CN101813679B - Method for detecting preparation success of artificial antigens - Google Patents
Method for detecting preparation success of artificial antigens Download PDFInfo
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- CN101813679B CN101813679B CN 201010145055 CN201010145055A CN101813679B CN 101813679 B CN101813679 B CN 101813679B CN 201010145055 CN201010145055 CN 201010145055 CN 201010145055 A CN201010145055 A CN 201010145055A CN 101813679 B CN101813679 B CN 101813679B
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Abstract
The invention discloses a method for estimating preparation success of artificial antigens. The method comprises the following steps: a) weighing artificial antigens and carrier protein of the same weight, adding solvent 1 to 10 times the weight for dissolution, applying ultrasound for 1 minute, performing centrifugation at 12,000 rpm, taking supernatant and obtaining an artificial antigen test sample and a carrier protein test sample respectively; b) using an instrument combining ultra performance liquid chromatography and quadrupole-rod electro-spray mass spectrometry to detect the test samples respectively to obtain ultra performance liquid chromatograms and quadrupole-rod electro-spray mass spectrograms respectively; and c) comparing the differences between the ultra performance liquid chromatograms of the artificial antigens and the carrier protein, as well as the corresponding mass spectrograms thereof to determine whether the preparation of the artificial antigens is successful. The method has the advantages of simple sample pretreatment, strong universality, no special requirement for the physical-chemical characteristics of hapten, simple instrument operation conditions, suitability for large-scale popularization and application, and the like.
Description
Technical field
The present invention relates to a kind of method of identifying artificial antigen, especially adopt Ultra Performance Liquid Chromatography and level Four bar electro spraying ionization-mass spectrometry method to judge the method whether artificial antigen prepares.
Background technology
Convenient, succinct, quick and sensitive analytical technology and detection means are to ensure food safety and the important leverage of environmental monitoring.The unreasonable use of Pesticides and veterinary drugs even abuse cause the food agricultural produce medicament residue to exceed standard, become one of outstanding problem of food security, that reliable, sensitive, the quick and practical express-analysis detection technique of exploitation is monitored beyond doubt and controlled is residual, ensure people's health and avoid the important technical of international trade dispute.Immuno analytical method (IA) particularly enzyme-linked immunosorbent analytical technique (ELISA) as a Fast Detection Technique, high specificity with its antigen-antibody reaction, assay method is simple, quick, highly sensitive, expense is low and be suitable for the advantages such as on-the-spot batch samples screening, has showed widely application prospect in the analysis of agricultural chemicals residue of veterinary drug and environment measuring.
Immunoassay is the detection method of utilizing the association reaction of antigen and antibody, and all being small-molecule substance, most of Pesticides and veterinary drugs (also are haptens, hapten), relative molecular mass is below 1000Da, its lack t cell epitope and directly the induced animal body produce specific antibody, but it is coupled to covalent is prepared into artificial antigen on the high molecular weight protein carrier and (also claims: conjugate), can come by its T cell propagation and the differentiation of indirect induction B cell, produce specific antibody, this also is the basic model that little molecular immune is analyzed.In this technology, identify artificial antigen (hapten-carrier protein conjugate) whether coupling be successfully prepared to be prerequisite and the key of setting up immune analysis method.
The main authentication method of artificial antigen has ultraviolet spectrophotometry (UV), infrared spectrophotometer (IR), SDS-page electrophoresis and substance assistant laser desorpted time-of-flight mass spectrometry (TOFMS) (MALDI-TOF) etc. at present.But the versatility of these methods is not strong, and restrictive condition is more.For example UV, only has when haptens and carrier protein have different chromophories and just can use to identify the synthetic of artificial antigen by the ultraviolet absorption spectrum of difference hapten-carrier protein conjugate (artificial antigen) and carrier protein; If the purifying of artificial antigen is incomplete, unreacted haptens or other small molecular weight impurities will certainly affect the reliability of the detection qualification result of artificial antigen; This method is helpless especially to the haptens that does not contain ultraviolet absorption group.Also there is similar shortcoming in the method for IR.Thereby electrophoretic techniques is to utilize the ion in the solution to move the technology that reaches separation under certain electric field action, electrophoresis biomacromolecule discriminating in use very extensive; In the evaluation of artificial antigen, the present main electrophoresis of using is SDS-polyacrylamide gel electrophoresis method, SDS-page electrophoresis, is to judge the different of pillar location that gel occurs whether synthesizing of artificial antigen be successful with its carrier protein by comparing artificial antigen; More precisely be exactly to judge according to the difference of the molecular weight of artificial antigen and carrier protein; Shortcoming is if carrier coupling haptens is less, makes the molecular weight increase of artificial antigen not obvious, uses the SDS-page method to show that the solidifying pillar location difference that goes up is just not obvious; Bibliographical information only has as larger the haptens (〉 800Da of molecular weight) (＜20000Da) artificial antigen of coupling preparation is used this method to identify and just has preferably identification result with the carrier protein of molecular weight; Substance assistant laser desorpted time-of-flight mass spectrometry (TOFMS) (Qi Xiaohua etc. the living beings spectrometry is to the Fast Measurement of artificial antigen coupling ratio. the inspection and quarantine science, 2007 supplementary issues) be the present authentication method of the most effective artificial antigen, shortcoming is need to select to be fit to Ionized matrix when the preparation of testing sample, if it is improper to select, can cause the poor repeatability of testing result; The laser desorption time-of-flight mass spectrometer is expensive simultaneously, and operating conditions is harsh, is not suitable for large-scale promotion application.
Because artificial antigen complex structure, it is to be connected to carrier protein such as bovine serum albumin(BSA), human serum albumins such as amino, carboxyl etc. by specific reaction by the special groups on the little molecule, be prepared on the specific amino acid residue such as ovalbumin, the binding site of little molecule and protein albumen is many, the analysis difficulty is large, also lacks the analyzing detecting method of a cover highly versatile at present.
Summary of the invention
The objective of the invention is to provide for the deficiencies in the prior art a kind of new, fast, effectively reach reliable method to judge whether artificial antigen prepares.
The object of the present invention is achieved like this:
A kind of method of judging whether artificial antigen prepares, the method comprises following concrete steps:
A, take by weighing artificial antigen and the carrier protein of identical weight respectively, add 1～10 times of weight dissolution with solvents, ultrasonic 1min after 12000rpm is centrifugal, gets supernatant, obtains respectively artificial antigen specimen and carrier protein specimen;
B, usefulness Ultra Performance Liquid Chromatography quadrupole rod electro spraying ionization-mass spectrometry instrument detect specimen respectively, obtain respectively Ultra Performance Liquid Chromatography figure and quadrupole rod electrospray ionization mass spectrum figure;
The difference of the Ultra Performance Liquid Chromatography figure of c, comparison gained artificial antigen and carrier protein and corresponding mass spectrogram thereof determines whether artificial antigen prepares;
Wherein: when with Ultra Performance Liquid Chromatography quadrupole rod electro spraying ionization-mass spectrometry instrument specimen being detected, its Ultra Performance Liquid Chromatography condition is:
Mobile phase:
A phase: acetonitrile
B phase: 0.01: 100～0.1: 100 trifluoroacetic acid aqueous solution of volume ratio
Linear gradient: be increased to 70%～90% at 0～10min A by 10%～30% linearity, corresponding B reduces to 30%～10% by 70%～90% linearity
Liquid-phase chromatographic column is: ACQUITY UPLC C4,2.1mm * 150mm, 1.7um.
Its quadrupole rod electrospray ionization mass spectrum condition is:
Ion gun: electric spray ion source
Ionization mode: positive ion ionization
Detection mode: full scan;
Capillary voltage: 2.5～3KV
Taper hole voltage; 20～35V
Quality of scanning scope: m/z 1000～1600 amu
Ion source temperature: 100～150 ℃
Desolventizing temperature: 250～300 ℃
Desolventizing gas: nitrogen
Desolventizing gas velocity: 400～700L/hr.
Described solvent is the potpourri of water or organic solvent and water; Organic solvent is ethanol, methyl alcohol, acetonitrile or acetone.
Described carrier protein is bovine serum albumin(BSA).
The volume ratio of described organic solvent and water is 1:1.
Test comprises with the preparation method of artificial antigen samples among the present invention: (hapten compound and the nitrite reaction that contain aromatic amine form diazo salt to the diazotising method, then be directly connected to the ortho position of the amino acid residue of phenolic hydroxy group in the carrier protein molecule, namely obtain with the continuous artificial antigen of azo bond), (carboxyl in the little molecule of haptens generates the reactive intermediate mixed acid anhydride with the isobutyl chlorocarbonate reaction to mixed anhydride method in the presence of tertiary amine, then with protein carrier on primary amino radical reaction, form amido link and prepare artificial antigen) and carbodlimide method (amino on the haptens or carboxylic acid and carbodiimide reaction generation one intermediate, again with carrier protein on amino or carboxyl formation amido link and prepare artificial antigen) etc.
Advantage of the present invention is: sample pre-treatments is simple, the method highly versatile, and without special requirement, the instrumentation condition is simple to haptenic physicochemical characteristics, is fit to apply on a large scale.
Description of drawings
Fig. 1 is the Ultra Performance Liquid Chromatography figure of the embodiment of the invention 1;
Fig. 2 is the electrospray ionization mass spectrum figure of the embodiment of the invention 1;
Fig. 3 is the Ultra Performance Liquid Chromatography figure of the embodiment of the invention 2;
Fig. 4 is the electrospray ionization mass spectrum figure of the embodiment of the invention 2;
Fig. 5 is the Ultra Performance Liquid Chromatography figure of the embodiment of the invention 3;
Fig. 6 is the electrospray ionization mass spectrum figure of the embodiment of the invention 3;
Fig. 7 is the Ultra Performance Liquid Chromatography figure of the embodiment of the invention 4;
Fig. 8 is the electrospray ionization mass spectrum figure of the embodiment of the invention 4.
Embodiment
Embodiment 1
Prepare aniline artificial antigen (aniline bovine serum albumin(BSA) conjugate) by following process route:
1 mg aniline, 10 mL, 1 mol/L hydrochloric acid are added in the 25 mL there-necked flasks, stir, the ice bath cooling; In 0～5 ℃ of 1% NaNO that drips precooling
2Aqueous solution to KI-starch paper becomes blue, continues lucifuge, insulated and stirred 30 min, makes diazonium salt solution.
Other gets 100 mg bovine serum albumin(BSA)s (BSA), 10 mL borax-borate buffer (0.2 mol/L, pH 8.7) place in another 50 mL there-necked flask, ice bath, the above-mentioned diazonium salt solution of the lower dropping of stirring maintain about 8 with 5% sodium hydroxide solution conditioned reaction liquid pH value in the dropping process.Dropwise lucifuge, insulation reaction 24 h.Stop reaction, reactant liquor is packed in the good bag filter of pre-service, 4 ℃ lower to distill water dialysis three days, changes liquid every day four times.Vacuum freeze drying makes the cotton-shaped artificial antigen of brown, and this artificial antigen is denoted as AN-BSA, and-20 ℃ save backup.
Take by weighing respectively this artificial antigen and bovine serum albumin(BSA) 5mg, add water 5 mg, ultrasonic 1min, after 12000rpm was centrifugal, getting supernatant was artificial antigen sample to be tested and bovine serum albumin(BSA) sample to be tested.
Sample to be tested detects with Ultra Performance Liquid Chromatography quadrupole rod electro spraying ionization-mass spectrometry instrument:
The Ultra Performance Liquid Chromatography condition:
Ultra Performance Liquid Chromatography post: ACQUITY UPLC C4,2.1mm * 150mm, 1.7um
Mobile phase: A phase: acetonitrile; B phase: 0.01: 100 trifluoroacetic acid aqueous solution of volume ratio
Linear gradient: be increased to 90% at 0～10min A by 10% linearity, corresponding B reduces to 10% by 90% linearity
Flow velocity: 0.2mL/min
Column temperature: 30 ℃
Quadrupole rod electrospray ionization mass spectrum condition:
Ion gun: electric spray ion source
Ionization mode: positively ionized
Capillary voltage: 3KV
Taper hole voltage; 30V
Detection mode: full scan
Quality of scanning scope: m/z 1000～1600 amu
Ion source temperature: 100 ℃
Desolventizing temperature: 300 ℃
Desolventizing gas: nitrogen
Desolventizing gas velocity: 700L/hr
Obtain respectively the Ultra Performance Liquid Chromatography figure (upper figure is that aniline artificial antigen, figure below are bovine serum albumin(BSA) among Fig. 1) of aniline artificial antigen, bovine serum albumin(BSA) and corresponding quadrupole rod electrospray ionization mass spectrum figure (upper figure is that aniline artificial antigen, figure below are bovine serum albumin(BSA) among Fig. 2).
According to the difference of the difference (Fig. 1) of the retention time of chromatographic peak among the Ultra Performance Liquid Chromatography figure of aniline artificial antigen and bovine serum albumin(BSA) and corresponding mass spectrogram (Fig. 2), determine that this artificial antigen prepares.
Embodiment 2
Prepare the sulfaquinoxaline artificial antigen by following process route:
15 mg sulfaquinoxalines, 10 mL 1mol/L hydrochloric acid are added in the 50 mL there-necked flasks, stir, the ice bath cooling.Drip 1% sodium nitrite in aqueous solution of precooling in 0～5 ℃ of scope, until potassium iodide-starch paper becomes blue, lucifuge, insulation reaction 30 min make diazonium salt solution.
300 mg BSA, 20 mL borax-borate buffer solutions (0.2 mol/L, pH 8.7) are placed in another 50 mL there-necked flask, stirred, the ice bath cooling.In 4 ℃ of above-mentioned diazonium salt solutions of lower dropping, maintain about 8 with 5% sodium hydroxide solution conditioned reaction liquid pH value in the dropping process, dropwise lucifuge, insulation reaction 24 h.Then, reactant liquor is packed in the good bag filter of pre-service, with distill water dialysis 3 days, change liquid every day 4 times, vacuum freeze drying makes the cotton-shaped artificial antigen of brown, and this artificial antigen is denoted as SQ-BSA, and-20 ℃ save backup.
Take by weighing respectively this artificial antigen and bovine serum albumin(BSA) 5mg, add volume ratio and be 1: 1 acetonitrile solution 50mg, ultrasonic 1min, after 12000rpm was centrifugal, getting supernatant was artificial antigen sample to be tested and bovine serum albumin(BSA) sample to be tested.
Sample to be tested detects with Ultra Performance Liquid Chromatography quadrupole rod electro spraying ionization-mass spectrometry instrument:
The Ultra Performance Liquid Chromatography condition:
Ultra Performance Liquid Chromatography post: ACQUITY UPLC C4,2.1mm * 150mm, 1.7um
Mobile phase: A phase: acetonitrile; B phase: 0.05: 100 trifluoroacetic acid aqueous solution of volume ratio
Linear gradient: be increased to 80% at 0～10min A by 20% linearity, corresponding B reduces to 20% by 80% linearity
Flow velocity: 0.2mL/min
Column temperature: 30 ℃
Quadrupole rod electrospray ionization mass spectrum condition:
Ion gun: electric spray ion source
Ionization mode: positively ionized
Capillary voltage: 3KV
Taper hole voltage; 30V
Detection mode: full scan
Quality of scanning scope: m/z 1000～1600 amu
Ion source temperature: 100 ℃
Desolventizing temperature: 300 ℃
Desolventizing gas: nitrogen
Desolventizing gas velocity: 700L/hr
Obtain respectively the Ultra Performance Liquid Chromatography figure (upper figure is that sulfaquinoxaline artificial antigen, figure below are bovine serum albumin(BSA) among Fig. 3) of sulfaquinoxaline artificial antigen, bovine serum albumin(BSA) and corresponding quadrupole rod electrospray ionization mass spectrum figure (upper figure is that sulfaquinoxaline artificial antigen, figure below are bovine serum albumin(BSA) among Fig. 4).
According to the difference of the difference (Fig. 3) of the retention time of chromatographic peak among the Ultra Performance Liquid Chromatography figure of sulfaquinoxaline artificial antigen and bovine serum albumin(BSA) and corresponding mass spectrogram (Fig. 4), determine that this artificial antigen prepares.
Embodiment 3
Prepare the Clenbuterol artificial antigen by following process route:
Preparation method with sulfaquinoxaline artificial antigen among the embodiment 2 makes the Clenbuterol artificial antigen, is denoted as CL-BSA, and-20 ℃ save backup.
Take by weighing respectively this artificial antigen and bovine serum albumin(BSA) 5mg, add volume ratio and be 1: 1 ethanol water 10mg, ultrasonic 1min, after 12000rpm was centrifugal, getting supernatant was artificial antigen sample to be tested and bovine serum albumin(BSA) sample to be tested.
Sample to be tested detects with Ultra Performance Liquid Chromatography quadrupole rod electro spraying ionization-mass spectrometry instrument:
The Ultra Performance Liquid Chromatography condition:
Ultra Performance Liquid Chromatography post: ACQUITY UPLC C4,2.1mm * 150mm, 1.7um
Mobile phase: A phase: acetonitrile; B phase: 0.1: 100 trifluoroacetic acid aqueous solution of volume ratio
Linear gradient: be increased to 70% at 0～10min A by 30% linearity, corresponding B reduces to 30% by 70% linearity
Flow velocity: 0.2mL/min
Column temperature: 30 ℃
Quadrupole rod electrospray ionization mass spectrum condition:
Ion gun: electric spray ion source
Ionization mode: positively ionized
Capillary voltage: 3KV
Taper hole voltage; 30V
Detection mode: full scan
Quality of scanning scope: m/z 1000～1600 amu
Ion source temperature: 100 ℃
Desolventizing temperature: 300 ℃
Desolventizing gas: nitrogen
Desolventizing gas velocity: 700L/hr
Obtain respectively the Ultra Performance Liquid Chromatography figure (upper figure is that Clenbuterol artificial antigen, figure below are bovine serum albumin(BSA) among Fig. 5) of Clenbuterol artificial antigen, bovine serum albumin(BSA) and corresponding quadrupole rod electrospray ionization mass spectrum figure (upper figure is that Clenbuterol artificial antigen, figure below are bovine serum albumin(BSA) among Fig. 6).
According to the difference of the difference (Fig. 5) of the retention time of chromatographic peak among the Ultra Performance Liquid Chromatography figure of Clenbuterol artificial antigen and bovine serum albumin(BSA) and corresponding mass spectrogram (Fig. 6), determine that this artificial antigen prepares.
Embodiment 4
Prepare the benzoic acid artificial antigen by following process route:
Take by weighing the 0.12g benzoic acid, be dissolved in 3mL Isosorbide-5-Nitrae-dioxane, restrain tri-n-butylamine and 0.14g isobutyl chlorocarbonates and stir 30min in 4 ℃ of lower addings 0.19, this is A liquid; Take by weighing the 0.3g bovine serum albumin(BSA) and be dissolved in the 50mL carbonate buffer solution (pH9.6,0.2mol/L), this is B liquid.Under 4 ℃, slowly drop to A liquid in the B liquid and stir 24h, reactant liquor changes in the bag filter, uses phosphate normal saline dialysis 4 days, changes phosphate normal saline dialysis liquid every day twice, it is the benzoic acid artificial antigen that the product freeze drying gets white powder, and note is BJS-BSA.
Take by weighing respectively this BJS-BSA and carrier bovine serum albumin(BSA) 5mg, add volume ratio and be 1: 1 aqueous acetone solution 5mg, ultrasonic 1min, after 12000rpm was centrifugal, getting supernatant was artificial antigen sample to be tested and bovine serum albumin(BSA) sample to be tested.
Sample to be tested detects with Ultra Performance Liquid Chromatography quadrupole rod electro spraying ionization-mass spectrometry instrument:
The Ultra Performance Liquid Chromatography condition:
Ultra Performance Liquid Chromatography post: ACQUITY UPLC C4,2.1mm * 150mm, 1,7um
Mobile phase: A phase: acetonitrile; B phase: 0.1: 100 trifluoroacetic acid aqueous solution of volume ratio
Linear tonsure: be increased to 70% at 0～10min A by 30% linearity, corresponding B reduces to 30% by 70% linearity
Flow velocity: 0.2mL/min
Column temperature: 30 ℃
Quadrupole rod electrospray ionization mass spectrum condition:
Ion gun: electric spray ion source
Ionization mode: positively ionized
Capillary voltage: 2.5KV
Taper hole voltage; 35V
Detection mode: full scan
Quality of scanning scope: m/z 1000～1600 amu
Ion source temperature: 150 ℃
Desolventizing temperature: 250 ℃
Desolventizing gas: nitrogen
Desolventizing gas velocity: 400L/hr
Obtain respectively the Ultra Performance Liquid Chromatography figure (upper figure is that benzoic acid artificial antigen, figure below are bovine serum albumin(BSA) among Fig. 7) of benzoic acid artificial antigen, bovine serum albumin(BSA) and corresponding quadrupole rod electrospray ionization mass spectrum figure (upper figure is that benzoic acid artificial antigen, figure below are bovine serum albumin(BSA) among Fig. 8).
Result's demonstration does not go out the peak in whole Ultra Performance Liquid Chromatography figure, once a baseline illustrated under this testing conditions, and artificial antigen does not prepare benzoic acid.
Claims (2)
1. method of judging whether artificial antigen prepares is characterized in that the method comprises following concrete steps:
A, take by weighing artificial antigen and the carrier protein of identical weight respectively, add 1～10 times of weight dissolution with solvents, ultrasonic 1min after 12000rpm is centrifugal, gets supernatant, obtains respectively artificial antigen specimen and carrier protein specimen;
B, usefulness Ultra Performance Liquid Chromatography quadrupole rod electro spraying ionization-mass spectrometry instrument detect specimen respectively, obtain respectively its Ultra Performance Liquid Chromatography figure and corresponding quadrupole rod electrospray ionization mass spectrum figure;
The difference of the Ultra Performance Liquid Chromatography figure of c, comparison gained artificial antigen and carrier protein and corresponding mass spectrogram thereof determines whether artificial antigen prepares;
Wherein: when with Ultra Performance Liquid Chromatography quadrupole rod electro spraying ionization-mass spectrometry instrument specimen being detected, its Ultra Performance Liquid Chromatography condition is:
Mobile phase:
A phase: acetonitrile
B phase: 0.01: 100～0.1: 100 trifluoroacetic acid aqueous solution of volume ratio
Linear gradient: be increased to 70%～90% at 0～10min A by 10%～30% linearity, corresponding B reduces to 30%～10% by 70%～90% linearity
Liquid-phase chromatographic column is: ACQUITY UPLC C4,2.1mm * 150mm, 1.7um;
Its quadrupole rod electrospray ionization mass spectrum condition is:
Ion gun: electric spray ion source
Ionization mode: positive ion ionization
Detection mode: full scan;
Capillary voltage: 2.5～3KV
Taper hole voltage; 20～35V
Quality of scanning scope: m/z 1000～1600 amu
Ion source temperature: 100～150 ℃
Desolventizing temperature: 250～300 ℃
Desolventizing gas: nitrogen
Desolventizing gas velocity: 400～700L/hr;
Described solvent is the potpourri of water or organic solvent and water; Organic solvent is ethanol, methyl alcohol, acetonitrile or acetone; The volume ratio of organic solvent and water is 1:1.
2. described method according to claim 1 is characterized in that described carrier protein is bovine serum albumin(BSA).
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| CN102507296B (en) * | 2011-11-04 | 2013-07-10 | 北京大学 | Trifluoroacetic acid removing device and application thereof |
| CN103588661A (en) * | 2012-08-17 | 2014-02-19 | 北京维德维康生物技术有限公司 | Preparation of hapten and artificial antigen of nicarbazin |
| CN110007023B (en) * | 2019-04-15 | 2021-08-13 | 陕西科技大学 | High-resolution mass spectrum screening method for sulfonamides in fish body and analysis method for interaction of sulfonamides and protein macromolecules |
| CN113372295A (en) * | 2021-07-08 | 2021-09-10 | 河北大学 | Phenothiazine hapten, complete antigen, preparation method and application thereof |
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| CN1700001A (en) * | 2005-02-24 | 2005-11-23 | 黄永 | Organic chlorine pesticide benzoepin artificial antigen and antibody, their preparation and use thereof |
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| CN1700001A (en) * | 2005-02-24 | 2005-11-23 | 黄永 | Organic chlorine pesticide benzoepin artificial antigen and antibody, their preparation and use thereof |
Non-Patent Citations (4)
| Title |
|---|
| 余伟等.苏丹红I人工抗原的合成.《卫生研究》.2008,第37卷(第03期),362-364、376. |
| 动物源食品中磺胺二甲嘧啶人工抗原的合成研究;胥传来等;《食品科学》;20050715;第26卷(第07期);118-121 * |
| 胥传来等.动物源食品中磺胺二甲嘧啶人工抗原的合成研究.《食品科学》.2005,第26卷(第07期),118-121. |
| 苏丹红I人工抗原的合成;余伟等;《卫生研究》;20080530;第37卷(第03期);362-364、376 * |
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