CN106226512A - A kind of test kit, the preparation method of test kit and utilize the detection method of peripheral blood glycocholic acid that this test kit realizes - Google Patents

A kind of test kit, the preparation method of test kit and utilize the detection method of peripheral blood glycocholic acid that this test kit realizes Download PDF

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CN106226512A
CN106226512A CN201610620346.0A CN201610620346A CN106226512A CN 106226512 A CN106226512 A CN 106226512A CN 201610620346 A CN201610620346 A CN 201610620346A CN 106226512 A CN106226512 A CN 106226512A
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glycocholic acid
antibody
taurine
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胡清
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HANGZHOU BOPU MEDICAL TECHNOLOGY CO LTD
HANGZHOU LI'AN BIOLOGICAL TECHNOLOGY Co Ltd
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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

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Abstract

The invention discloses a kind of test kit, including reagent R1, reagent R2, described reagent R1 contains: trishydroxymethylaminomethane (Tris HCL) buffer, anti-taurine antibody, anti-glycocholic acid antibody, glucose 6 phosphoric acid (G6P)；Described reagent R2 contains: trishydroxymethylaminomethane (Tris HCL) buffer, glycocholic acid G 6 PD conjugate, NADP, BSA.The present invention discloses the preparation method of test kit, the most anti-taurine antibody, the preparation method of anti-glycocholic acid antibody.Meanwhile, invention also discloses the detection method of the peripheral blood glycocholic acid utilizing this test kit to realize.The present invention can close the interference of taurocholic acid, and detection sensitivity is up to the advantages such as 0.1ug/mL, high specificity, detection repeatability height and good stability.

Description

A kind of test kit, the preparation method of test kit and utilize the periphery that this test kit realizes The detection method of blood glycocholic acid
Technical field
The present invention relates to technical field of immunological detection, a kind of test kit, the preparation method of test kit, Yi Jili By the detection method of the peripheral blood glycocholic acid that this test kit realizes.
Background technology
Serum glycocholic acid (Cholyglycine, CG) is cholesterol metabolite in hepatocyte, is in cholic acid One of key component.The physiological intestinal of the cholic acid participated in-liver circulation.In hepatocyte, cholesterol through a series of enzymatic reactions, It is transformed into primary cholic acid, enters gallbladder through bile capillary and bile duct, enter duodenum in company with bile, help food digestion； 95% cholic acid heavily absorbs at terminal ileum, and trans-portal vein returns liver, by hepatocyte picked-up recycling；Not by re-absorbed gallbladder Juice acid forms cholane acid derivative under Enterobacter cloaca effect, feces discharge, and overflows into the total amount of body circulation less than 1%.In positive reason Under condition, in peripheral blood, cholic acid content is little；Adult normal is the most on an empty stomach or after the meal, its Bile Acid in Serum concentration is stable in low-level. When hepatocyte is impaired, hepatocyte picked-up cholic acid ability declines, and causes cholic acid content in blood to increase；During cholestegnosis, liver is arranged Let out cholic acid generation obstacle, and the sanguimotor cholic acid content that backflows increases, and also makes blood cholic acid content increase.Therefore, serum is measured Cholic acid is one of sensitive indicator evaluating hepatocyte function and liver and gall system material circulatory function thereof.But cholic acid forms in serum Dividing complexity, heterogeneous obvious, total bile acid measures and tends not to sensitivity reflection hepatocyte injury or cholestasis.Glycocholic acid is One of key component in cholic acid, and character and stable content, can sensitive reflection hepatocyte injury or cholestasis.
Glycocholic acid is one of conjunction type cholic acid of being combined into glycine of cholic acid.In serum, cholic acid complex component is multiple Miscellaneous.Conjucated bile acids (glycocholic acid, liver goose glycocholeic acid) and free cholic acid is included 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, ursodeoxycholic acid). Molecular formula C26H43NO6 of CG, its molecular weight 465.5；Belong to small haptens material.
It is reflection intrahepatic cholestasis of pregnancy (Intrahepatic cholestasis of that glycocholic acid increases Pregnancy, ICP) sensitive indicator.During normal pregnancy, due to the increasing of progesterone level in blood, reduce smooth muscle Power, during causing gestation, gallbladder tension reduces and emptying suppression, makes liver that the picked-up of bile and excretion are occurred obstacle, causes gallbladder Juice alluvial in various degree.Therefore, part gravid woman's serum glycocholic acid value may occur in which that physiological raises, the most significantly Intrahepatic cholestasis of pregnancy symptom.When in anemia of pregnant woman's lobules of liver, central area and blood capillary with the presence of cholestasis and gallbladder bolt, Bile excretion obstacle causes cholic acid accumulation in peripheral circulation, causes glycocholic acid content in blood to increase, and produces skin scabies The symptom such as itch, then make amniotic fluid-pollution rate, early productivity, fetal distress in uterus rate and cesarean delivery rate increase.But to ICP anemia of pregnant woman when Termination of pregnancy, is still many doctors and is paid close attention to and stubborn problem.Premature end gestation then can increase peri-natal infant because of premature labor Prevalence and case fatality rate, if but not timely termination of pregnancy, there is the most again the danger of fetal distress in uterus, even Intrauterine Fetal Death. In ICP practice guidelines in 2015, clear and definite serum Ievel of total bile acids changes is the topmost Laboratory evidence of ICP, Serum And Bile sour water Flat mensuration mainly includes TOTAL BILE ACID TBA and glycocholic acid.Previously (ICP practice guidelines in 2011) is by TOTAL BILE ACID TBA and sweet ammonia gallbladder Acid is classified as of equal importance.
Detection serum glycocholic acid level is a key issue accurately and rapidly.The mensuration blood of domestic and foreign literature report Clear glycocholic acid horizontal process has: radioimmunoassay, efficient liquid phase (ultraviolet/fluorescence) mensurations, gas phase and liquid phase-mass spectrum, homogeneously Enzyme exempts from method etc..Radioimmunoassay needs to put exempts from facility, has again radioactive pollution；Efficiently liquid phase (ultraviolet) mensuration, gas phase/liquid Phase-mass spectrum is required to equipment costly, is the confirmation method measuring serum glycocholic acid level, but is not suitable for clinical laboratory Conventional sense.Despite measuring the glycocholic acid method in sample with immunoturbidimetry, i.e. with anti-glycocholic acid antibody latex , there is agglutination in grain and the glycocholic acid antigen-reactive in sample, and in the change of 600nm its absorbance of wavelength detecting, it becomes Change degree is directly proportional to the glycocholic acid content in sample, energy Aulomatizeted Detect, but its specificity needs to be further characterized by.Homogeneously Immunoenzyme techniques (Homogeneous Enzyme Immunoassay, HEIA) is mainly used in the mensuration of small-molecule substance, is one Plant competitive reaction based on liquid phase homogeneous system, and have the spy of " antigen and antibody " and " enzyme and substrate " two kinds of systems concurrently Point, has the advantages such as detection speed fast, the most accurate, precision height, high specificity；Can be applicable to various types of biochemistry simultaneously Analyser, it is achieved the high flux of sample and automatic assay, but its detection specificity depends on the specificity of antibody used.
Under normal circumstances, serum also exists glycocholic acid and taurocholic acid simultaneously.Glycocholic acid and taurocholic acid structure The most similar, specific cross reaction can be there is with the corresponding antibodies of immune animal induction, the most anti-glycocholic acid antibody can be with There is specific combination in taurocholic acid.When using immunologic detection method based on antibody, easily there is cross reaction, cause sweet It is inaccurate that ammonia cholic acid measures.
Summary of the invention
Technical problem solved by the invention is to provide the preparation method of a kind of test kit, test kit, and utilization should The detection method of the peripheral blood glycocholic acid that test kit realizes, thus solve the problem in above-mentioned background technology.
Technical problem solved by the invention realizes by the following technical solutions:
A kind of test kit, including reagent R1, reagent R2,
Described reagent R1 contains: Tris-HCL (trishydroxymethylaminomethane) buffer, anti-taurine antibody, anti-sweet ammonia gallbladder Acid antibody, G6P (G-6-P)；
Described reagent R2 contains: Tris-HCL (trishydroxymethylaminomethane) buffer, glycocholic acid-G-6-PD conjugate (Hangzhou Bopu Medical Technology Co., Ltd.), NADP (codehydrogenase Ⅱ), BSA (bovine serum albumin).
In the present invention, a kind of test kit, also include titer,
Described titer is: taking concentration is 80 μ g/mL glycocholic acid, with including 5g/L BSA, 50mM trihydroxy methyl amino Methane (Tris-HCL) buffer becomes series concentration solution as doubling dilution.
In the present invention, a kind of test kit, also include quality-control product,
Described quality-control product is: takes glycocholic acid sodium salt (purity 97%) 1.000mg and is dissolved in 6.25mL trihydroxy methyl amino Methane (Tris-HCL) buffer 50mM, pH6.8, include 5g/L BSA；Glycocholic acid concentration is 160 μ g/mL, the most again with strong Health people's fresh serum makees 1:8 and 1:32 dilution, i.e. glycocholic acid concentration is respectively 20 μ g/mL and 5 μ g/mL.
Further, described reagent R1 contains: 1-10 μ g/mL anti-taurine IgG antibody, and 10-100 μ g/mL resists 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 of pH6.5-8.0；
Described reagent R2 contains: 0.2-3U/mL glycocholic acid-G6PDH conjugate, 50-1000ug/mLNADP, 0.1-1% BSA, solvent is the Tris-HCL buffer of pH6.5-8.0.
Described 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 trishydroxymethylaminomethane (Tris-HCL) buffer 50mM, pH6.8, includes 5g/L BSA；Sweet Ammonia Bile acid concentrations is 80 μ g/mL, and the most again with including 5g/L BSA, 50mM, trishydroxymethylaminomethane (Tris-HCL) buffers Liquid as 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..
Described quality-control product is formulated as follows: accurately weigh Sigma Co., USA's glycocholic acid sodium salt (purity 97%) 1.000mg It is dissolved in 6.25mL trishydroxymethylaminomethane (Tris-HCL) buffer 50mM, pH6.8, includes 5g/L BSA；Glycocholic acid Concentration is 160 μ g/mL, makees 1:8 and 1:32 dilution with Healthy People fresh serum the most again, i.e. glycocholic acid concentration is respectively 20 μ G/mL and 5 μ g/mL.
Test kit as above, wherein, described anti-taurine antibody includes anti-taurine monoclonal IgG antibody or anti-cattle Sulfonic acid polyclone IgG antibody.
Described anti-taurine monoclonal IgG antibody includes the one in IgG1, IgG2a, IgG2b, IgG3 or IgG4；Described Anti-taurine polyclone IgG antibody comprises F (ab) ' 2 specific Ab fragments.
Described anti-glycocholic acid antibody includes that anti-glycocholic acid monoclonal IgG antibody or anti-glycocholic acid polyclone IgG resist Body.
Described anti-glycocholic acid monoclonal IgG antibody is the one in IgG1, IgG2a, IgG2b, IgG3 or IgG4；Described Anti-glycocholic acid polyclone IgG antibody specific Ab fragments Han F (ab) ' 2.
The preparation of test kit, it is critical only that anti-taurine antibody and the preparation of anti-glycocholic acid antibody.
The preparation method of anti-taurine antibody is as follows: utilize-the NH on taurine2Enter with-the COOH on protein molecule Row coupling；Recycle these conjugate immunity things and carry out immune mouse or rabbit as immunogen, finally obtain specific anti- Taurine antibody.
Wherein, anti-taurine monoclonal IgG antibody is mouse monoclonal IgG type antibody, and anti-taurine polyclone IgG resists Body is rabbit source property polyclone IgG antibody.
In above-mentioned steps, taurine is 1:20-200 with the mol ratio of protein or poly-D-lysine, is excellent with 1:100 Choosing.
The preparation method of anti-glycocholic acid antibody is as follows: utilize-COOH and the protein or many of glycocholic acid branch terminals -NH2 on polylysine molecule carries out coupling；Recycle these conjugate immunity things and carry out immune mouse or family as immunogen Rabbit, finally obtains specific anti-glycocholic acid antibody.
Wherein, anti-glycocholic acid monoclonal IgG antibody is mouse monoclonal IgG type antibody, anti-glycocholic acid polyclone IgG antibody is rabbit source property polyclone IgG antibody.
In above-mentioned steps, glycocholic acid is 1:20-200 with the mol ratio of protein or poly-D-lysine, is excellent with 1:100 Choosing.
When preparing anti-glycocholic acid monoclonal IgG antibody, respectively with the sweet ammonia deoxidation of the analog 10 μ g/mL of glycocholic acid Cholic acid, deoxycholic acid, cholic acid, chenocholic acid, chenodeoxy cholic acid, lithocholic acid, taurocholic acid, tauroursodeoxycholic acid carry out blocking detection Screening, retains cross reacting rate < the antibody cloning hole of 10%.
Prepare trishydroxymethylaminomethane (Tris-HCL) buffer, G6P, Tween-20, MgCl again2, NaN3, i.e. obtain Reagent R1.
Prepare simultaneously trishydroxymethylaminomethane (Tris-HCL) buffer, NADP, glycocholic acid-G6PDH conjugate, BSA i.e. obtains reagent R2, and compound method does not repeats.
Utilize the detection method of the peripheral blood glycocholic acid that this test kit realizes, use immunoassay, exempt from homogeneous enzyme Epidemic disease algoscopy is preferential, first uses anti-taurine antibody to close the interference of taurocholic acid that may be present in sample, then with anti-sweet 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, comprises the steps:
Reagent R1 is mixed with testing sample, 25-37 DEG C of reaction 3-5min, add reagent R2,25-37 DEG C of reaction 5- 10min, light absorption value at detection 340nm, obtain glycocholic acid concentration in testing sample according to standard curve；
In above-mentioned detection method, the drafting of standard curve is as follows: by glycocholic acid with containing volumetric concentration 0.25% Ox blood serum Albuminous buffer is diluted to 0 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 40 μ g/mL, the diluent of 80 μ g/mL, with Buffer containing described bovine serum albumin is blank, by the glycocholic acid diluent of described variable concentrations and reagent R1 with Mixing, 25-37 DEG C of reaction 5-10min, it is subsequently adding reagent R2,25-37 DEG C of reaction 5-10min, detects light absorption value at 340nm, With light absorption value as vertical coordinate, in diluent, glycocholic acid concentration makes standard curve as abscissa.
Owing to have employed above technical scheme, the method have the advantages that
The present invention first closes taurocholic acid with anti-taurine antibody and participates in reaction, 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 test kit, utilizes test kit, it is possible to first close cattle with anti-taurine antibody Sulphur cholic acid participates in reaction, then by the method for anti-glycocholic acid antibody test peripheral blood glycocholic acid.It is a kind of based on specificity The glycocholic acid detection method that monoclonal or polyclonal antibody are set up.Meanwhile, the preparation method of test kit of the present invention includes sweet ammonia Cholic acid and taurine specific monoclonal or the preparation method of polyclonal antibody, and (include serum, blood plasma and complete for peripheral blood Blood) etc. the detection of glycocholic acid in body fluid.
Further, first the present invention is utilized respectively-COOH and protein or the poly of glycocholic acid branch terminals -NH2 on lysine molecule carries out coupling；-the NH2 on taurine is utilized to carry out coupling with the-COOH on protein molecule；Connect And utilize these conjugate immunity things to carry out immune mouse or rabbit as immunogen, finally obtain specific anti-glycocholic acid Monoclonal or polyclonal antibody and anti-taurine monoclonal or polyclonal antibody.And by above-mentioned specific anti-glycocholic acid Dan Ke Grand or polyclonal antibody is prepared as corresponding test kit with anti-taurine monoclonal or polyclonal antibody.When carrying out homogeneous enzyme immunoassay During mensuration, first combine the taurocholic acid in sample with anti-taurine monoclonal or polyclonal antibody, to close the dry of taurocholic acid Disturb, detect the glycocholic acid content in sample with anti-glycocholic acid monoclonal or polyclonal antibody the most again.
The principle of the present invention homogeneous EIA method for detecting glycocholic acid in peripheral blood is based on sweet ammonia gallbladder Acid is specific binding with its specific antibody.
Compared with prior art, the present invention has the advantages that: sweet ammonia gallbladder in detection peripheral blood of the present invention The method of acid can carry out large sample Aulomatizeted Detect analysis on the large automatic instruments such as biochemistry analyzer.Method has detection Highly sensitive, high specificity, detects the advantages such as repeatability height and good stability.The inventive method detection sensitivity 0.1ug/mL.
Accompanying drawing explanation
Fig. 1 is glycocholic acid and protein and/or poly-D-lysine coupling schematic diagram；
Fig. 2 is taurine and protein and/or poly-D-lysine coupling schematic diagram；
Fig. 3 is that glycocholic acid measures reaction normal curve.
Detailed description of the invention
For the technological means making the present invention realize, creation characteristic, reach purpose and be easy to understand with effect, below knot Close specific embodiment, the present invention is expanded on further.
The test kit that the present embodiment provides, including reagent R1, reagent R2.
Wherein, described reagent R1 contains: 1 μ g/mL anti-taurine monoclonal IgG antibody, 10 μ g/mL anti-glycocholic acid cattle sulphur Acid 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；
Described reagent R2 contains: 0.2U/mL glycocholic acid-G6PDH conjugate, 50ug/mL NADP, 0.1%BSA, solvent Tris-HCL buffer for pH6.5.
It addition, test kit can include titer.And titer can also be prepared voluntarily when test kit uses.
Described 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 trishydroxymethylaminomethane (Tris-HCL) buffer 50mM, pH6.8, includes 5g/L BSA；Sweet Ammonia Bile acid concentrations is 80 μ g/mL, and the most again with including 5g/L BSA, 50mM, trishydroxymethylaminomethane (Tris-HCL) buffers Liquid as 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, test kit can include quality-control product.And quality-control product can also be prepared voluntarily when test kit uses.
Described quality-control product is formulated as follows: accurately weigh Sigma Co., USA's glycocholic acid sodium salt (purity 97%) 1.000mg It is dissolved in 6.25mL trishydroxymethylaminomethane (Tris-HCL) buffer 50mM, pH6.8, includes 5g/L BSA；Glycocholic acid Concentration is 160 μ g/mL, makees 1:8 and 1:32 dilution with Healthy People fresh serum the most again, i.e. glycocholic acid concentration is respectively 20 μ G/mL and 5 μ g/mL.
The present embodiment essentially describes anti-taurine antibody, the preparation method of anti-glycocholic acid antibody.And the system of test kit Standby, key is that anti-taurine antibody and the preparation of anti-glycocholic acid antibody.
The preparation method of anti-taurine antibody is as follows: utilize-the NH2 on taurine to enter with-the COOH on protein molecule Row coupling；Recycle these conjugate immunity things and carry out immune mouse or rabbit as immunogen, finally obtain specific anti- Taurine antibody.Wherein, anti-taurine monoclonal IgG antibody is mouse monoclonal IgG type antibody, anti-taurine polyclone IgG antibody is rabbit source property polyclone IgG antibody.In above-mentioned steps, taurine with the mol ratio of protein or poly-D-lysine is 1:20-200, is preferred with 1:100.
Prepare trishydroxymethylaminomethane (Tris-HCL) buffer again, glycocholic acid-G-6-PD conjugate is i.e. tried Agent R1, compound method does not repeats.
The preparation method of anti-glycocholic acid antibody is as follows: utilize-COOH and the protein or many of glycocholic acid branch terminals -NH2 on polylysine molecule carries out coupling；Recycle these conjugate immunity things and carry out immune mouse or family as immunogen Rabbit, finally obtains specific anti-glycocholic acid antibody.Wherein, anti-glycocholic acid monoclonal IgG antibody is mouse monoclonal IgG type antibody, anti-glycocholic acid polyclone IgG antibody is rabbit source property polyclone IgG antibody.In above-mentioned steps, glycocholic acid with The mol ratio of protein or poly-D-lysine is 1:20-200, is preferred with 1:100.Prepare anti-glycocholic acid monoclonal IgG to resist During body, respectively with the glycodesoxycholic acid of analog 10 μ g/mL of glycocholic acid, deoxycholic acid, cholic acid, chenocholic acid, goose deoxidation Cholic acid, lithocholic acid, taurocholic acid, tauroursodeoxycholic acid carry out blocking detection screening, retain the cross reacting rate < antibody gram of 10% Grand hole.
Prepare trishydroxymethylaminomethane (Tris-HCL) buffer, NADPH, G-6-P, BSA again and get final product To reagent R2, compound method does not repeats.
Utilize the detection method of the peripheral blood glycocholic acid that this test kit realizes, use immunoassay, exempt from homogeneous enzyme Epidemic disease algoscopy is preferential, first uses anti-taurine antibody to close the interference of taurocholic acid that may be present in sample, then with anti-sweet 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, comprises the steps:
Reagent R1 is mixed with testing sample, 25-37 DEG C of reaction 3-5min, add reagent R2,25-37 DEG C of reaction 5- 10min, light absorption value at detection 340nm, obtain glycocholic acid concentration in testing sample according to standard curve；
In above-mentioned detection method, the drafting of standard curve is as follows: by glycocholic acid with containing volumetric concentration 0.25% Ox blood serum Albuminous buffer is diluted to 0 μ g/mL, 1.25 μ g/mL, 2.5 μ g/mL, 10 μ g/mL, 20 μ g/mL, the dilution of 40 μ g/mL Liquid, with the buffer containing described bovine serum albumin as blank, by glycocholic acid diluent and the examination of described variable concentrations Agent R1 with mix, 25-37 DEG C of reaction 5-10min, be subsequently adding reagent R2,25-37 DEG C of reaction 5-10min, detection 340nm place's suction Light value, with light absorption value as vertical coordinate, in diluent, glycocholic acid concentration makes standard curve as abscissa.
More detailed preparation method, detection method, step is as follows.
1. protein conjugate prepares (as depicted in figs. 1 and 2)
Prepared by 1.1 glycocholic acid-protein conjugate
Weigh 100mg glycocholic acid (Sigma Co., USA) and 10mg bovine serum albumin (BSA) or hemocyanin Or ovalbumin (OVA) or poly-D-lysine (Roche company of Germany) are dissolved in the phosphate-buffered of pH7.6,100mM (KLH) In liquid, it is stirred at room temperature 15 minutes, adds 125mg water-soluble carbodiimide powder (Shanghai Aladdin company), room temperature lucifuge temperature With stirring 180 minutes, load bag filter (freely penetrating molecular size < 14KD), put 4 DEG C of dialysis, within every 6 hours, change liquid 1 time, continuously 6-8 time.
1.2 glycocholic acid protein conjugate coupling efficiencies are identified
Taking the glycocholic acid-protein conjugate pH7.6 of 1.1 couplings, the phosphate buffer of 100mM is diluted to concentration < 1mg/mL, respectively with the absorbance of spectrophotometric determination wavelength 210nm, 280nm, calculates coupling efficiency by following equation, when Glycocholic acid: protein > 20:1 time, it is qualified to be judged to.
Coupling efficiency=A210х69000/A280х483
Prepared by 1.3 taurines-protein conjugate
Weigh 100mg taurine (Sigma Co., USA) and 10mg bovine serum albumin or hemocyanin or ovalbumin (Roche company of Germany) is dissolved in the phosphate buffer of pH7.6,100mM, is stirred at room temperature 15 minutes, adds 125mg water Soluble carbodiimide powder (Shanghai Aladdin company), room temperature lucifuge gentle agitation 180-240 minute, load bag filter (freely Penetrating molecular size < 14KD), put 4 DEG C of dialysis, within every 6 hours, change liquid 1 time, continuous 6-8 time.
The most anti-glycocholic acid monoclonal IgG type preparation method for antibody
2.1 animal immune
With the glycocholic acid-protein conjugate of above-mentioned steps 1.1 preparation, it is preferred with glycocholic acid-hemocyanin, with The concentration of 100 μ g glycocholic acid-protein conjugate/time/mice mixes with the Split completely of equivalent, and subcutaneous multiple spot is noted Penetrate immunity Balb/c female mice (5-6 week old, body weight 18-20g), the 1st time with the 2nd minor tick 10-14 days, the most every minor tick 7 days, continuous 5 times, 2-5 glycocholeic acid-protein conjugate and the incomplete freund adjuvant emulsifying mixing of equivalent, it is spaced 1 100 μ g (the 100 μ L volume) glycocholic acid-poly-D-lysine conjugate using tail vein/lumbar injection aseptic after week is (quiet with tail Arteries and veins is preferred), with booster immunization 1 time, after 3 days, cell merges.
The preparation of 2.2 hybridomies
Carry out according to regular growth fusion method.Take the splenocyte of step 2.1 immune mouse and SP2/0 myeloma cell by Volume ratio 1:6 merges under the effect of 50%PEG (MW400-800), and fused cell is first at hypoxanthine-methotrexate-thymus In pyrimidine (HAT) Selective agar medium (Hyclone company of the U.S.), at 37 DEG C, 5%CO2And cultivate under the conditions of saturated humidity, 2 weeks After be changed to hypoxanthine-thymus pyrimidine (HT) culture medium (Hyclone company of the U.S.), continue at 37 DEG C, 5%CO2And it is saturated wet Cultivate under the conditions of degree.In time cloning Kong Changzhi 1/3-1, take culture supernatant and carry out antibody test screening.
2.3 specificity clone's hole sizer choosings
Positive hole is expressed with ELISA method screening antibodies.Glycocholic acid-protein is diluted even with the carbonate buffer solution of 50mM Connection thing (be preferred with glycocholic acid-OVA), to after 10 μ g/mL, is coated with 100 μ L/ holes and reacts micropore 4 DEG C overnight or 37 DEG C 120min, washs 3 times with the phosphate buffer (PBS) containing 0.05%Tween-20 or Tris-HCL buffer (PBS), uses 10% calf serum room temperature closes 30min, adds 100 μ l above-mentioned steps 2.2 culture supernatant room temperature reaction 60min, with containing Phosphate buffer (PBS) or the Tris-HCL buffer (PBS) of 0.05%Tween-20 wash 3 times, finally add 1: Goat anti-mouse igg-HRP room temperature reaction the 60min of 2000 dilutions, with the phosphate buffer (PBS) containing 0.05%Tween-20 Or Tris-HCL buffer (PBS) washs 3 times, add the colour developing of tetramethyl biphenyl ammonia (TMB) substrate and observe.All there is obvious blue Responder is positive, is otherwise negative.Retain the cell clone hole responded with glycocholic acid-protein conjugate, then use Limiting dilution assay carries out 3 time cloning screenings, builds the hybridoma amplification culture again of strain, frozen, is used for preparing ascites.
The preparation and purification of 2.4 ascites
After the hybridoma amplification culture of stably excreting monoclonal antibody, with 0.5 × 104～5 × 105Individual/only inoculation To in advance with 0.5mL norphytane or the abdominal cavity of liquid paraffin sensitization 7-10 days Balb/c mice, observe in 7-12 days, collect abdomen Water, measures titer after being centrifuged.Preserve or cryopreservation through 50% saturated ammonium sulphate.Ascites uses 50% saturated ammonium sulfate Precipitation-caprylic acid precipitates, or with the 50% saturated ammonium sulphate-rProteinA-Sepharose affine layer of Fast Flow Analysis purification, 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 reaction with glycocholic acid-protein conjugate, and not with non-glycocholic acid- Protein conjugate generation nonspecific reaction.
The most anti-glycocholic acid-protein conjugate polyclone IgG antibody preparation method
With above-mentioned steps 1.1 preparation glycocholic acid-protein conjugate, with 10mg glycocholic acid-protein conjugate/ Secondary/only to mix with equivalent Split completely, it is preferred with glycocholic acid-hemocyanin, the immunity of rabbit subcutaneous multi-point injection.Often 1-2 week 1 time, continuous 5 times, use after being spaced 1 week and separate carotid artery, collection blood about 80-120 milliliter, room temperature placement 2 hours, 4 DEG C 10000rpm is centrifuged 30min, separates and collects serum.Above-mentioned serum uses glycocholic acid-protein conjugate (with sweet ammonia gallbladder again Acid-OVA is preferred), the specific IgG type antibody of the anti-glycocholic acid of affinity chromatograph column purification, finally detects with 8%SDS-PAGE Purity, need to reach 90%.Obtain rabbit source property polyclone IgG antibody.
For obtaining the F (ab) ' of anti-glycocholic acid2Specific Ab fragments, the rabbit source property polyclone IgG antibody of above-mentioned purification Hold with the Fc ' of the pepsin excision IgG of 0.1% (mass ratio) in the 50mM acetate buffer of pH 4.5 further, 37 DEG C reaction 12 hours, then adjust or uncomfortable pH value in the case of, with saturated ammonium sulfate and/or SPA and/or glycocholic acid-egg White matter conjugate affinity chromatograph method purification F (ab) '2Antibody fragment.Obtain the F (ab) ' of specific anti-glycocholic acid2Special Property antibody fragment.
Anti-glycocholic acid Anti-TNF-α physical ability prepared by the present embodiment and the glycocholic acid specific binding reaction of generation, and not With non-glycocholic acid-protein conjugate generation nonspecific reaction.
The most anti-taurine monoclonal IgG type preparation method for antibody
4.1 animal immune
With the taurine-protein conjugate of above-mentioned steps 1.2 preparation, it is preferred with taurine-hemocyanin, with 100 μ The concentration of g taurine-protein conjugate/time/mice mixes with the Split completely of equivalent, the immunity of subcutaneous multi-point injection Balb/c female mice (5-6 week old, body weight 18-20g), the 1st time with the 2nd minor tick 10-14 days, the most every minor tick 7 days, even Continuous 5 times, 2-5 hypotaurine-protein conjugate and the incomplete freund adjuvant emulsifying mixing of equivalent, use after being spaced 1 week Taurine-poly-D-lysine conjugate (being preferred with tail vein) that tail vein/lumbar injection 100 μ g (100 μ L volume) is aseptic, With booster immunization 1 time, after 3 days, cell merges.
The preparation of 4.2 hybridomies
Carry out according to regular growth fusion method.Take the splenocyte of step 2.1 immune mouse and SP2/0 myeloma cell by Volume ratio 1:6 merges under the effect of 50%PEG (MW400-800), and fused cell is first at hypoxanthine-methotrexate-thymus In pyrimidine (HAT) Selective agar medium, at 37 DEG C, 5%CO2And cultivate under the conditions of saturated humidity, it is changed to hypoxanthine-breast after 2 weeks Gland pyrimidine (HT) culture medium, continues at 37 DEG C, 5%CO2And cultivate under the conditions of saturated humidity.In time cloning Kong Changzhi 1/3-1, take Culture supernatant carries out antibody test screening.
4.3 specificity clone's hole sizer choosings
Positive hole is expressed with ELISA method screening antibodies.Taurine-protein molecule is diluted with the carbonate buffer solution of 50mM Thing (being preferred with taurine-OVA), to after 10 μ g/mL, is coated reaction 4 DEG C of overnight or 37 DEG C of 120min of micropore with 100 μ L/ holes, Wash 3 times with the phosphate buffer (PBS) containing 0.05%Tween-20 or Tris-HCL buffer (PBS), use 10% calf Serum room temperature closes 30min, adds 100 μ l above-mentioned steps 2.2 culture supernatant room temperature reaction 60min, with containing 0.05% Phosphate buffer (PBS) or the Tris-HCL buffer (PBS) of Tween-20 wash 3 times, finally add 1:2000 dilution Goat anti-mouse igg-HRP room temperature reaction 60min, with the phosphate buffer (PBS) containing 0.05%Tween-20 or Tris- HCL buffer (PBS) washs 3 times, adds the colour developing of tetramethyl biphenyl ammonia (TMB) substrate and observes.All there is obvious blue responder For the positive, it is otherwise negative.Retain the cell clone hole responded with taurine-protein conjugate, then use limiting dilution Method carries out 3 time cloning screenings, builds the hybridoma amplification culture again of strain, frozen, is used for preparing ascites.
The preparation and purification of 4.4 ascites
After the hybridoma amplification culture of stably excreting monoclonal antibody, with 0.5 × 104～5 × 105Individual/only inoculation To in advance with 0.5mL norphytane or the abdominal cavity of liquid paraffin sensitization 7-10 days Balb/c mice, observe in 7-12 days, collect abdomen Water, measures titer after being centrifuged.Preserve or cryopreservation through 50% saturated ammonium sulphate.Ascites uses 50% saturated ammonium sulfate Precipitation-caprylic acid precipitates, or with the 50% saturated ammonium sulphate-rProteinA-Sepharose affine layer of Fast Flow Analysis purification, 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 reaction with taurine-protein conjugate, and not with non-taurine-protein Conjugate generation nonspecific reaction.
The most anti-taurine-protein conjugate polyclone IgG antibody preparation method
With above-mentioned steps 1.1 preparation taurine-protein conjugate, with 10mg taurine-protein conjugate/time/ Only mix with equivalent Split completely, the immunity of rabbit subcutaneous multi-point injection.Every 1-2 week 1 time, continuous 5 times, adopt after being spaced 1 week With separating carotid artery, collecting blood about 80-120 milliliter, room temperature is placed 2 hours, and 4 DEG C of 10000rpm are centrifuged 30min, separate and collect Serum.The specific IgG type antibody of taurine-anti-taurine of protein conjugate affinity chromatograph column purification used again by above-mentioned serum, Finally detect purity with 8%SDS-PAGE, 90% need to be reached.Obtain rabbit source property polyclone IgG antibody.
For obtaining the F (ab) ' of anti-taurine2Specific Ab fragments, the rabbit source property polyclone IgG antibody of above-mentioned purification is entered The Fc ' that one step excises IgG with the pepsin of 0.1% (mass ratio) in the 50mM acetate buffer of pH 4.5 holds, 37 DEG C React 12 hours, then adjusting or in the case of uncomfortable pH value, with saturated ammonium sulfate and/or SPA and/or taurine-protein Conjugate affinity chromatograph method purification F (ab) '2Antibody fragment.Obtain specific anti-taurine F (ab) '2Specific antibody sheet Section.
Anti-taurine Anti-TNF-α physical ability prepared by the present embodiment occurs specific binding with taurine-protein conjugate Reaction, and not with non-taurine-protein conjugate generation nonspecific reaction.
6. homogeneous enzyme immunoassay method measures serum glycocholic acid content
Glucose-6-phosphate dehydrogenase (G6PD) (G-6-PD) method is used to carry out.
6.1 main agents constituents
Reagent R1:10ug/mL anti-taurine polyclone IgG antibody (comprises F (ab) ' 2 specific Ab fragments), 100 μ g/ ML anti-glycocholic acid taurine polyclone IgG antibody (comprises F (ab) ' 2 specific Ab fragments), 500 μ g/mL G6P, and 0.5% Tween-20,10mM MgCl2, 0.1%NaN3, solvent is the Tris-HCL buffer of pH8.0；
Reagent R2:3U/mL glycocholic acid-G6PDH conjugate, 1000ug/mL NADP, 1%BSA, solvent is pH8.0's Tris-HCL buffer.
Titer is prepared: accurately weighs Sigma Co., USA's glycocholic acid sodium salt (purity 97%) 1.000mg and is dissolved in 12.5mL trishydroxymethylaminomethane (Tris-HCL) buffer 50mM, pH6.8, include 5g/LBSA；Glycocholic acid concentration is 80 μ g/mL, the most again with including 5g/L BSA, 50mM, trishydroxymethylaminomethane (Tris-HCL) buffer as doubling dilution is 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 weighs Sigma Co., USA's glycocholic acid sodium salt (purity 97%) 1.000mg and is dissolved in 6.25mL trishydroxymethylaminomethane (Tris-HCL) buffer 50mM, pH6.8, include 5g/LBSA；Glycocholic acid concentration is 160 μ g/mL, make 1:8 and 1:32 dilution with Healthy People fresh serum the most again, i.e. glycocholic acid concentration is respectively 20 μ g/mL and 5 μg/mL。
6.2 detecting step
Response type: performance rate method temperature: 37 DEG C of cuvette optical path: 0.6cm
Master/slave wavelength: 340nm unit: U/ml the Direction of Reaction: rise
6.3 results determine:
Calculate the difference (A calibration-A is blank) of calibration object absorbance, set up suitable mathematical modulo (non-linear) such as Logit- Log etc., fit to the calibration curve (such as Fig. 3) of multiple spot calibration.Reactions change amplitude according to calibration curve, the range of linearity, phase Close coefficient and reaction repeatability determines serum glycocholic acid content.
The most anti-glycocholic acid-protein conjugate antibody crossing-over rate measures
7.1 glycocholic acid analog preparations
Glycocholic acid analog includes glycodesoxycholic acid, deoxycholic acid, cholic acid, chenocholic acid, chenodeoxy cholic acid, stone gallbladder Acid, taurocholic acid, tauroursodeoxycholic acid etc. are purchased from (Shanghai Aladdin company).Above-mentioned each cholate is all dissolved in and comprises 0.1% In trishydroxymethylaminomethane (Tris-HCL) buffer (100mM, pH8.0) of Tween-80, concentration is 40 μ g/mL.
7.2 main agents constituents
Reagent R1:1ug/mL anti-taurine monoclonal IgG antibody, 10ug/mL anti-glycocholic acid taurine monoclonal antibody, 50ug/mL G6P, 0.01%Tween-20,0.3mM MgCl2, 0.01%NaN3, solvent is the Tris-HCL buffering of pH6.5 Liquid；
Reagent R2:0.2U/mL glycocholic acid-G6PDH conjugate, 50ug/mL NADP, 0.1%BSA, solvent is pH6.5 Tris-HCL buffer.
Titer is prepared: accurately weighs Sigma Co., USA's glycocholic acid sodium salt (purity 97%) 1.000mg and is dissolved in 12.5mL trishydroxymethylaminomethane (Tris-HCL) buffer 50mM, pH6.8, include 5g/LBSA；Glycocholic acid concentration is 80 μ g/mL, but again with including 5g/L BSA, 50mM, trishydroxymethylaminomethane (Tris-HCL) buffer as doubling dilution is 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 weighs Sigma Co., USA's glycocholic acid sodium salt (purity 97%) 1.000mg and is dissolved in 6.25mL trishydroxymethylaminomethane (Tris-HCL) buffer 50mM, pH6.8, include 5g/LBSA；Glycocholic acid concentration is 160 μ g/mL, make 1:8 and 1:32 dilution with Healthy People fresh serum the most again, i.e. glycocholic acid concentration is respectively 20 μ g/mL and 5 μg/mL。
7.3 detecting step
Response type: performance rate method temperature: 37 DEG C of cuvette optical path: 0.6cm
Master/slave wavelength: 340nm unit: U/ml the Direction of Reaction: rise
7.4 results determine:
Calculating the difference (A calibration-A is blank) of calibration object absorbance, with absorbance as vertical coordinate, concentration is abscissa, builds The calibration curve of vertical multiple spot calibration, measures the concentration of known various analogs in table 1, measures concentration and concentration known (40ug/ ML) ratio is suppression ratio.The results are shown in Table 1.
Table 1
The ultimate principle of the present invention and principal character and advantages of the present invention have more than been shown and described.The technology of the industry Personnel, it should be appreciated that the present invention is not restricted to the described embodiments, simply illustrating this described in above-described embodiment and description The principle of invention, without departing from the spirit and scope of the present invention, the present invention also has various changes and modifications, and these become Change and improvement both falls within scope of the claimed invention.Claimed scope by appending claims and Equivalent defines.

Claims (10)

1. a test kit, it is characterised in that: include reagent R1, reagent R2,
Described reagent R1 contains: Tris-HCL (trishydroxymethylaminomethane) buffer, anti-taurine antibody, and anti-glycocholic acid resists Body, G6P (G-6-P)；
Described reagent R2 contains: Tris-HCL (trishydroxymethylaminomethane) buffer, glycocholic acid-G-6-PD conjugate (Hangzhoupro Zhou Bopu Pharmaceutical Technology Co., Ltd), NADP (codehydrogenase Ⅱ), BSA (bovine serum albumin).
2. a kind of test kit as claimed in claim 1, it is characterised in that: also include titer,
Described titer is: taking concentration is 80 μ g/mL glycocholic acid, with including 5g/L BSA, 50mM trishydroxymethylaminomethane (Tris-HCL) buffer becomes series concentration solution as doubling dilution.
3. a kind of test kit as claimed in claim 2, it is characterised in that: also include quality-control product,
Described quality-control product is: takes glycocholic acid sodium salt (purity 97%) 1.000mg and is dissolved in 6.25mL trishydroxymethylaminomethane (Tris-HCL) buffer 50mM, pH6.8, includes 5g/L BSA；Glycocholic acid concentration is 160 μ g/mL, uses Healthy People the most again Fresh serum makees 1:8 and 1:32 dilution.
4. a kind of test kit as claimed in claim 3, it is characterised in that: described anti-taurine antibody includes anti-taurine Dan Ke Grand IgG antibody or anti-taurine polyclone IgG antibody.
5. a kind of test kit as claimed in claim 4, it is characterised in that: described anti-taurine monoclonal IgG antibody includes One in IgG1, IgG2a, IgG2b, IgG3 or IgG4；It is special that described anti-taurine polyclone IgG antibody comprises F (ab) ' 2 Property antibody fragment.
6. a kind of test kit as claimed in claim 3, it is characterised in that: described anti-glycocholic acid antibody includes anti-glycocholic acid Monoclonal IgG antibody or anti-glycocholic acid polyclone IgG antibody.
7. a kind of test kit as claimed in claim 6, it is characterised in that: described anti-glycocholic acid monoclonal IgG antibody is One in IgG1, IgG2a, IgG2b, IgG3 or IgG4；Described anti-glycocholic acid polyclone IgG antibody is special containing F (ab) ' 2 Property antibody fragment.
8. the method preparing test kit as claimed in claim 1, it is characterised in that: the preparation method of anti-taurine antibody therein As follows: to utilize-the NH on taurine2Coupling is carried out with-the COOH on protein molecule；Recycle these conjugate immunity things to make Carry out immune mouse or rabbit for immunogen, finally obtain specific anti-taurine antibody, taurine and protein or poly The mol ratio of lysine is 1:20-200, is preferred with 1:100；The preparation method of anti-glycocholic acid antibody therein is as follows: profit Coupling is carried out with-the NH2 on protein or poly-D-lysine molecule with the-COOH of glycocholic acid branch terminals；Recycle these Conjugate immunity thing carries out immune mouse or rabbit as immunogen, finally obtains specific anti-glycocholic acid antibody, sweet ammonia Cholic acid is 1:20-200 with the mol ratio of protein or poly-D-lysine, is preferred with 1:100.
9. utilize the detection method of the peripheral blood glycocholic acid that test kit as claimed in claim 1 realizes, it is characterised in that: use Immunoassay, is preferential with homogeneous EIA method, first uses anti-taurine antibody to close cattle that may be present in sample The interference of sulphur cholic acid, then the detection of glycocholic acid is carried out with anti-glycocholic acid antibody；The working concentration of anti-taurine antibody is 1- 10 μ g/mL, are preferred with 5 μ g/mL.
10. utilize the detection method of the peripheral blood glycocholic acid that test kit as claimed in claim 1 realizes, it is characterised in that: bag Include following steps:
Reagent R1 is mixed with testing sample, 25-37 DEG C of reaction 3-5min, adds reagent R2,25-37 DEG C of reaction 5-10min, Light absorption value at detection 340nm, obtains glycocholic acid concentration in testing sample according to standard curve；
Described reagent R1 quality group becomes:
1-10 μ g/mL anti-taurine monoclonal IgG antibody (including IgG1, IgG2a, IgG2b, IgG3 or IgG4) or polyclone IgG antibody (comprises F (ab) ' 2 specific Ab fragments),
10-100 μ g/mL anti-glycocholic acid taurine monoclonal IgG antibody (including IgG1, IgG2a, IgG2b, IgG3 or IgG4) Or polyclone IgG antibody (comprising F (ab) ' 2 specific Ab fragments),
50-500 μ g/mL G6P,
0.01-0.5%Tween-20,
0.3-10mM MgCl2,
0.01-0.1%NaN3,
Solvent is pH6.5-8.0Tris-HCL buffer；
Described reagent R2 quality group becomes:
0.2-3U/mL glycocholic acid-G6PDH conjugate,
50-1000ug/mL NADP,
0.1-1%BSA
Solvent is pH6.5-8.0Tris-HCL buffer.
In above-mentioned detection method, the drafting of standard curve is as follows: by glycocholic acid with containing volumetric concentration 0.25% bovine serum albumin White buffer is diluted to 0 μ g/mL, 1.25 μ g/mL, 2.5 μ g/mL, 10 μ g/mL, 20 μ g/mL, the diluent of 40 μ g/mL, with Buffer containing described bovine serum albumin is blank, by the glycocholic acid diluent of described variable concentrations and reagent R1 with Mixing, 25-37 DEG C of reaction 5-10min, it is subsequently adding reagent R2,25-37 DEG C of reaction 5-10min, detects light absorption value at 340nm, With light absorption value as vertical coordinate, in diluent, glycocholic acid concentration makes standard curve as abscissa.
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