CN108982860A - A kind of reagent using latex immunoturbidimetry technology measurement human serum content of glycocholic acid - Google Patents
A kind of reagent using latex immunoturbidimetry technology measurement human serum content of glycocholic acid Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses a kind of reagents using latex immunoturbidimetry technology measurement human serum content of glycocholic acid, including reagent 1 and reagent 2, reagent 1 is by 0.04% latex microsphere-BSA- glycocholic acid conjugate, and add adjuvant 100mM, the phosphate buffer of pH8.0,0.9% sodium chloride solution, 0.1% BSA and stabilizer are formulated；Reagent 2 is the anti-glycocholic acid monoclonal antibody of mouse by 5% and adds adjuvant 15mM, the phosphate buffer of pH7.4,0.9% sodium chloride solution, 0.1% BSA, and 0.1% surfactant and stabilizer is formulated.Latex microsphere is introduced into reagent by the present invention, due to the presence of latex microsphere, is significantly increased the sensitivity of the detection reagent, is met the requirement of clinical use.It is compared with homogeneous enzyme immunoassay method, the present invention has great advantage on reagent stability, and under acceleration conditions, stability time is at least the 2 times or more of homogeneous enzyme immunoassay method reagent.
Description
Technical field
The present invention relates to biological detection reagents, sweet more particularly, to a kind of application latex immunoturbidimetry technology measurement human serum
The reagent of cholic acid content.
Background technique
Serum CG (Cholyglycine, CG, molecular weight 465.6) belongs to one kind of bile acid.Bile acid includes
The cholanic acid of one major class mainly has cholic acid (CA), chenodesoxycholic acid (CDCA), deoxycholic acid (DCA) in bile, and there are also a small amount of
Lithocholic acid (LCA) and micro ursodesoxycholic acid (UDCA), various cholanic acids all may be combined into combination with glycine, taurine
Form, this is principal mode of the hepatic secretion into bile.Various bile acids, whether free state or reference state, molecule
Inside is all not only (hydroxyl, carboxyl, sulfonyl) containing hydrophilic radical, but also (methyl and hydrocarbon core) containing hydrophobic grouping, therefore bile acid is vertical
Body configuration has hydrophilic and hydrophobic two sides, thus bile acid is made to show very strong interfacial activity.It can reduce rouge, water two
Surface tension between phase promotes lipid to form mixed micelles, this is to gallbladder in the digestion and absorption of lipid material and maintenance bile
The dissolution of sterol all plays an important role.
Serum CG synthesizes in liver, is stored in gall-bladder, by intestinal absorption, overwhelming majority warp in digestion process
Portal vein is reuptaked by liver, finally constitutes liver-gallbladder-intestines-vein-liver circulation.When liver is by outside damage or disease
When disease influences function, liver declines the reabsorption of Serum CG, will lead to Serum CG raising；When gall-bladder blocks
When, the glycocholic acid in liver cannot be excreted in enteron aisle in time, caused to backflow into hematological system, can also be caused Serum CG liter
It is high；When intestinal absorption ability is damaged, the content of Serum CG also will affect.Therefore, the high and low energy of Serum CG content
Whether the physiological function for directly feeding back liver and gall is normal.
Clinically often judge whether pregnant woman suffers from Intrahepatic Cholestasis with the measurement of Serum CG.Normal condition
Under, the organs such as each internal organ such as heart, lung, stomach and liver can be oppressed in the increase of latter half of gestation, entire placenta, cause a system
The problem of column such as edema of lower extremity, be short of breath.And will affect the secretion of liver bile to the compressing of liver, so as to cause serum
Glycocholic acid increases.Therefore, the reference range of pregnancy serum glycocholic acid is significantly different from normal person.Meanwhile the sweet gallbladder of pregnancy serum
The measurement of acid can directly feed back the degree of liver inner bile siltation.Intrahepatic Cholestasis often has family history, has certain heredity special
Sign, pregnant woman's own situation with Intrahepatic Cholestasis is better, mainly will appear pruitus and slight jaundice, but give a birth it
Symptom rapidly disappears afterwards, but has large effect to fetus, easily cause premature labor, the infant of low-birth weight, fetal distress in uterus it is dead and
The symptoms such as asphyxia neonatorum.Therefore detection content of glycocholic acid promptly and accurately especially has timely pregnant woman to the health of fetus
Diagnostic significance.
Clinic mainly has radio immunoassay, enzyme-linked immunization, chemiluminescence to exempt from the diagnosis of Serum CG at present
Epidemic disease analytic approach and homogeneous enzyme immunoassay analytic approach etc..
Radio immunoassay is using anti-in the glycocholic acid competition reagent in radioactive element label glycocholic acid, with sample
Body, by analyzing the height of reflexive constituent content in the glycocholic acid to react with antibody come the examination of glycocholic acid in feedback samples
Agent concentration.This method accuracy is higher, and label glycocholic acid is not significantly different with binding performance of the normal glycocholic acid to antibody.But it is anti-
The effect phase of penetrating property element is shorter, and later period waste is difficult to handle, and the method is gradually by market.
Enzyme-linked immunization detects glycocholic acid using the method for double-antibody sandwich, and is increased by the effect of enzyme-linked amplification
Reagent sensitivity has the advantages that high sensitivity.But the method reaction time is longer, and result is sxemiquantitative, is not able to satisfy market
The demand of upper extensive screening.
Chemiluminescence immunoassay principle is similar with enzyme-linked immunization, replaces enzyme-linked amplification by chemiluminescent method
To increase reagent sensitivity.This method is improved compared with enzyme linked immunological sensitivity, and accuracy also correspondinglys increase, and result is quantitative result, but
So far, this method cost is still higher, and marketing has certain pressure.
Homogeneous enzyme immunoassay analytic approach marks glycocholic acid using specific enzyme, and by conjunction with special glycocholic acid antibody
Enzymatic activity is influenced, when marking glycocholic acid and normal glycocholic acid together with antibody response, the content height shadow of normal glycocholic acid
The whole enzymatic activity of label glycocholic acid is rung, therefore the concentration of normal glycocholic acid in sample can be measured.The existing enzyme linked immunological of this method
The advantages of method high sensitivity, also has biochemical reagents can be the advantages of fast high-flux on full automatic biochemical apparatus detects.But by
It is poor in current label enzyme stability, it is not able to satisfy the requirement of clinical use.
Summary of the invention
It is an object of the invention to provide a kind of application latex immunoturbidimetry for defect present in the above-mentioned prior art
Technology measures the reagent of human serum content of glycocholic acid.
To achieve the above object, the present invention can take following technical proposals:
Reagent of the present invention using latex immunoturbidimetry technology measurement human serum content of glycocholic acid, including reagent 1 and examination
Agent 2, in which:
Reagent 1 is and to add adjuvant 100mM by 0.04% latex microsphere-BSA- glycocholic acid conjugate, and the phosphate of pH8.0 is slow
Fliud flushing, 0.9% sodium chloride solution, 0.1% BSA and stabilizer are formulated；
Reagent 2 is the anti-glycocholic acid monoclonal antibody of mouse by 5% and adds adjuvant 15mM, the phosphate buffer of pH7.4,0.9%
Sodium chloride solution, 0.1% BSA, 0.1% surfactant and stabilizer be formulated.
In the reagent 1 latex microsphere-BSA- glycocholic acid conjugate the preparation method is as follows: glycocholic acid is activated first,
Then the glycocholic acid of activation and BSA are coupled, obtain glycocholic acid-BSA conjugate solution for standby；By the carboxyl of 100 nm of partial size
The latex microsphere solution of activation is made in latex microsphere activation, under agitation, glycocholic acid-BSA obtained in advance is added dropwise
In conjugate solution, Seal treatment is carried out after being persistently stirred to react, and after ultrasound is resuspended, it is even to obtain present latex particulate-glycocholic acid-BSA
Join object.
Surfactant in the reagent 2 is one of Triton X-100,20 Tween, Brij 35 or appoints
Two kinds of combinations.
Stabilizer used in the reagent 1 and reagent 2 is casein, in gelatin, calf serum, mannitol, sucrose
One of or two kinds and two or more combinations.
Advantages of the present invention are as follows:
Through the conjugate of glycocholic acid and BSA under suitable buffer matrix, it is coupled (simultaneously with the latex microsphere of certain partial size
Add a variety of auxiliary) reagent 1 is made, since glycocholic acid is small-molecule substance, belongs to haptens, be unable to direct immunization animal,
It can only be immunoreacted with an antibody, so free glycocholic acid cannot form turbidity with antibody response and react；But sweet gallbladder
With after BSA coupling, each BSA can be coupled acid with several CG, this conjugate can have multiple antigen sites, can be with
Multiple antibody are immunoreacted.
Main component in reagent 2 is the anti-glycocholic acid monoclonal antibody of mouse (and adding various adjuvants), monoclonal antibody pair
The high specificity in site can be improved the detection accuracy of reagent, exclude other bile acids such as cholic acid, chenodesoxycholic acid, deoxidation
The interference of cholic acid etc..
When detection, the glycocholic acid in sample is first at war with the latex microsphere-BSA- glycocholic acid conjugate in reagent 1,
Then again with the antibody response in reagent 2, due to latex microsphere-BSA- glycocholic acid conjugate can generate with antibody response it is immune
Than turbid reaction, and free glycocholic acid can not generate immunoturbidimetry with antibody response and react.Therefore, dissociate glycocholic acid in sample
Content and final degree of reaction are in inverse relation.According to corresponding calibration curve, we can detect containing for glycocholic acid in sample
Amount.
Latex microsphere is introduced into reaction by the present invention, due to the presence of latex microsphere, significantly increases detection examination
The sensitivity of agent meets the requirement of clinical use.
The present invention exempts from, compared with enzyme the methods of is exempted from putting, and has great odds for effectiveness, by means of the full-automatic biochemical of high speed
Instrument can achieve 2000 or more per hour detection sample numbers.It is compared with homogeneous enzyme immunoassay method, on reagent stability
With great advantage, under acceleration conditions, stability time is at least the 2 times or more of homogeneous enzyme immunoassay method reagent.
Detailed description of the invention
Fig. 1 is to existing radioimmunology and the detection of homogeneous enzyme immunoassay method using reagent of the present invention with the related of a sample
Property diagram.
Specific embodiment
Embodiment 1 is prepared the reagent of measurement human serum content of glycocholic acid by the method for the present invention
One, reagent preparation 1
1, latex microsphere-BSA- glycocholic acid conjugate is prepared
1) activation of glycocholic acid
400mg 1- (3- dimethyl aminopropyl) -3- ethyl carbodiimide (EDC) is accurately weighed, 100mL MES buffer is dissolved in
In (20mmol/L, pH=6.0), obtain EDC solution；200mg glycocholic acid, 400mg Sulfo-NHS are accurately weighed, 100mL is dissolved in
In MES buffer (20mmol/L, pH=6.0), glycocholic acid solution is obtained；Under agitation, EDC solution is slowly added into
In glycocholic acid solution；It is persistently stirred to react 30 minutes after adding, the glycocholic acid solution activated.
2) glycocholic acid and BSA are coupled
100mg BSA is accurately weighed, (20mmol/L, pH=6.0) is dissolved in 100mL MES buffer, obtains BSA solution；It is stirring
In the case of mixing, the glycocholic acid solution of activation is slowly added into BSA solution；It is persistently stirred to react 4 hours after adding
Obtain glycocholic acid-BSA conjugate；After the free glycocholic acid of the methods of column chromatography, dialysis removal, it can be used for even in next step
Connection reaction.
3) activation of latex microsphere
100mg 1- (3- dimethyl aminopropyl) -3- ethyl carbodiimide (EDC) is accurately weighed, is dissolved in 50mL MES buffer
(20mmol/L, pH=6.0), obtain EDC solution；It is accurate measure 50mL latex microsphere solution (carboxylated latex microballoon, partial size 100nm,
Content 10%), (20mmol/L, pH=6.0) are dissolved in 250mL MES buffer, latex microsphere solution is obtained；In stirring state
Under, EDC solution is slowly added into latex microsphere solution；It is persistently stirred to react 30 minutes after adding, the glue activated
Newborn microspheres solution.
4) coupling of glycocholic acid-BSA conjugate and latex microsphere
The 2) glycocholic acid-BSA conjugate that step obtains be diluted with MES buffer (20mmol/L, pH=6.0), obtain most
The glycocholic acid of final concentration of 0.5mg/mL-BSA conjugate solution；Under agitation, the latex microsphere of the 3) step activation is molten
Liquid is slowly added into glycocholic acid-BSA conjugate solution；It is persistently stirred to react 4 hours after adding；Add BSA solution into
Row Seal treatment 1 hour；It is then centrifuged for removing extra EDC, glycocholic acid-BSA conjugate etc., phosphorus is added in the latex microsphere of precipitating
In acid buffer (100mmol/L, pH=8.0), ultrasound is resuspended, i.e. acquisition latex microsphere-BSA- glycocholic acid conjugate.
2, reagent preparation 1
In 800mL deionized water, 32.22g disodium hydrogen phosphate (Na is added2HPO4.12H2O), 1.36g potassium dihydrogen phosphate
(KH2PO4), 9g sodium chloride (NaCl), mix dissolution, adjust pH be 8.0, supply water to 1L, this is phosphate buffer；Use this
Resulting latex microsphere-BSA- glycocholic acid the conjugate of phosphate buffer dilution step 1 simultaneously makes its ultimate density 0.04%, adds
Add 0.1% BSA and stabilizer, as reagent 1.Final prepare contains 100mM in the reagent 1 completed, the phosphate of pH8.0 is slow
Fliud flushing, 0.04% latex microsphere-BSA- glycocholic acid conjugate, 0.9% sodium chloride, 0.1% BSA and stabilizer.
Two, reagent preparation 2
In 800mL deionized water, 4.56g disodium hydrogen phosphate (Na is added2HPO4.12H2O), 0.31g potassium dihydrogen phosphate
(KH2PO4), 9g sodium chloride (NaCl), mix dissolution, adjust pH be 7.4, supply water to 1L, this is 15mM, the phosphate of pH7.4
Buffer；The anti-glycocholic acid monoclonal antibody of mouse of addition 5% in this phosphate buffer, 0.1% BSA, 0.1% surface are living
Property agent and stabilizer are reagent 2.
2 detection method of embodiment and equipment
The present invention needs the serum or blood plasma of fresh no haemolysis to the detection of glycocholic acid, to the reaction noisy sample of absorbance
If haemolysis or rouge are turbid, final result may be will affect, it is proposed that take a blood sample again.
Reagent of the invention is applied to the biochemical instruments of various models, such as Hitachi Hitachi7080/7600P, Olympus
Olympus AU400, Beckman Beckman Coulter LX20/AU680/AU5800, Toshiba Toshiba 120FR, Abbott Laboratories
Abbott C8000, Siemens's SIEMENS ADVIA1800/ADVIA2400/Dimension RxL Max Biochemical Analyzer
Deng.
Specific detection parameters setting is as shown in the table, and the different instrument of the read point basis of time of A1 and A2 carries out corresponding
Setting:
3 correlation of embodiment compares
The reagent made from embodiment 1 is obtained with conventional use of radioimmunology and the detection of homogeneous enzyme immunoassay method with a sample
The correlation data obtained see the table below 1 and Fig. 1:
From table 1 we can see that institute's measured data of the present invention and radioimmunology acquired results are close, and homogeneous enzyme immunoassay method institute
Result is surveyed in low value stage and radioimmunology acquired results good relationship, but the high level stage is significantly relatively low, so it is linear
It will be lower than the present invention and radioimmunology reagent.
Fig. 1 then more intuitively embodies the comparison of three kinds of reagents, institute's measured data of the present invention and radioimmunology institute
The Trendline of result is obtained close to straight line, shows that the two is linear suitable in 0~60 mg/L, accuracy is also suitable；Homogeneous enzyme immunoassay
Method measured result and the Trendline of radioimmunology acquired results are curve, close to straight line in 0~40 mg/L, are higher than 40
There is apparent bending after mg/L.
Claims (4)
1. a kind of reagent using latex immunoturbidimetry technology measurement human serum content of glycocholic acid, it is characterised in that: including reagent 1
With reagent 2, in which:
Reagent 1 is and to add adjuvant 100mM by 0.04% latex microsphere-BSA- glycocholic acid conjugate, and the phosphate of pH8.0 is slow
Fliud flushing, 0.9% sodium chloride solution, 0.1% BSA and stabilizer are formulated；
Reagent 2 is the anti-glycocholic acid monoclonal antibody of mouse by 5% and adds adjuvant 15mM, the phosphate buffer of pH7.4,0.9%
Sodium chloride solution, 0.1% BSA, 0.1% surfactant and stabilizer be formulated.
2. the reagent according to claim 1 using latex immunoturbidimetry technology measurement human serum content of glycocholic acid, special
Sign is: latex microsphere-BSA- glycocholic acid conjugate the preparation method is as follows: first activating glycocholic acid, so in the reagent 1
The glycocholic acid of activation and BSA are coupled afterwards, obtain glycocholic acid-BSA conjugate solution for standby；By the carboxyl glue of 100 nm of partial size
The latex microsphere solution of activation is made in newborn microballoon activation, under agitation, it is even that glycocholic acid-BSA obtained in advance is added dropwise
Join in object solution, Seal treatment is carried out after being persistently stirred to react, after ultrasound is resuspended, obtains present latex particulate-glycocholic acid-BSA coupling
Object.
3. the reagent according to claim 1 using latex immunoturbidimetry technology measurement human serum content of glycocholic acid, special
Sign is: the surfactant in the reagent 2 is one of Triton X-100,20 Tween, Brij 35 or wantonly two
Kind combination.
4. the reagent according to claim 1 using latex immunoturbidimetry technology measurement human serum content of glycocholic acid, special
Sign is: stabilizer used in the reagent 1 and reagent 2 is casein, gelatin, calf serum, mannitol, its in sucrose
In one or two kinds of and two or more combinations.
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| CN109884317A (en) * | 2019-03-12 | 2019-06-14 | 杭州博谱医药科技有限公司 | Application of the oxidized form Thio-NAD+ I in homogeneous enzyme immunoassay diagnostic reagent |
| CN110007092A (en) * | 2019-04-08 | 2019-07-12 | 杭州博谱医药科技有限公司 | The kit of CMPF content in a kind of latex microsphere-albumen-CMPF conjugate and preparation method thereof and measurement serum |
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| CN112285345A (en) * | 2020-08-31 | 2021-01-29 | 北京九强生物技术股份有限公司 | Glycocholic acid detection kit |
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