1 with
Then mixing, 25-37 DEG C of reaction 5-10min are added reagent R2,25-37 DEG C of reaction 5-10min and detect light absorption value at 340nm,
Using light absorption value as ordinate, glycocholic acid concentration makes standard curve as abscissa using in dilution.
As a result of above technical scheme, the invention has the advantages that：
The present invention first participates in reaction with anti-taurine antibody closing taurocholate, then with outside anti-glycocholic acid antibody test
The method of all blood glycocholic acid effectively reduces the specificity of detection glycocholic acid.
Further, the present invention provides a kind of kit, using kit, first can close ox with anti-taurine antibody
Sulphur cholic acid participates in reaction, then with the method for anti-glycocholic acid antibody test peripheral blood glycocholic acid.It is that one kind being based on specificity
The glycocholic acid detection method that monoclonal or polyclonal antibody are established.Meanwhile the preparation method of kit of the present invention includes sweet ammonia
The preparation method of cholic acid and taurine specific monoclonal or polyclonal antibody, and for peripheral blood (including serum, blood plasma and complete
Blood) etc. the detection of glycocholic acid in body fluid.
Further, the present invention is utilized respectively the-COOH and protein or poly of glycocholic acid branch terminals first
- NH2 on lysine molecule is coupled；It is coupled using-NH2 and-the COOH on protein molecule on taurine；It connects
It and object is immunized using these conjugates carries out immune mouse or rabbit as immunogene, finally obtain specific anti-glycocholic acid
Monoclonal or polyclonal antibody and anti-taurine monoclonal or polyclonal antibody.And by the anti-glycocholic acid Dan Ke of above-mentioned specificity
Grand or polyclonal antibody is prepared into corresponding kit with anti-taurine monoclonal or polyclonal antibody.When progress homogeneous enzyme immunoassay
When measurement, first with the taurocholate in anti-taurine monoclonal or polyclonal antibody combination sample, to close the dry of taurocholate
It disturbs, then again with the glycocholic acid content in anti-glycocholic acid monoclonal or polyclonal antibody detection sample.
The principle for the homogeneous EIA method that the present invention is used to detect glycocholic acid in peripheral blood is to be based on sweet ammonia courage
The specific binding of acid and its specific antibody.
Compared with prior art, advantageous effect of the present invention is mainly reflected in：Sweet ammonia courage in detection peripheral blood of the present invention
The method of acid can carry out large sample automatic detection analysis on the large automatics instrument such as Biochemical Analyzer.Method has detection
High sensitivity, high specificity detect the advantages that repeatability is high and stability is good.The method of the present invention detection sensitivity 0.1ug/mL.
Description of the drawings
Fig. 1 is that glycocholic acid is coupled schematic diagram with protein and/or poly-D-lysine；
Fig. 2 is that taurine is coupled schematic diagram with protein and/or poly-D-lysine；
Fig. 3 is that glycocholic acid measures reaction normal curve.
Specific implementation mode
In order to make the technical means, the creative features, the aims and the efficiencies achieved by the present invention be easy to understand, tie below
Specific embodiment is closed, the present invention is further explained.
Kit provided in this embodiment, including reagent R1, reagent R2.
Wherein, the reagent R1 contains：The anti-taurine monoclonal IgG antibodies of 1 μ g/mL, the anti-glycocholic acid ox sulphurs of 10 μ g/mL
Sour monoclonal antibody, 50 μ g/mL G6P, 0.01%Tween-20,0.3mM MgCl2, 0.01%NaN3, solvent is pH6.5's
Tris-HCL buffer solutions；
The reagent R2 contains：0.2U/mL glycocholic acid-G6PDH conjugates, 50ug/mL NADP, 0.1%BSA, solvent
For the Tris-HCL buffer solutions of pH6.5.
In addition, kit may include titer.And titer can also be in kit in use, voluntarily preparing.
The titer process for preparation is as follows：Accurately weigh Sigma Co., USA's glycocholic acid sodium salt (purity 97%)
1.000mg is dissolved in 12.5mL trishydroxymethylaminomethanes (Tris-HCL) buffer solution 50mM, pH6.8, includes 5g/L BSA；It is sweet
Ammonia Bile acid concentrations are 80 μ g/mL, and then again with 5g/L BSA, 50mM is included, trishydroxymethylaminomethane (Tris-HCL) buffers
Liquid make doubling dilution be 40 μ g/mL, 20 μ g/mL, 10 μ g/mL, 5 μ g/mL, 2.5 μ g/mL, 1.25 μ g/mL, 0.625 μ g/mL,
0.3125 μ g/mL, 0.16 μ g/mL and 0.08 μ g/mL etc..
Equally, kit may include quality-control product.And quality-control product can also be in kit in use, voluntarily preparing.
The quality-control product is formulated as follows：Accurately weigh Sigma Co., USA glycocholic acid sodium salt (purity 97%) 1.000mg
It is dissolved in 6.25mL trishydroxymethylaminomethanes (Tris-HCL) buffer solution 50mM, pH6.8, includes 5g/L BSA；Glycocholic acid
Then a concentration of 160 μ g/mL use Healthy People fresh serum to make 1 again:8 and 1:32 dilutions, i.e., glycocholic acid concentration is respectively 20 μ
G/mL and 5 μ g/mL.
The present embodiment essentially describes the preparation method of anti-taurine antibody, anti-glycocholic acid antibody.And the system of kit
Standby, key is that the preparation of anti-taurine antibody and anti-glycocholic acid antibody.
The preparation method of anti-taurine antibody is as follows：Using-the COOH on-NH2 and the protein molecule on taurine into
Row coupling；It recycles these conjugates that object is immunized and carries out immune mouse or rabbit as immunogene, finally obtain the anti-of specificity
Taurine antibody.Wherein, anti-taurine monoclonal IgG antibody is mouse monoclonal IgG type antibody, and anti-taurine is polyclonal
IgG antibody is the polyclonal IgG antibody of rabbit source property.In above-mentioned steps, the molar ratio of taurine and protein or poly-D-lysine is
1:20-200, with 1:100 be preferred.
Trishydroxymethylaminomethane (Tris-HCL) buffer solution is prepared again, glycocholic acid-G-6-PD conjugates are tried
Agent R1, preparation method do not repeat.
The preparation method of anti-glycocholic acid antibody is as follows：Utilize-the COOH and protein or more of glycocholic acid branch terminals
- NH2 on polylysine molecule is coupled；It recycles these conjugates that object is immunized and carries out immune mouse or family as immunogene
Rabbit finally obtains the anti-glycocholic acid antibody of specificity.Wherein, anti-glycocholic acid monoclonal IgG antibody is mouse monoclonal
IgG type antibody, the anti-polyclonal IgG antibody of glycocholic acid are the polyclonal IgG antibody of rabbit source property.In above-mentioned steps, glycocholic acid with
The molar ratio of protein or poly-D-lysine is 1:20-200, with 1:100 be preferred.It is anti-to prepare anti-glycocholic acid monoclonal IgG
When body, respectively with the glycodesoxycholic acid of the 10 μ g/mL of analog of glycocholic acid, deoxycholic acid, cholic acid, chenocholic acid, goose deoxidation
Cholic acid, lithocholic acid, taurocholate, tauroursodeoxycholic acid carry out blocking detection screening, retain cross reacting rate<10% antibody gram
Grand hole.
Prepare trishydroxymethylaminomethane (Tris-HCL) buffer solution, NADPH, G-6-P, BSA again to obtain the final product
To reagent R2, preparation method does not repeat.
The detection method for the peripheral blood glycocholic acid realized using the kit is exempted from using immunoassay with homogeneous enzyme
Epidemic disease measuring method is preferential, the interference of taurocholate that may be present in sample is first closed using anti-taurine antibody, then sweet with resisting
Ammonia cholic acid antibody carries out the detection of glycocholic acid.The working concentration of anti-taurine antibody is 1-10 μ g/mL, is excellent with 5 μ g/mL
Choosing.
Detection method includes the following steps：
Reagent R1 is mixed, 25-37 DEG C of reaction 3-5min with sample to be tested, adds reagent R2,25-37 DEG C of reaction 5-
10min detects light absorption value at 340nm, and glycocholic acid concentration in sample to be tested is obtained according to standard curve；
In above-mentioned detection method, the drafting of standard curve is as follows：By glycocholic acid 0.25% cow's serum containing volumetric concentration
The buffer solution of albumin is diluted to the dilution of 0 μ g/mL, 1.25 μ g/mL, 2.5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 40 μ g/mL
Liquid, using the buffer solution containing the bovine serum albumin(BSA) as blank control, by the glycocholic acid dilution of the various concentration and examination
Agent R1 with mix, then reagent R2,25-37 DEG C of reaction 5-10min, detection 340nm place's suctions is added in 25-37 DEG C of reaction 5-10min
Light value, using light absorption value as ordinate, glycocholic acid concentration makes standard curve as abscissa using in dilution.
More detailed preparation method, detection method, steps are as follows.
1. protein conjugate is prepared (as depicted in figs. 1 and 2)
It is prepared by 1.1 glycocholic acid-protein conjugate
Weigh 100mg glycocholic acid (Sigma Co., USA) and 10mg bovine serum albumin(BSA)s (BSA) or hemocyanin
(KLH) or ovalbumin (OVA) or poly-D-lysine (German Roche companies) are dissolved in pH7.6, the phosphate-buffered of 100mM
It in liquid, is stirred at room temperature 15 minutes, adds 125mg water-soluble carbodiimides powder (Shanghai Aladdin company), room temperature is protected from light temperature
With stirring 180 minutes, it is packed into bag filter (freely penetrating molecular size<14KD), 4 DEG C of dialysis are set, change liquid 1 time within every 6 hours, continuously
6-8 times.
1.2 glycocholic acid protein conjugate coupling efficiencies are identified
The phosphate buffer of glycocholic acid-protein conjugate pH7.6,100mM of 1.1 couplings is taken to be diluted to concentration
<1mg/mL uses the absorbance of spectrophotometric determination wavelength 210nm, 280nm, coupling efficiency is calculated according to the following formula respectively, when
Glycocholic acid:Protein>20:When 1, it is judged to qualification.
Coupling efficiency=A210х69000/A280х483
It is prepared by 1.3 taurines-protein conjugate
Weigh 100mg taurines (Sigma Co., USA) and 10mg bovine serum albumin(BSA)s or hemocyanin or ovalbumin
(German Roche companies) is dissolved in pH7.6, in the phosphate buffer of 100mM, is stirred at room temperature 15 minutes, adds 125mg water
Soluble carbodiimide powder (Shanghai Aladdin company), room temperature is protected from light gentle agitation 180-240 minutes, is packed into bag filter (freely
Penetrating molecular size<14KD), 4 DEG C of dialysis are set, change liquid 1 time within every 6 hours, it is 6-8 times continuous.
2. anti-glycocholic acid monoclonal IgG type preparation method for antibody
2.1 animal immune
With above-mentioned steps 1.1 prepare glycocholic acid-protein conjugate, be with glycocholic acid-hemocyanin it is preferred, with
100 μ g glycocholic acid-concentration of protein conjugate/time/mouse and the Split completely mixing of equivalent, subcutaneous multiple spot note
Penetrate immune Balb/c female mices (5-6 week old, weight 18-20g), the 1st time with the 2nd minor tick 10-14 days, later every minor tick
7 days, continuous 5 times, the incomplete freund adjuvant emulsification mixing of 2-5 glycocholeic acids-protein conjugate and equivalent, interval 1
100 μ g (the 100 μ L volumes) glycocholic acid-poly-D-lysine conjugate for using tail vein/intraperitoneal injection sterile after week is (quiet with tail
Arteries and veins is preferred), with cell fusion after booster immunization 1 time, 3 days.
2.2 the preparation of hybridoma
It is carried out according to regular growth fusion method.Take step 2.1 be immunized the splenocyte of mouse and SP2/0 myeloma cell by
Volume ratio 1:6 merge under the action of 50%PEG (MW400-800), and fused cell is first in hypoxanthine-methotrexate-thymus gland
In pyrimidine (HAT) Selective agar medium (Hyclone companies of the U.S.), in 37 DEG C, 5%CO2And it is cultivated under the conditions of saturated humidity, 2 weeks
After be changed to hypoxanthine-thymidine (HT) culture medium (Hyclone companies of the U.S.), continue in 37 DEG C, 5%CO2And saturation is wet
It is cultivated under the conditions of degree.When cloning Kong Changzhi 1/3-1, culture supernatant is taken to carry out antibody test screening.
2.3 specificity clone hole screenings
Positive hole is expressed with ELISA method screening antibodies.It is even with carbonate buffer solution dilution glycocholic acid-protein of 50mM
After joining object (be preferred with glycocholic acid-OVA) to 10 μ g/mL, it is overnight or 37 DEG C that 4 DEG C of micropore is reacted with 100 holes μ L/ coating
120min, with containing 0.05%Tween-20 phosphate buffer (PBS) or Tris-HCL buffer solutions (PBS) wash 3 times, use
10% calf serum room temperature close 30min, add 100 μ l above-mentioned steps, 2.2 culture supernatant room temperature reaction 60min, with containing
The phosphate buffer (PBS) or Tris-HCL buffer solutions (PBS) of 0.05%Tween-20 washs 3 times, finally adds 1:
2000 diluted goat anti-mouse igg-HRP react at room temperature 60min, with the phosphate buffer (PBS) containing 0.05%Tween-20
Or Tris-HCL buffer solutions (PBS) wash 3 times, and the colour developing observation of tetramethyl biphenyl ammonia (TMB) substrate is added.It is all obvious blue occur
Reactor is the positive, is otherwise feminine gender.Reservation has the cell clone hole reacted with glycocholic acid-protein conjugate, then uses
Limiting dilution assay carries out 3 time cloning screenings, and the hybridoma for building strain expands culture, freezes again, is used to prepare ascites.
The preparation and purification of 2.4 ascites
After the hybridoma of stably excreting monoclonal antibody is expanded culture, with 0.5 × 104~5 × 105It is a/to be only inoculated with
To the abdominal cavity for using 7-10 days Balb/c mouse of 0.5mL norphytanes or atoleine sensitization in advance, observation, collects abdomen in 7-12 days
Water measures potency after centrifugation.Through the preservation of 50% saturated ammonium sulphate or cryopreservation.Ascites uses 50% saturated ammonium sulfate
Precipitation-caprylic acid precipitation, or with the 50% affine layers of saturated ammonium sulphate-rProteinA-Sepharose Fast Flow
Analysis purifying, finally detects purity with SDS-PAGE, need to reach 90%.Anti- glycocholic acid-protein conjugate prepared by this step
Monoclonal antibody specific can occur specific binding with glycocholic acid-protein conjugate and react, without with non-glycocholic acid-
Nonspecific reaction occurs for protein conjugate.
3. the anti-polyclonal IgG antibody preparation method of glycocholic acid-protein conjugate
With above-mentioned steps 1.1 prepare glycocholic acid-protein conjugate, with 10mg glycocholic acid-protein conjugate/
Secondary/only with equivalent Split completely mixing, it is preferred with glycocholic acid-hemocyanin, the subcutaneous multi-point injection of rabbit is immune.Often
1-2 weeks 1 time, continuous 5 times, interval, using separation arteria carotis, collects about 80-120 milliliters of blood, is placed at room temperature for 2 hours after 1 week, and 4
DEG C 10000rpm centrifuges 30min, separates and collects serum.Above-mentioned serum uses glycocholic acid-protein conjugate (with sweet ammonia courage again
Acid-OVA is preferred), the specific IgG type antibody of the anti-glycocholic acid of affinity chromatography column purification is finally detected with 8%SDS-PAGE
Purity need to reach 90%.Obtain the polyclonal IgG antibody of rabbit source property.
To obtain the F (ab) ' of anti-glycocholic acid2Specific Ab fragments, the polyclonal IgG antibody of rabbit source property of above-mentioned purifying
The Fc ' for further cutting off IgG with the pepsin of 0.1% (mass ratio) in the 50mM acetate buffers of pH 4.5 is held, and 37
DEG C reaction 12 hours, then adjusting or in the case of uncomfortable pH value, with saturated ammonium sulfate and/or SPA and/or glycocholic acid-egg
White matter conjugate affinity chromatography method purifies F (ab) '2Antibody fragment.Obtain the F (ab) ' of the anti-glycocholic acid of specificity2Specifically
Property antibody fragment.
Anti- glycocholic acid Anti-TNF-α physical efficiency manufactured in the present embodiment occurs specific binding with glycocholic acid and reacts, without
Nonspecific reaction occurs with non-glycocholic acid-protein conjugate.
4. anti-taurine monoclonal IgG type preparation method for antibody
4.1 animal immune
It is preferred with taurine-hemocyanin, with 100 μ with taurine-protein conjugate prepared by above-mentioned steps 1.2
The concentration of g taurines-protein conjugate/time/mouse and the Split completely mixing of equivalent, subcutaneous multi-point injection are immune
Balb/c female mices (5-6 week old, weight 18-20g), the 1st time with the 2nd minor tick 10-14 days, later per minor tick 7 days, company
5 times continuous, the incomplete freund adjuvant of 2-5 hypotaurines-protein conjugate and equivalent emulsifies mixing, and interval uses after 1 week
Taurine-poly-D-lysine conjugate (being preferred with tail vein) sterile 100 μ g of tail vein/intraperitoneal injection (100 μ L volumes),
With cell fusion after booster immunization 1 time, 3 days.
The preparation of 4.2 hybridomas
It is carried out according to regular growth fusion method.Take step 2.1 be immunized the splenocyte of mouse and SP2/0 myeloma cell by
Volume ratio 1:6 merge under the action of 50%PEG (MW400-800), and fused cell is first in hypoxanthine-methotrexate-thymus gland
In pyrimidine (HAT) Selective agar medium, in 37 DEG C, 5%CO2And cultivated under the conditions of saturated humidity, hypoxanthine-chest is changed to after 2 weeks
Gland pyrimidine (HT) culture medium continues in 37 DEG C, 5%CO2And it is cultivated under the conditions of saturated humidity.When cloning Kong Changzhi 1/3-1, take
Culture supernatant carries out antibody test screening.
4.3 specificity clone hole screenings
Positive hole is expressed with ELISA method screening antibodies.With carbonate buffer solution dilution taurine-protein coupling of 50mM
After object (being preferred with taurine-OVA) to 10 μ g/mL, 4 DEG C of micropore of reaction is coated with overnight or 37 DEG C of 120min with 100 holes μ L/,
With containing 0.05%Tween-20 phosphate buffer (PBS) or Tris-HCL buffer solutions (PBS) wash 3 times, with 10% calf
Serum room temperature closes 30min, 100 μ l above-mentioned steps, 2.2 culture supernatant room temperature reaction 60min is added, with containing 0.05%
The phosphate buffer (PBS) or Tris-HCL buffer solutions (PBS) of Tween-20 washs 3 times, finally adds 1:2000 dilutions
Goat anti-mouse igg-HRP react at room temperature 60min, with phosphate buffer (PBS) or Tris- containing 0.05%Tween-20
HCL buffer solutions (PBS) wash 3 times, and the colour developing observation of tetramethyl biphenyl ammonia (TMB) substrate is added.It is all obvious blue reactor occur
It is otherwise feminine gender for the positive.Reservation has the cell clone hole reacted with taurine-protein conjugate, then uses limiting dilution
Method carries out 3 time cloning screenings, and the hybridoma for building strain expands culture, freezes again, is used to prepare ascites.
The preparation and purification of 4.4 ascites
After the hybridoma of stably excreting monoclonal antibody is expanded culture, with 0.5 × 104~5 × 105It is a/to be only inoculated with
To the abdominal cavity for using 7-10 days Balb/c mouse of 0.5mL norphytanes or atoleine sensitization in advance, observation, collects abdomen in 7-12 days
Water measures potency after centrifugation.Through the preservation of 50% saturated ammonium sulphate or cryopreservation.Ascites uses 50% saturated ammonium sulfate
Precipitation-caprylic acid precipitation, or with the 50% affine layers of saturated ammonium sulphate-rProteinA-Sepharose Fast Flow
Analysis purifying, finally detects purity with SDS-PAGE, need to reach 90%.Anti- taurine prepared by this step-protein conjugate is special
Specific monoclonal antibodies can occur specific binding with taurine-protein conjugate and react, without with non-taurine-protein
Nonspecific reaction occurs for conjugate.
5. the anti-polyclonal IgG antibody preparation method of taurine-protein conjugate
With above-mentioned steps 1.1 prepare taurine-protein conjugate, with 10mg taurines-protein conjugate/time/
Only with equivalent Split completely mixing, the subcutaneous multi-point injection of rabbit is immune.Per 1-2 weeks 1 time, continuous 5 times, interval is adopted after 1 week
With separation arteria carotis, about 80-120 milliliters of blood is collected, is placed at room temperature for 2 hours, 4 DEG C of 10000rpm centrifuge 30min, separate and collect
Serum.Above-mentioned serum uses the specific IgG type antibody of taurine-anti-taurine of protein conjugate affinity chromatography column purification again,
Purity finally is detected with 8%SDS-PAGE, 90% need to be reached.Obtain the polyclonal IgG antibody of rabbit source property.
To obtain the F (ab) ' of anti-taurine2Specific Ab fragments, the polyclonal IgG antibody of rabbit source property of above-mentioned purifying into
The Fc ' that one step cuts off IgG in the 50mM acetate buffers of pH 4.5 with the pepsin of 0.1% (mass ratio) is held, 37 DEG C
Reaction 12 hours, then in the case where adjusting or uncomfortable pH value, with saturated ammonium sulfate and/or SPA and/or taurine-protein
Conjugate affinity chromatography method purifies F (ab) '2Antibody fragment.Obtain the anti-taurine F (ab) of specificity '2Specific antibody piece
Section.
Anti- taurine Anti-TNF-α physical efficiency manufactured in the present embodiment is specifically bound with taurine-protein conjugate
Reaction, without nonspecific reaction occurs with non-taurine-protein conjugate.
6. homogeneous enzyme immunoassay method measures serum glycocholic acid content
It is carried out using glucose-6-phosphate dehydrogenase (G6PD) (G-6-PD) method.
6.1 main agents constituents
Reagent R1：The polyclonal IgG antibody of the anti-taurines of 10ug/mL (including ' 2 F (ab) specific Ab fragments), 100 μ g/
The anti-polyclonal IgG antibodies of glycocholic acid taurine of mL (include ' 2 F (ab) specific Ab fragments), 500 μ g/mL G6P, 0.5%
Tween-20,10mM MgCl2, 0.1%NaN3, solvent is the Tris-HCL buffer solutions of pH8.0；
Reagent R2：3U/mL glycocholic acid-G6PDH conjugates, 1000ug/mL NADP, 1%BSA, solvent is pH8.0's
Tris-HCL buffer solutions.
Titer is prepared：Accurately Sigma Co., USA glycocholic acid sodium salt (purity 97%) 1.000mg is weighed to be dissolved in
12.5mL trishydroxymethylaminomethanes (Tris-HCL) buffer solution 50mM, pH6.8, includes 5g/LBSA；Glycocholic acid a concentration of 80
μ g/mL, then again with including 5g/L BSA, 50mM, trishydroxymethylaminomethane (Tris-HCL) buffer solution is as doubling dilution
40μg/mL、20μg/mL、10μg/mL、5μg/mL、2.5μg/mL、1.25μg/mL、0.625μg/mL、0.3125μg/mL、0.16
μ g/mL and 0.08 μ g/mL etc. take and suitably do calibration curve.
Quality-control product is prepared：Accurately Sigma Co., USA glycocholic acid sodium salt (purity 97%) 1.000mg is weighed to be dissolved in
6.25mL trishydroxymethylaminomethanes (Tris-HCL) buffer solution 50mM, pH6.8, includes 5g/LBSA；Glycocholic acid is a concentration of
Then 160 μ g/mL use Healthy People fresh serum to make 1 again:8 and 1:32 dilutions, i.e., glycocholic acid concentration is respectively 20 μ g/mL and 5
μg/mL。
6.2 detecting step
Reaction type：Performance rate method temperature：37 DEG C of cuvette optical paths：0.6cm
Master/slave wavelength：340nm units：U/ml the Direction of Reaction：Rise
6.3 results determine：
The difference (A calibration-A blank) for calculating calibration object absorbance, establishes suitable mathematical modulo (non-linear) such as Logit-
Log etc. is fitted to the calibration curve (such as Fig. 3) of multiple spot calibration.According to reactions change amplitude, the range of linearity, the phase of calibration curve
Relationship number and reaction repeatability determine serum glycocholic acid content.
7. anti-glycocholic acid-protein conjugate antibody crossing-over rate measures
7.1 glycocholic acid analogs are prepared
Glycocholic acid analog includes glycodesoxycholic acid, deoxycholic acid, cholic acid, chenocholic acid, chenodesoxycholic acid, stone courage
Acid, taurocholate, tauroursodeoxycholic acid etc. are purchased from (Shanghai Aladdin company).Above-mentioned each cholate is dissolved in comprising 0.1%
In trishydroxymethylaminomethane (Tris-HCL) buffer solution (100mM, pH8.0) of Tween-80, concentration is 40 μ g/mL.
7.2 main agents constituents
Reagent R1：The anti-taurine monoclonal IgG antibodies of 1ug/mL, the anti-glycocholic acid taurine monoclonal antibodies of 10ug/mL,
50ug/mL G6P, 0.01%Tween-20,0.3mM MgCl2, 0.01%NaN3, the Tris-HCL that solvent is pH6.5 are buffered
Liquid；
Reagent R2：0.2U/mL glycocholic acid-G6PDH conjugates, 50ug/mL NADP, 0.1%BSA, solvent pH6.5
Tris-HCL buffer solutions.
Titer is prepared：Accurately Sigma Co., USA glycocholic acid sodium salt (purity 97%) 1.000mg is weighed to be dissolved in
12.5mL trishydroxymethylaminomethanes (Tris-HCL) buffer solution 50mM, pH6.8, includes 5g/LBSA；Glycocholic acid a concentration of 80
μ g/mL, however again with including 5g/L BSA, 50mM, trishydroxymethylaminomethane (Tris-HCL) buffer solution is as doubling dilution
40μg/mL、20μg/mL、10μg/mL、5μg/mL、2.5μg/mL、1.25μg/mL、0.625μg/mL、0.3125μg/mL、0.16
μ g/mL and 0.08 μ g/mL etc. take and suitably do calibration curve.
Quality-control product is prepared：Accurately Sigma Co., USA glycocholic acid sodium salt (purity 97%) 1.000mg is weighed to be dissolved in
6.25mL trishydroxymethylaminomethanes (Tris-HCL) buffer solution 50mM, pH6.8, includes 5g/LBSA；Glycocholic acid is a concentration of
Then 160 μ g/mL use Healthy People fresh serum to make 1 again:8 and 1:32 dilutions, i.e., glycocholic acid concentration is respectively 20 μ g/mL and 5
μg/mL。
7.3 detecting step
Reaction type：Performance rate method temperature：37 DEG C of cuvette optical paths：0.6cm
Master/slave wavelength：340nm units：U/ml the Direction of Reaction：Rise
7.4 results determine：
The difference (A calibration-A blank) of calibration object absorbance is calculated, using absorbance as ordinate, a concentration of abscissa is built
The calibration curve of vertical multiple spot calibration, measures the concentration of known various analogs in table 1, measured concentration and known concentration (40ug/
ML) ratio is inhibiting rate.It the results are shown in Table 1.
Table 1
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (8)
1. a kind of kit, it is characterised in that：Including reagent R1, reagent R2, the reagent R1 contains：Trishydroxymethylaminomethane
Buffer solution, anti-taurine antibody, anti-glycocholic acid antibody, G6P (G-6-P)；
The reagent R2 contains：TRIS buffer, glycocholic acid-G-6-PD conjugates, NADP (codehydrogenase Ⅱ),
BSA (bovine serum albumin(BSA)).
2. a kind of kit as described in claim 1, it is characterised in that：Further include titer,
The titer is：A concentration of 80 μ g/mL glycocholic acid is taken, with including 5g/L BSA, 50mM trishydroxymethylaminomethanes
Buffer solution makees doubling dilution into series concentration solution.
3. a kind of kit as claimed in claim 2, it is characterised in that：Further include quality-control product,
The quality-control product is：The glycocholic acid sodium salt 1.000mg of purity 97% is taken to be dissolved in 6.25mL trishydroxymethylaminomethanes
Buffer solution 50mM, pH6.8 include 5g/L BSA；Then a concentration of 160 μ g/mL of glycocholic acid use Healthy People fresh serum to make again
1:8 and 1:32 dilutions.
4. a kind of kit as claimed in claim 3, it is characterised in that：The anti-taurine antibody includes anti-taurine Dan Ke
Grand IgG antibody or the polyclonal IgG antibody of anti-taurine.
5. a kind of kit as claimed in claim 4, it is characterised in that：The anti-taurine monoclonal IgG antibody includes
One kind in IgG1, IgG2a, IgG2b, IgG3 or IgG4；The polyclonal IgG antibody of anti-taurine includes that ' 2 F (ab) are special
Property antibody fragment.
6. a kind of kit as claimed in claim 3, it is characterised in that：The anti-glycocholic acid antibody includes anti-glycocholic acid
Monoclonal IgG antibody or the polyclonal IgG antibody of anti-glycocholic acid.
7. a kind of kit as claimed in claim 6, it is characterised in that：The anti-glycocholic acid monoclonal IgG antibody is
One kind in IgG1, IgG2a, IgG2b, IgG3 or IgG4；The polyclonal IgG antibody of anti-glycocholic acid is special containing ' 2 F (ab)
Property antibody fragment.
8. the method for preparing kit as described in claim 1, it is characterised in that：The preparation method of anti-taurine antibody therein
It is as follows：Utilize-the NH on taurine2It is coupled with-the COOH on protein molecule；Recycle these conjugates as immune
Original carries out immune mouse or rabbit, finally obtains the anti-taurine antibody of specificity, and the molar ratio of taurine and protein is 1:
20-200；The preparation method of anti-glycocholic acid antibody therein is as follows：Utilize-the COOH and protein of glycocholic acid branch terminals
- NH2 on molecule is coupled；It recycles these conjugates to carry out immune mouse or rabbit as immunogene, finally obtains special
The molar ratio of anisotropic anti-glycocholic acid antibody, glycocholic acid and protein is 1:20-200.
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| 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 |
| CN109884317B (en) * | 2019-03-12 | 2022-03-08 | 杭州博谱医药科技有限公司 | Application of oxidized thio-coenzyme I in homogeneous enzyme immunodiagnosis reagent |
| 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 |
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| US4550163A (en) * | 1979-02-05 | 1985-10-29 | Abbott Laboratories | Ligand analog-irreversible enzyme inhibitor conjugates |
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| JPH0737982B2 (en) * | 1992-12-14 | 1995-04-26 | 元成 狩野 | Anti-bile acid antibody and method for examining bile acid in feces using the same |
| 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 |
| CN105301255B (en) * | 2014-04-18 | 2016-09-07 | 安徽大千生物工程有限公司 | A kind of kit preparation method measuring human body content of glycocholic acid |
| CN104459113B (en) * | 2014-12-05 | 2016-03-16 | 重庆中元生物技术有限公司 | A kind of glycocholic acid detection kit |
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| CN105481977A (en) * | 2015-10-30 | 2016-04-13 | 北京华信缘生物技术有限公司 | Glycocholic-acid immunodetection reagent based on anti-glycocholic acid specific antibody and preparation method thereof |
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