CN107796937A - The FPIA method and its application of glycocholic acid in a kind of detection serum - Google Patents

The FPIA method and its application of glycocholic acid in a kind of detection serum Download PDF

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CN107796937A
CN107796937A CN201710794610.7A CN201710794610A CN107796937A CN 107796937 A CN107796937 A CN 107796937A CN 201710794610 A CN201710794610 A CN 201710794610A CN 107796937 A CN107796937 A CN 107796937A
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glycocholic acid
acid
serum
glycocholic
concentration
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赵肃清
沈定
崔锡平
陈莹珊
何绮怡
吕瑞
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Guangdong Nanyue Pharmaceutical Co ltd
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Guangdong University of Technology
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    • 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
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    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"

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Abstract

The invention belongs to immuno analytical method detection field, discloses a kind of FPIA method for detecting glycocholic acid in serum.This method includes：Fluorescent tracing thing is mixed with the glycocholic acid standard solution of known various concentrations, glycocholic acid Anti-TNF-α liquid solution mixing incubation is added and is at war with reaction, measures the fluorescence polarization value of the system；Ratio by the fluorescence polarization value measured and the fluorescence polarization value of blank group is inhibiting rate, using inhibiting rate as ordinate, it is known that the concentration of glycocholic acid standard items is abscissa, draws standard curve；The glycocholic acid standard items of concentration known are replaced with testing sample, under the same conditions, measure the fluorescence polarization value of system after being incubated, further according to standard curve, the concentration of glycocholic acid in the testing sample is calculated.This method is contrasted with ELISA glycocholic acid kits, the results showed that both approaches have preferable similitude.Cross reaction result shows that the polyclonal antibody has very high specificity to glycocholic acid.

Description

The FPIA method and its application of glycocholic acid in a kind of detection serum
Technical field
The invention belongs to immuno analytical method detection field, and in particular to a kind of fluorescence polarization for detecting glycocholic acid in serum Immunoassay method and its application.
Background technology
Glycocholic acid is the mating type cholic acid being combined into by cholic acid and glycine, is one of main component of bile acid. In liver cell, cholesterol passes through extremely complex enzymatic reaction, is transformed into primary bile acid, includes cholic acid (CA) and CDCA Sour (CDCA), there are three hydroxyls (C3, C7, C12) in the steroids core of cholic acid, the hydroxyl of side chain terminal is with peptide bond and glycine knot Synthesize glycocholic acid, molecular weight 466.3.After glycocholic acid is synthesized by liver cell, gall-bladder, companion are discharged into through bile capillaries, bile duct Bile enters duodenum, helps fatty in food digest and assimilate.Most of bile acid is in ileum and colon by reabsorption, warp Portal vein returns liver again, is absorbed and recycled by liver cell, and re-absorbed glycocholic acid enters enter liver-intestines and circulated again, by this mechanism, Body can make full use of glycocholic acid.Under normal circumstances, content of glycocholic acid is little in peripheral blood, normal person no matter on an empty stomach or meal Afterwards, its glycocholic acid concentration is stable in low-level.When human hepatocyte is impaired or during cholestatis, glycocholic acid will be caused to be metabolized And disturbance of circulation, decline the ability of liver cell intake glycocholic acid, cause content of glycocholic acid in blood to raise, and content of glycocholic acid Height it is related to the order of severity of hepatocellular damage and bile acid biosynthesis obstacle.There is experimental data to show oxyhepatitis, primary Property the patients serum such as liver cancer, hepatic sclerosis in content of glycocholic acid obviously higher than normal person, and presentation property increases.Content of glycocholic acid is also It is the important indicator of a variety of biliary tract diseases of reflection, pregnant women's intrahepatic cholestasis, alcoholic liver damage etc..Cause This, glycocholic acid detects the Clinical laboratory test important as one, can be clinical diagnosis, treatment, the therapeutic evaluation of a variety of diseases And prognosis evaluation provides important evidence, using huge with promotion prospect.
The detection method on glycocholic acid is mainly instrumental method at present, such as efficient liquid phase-tandem mass spectrometry and superelevation Effect liquid phase chromatogram mass spectrometry method, these methods need large-scale instrument and complicated sample pre-treatments；On the other hand it is immunization, it is main To include radioimmunology (RIA), Chemiluminescence immunoassay and Immunity transmission turbidity.Although these method degrees of accuracy are high, again Renaturation is good, the advantages that suitable for quantitative detection, but these detection technique complex operations, it is heterogeneous reaction system mostly, Need repeatedly to be incubated and wash, time-consuming, cost is high, it is impossible to meets the requirement of high flux quick detection；Instrument and equipment price is held high Expensive, the shortcomings of operating procedure flow is more, time-consuming, it is unfavorable for Routine Test Lab development, clinical practice limitation is obvious.
Immunofluorescence technique is chemically to make fluorescein-labeled antibody (or antigen) and the phase in tissue or cell Answer antigen (or antibody) to combine, carry out the method that qualitative positioning checks antigen or antibody.This method has high specific, sensitive Property, it is convenient the advantages that, be widely used in small molecule detection field.
FPIA method (FPIA) is a kind of simple, quick, efficient and reliable analytical technology, and system is complete Entirely in homogeneous phase solution complete reaction, it is not necessary to separation and washing operation, have high sensitivity, high specificity, it is simple to operate, into Originally the advantages that high flux screening that is cheap, being easy to automation, clinic, the detection of the field such as food and environment small molecular are increasingly becoming The common technology of analysis.Moreover, the FPIA method of glycocholic acid has not been reported in serum, there is wide open Make an offer value.
The content of the invention
In order to overcome glycocholic acid immunoassay technology in the prior art, complex operation, the shortcomings that time-consuming and deficiency, this hair FPIA method of the bright primary and foremost purpose in the glycocholic acid in a kind of detection serum is provided.This method entirely detects Process only needs a step competitive reaction, simple to operate, high-efficient simple.
It is an object of the invention to provide a kind of FPIA method of glycocholic acid in above-mentioned detection serum Using.
The purpose of the present invention is achieved through the following technical solutions：
The FPIA method of glycocholic acid, comprises the following steps in a kind of detection serum：
(1) fluorescent tracing thing is mixed with a series of glycocholic acid standard solution of concentration knowns, it is more adds glycocholic acid Clonal antibody solution mixing incubation is at war with reaction, measures the fluorescence polarization value of the system；The fluorescence polarization value mP that will be measured With the fluorescence polarization value mP of blank group0Ratio be inhibiting rate, with inhibiting rate mP/mP0For ordinate, it is known that glycocholic acid standard The concentration of product is abscissa, draws standard curve：Y=0.80675/ [(1+ (x/682.21468)0.96872]+0.1536；
(2) fluorescent tracing thing is mixed with testing sample, adds the mixing of glycocholic acid Anti-TNF-α liquid solution and be incubated progress Competitive reaction, the fluorescence polarization value of the system is measured, further according to standard curve, the dense of glycocholic acid in the testing sample is calculated Degree.
Step (1) the fluorescent tracing thing is the conjugate of glycocholic acid haptens and fluorescein：
The glycocholic acid haptens is the N- (3,7,12- trihydroxy -24- carbonyls by cholic acid and 4-Aminobutanoicacid reaction generation Base cholane -24- bases)-Gamma Amino Butyric Acid is as glycocholic acid haptens；
The fluorescein is fluorescein isothiocynate ethylenediamine (EDF).
The specific preparation process of step (1) the glycocholic acid polyclonal antibody is according to Application No. 201610727381.2 Chinese patent application in described step prepared.
A series of solute of the glycocholic acid standard solution of step (1) concentration knowns is glycocholic acid standard items, solvent For phosphate buffer solution；
In the phosphate buffer solution, NaH2PO4·2H2O molal weight is 1-8mmol/L, Na2HPO4·12H2O Molal weight be 8-15mmol/L, the molal weight of sodium chloride is 10-20mmol/L, pH value 7.0-8.5；
A series of concentration knowns are 106Ng/mL, 105Ng/mL, 104Ng/mL, 103Ng/mL, 100ng/mL, 10ng/ ML and 1ng/mL totally 7 gradient concentrations.
The working concentration of step (1) the fluorescent tracing thing is to be used as it using the intensity of polarization light of 10-15 times of blank solution Working concentration, blank solution select borate buffer solution；
In the borate buffer solution, the molal weight of Boratex is 4-30mmol/L, and the molal weight of sodium chloride is 1-10mmol/L, pH value 7.0-9.0.
The dilution factor of step (1) the glycocholic acid polyclonal antibody is 1:1000-1:3000.
Step (1) volume for adding fluorescent tracing thing, the volume of glycocholic acid standard solution, glycocholic acid Anti-TNF-α The volume of liquid solution is respectively 400-500 μ L, 10-50 μ L, 400-500 μ L；The incubation time is 1-8min, competitive reaction temperature Spend for 20-30 DEG C.
Step (2) volume for adding fluorescent tracing thing, the volume of testing sample, glycocholic acid Anti-TNF-α liquid solution Volume is respectively 400-500 μ L, 10-50 μ L, 400-500 μ L；The incubation time is 1-8min, and competitive reaction temperature is 20- 30℃。
Step (2) described testing sample is human serum.
In above-mentioned detection serum the FPIA method of glycocholic acid can be applied to detect hepatitis serum and Healthy Human Serum.
Testing result of the present invention shows：The content of glycocholic acid is far longer than sweet courage in Healthy Human Serum in hepatitis serum The content of acid, its positive rate is 96.9%, so glycocholic acid can be as a clinical detection index of hepatitis.
The present invention is had the following advantages relative to prior art and effect：The invention provides a kind of homogeneous, Gao Te The opposite sex, simple and rapid glycocholic acid FPIA method；Detection model of this method in cushioning liquid standard curve Enclose for 0.2-2.8 μ g mL-1, IC50Value is 0.68 μ g mL-1, R2=0.9984；This method is carried out with ELISA glycocholic acid kit Contrast, the results showed that both approaches have preferable similitude, and it is reliable and accurate to show this method；Cross reaction result table The bright polyclonal antibody has very high specificity to glycocholic acid；Testing result of the present invention shows：Sweet courage in hepatitis serum The content of acid is far longer than the content of glycocholic acid in Healthy Human Serum, and glycocholic acid can refer to as a clinical detection of hepatitis Mark；This method is quick, easy high flux, and disclosure satisfy that the detection limitation requirement of glycocholic acid, is very suitable for sweet acid in serum Detection need.
Brief description of the drawings
Fig. 1 is the fluorescence intensity figure that three kinds of fluorescent tracing things are combined with glycocholic acid polyclonal antibody.
Fig. 2 is the dilution curve figure of glycocholic acid polyclonal antibody.
Fig. 3 is glycocholic acid Fluorescence Polarised Immunoassay canonical plotting.
Fig. 4 is that fluorescence polarization immunoassay detects glycocholic acid in sample and contains spirogram.
Fig. 5 is the comparison diagram of FPIA method and ELISA kit.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited In this.
Test method used is conventional method as not having specified otherwise in following embodiments；Institute in following embodiments Reagent, biomaterial etc., such as there is no specified otherwise, commercially obtain.
Fluorescence polarization detector used in following embodiments is Portable fluorescence polarimeter (SENTARY200), sweet courage The specific preparation process of sour polyclonal antibody is described in the Chinese patent application according to Application No. 201610727381.2 The preparation process of glycocholic acid polyclonal antibody prepared.
Preparation of the embodiment 1. based on the fluorescent tracing thing of glycocholic acid haptens after transformation.
Step 1. fluorescein EDF synthesis
20ml methanol triethylamine solutions are first prepared, wherein triethylamine concentration is 10ml/L；By 70ul ethylenediamines and 35.1mgFITC fluoresceins are dissolved in 15ml methanol triethylamine solution and 3ml methanol triethylamine solutions respectively；It will contain FITC's Solution is gradually added drop-wise in the triethylamine solution of ethylenediamine, normal temperature lucifuge stirring 5h, finally reacting liquid filtering, then uses first repeatedly Alcohol rinses filter paper, treats Filter Paper Dry, the reddish yellow solid on filter paper is scraped off, obtains fluorescein EDF.
The transformation of step 2. glycocholic acid haptens
Weigh 1.5g cholic acid to be added in 50ml flasks, add the anhydrous tetrahydrofurans of 3ml, the second of 509ul tri- thereto successively Amine and 464ul isobutyl chlorocarbonates, are stirred at room temperature 30min, after solution becomes clarification, add 379mg 4-Aminobutanoicacids, after It is continuous that reaction 3 days is stirred at room temperature.After the completion of reaction, a small amount of deionized water is added, decompression rotary evaporation removes tetrahydrofuran, then With the pH of hydrochloric acid conditioning solution to acidity, add substantial amounts of ethyl acetate and repeatedly extracted, according to a small amount of multiple principle, received Collect ethyl acetate layer, decompression rotary evaporation obtains crude product.Finally with dichloromethane：Methanol：Acetic acid=10：1：0.01 is expansion Above-mentioned crude product is carried out separating-purifying by agent by silicagel column, obtains glycocholic acid haptens.
The synthesis of step 3. fluorescent tracing thing
Weigh 2mg glycocholic acid haptens and be added to 500ul and contain 1mgN- HOSu NHSs (NHS) and 2mg dicyclohexyls In the DMF solution of carbodiimide (DCC), normal temperature lucifuge is stirred overnight；The above-mentioned solution of 200ul is taken to add 1mgEDF, normal temperature lucifuge Stir 3h；Take above-mentioned reaction solution thin-layered chromatography (TLC) separation of 40ul, solvent CHCl3, scrape Rf=on silica gel plate 0.82,0.58,0.36 three yellow band, eluted respectively with 300ul methanol, obtain three kinds of fluorescent tracing things.
The screening of the optimal fluorescent tracing thing of embodiment 2.
The glycocholic acid polyclonal antibody of three kinds of fluorescent tracing things of above-mentioned synthesis and same concentration is combined respectively, it is first It is corresponding when the working concentration of three kinds of fluorescent tracing things first being set as into fluorescence intensity is 10 times of the fluorescence intensity of cushioning liquid The concentration of fluorescent tracing thing, then tracer and each 500ul of antibody are well mixed, the maximum changing value δ mP of fluorescence intensity are measured, The fluorescence intensity change value of wherein Rf=0.36 tracer and antibody binding is maximum, so, selected Rf=0.36 tracer For optimal fluorescent tracing thing.Accompanying drawing 1 is the fluorescence intensity that three kinds of fluorescent tracing things are combined with glycocholic acid polyclonal antibody.
The determination of the working concentration of the glycocholic acid polyclonal antibody of embodiment 3.
The working concentration of optimal fluorescent tracing thing is set as that fluorescence intensity is the 10 of the fluorescence intensity of cushioning liquid first Times when corresponding fluorescent tracing thing concentration, then by glycocholic acid antibody borate buffer solution according to 1/1000,1/2000,1/ 4000,1/8000,1/16000,1/32000 doubling dilutions, then tracer and each 500ul of antibody are well mixed, measurement is glimmering Luminous intensity, draws antibody binding curve, and accompanying drawing 2 is the dilution curve of glycocholic acid polyclonal antibody.So selected glycocholic acid antibody Extension rate be 1/2000, the as working concentration of glycocholic acid polyclonal antibody.
The foundation of the FPIA method of embodiment 4.
Step 1. competitive inhibition reaction：10 are prepared respectively with phosphate buffer6Ng/mL, 105Ng/mL, 104Ng/mL, 103The glycocholic acid standard items of ng/mL, 100ng/mL, 10ng/mL and 1ng/mL totally 7 gradient concentrations；Divide in quartzy small test tube Not Jia Ru 500ul fluorescent tracing thing working solutions and 50ul standard solutions, mix, add 500ul antibody working solutions, mixing is equal Even, normal temperature is incubated, measurement fluorescence intensity level (mP).Wherein fluorescence intensity level (the mP of blank group0) it is to replace standard solution Measured by phosphate buffer.
Step 2. draws standard curve：By the fluorescence intensity level measured and the ratio (mP/mP of blank value0) sat as vertical Mark, using the logarithm value of glycocholic acid standard concentration as abscissa, mapped in software Origin8.5 and establish the fitting of glycocholic acid Standard curve, as shown in Figure 3.
The standard curve that this method is established is y=0.80675/ [(1+ (x/682.21468)0.96872]+0.1536, it is examined Survey scope is 0.2-2.8 μ g mL-1, IC50Value is 0.68 μ g mL-1, R2=0.9984.This method is quick, easy high flux, and And the detection limitation requirement of glycocholic acid is disclosure satisfy that, the detection needs of glycocholic acid very in appropriate serum.
The measure of the cross reacting rate of embodiment 5.
Glycocholic acid 26S Proteasome Structure and Function analog (cholic acid, deoxycholic aicd, stone courage are determined using FPIA method Acid, chenodeoxycholic acid, urso, sweet ammonia urso, ergosterol) with antibody do cross reaction, will prepare 106Ng/mL, 105Ng/mL, 104Ng/mL, 103The class of ng/mL, 100ng/mL, 10ng/mL and 1ng/mL totally 7 gradient concentrations Competitive reaction is done like thing with antibody respectively.IC using glycocholic acid antibody to glycocholic acid50Value is with antibody to various analogs IC50The ratio of value is worth to cross reacting rate (CR%).Experimental result is as shown in table 1.
The cross reaction of the glycocholic acid polyclonal antibody of table 1 and related compound
The application of the sample detection of embodiment 6.
Using 383 hepatitis serum and 441 Healthy Human Serums as testing sample；
All samples 3000g under 4 DEG C of freezing conditions centrifuges 10min, takes supernatant liquor as testing sample.Wherein use Healthy Human Serum and hepatitis serum are diluted by phosphate buffer, 5min are then boiled in boiling water, then carry out fluorescence Polarization immunoassay analysis detection.Experimental result is as shown in Figure 4.
From fig. 4, it can be seen that glycocholic acid is apparently higher than Healthy People group, its positive rate in the content of hepatitis serum 96.9%, show that glycocholic acid can be as the clinical detection index of a hepatitis.From table 1, these analogue compounds are to this The interference of method detection glycocholic acid is seldom, shows that this method has high specificity.The glycocholic acid fluorescence that the present invention establishes is inclined The immunoassay method that shakes can meet the testing requirements of glycocholic acid in human serum, have a high specific, high sensitivity, and price is low It is honest and clean, it is simple and quick the advantages of, avoid traditional immunization method complex operation well, the shortcomings of time-consuming, can be good at transporting Used in the quick of glycocholic acid, high-throughout detection.
The FPIA method (FPIA) of embodiment 7. contrasts with enzyme linked immunosorbent assay (ELISA)
Same batch of sample is detected with FPIA method and enzyme linked immunosorbent assay respectively, by FPIA's Testing result is mapped in software Origin8.5 as ordinate as abscissa, ELISA testing result and establishes two kinds of sides The comparison diagram of method.As shown in Figure 5, the results showed that both approaches have preferable similitude.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

Claims (10)

  1. A kind of 1. FPIA method for detecting glycocholic acid in serum, it is characterised in that comprise the following steps：
    (1) fluorescent tracing thing is mixed with a series of glycocholic acid standard solution of concentration knowns, it is polyclonal adds glycocholic acid Antibody-solutions mixing incubation is at war with reaction, measures the fluorescence polarization value of the system；By the fluorescence polarization value mP measured and sky The fluorescence polarization value mP organized in vain0Ratio be inhibiting rate, with inhibiting rate mP/mP0For ordinate, it is known that glycocholic acid standard items Concentration is abscissa, draws standard curve；Y=0.80675/ [(1+ (x/682.21468)0.96872]+0.1536；
    (2) fluorescent tracing thing is mixed with testing sample, adds glycocholic acid Anti-TNF-α liquid solution mixing incubation and be at war with Reaction, the fluorescence polarization value of the system is measured, further according to standard curve, the concentration of glycocholic acid in the testing sample is calculated.
  2. 2. a kind of FPIA method for detecting glycocholic acid in serum according to claim 1, its feature exist In：Step (1) the fluorescent tracing thing is the conjugate of glycocholic acid haptens and fluorescein：
    The glycocholic acid haptens is the N- (3,7,12- trihydroxy -24- carbonyl courages by cholic acid and 4-Aminobutanoicacid reaction generation Alkane -24- bases)-Gamma Amino Butyric Acid is as glycocholic acid haptens；
    The fluorescein is fluorescein isothiocynate ethylenediamine.
  3. 3. a kind of FPIA method for detecting glycocholic acid in serum according to claim 1, its feature exist In：The specific preparation process of step (1) the glycocholic acid polyclonal antibody is according in Application No. 201610727381.2 In state's patent application prepared by described step.
  4. 4. a kind of FPIA method for detecting glycocholic acid in serum according to claim 1, its feature exist In：A series of solute of the glycocholic acid standard solution of step (1) concentration knowns is glycocholic acid standard items, and solvent is phosphoric acid Salt buffer solution；
    In the phosphate buffer solution, NaH2PO4·2H2O molal weight is 1-8mmol/L, Na2HPO4·12H2O's rubs Your quality is 8-15mmol/L, and the molal weight of sodium chloride is 10-20mmol/L, pH value 7.0-8.5；
    A series of concentration knowns are 106Ng/mL, 105Ng/mL, 104Ng/mL, 103Ng/mL, 100ng/mL, 10ng/mL and 1ng/mL totally 7 gradient concentrations.
  5. 5. a kind of FPIA method for detecting glycocholic acid in serum according to claim 1, its feature exist In：The working concentration of step (1) the fluorescent tracing thing is dense using the intensity of polarization light of 10-15 times of blank solution as its work Degree, blank solution select borate buffer solution；
    In the borate buffer solution, the molal weight of Boratex is 4-30mmol/L, and the molal weight of sodium chloride is 1- 10mmol/L, pH value 7.0-9.0.
  6. 6. a kind of FPIA method for detecting glycocholic acid in serum according to claim 1, its feature exist In：The dilution factor of step (1) the glycocholic acid polyclonal antibody is 1:1000-1:3000.
  7. 7. a kind of FPIA method for detecting glycocholic acid in serum according to claim 1, its feature exist In：Step (1) volume for adding fluorescent tracing thing, the volume of glycocholic acid standard solution, glycocholic acid polyclonal antibody are molten The volume of liquid is respectively 400-500 μ L, 10-50 μ L, 400-500 μ L；The incubation time is 1-8min, and competitive reaction temperature is 20-30℃。
  8. 8. a kind of FPIA method for detecting glycocholic acid in serum according to claim 1, its feature exist In：Step (2) volume for adding fluorescent tracing thing, the volume of testing sample, the volume of glycocholic acid Anti-TNF-α liquid solution Respectively 400-500 μ L, 10-50 μ L, 400-500 μ L；The incubation time is 1-8min, and competitive reaction temperature is 20-30 DEG C.
  9. 9. a kind of FPIA method for detecting glycocholic acid in serum according to claim 1, its feature exist In：Step (2) described testing sample is human serum.
  10. 10. a kind of FPIA method for detecting glycocholic acid in serum according to claim 1 is in detection liver Application in scorching patients serum and Healthy Human Serum.
CN201710794610.7A 2017-04-27 2017-09-06 The FPIA method and its application of glycocholic acid in a kind of detection serum Pending CN107796937A (en)

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Publication number Priority date Publication date Assignee Title
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
CN112285345B (en) * 2020-08-31 2024-05-24 北京九强生物技术股份有限公司 Glycocholic Acid Detection Kit

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