CN104478813A - 5-fluorouracil derivatives, 5-fluorouracil immunogens, antibodies for immunogens and 5-fluorouracil detection kit - Google Patents
5-fluorouracil derivatives, 5-fluorouracil immunogens, antibodies for immunogens and 5-fluorouracil detection kit Download PDFInfo
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- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical class FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 title claims abstract description 238
- 229960002949 fluorouracil Drugs 0.000 title claims abstract description 135
- 238000001514 detection method Methods 0.000 title claims abstract description 34
- 241001465754 Metazoa Species 0.000 claims abstract description 12
- -1 5 FU 5 fluorouracil derivative Chemical class 0.000 claims description 77
- 230000002163 immunogen Effects 0.000 claims description 70
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 48
- 239000000243 solution Substances 0.000 claims description 31
- 238000002360 preparation method Methods 0.000 claims description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 22
- 238000003756 stirring Methods 0.000 claims description 22
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 12
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- 238000012360 testing method Methods 0.000 claims description 11
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
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- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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- RWQNBRDOKXIBIV-UHFFFAOYSA-N Thymine Natural products CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 1
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- CZDYPVPMEAXLPK-UHFFFAOYSA-N tetramethylsilane Chemical compound C[Si](C)(C)C CZDYPVPMEAXLPK-UHFFFAOYSA-N 0.000 description 1
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
- C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
- C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
- C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
- C07D239/52—Two oxygen atoms
- C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
- C07D239/545—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals with other hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
- C07D239/553—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals with other hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms with halogen atoms or nitro radicals directly attached to ring carbon atoms, e.g. fluorouracil
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention provides 5-fluorouracil derivatives, 5-fluorouracil immunogens, antibody for the immunogens, and a 5-fluorouracil detection kit. The 5-fluorouracil derivatives provided by the invention have a structure shown in a formula (I) as shown in the specification, wherein R is -(CH2)n-COO- and n is an integer between 1 and 20. The 5-fluorouracil derivatives with the structural formula are combined with a specific carrier, so that the prepared 5-fluorouracil immunogens have high immunogenicity; and the antibodies generated by an animal immunized with the immunogens have relatively high sensitivity and specificity, and have high specific combining ability with 5-fluorouracil. Through use of a homogeneous enzyme immune detection technology, high-flux and fast detection of 5-fluorouracil by an full automatic biochemical analyzer can be achieved; and the detection is convenient to operate, high in sensitivity, high in specificity, accurate in result and the like; the 5-fluorouracil detection cost can be effectively reduced; and clinical promotion and application are facilitated.
Description
Technical field
The present invention relates to field of immunodetection, be specifically related to a kind of 5 FU 5 fluorouracil derivative, 5 FU 5 fluorouracil immunogen and antibody thereof and 5 FU 5 fluorouracil detection kit.
Background technology
5 FU 5 fluorouracil (5-Fluorouracil, 5-FU), its structural formula is as shown in formula III:
5 FU 5 fluorouracil is the analogue of uridylic, is one of most important chemotherapeutics in cancer therapy.Be generally used in the chemotherapy regimen of the various cancers such as colon and rectum carcinoma, cancer of the stomach, mammary cancer and carcinoma of the pancreas.This medicine determined curative effect, the thymine synthetase inactivation of synthetic DNA can be made, also can mix in RNA and cause lethal synthesis, 5-FU metabolism in vivo, remove relevant with many factors, pharmacokinetics has the feature of non-linear and easy saturability, and toxic reaction and curative effect have in individuality highly and interindividual variation.5-FU, usually with severe side effect, comprises bone marrow depression, mucositis, dermatitis, nausea,vomiting,diarrhea, cardiac toxic even dead.The clinical monitoring using 5-FU to carry out patient's Plasma Concentration for the treatment of all has important clinical meaning for 5-FU dose titration, curative effect optimization and minimizing serious adverse reaction.
At present, domestic and international monitoring 5-FU Plasma Concentration mainly uses the traditional methods such as high performance liquid chromatography (HPLC) method, but these methods are not suitable for clinical application, although domestic existing use latex enhancing immune turbidimetry, the 5 FU 5 fluorouracil that can be applicable to Biochemical Analyzer measures test kit listing, but far can not meet growing clinical detection demand.The 5 FU 5 fluorouracil detection reagent of good, highly sensitive, the high specificity of deficient in stability in the market, especially the measured Automated inspection reagent of matter, therefore, development & production quality reaches clinical requirement, practical, cost performance is high, and the 5 FU 5 fluorouracil that can be applicable to automatic clinical chemistry analyzer measures the focus that reagent has become domestic and international external diagnosis reagent industry.
Summary of the invention
Main purpose of the present invention is to provide a kind of 5 FU 5 fluorouracil derivative, 5 FU 5 fluorouracil immunogen and antibody thereof and 5 FU 5 fluorouracil detection kit, to provide a kind of good stability, high specificity, be applicable to the 5 FU 5 fluorouracil mensuration reagent that full-automatic biochemical is analyzed.
In order to achieve the above object, according to an aspect of the present invention, provide a kind of 5 FU 5 fluorouracil derivative, there is the structure shown in formula I:
Wherein, R is-(CH
2) n-COO-, n be integer between 1 to 20.The 5 FU 5 fluorouracil derivative with this structure possesses to be prepared into and has immunogenic immunogenic basic structure, provides architecture basics for preparing new 5 FU 5 fluorouracil detection reagent.In the present invention, the 5 FU 5 fluorouracil derivative preferably as n=1, now, R is-CH
2-COO-.
In accordance with a further aspect of the present invention, additionally provide a kind of 5 FU 5 fluorouracil immunogen, there is the structural formula shown in formula II:
Wherein, the R integer that to be linking group-(CH2) n-COO-, n be between 1 to 20, carrier is for having immunogenic protein or polypeptide.Comprise 5 FU 5 fluorouracil derivative molecular structure and the 5 FU 5 fluorouracil immunogen of the present invention with immunogenic protein or polypeptide, immunogenicity is high, immunne response can be produced by stimulating animal body, produce the anti-5 FU 5 fluorouracil specific antibody of high-titer, and there is very strong reactionogenicity, be suitable as the competitive reagent of vitro detection 5 FU 5 fluorouracil content.
Carrier in above-mentioned 5 FU 5 fluorouracil immunogen, because immunogenic carrier is generally protein or polypeptide, what other were enough large in theory possess immunogenic material also can as carrier, but select protein as carrier under normal circumstances.The most frequently used immunogenic carrier comprises serum protein, hemocyanin (KLH) and thyroglobulin.In the present invention, above-mentioned carrier includes but are not limited to any one in serum protein, hemocyanin or thyroglobulin.Serum protein, hemocyanin or thyroglobulin, as carrier, have relatively high immunogenicity, and above-mentioned 5 FU 5 fluorouracil immunogen can be made to have higher immunogenicity.In the present invention, be more preferably serum protein, serum protein wide material sources, simple and easy to get and immunogenicity is high.
According to another aspect of the present invention, provide the immunogenic preparation method of a kind of 5 FU 5 fluorouracil, this 5 FU 5 fluorouracil immunogen is formed by connecting by above-mentioned 5 FU 5 fluorouracil derivative and carrier, and carrier is for having immunogenic protein or polypeptide.The 5 FU 5 fluorouracil derivative prepared by aforesaid method with there is immunogenic protein or polypeptide links together, above-mentioned 5 FU 5 fluorouracil immunogen of the present invention can be obtained, simple to operate, and the immunogenic immunogenicity of the 5 FU 5 fluorouracil obtained is strong.
In the immunogenic preparation method of above-mentioned 5 FU 5 fluorouracil, additionally provide a kind of preparation method of above-mentioned 5 FU 5 fluorouracil derivative, above-mentioned 5 FU 5 fluorouracil derivative is by 5 FU 5 fluorouracil and Br-(CH
2) n-COOH substitution reaction occurs in the basic conditions and obtains.By with 5 FU 5 fluorouracil and Br-(CH
2) n-COOH is raw material, can prepare above-mentioned 5 FU 5 fluorouracil derivative through simple substitution reaction, obtain 5 FU 5 fluorouracil immunogen of the present invention provide basic substance for follow-up.Further, as n=1, the preparation process of above-mentioned 5 FU 5 fluorouracil derivative is as follows:
The preparation process of above-mentioned 5 FU 5 fluorouracil derivative selects 2-bromoacetic acid to be synthesis material, and obtaining linking group R is-CH
2the 5 FU 5 fluorouracil derivative of-COO-.2-bromoacetic acid and Br-(CH
2) n-COOH is only the difference of n numerical value, both are above-mentioned, and to prepare the method steps of 5 FU 5 fluorouracil derivative identical.
In the immunogenic preparation method of above-mentioned 5 FU 5 fluorouracil, 5 FU 5 fluorouracil derivative suitably can adjust according to the different of connected carrier from the step that carrier connects.In the present invention, this step comprises: step S1, prepares carrier soln and 5 FU 5 fluorouracil derivative solution; Step S2, drops in carrier soln by 5 FU 5 fluorouracil derivative solution, obtains 5 FU 5 fluorouracil immunogen crude product; Step S3, carries out purifying to immunogen crude product, obtains 5 FU 5 fluorouracil immunogen.Preparation process of the present invention can obtain target product by simple dropping, purification step, and preparation method is simple, technology stability is high, favorable repeatability.
Prepare in carrier Connection Step above-mentioned, step S1 comprises: step S11, by carrier solubilizes in the sodium phosphate buffer of 0.15 ~ 0.25M, pH 8.2 ~ 8.7, obtains carrier soln; Step S12, mixes the potassium phosphate buffer of 5 FU 5 fluorouracil derivative and 8 ~ 12mM, pH 4.8 ~ 5.3 and organic solvent, obtains mixture; Step S13, stirs mixture, obtains 5 FU 5 fluorouracil derivative solution.In the step preparing carrier soln, adopt phosphoric acid buffer in above-mentioned concentration and above-mentioned pH value range can solution carrier more up hill and dale, the structures and characteristics of carrier can be kept again stable and there is immunogenicity.And in the step preparing 5 FU 5 fluorouracil derivative solution, adopt concentration can keep the biologically stable of 5 FU 5 fluorouracil derivative solution at 8 ~ 12mM, the pH potassium phosphate buffer in 4.8 ~ 5.3 scopes; Adopt organic solvent can promote that it dissolves.
In above-mentioned steps S11, to the mass volume ratio not particular requirement of carrier and sodium phosphate buffer, as long as selected carrier solubilizes can be made according to the difference of the kind of selected carrier.In the present invention, the mass volume ratio (mg:ml) of preferred vector and sodium phosphate buffer is 3:1 ~ 5:1, and within the scope of this, carrier solubilizes obtains comparatively abundant; Be more preferably 4:1, carrier can dissolve more fully.
In above-mentioned steps S12, to the selection of organic solvent also without particular requirement, all organic solvents that can promote that 5 FU 5 fluorouracil derivative dissolves all are applicable to the present invention.In the present invention, preferred above-mentioned organic solvent comprises dimethyl formamide, ethanol, 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide and N-hydroxy thiosuccinimide.Above-mentioned organic solvent cooperatively interacts, to promoting that the linking group of the dissolving of 5 FU 5 fluorouracil derivative and activation 5 FU 5 fluorouracil derivative has collaborative promoter action.
In above-mentioned steps S12, the mass ratio of 5 FU 5 fluorouracil derivative and 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide and N-hydroxy thiosuccinimide is 1:0.8 ~ 1.1:0.20 ~ 0.30; The volume ratio of dimethyl formamide and ethanol and potassium phosphate buffer is 1:0.8 ~ 1.1:1.8 ~ 2.2; The mass volume ratio of the cumulative volume of preferred 5 FU 5 fluorouracil derivative and dimethyl formamide, ethanol and potassium phosphate buffer is 95 ~ 105:6 ~ 8.
In the preparation process of above-mentioned 5 FU 5 fluorouracil derivative solution, the mass ratio of 5 FU 5 fluorouracil derivative and 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide and N-hydroxy thiosuccinimide is controlled in the scope of 1:0.8 ~ 1.1:0.20 ~ 0.30; The volume ratio of dimethyl formamide and ethanol and potassium phosphate buffer is controlled in the scope of 1:0.8 ~ 1.1:1.8 ~ 2.2; And the mass volume ratio of the cumulative volume of 5 FU 5 fluorouracil derivative and dimethyl formamide, ethanol and potassium phosphate buffer is controlled in the scope of 95 ~ 105:6 ~ 8, three cooperatively interacts optimization, 5 FU 5 fluorouracil derivative can be made to dissolve more fully, and more efficiently activated by the linking group of 5 FU 5 fluorouracil derivative, the efficiency making linking group be connected with carrier is higher.
In above-mentioned steps S13, stir the dissolving that can promote 5 FU 5 fluorouracil derivative, stir and at room temperature carry out.The time of concrete stirring carries out Reasonable adjustment according to the number of the amount of dissolved 5 FU 5 fluorouracil derivative.In invention, the time of stirring is 20 ~ 40min.
In the immunogenic preparation method of above-mentioned 5 FU 5 fluorouracil of the present invention, to the mass ratio of 5 FU 5 fluorouracil derivative and carrier without particular requirement, as long as the 5 FU 5 fluorouracil immunogen with high immunogenicity can be connected to form efficiently.In the present invention, preferably both mass ratioes are 0.8 ~ 1.1:0.8 ~ 1.1, to be controlled by both mass ratioes above-mentioned, close in the scope of 1:1, can improve joint efficiency.
In the immunogenic preparation method of above-mentioned 5 FU 5 fluorouracil of the present invention, above-mentioned steps S2 comprises: step S21, drops in carrier soln, obtain mixing solutions by 5 FU 5 fluorouracil derivative solution; Step S22, carries out low temperature stirring to mixing solutions, obtains immunogen crude product; Wherein, the temperature that low temperature stirs is 2 ~ 8 DEG C, and the time that low temperature stirs is more than or equal to 24h.Dropping can promote the abundant reaction of 5 FU 5 fluorouracil derivative and carrier, and stirs at low temperatures, is that low temperature stability inferior is good in order to make the target product generated successively keep its distinctive materialization active.
In the step S3 of above-mentioned preparation method, as long as under the prerequisite ensureing 5 FU 5 fluorouracil immunogen physicochemical stability, any method that its purity can be made to improve all is applicable to the present invention.In the present invention, include but not limited to adopt the mode of dialysis to carry out purifying to 5 FU 5 fluorouracil immunogen crude product, obtain 5 FU 5 fluorouracil immunogen.The mode of dialysis is simple and purification effect is good.
According to a further aspect of the invention, provide a kind of antibody, this antibody obtains by after immunogen immune animal, and wherein immunogen is above-mentioned any one immunogen mentioned.Because above-mentioned immunogen has very strong immunogenicity, after making immune animal, the antibodies specific that stimulating animal body produces is strong, susceptibility is high, strong with the bonding force of 5 FU 5 fluorouracil.
In the present invention, " antibody " of indication not only refers to complete antibody protein molecule, also comprises the antibody polypeptides segment or derivatives thereof retaining complete antibody specific binding capacity.Antibody of the present invention can be for polyclonal antibody also can be monoclonal antibody, is preferably polyclonal antibody.
Antibody of the present invention can be prepared by prior art.The typical method obtaining polyclonal antibody uses single immunogen, and after adding or not adding adjuvant, carry out immunity at one or more position of animal, host animal comprises: rabbit, goat, mouse, sheep, cavy or horse.Persistent immunological carries out always, until antibody titer reaches the highest.Animal timing blood sampling obtains appropriate specific corrosioning anteserum, and antiserum(antisera) can purifying.Monoclonal antibody is prepared by somatocyte hybriding technology.
According to another aspect of the invention, additionally provide a kind of 5 FU 5 fluorouracil detection reagent, comprise the substrate of anti-5 FU 5 fluorouracil specific antibody, 5 FU 5 fluorouracil enzyme mark conjugate and enzyme, wherein, anti-5 FU 5 fluorouracil specific antibody is any one anti-5 FU 5 fluorouracil specific antibody above-mentioned; 5 FU 5 fluorouracil enzyme mark conjugate is formed by enzyme and hapten conjugation, and haptens is above-mentioned 5 FU 5 fluorouracil derivative.Mentioned reagent of the present invention, due to the high specificity of anti-5 FU 5 fluorouracil specific antibody, strong with the bonding force of 5 FU 5 fluorouracil, detection sensitivity is far above corresponding product of the prior art.Preferably, above-mentioned enzyme mark conjugate is glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate; The substrate of above-mentioned enzyme is G-6-P.The 5 FU 5 fluorouracil content adopting glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate and adopt G-6-P can determine as the detection reagent of the substrate of enzyme in sample easily and accurately, is applicable to high-throughout Aulomatizeted Detect.
According to another aspect of the invention, additionally provide a kind of 5 FU 5 fluorouracil detection kit, containing above-mentioned anti-5 FU 5 fluorouracil specific antibody and detect anti-5 FU 5 fluorouracil specific antibody and be combined with 5 FU 5 fluorouracil and the indicator of the mixture formed.Indicator is selected from enzyme reagent, radio isotope reagent, fluorescent reagent, luminescence reagent.Preferably, indicator is made up of the substrate of 5 FU 5 fluorouracil enzyme mark conjugate and enzyme, can determine the 5 FU 5 fluorouracil content in sample easily and accurately, be applicable to high-throughout Aulomatizeted Detect.
It should be noted that, 5 FU 5 fluorouracil derivative of the present invention and anti-5 FU 5 fluorouracil antibody can be applied to separately in the detection of 5 FU 5 fluorouracil, also can apply simultaneously, specifically can carry out choose reasonable according to the difference of testing goal.
Apply technical scheme of the present invention, the preparation-obtained 5 FU 5 fluorouracil immunogen of 5 FU 5 fluorouracil derivative provided by the present invention, have high immunogenicity, the antibodies specific that the animal of institute's immune induction produces is high, strong with the specific combination ability of 5 FU 5 fluorouracil.Can realize on automatic clinical chemistry analyzer the high-throughput of 5 FU 5 fluorouracil, rapid detection by using homogeneous enzyme immunoassay detection technique, and there is the advantages such as easy and simple to handle, highly sensitive, high specificity, result are accurate, can also effectively reduce 5 FU 5 fluorouracil testing cost, be conducive to clinical expansion and use.
Accompanying drawing explanation
The Figure of description forming a application's part is used to provide a further understanding of the present invention, and schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 shows the ELISA detection reaction curve according to 5 FU 5 fluorouracil in embodiments of the invention six; And
Fig. 2 shows according to 5 FU 5 fluorouracil homogeneous enzyme immunoassay response curve in embodiments of the invention seven.
Embodiment:
It should be noted that, when not conflicting, the embodiment in the application and the feature in embodiment can combine mutually.Below with reference to the accompanying drawings and describe the present invention in detail in conjunction with the embodiments.
Embodiment one: the synthesis of 5 FU 5 fluorouracil derivative and structural confirmation thereof
The 5 FU 5 fluorouracil derivatives chemical structure used in following examples is as shown in formula IV:
Shown in this formula IV, the synthetic route of 5 FU 5 fluorouracil derivative is as follows:
The synthesis step of the 5 FU 5 fluorouracil derivative shown in formula IV is as follows:
1) taking 5g (38.5mmol) compound 15-Fluracil, 6.95g (50.3mmol) 2-bromoacetic acid and 4.48g (80.0mmol) KOH is dissolved in 50mL water jointly, is then stirred at 60 DEG C by this solution and spends the night; By this reactant by HCl adjust ph to pH=5, then filter; Vacuum-drying is carried out, the final 5 FU 5 fluorouracil derivative obtaining 2g white solid, productive rate 27.6% after the solid matter water used wash that filtration is obtained.
2) Structural Identification is carried out to above-mentioned gained purified product:
A. utilize Varian III plus 300MHz to carry out NMR (Nuclear Magnetic Resonance) spectrum scanning to above-claimed cpd, adopt tetramethylsilane (TMS) as interior mark.Result is as follows: 1H-NMR (400MHz, DMSO-d6): δ 4.37 (s, 2H), 8.09-8.11 (d, 1H), 11.91-11.92 (d, 1H), 13.23 (br, 1H).Be characterized by the 5 FU 5 fluorouracil derivative shown in formula IV.
B. utilize Chromatography/Mass Spectrometry technology (LCMS) to carry out Analysis and Identification to the derivative obtained, determine that this final gained compound is for the 5 FU 5 fluorouracil derivative shown in formula IV.
In the present embodiment, be when n=1, selected 2-bromoacetic acid to be synthesis material, therefore the link radicals R of the final product 5 FU 5 fluorouracil derivative of gained is-CH
2-COO-; When n gets the integer between other 2-20, select other and 2-bromoacetic acid relative to the organic acid that replaces of the bromine of n test time, except n value difference, synthetic method is completely the same.The 5 FU 5 fluorouracil derivative prepared detects and lcms analysis qualification through above-mentioned nuclear-magnetism equally, all meets the 5 FU 5 fluorouracil derivative corresponding with n.
The immunogenic synthesis of embodiment two: BSA-5-Fluorouracil derivative
BSA-5-Fluracil immunogen by the 5 FU 5 fluorouracil derivative shown in bovine serum albumin (Bovine Serum Albumin, BSA) Yu formula I-(CH
2) n-COO-group is formed by connecting, in the present embodiment, describe this immunogenic synthetic method in detail for n=1, concrete steps are as follows:
Bovine serum albumin (200mg) is dissolved in 50ml 0.2M, in the phosphoric acid buffer of pH 8.5;
Following chemical is joined stirring and dissolving in small beaker: 5 FU 5 fluorouracil derivative, the 3.5ml dimethyl formamide (dimethylformamide of 200mg synthesis, DMF), 3.5ml ethanol, 7.0ml 10mM, the potassium phosphate buffer of pH 5.0,220mg 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 50mg N-hydroxy thiosuccinimide (N-hydroxysulfosuccinimide, Sulfo-NHS), by these chemical at room temperature stirring and dissolving reaction 30min;
The solution dissolved is dropped in BSA solution, and stirs at 4 DEG C and spend the night, obtain antigen; Synthetic antigen is carried out purifying through dialysis, obtains BSA-5-Fluracil immunogen.
The immunogenic synthesis of embodiment three: KLH-5-Fluorouracil derivative
KLH-5-Fluracil immunogen by the 5 FU 5 fluorouracil derivative shown in hemocyanin (KLH) Yu formula I-(CH
2) n-COO-group is formed by connecting, in the present embodiment, describe this immunogenic synthetic method in detail for n=2, concrete steps are as follows:
Hemocyanin (180mg) is dissolved in 60ml 0.15M, in the phosphoric acid buffer of pH 8.7;
Following chemical is joined stirring and dissolving in small beaker: 5 FU 5 fluorouracil derivative, the 3.5ml dimethyl formamide (dimethylformamide of 200mg synthesis, DMF), 2.8ml ethanol, 6.3ml 12mM, the potassium phosphate buffer of pH 4.8,160mg 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 40mg N-hydroxy thiosuccinimide (N-hydroxysulfosuccinimide, Sulfo-NHS), by these chemical at room temperature stirring and dissolving reaction 25min;
The solution dissolved is dropped in KLH solution, and stirs at 2 DEG C and spend the night, obtain antigen; Synthetic antigen is carried out purifying through dialysis, obtains KLH-5-Fluracil immunogen.
Embodiment four: thyroglobulin-immunogenic synthesis of 5 FU 5 fluorouracil derivative
Thyroglobulin-5 FU 5 fluorouracil immunogen by the 5 FU 5 fluorouracil derivative shown in thyroglobulin and formula I-(CH
2) n-COO-group is formed by connecting, in the present embodiment, describe this immunogenic synthetic method in detail for n=5, concrete steps are as follows:
Thyroglobulin (220mg) is dissolved in 49ml 0.25M, in the phosphoric acid buffer of pH 8.2;
Following chemical is joined stirring and dissolving in small beaker: 5 FU 5 fluorouracil derivative, the 3.5ml dimethyl formamide (dimethylformamide of 180mg synthesis, DMF), 3.85ml ethanol, 7.7ml 8mM, the potassium phosphate buffer of pH 5.3,245mg 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 60mg N-hydroxy thiosuccinimide (N-hydroxysulfosuccinimide, Sulfo-NHS), by these chemical at room temperature stirring and dissolving reaction 40min;
The solution dissolved is dropped in thyroglobulin solution, and stirs at 8 DEG C and spend the night, obtain antigen; Synthetic antigen is carried out purifying through dialysis, obtains thyroglobulin-5 FU 5 fluorouracil immunogen.
Similar, when n gets other integers in 1 ~ 20 scope, suitably adjust above-mentioned parameter, use the same method the 5 FU 5 fluorouracil immunogen can prepared as shown in formula II.Certainly, carrier, still for having immunogenic protein, can be serum protein, hemocyanin (KLH) and thyroglobulin.Preferably, carrier is bovine serum albumin.
Linking group R is only provided to be-(CH2) n-COO-in the above embodiment of the present invention, and n=1,2 and 55 FU 5 fluorouracil derivative synthetic example and carried out relevant subsequent experiment, because linking group mainly plays the ligation of small molecule derivative and carrier, and linking group structure of the present invention is simple, unbranched, interference can not be caused to micromolecular 5-FU, connect in specific connection site, carry out being connected formed 5 FU 5 fluorouracil immunogen with specific carrier, there is the advantage that immunogenicity is strong.Therefore, through experimental verification, when n gets other integers any between 1 to 20, experimental result there is no significant difference, the 5 FU 5 fluorouracil immunogen using the 5 FU 5 fluorouracil derivative of different n value to prepare all possesses strong immunogenicity, and the specific antibody of corresponding preparation all has excellent properties.
Embodiment five: the preparation of anti-5 FU 5 fluorouracil specific antibody
Above-mentioned obtained BSA-5-Fluracil immunogen is adopted ordinary method inoculation experiments animal rabbit, get antiserum(antisera) after booster immunization, concrete steps are as follows:
With PBS, the BSA-5-Fluracil immunogen of above-mentioned synthesis is diluted to 1.0mg/ml, obtains antigenic solution, then mix with 1.0ml Freund's complete adjuvant with 1.0ml antigenic solution, experimental animal rabbit is injected.
After 2 ~ 3 weeks, then with the identical antigenic solution of 1.0ml and Freund's incomplete adjuvant, above-mentioned experimental animal rabbit is injected once, afterwards every surrounding injection once, amount to injection 4 times.
Get blood to above-mentioned experimental animal rabbit, separation and purification obtains antiserum(antisera).Utilize conventional antibody titer measuring method, with the blank serum without antibody in contrast, ELISA detection is carried out after antiserum(antisera) being diluted certain multiple, final detection obtains tiring as 1:30000-1:50000 of anti-5 FU 5 fluorouracil specific antibody of the present invention, shows the high specificity of the antibody prepared by the present invention, highly sensitive.
Embodiment six: 5 FU 5 fluorouracil ELISA checks
Adopt antibody obtained in above-described embodiment five to carry out the ELISA inspection of 5 FU 5 fluorouracil, this inspection utilizes competitive immunization analytical method to measure the 5 FU 5 fluorouracil content in liquid sample.
5 FU 5 fluorouracil derivative (HRP-5-Fluorouracil derivative enzyme conjugates) competition binding shown in 5 FU 5 fluorouracil in sample and the formula IV of coupling is coated on the limited site in elisa plate on antibody.If be not almost with or without 5 FU 5 fluorouracil in liquid sample, the 5 FU 5 fluorouracil derivative of HRP enzyme coupling will with the antibodies in enzyme plate.Contrary, if containing 5 FU 5 fluorouracil that is a large amount of or some amount in liquid sample, so enzyme-5 FU 5 fluorouracil derivative conjugate will reduce the combination with antibody, thus color signal is weakened.Therefore, the 5 FU 5 fluorouracil content in the absorbancy produced and liquid sample is checked to be inversely proportional to.Specific experiment step is as follows:
The foundation of 1.5-Fluracil ELISA examination criteria curve
(1) preparation of standard substance
5 FU 5 fluorouracil powder (being purchased from Sigma company) is dissolved in methanol solution, is prepared into the storage liquid of 1mg/ml.Storage liquid being diluted successively with ELISA damping fluid is the standardized solution of 250.00ng/mL, 200.00ng/mL, 150.00ng/mL, 100.00ng/mL, 50.00ng/mL and 0.00ng/mL.Wherein, ELISA damping fluid contains 50.0mM Tris, the BSA of 145mM NaCl and 0.25%.
(2) the ELISA method of inspection preparation standard curve of 5 FU 5 fluorouracil is utilized
With PBS, anti-5 FU 5 fluorouracil antibody dilution prepared in embodiment five is become the final concentration solution of 1:8000,100 μ L/ holes are coated on 96 hole elisa plates, place 12-24h for 4 DEG C; After the above-mentioned 96 hole elisa plates being coated with anti-5 FU 5 fluorouracil antibody being washed 3 times with PBS, add the BSA solution of 0.5% of 200 μ L/ holes, close for 4 DEG C and place 8-16h.Then wash 3 times with PBS, add the standard substance in 20 μ L/ holes.Add the HRP-5-Fluracil conjugate of 100 μ L/ hole working concentrations again; After incubated at room temperature 30min, PBS washes plate 5 times; Then every hole adds 100 μ L tmb substrates, incubated at room 30min.Every hole adds 100 μ L stop buffers (2M sulfuric acid) again.Measure the light absorption value of 450nm.The light absorption value calibration of the 450nm corresponding to each standard substance, production standard curve, result as shown in Figure 1.
2. the detection of 5 FU 5 fluorouracil content in testing sample
(1) testing sample is made
Preparation method: 5 FU 5 fluorouracil powder (being purchased from Sigma company) is dissolved in the storage liquid that methanol solution makes 1 μ g/mL, and this storage liquid is diluted in blank whole blood, 0.00 is respectively to final concentration, 20.00,100.00,200.00ng/mL, forms whole blood sample that is blank, basic, normal, high concentration.This blank whole blood is not containing the healthy human blood of 5 FU 5 fluorouracil.
(2) testing method
Utilize the ELISA method of inspection of above-mentioned 5 FU 5 fluorouracil, the whole blood sample of above-mentioned blank, basic, normal, high concentration replaced standard substance, test above-mentioned blank, basic, normal, high concentration whole blood sample at the light absorption value of 450nm.
(3) test result
The typical curve that the 5 FU 5 fluorouracil ELISA of contrast shown in Fig. 1 checks, calculate 5 FU 5 fluorouracil content in each sample, and 3 multiple holes mensuration are carried out to each sample, the actual content according to 5 FU 5 fluorouracil in above-mentioned sample calculates the rate of recovery, and result is as shown in table 1.
The ELISA of table 1:5-Fluracil detects recovery experiment
From result in table 1: the 5 FU 5 fluorouracil rate of recovery adopting 5 FU 5 fluorouracil ELISA detection reagent of the present invention to measure in different concns sample is all higher, equal > 90%, illustrate that anti-5 FU 5 fluorouracil specific antibody of the present invention may be used for the detection of 5 FU 5 fluorouracil in sample, and result precision is high.And, from the Dilution ratio of antibody and the minimum concentration value of detection sample, show that antibody of the present invention is highly sensitive.
Embodiment seven: 5 FU 5 fluorouracil homogeneous enzyme immunoassay is checked
Obtained antibody is adopted to carry out homogeneous enzyme immunoassay inspection (Homogeneous Enzyme Immunoassay) of 5 FU 5 fluorouracil.
This inspection is a kind of competitive reaction, does not need to be separated by solid phase in reaction system with the 5 FU 5 fluorouracil of antibodies with free 5 FU 5 fluorouracil.The ultimate principle of this inspection is, 5 FU 5 fluorouracil free in liquid sample and the binding site of 5 FU 5 fluorouracil derivative to specific antibody be coupled on glucose-6-phosphate dehydrogenase (G6PD) (Glucose-6-Phosphate Dehydrogenase, G6PDH) are at war with.The 5 FU 5 fluorouracil enzyme conjugates of the emulative replacement of the 5 FU 5 fluorouracil in liquid sample and antibodies, and make it discharge from the binding site of antibody, thus make enzyme activity recovery.Therefore, in liquid sample, the content of 5 FU 5 fluorouracil is more, and free 5 FU 5 fluorouracil derivative-G6PDH enzyme conjugates is more, thus can obtain stronger signal.Specific experiment step is as follows:
1. obtain typical curve: arrange auspicious BS200 automatic clinical chemistry analyzer reaction parameter (see table 2) advanced in years, operating process is: first reagent adding A, then adds standard substance, finally adds reagent B.After adding reagent B, measure the OD of different time points
340light absorption value, calculates speed of reaction during different standards product concentration, needs the volume ratio constantly adjusting reagent A and reagent B in actual mechanical process, adjusts light-metering point simultaneously, finally draws comparatively ideal reaction normal graphic representation, as shown in Figure 2.
Table 2: step auspicious BS200 automatic clinical chemistry analyzer reaction parameter
2. by typical curve that homogeneous enzyme immunoassay detection reagent of the present invention obtains, replication basic, normal, high concentration Quality Control sample 10 times, above-mentioned Quality Control sample is: be dissolved in human serum by 5 FU 5 fluorouracil standard substance, is respectively 0.80,5.00 to concentration, 15.00 μ g/ml.Detection data and data analysis are in table 3.
Table 3: sample determination and precision and rate of recovery assessment
3. detected result: the accuracy that homogeneous enzyme immunoassay detection reagent of the present invention measures is high, and the rate of recovery reaches 95%-105%, and precision is high, and CV is all lower than 5%.
Embodiment eight: interfering effects of drug is tested
Choose 62 kinds of common compounds and medicine carries out Interference Detection, adjustment concentration to 10.0 μ g/ml, utilizes homogeneous enzyme immunoassay method to measure, obtains the concentration of respective substance according to typical curve.62 kinds of common compounds and medicine name and measurement result are specifically see table 4.
Table 4: common interference drug determination result
Measurement result: the concentration that above-mentioned 62 kinds of common compounds and medicine are equivalent to 5 FU 5 fluorouracil is all less than 0.1 μ g/ml.Visible, antibody of the present invention is the specific antibody of anti-5 FU 5 fluorouracil.
From above description, can find out, the preparation-obtained 5 FU 5 fluorouracil immunogen of 5 FU 5 fluorouracil derivative provided by the present invention, there is high immunogenicity, the antibodies specific that the animal of institute's immune induction produces is high, strong with the specific combination ability of 5 FU 5 fluorouracil, in detecting in vitro, good stability and highly sensitive, use homogeneous enzyme immunoassay detection technique can be implemented in the high-throughput to 5 FU 5 fluorouracil on automatic clinical chemistry analyzer, rapid detection, and have easy and simple to handle, highly sensitive, high specificity, the advantages such as result is accurate, can also effectively reduce 5 FU 5 fluorouracil testing cost, be conducive to clinical expansion to use.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (12)
1. a 5 FU 5 fluorouracil derivative, is characterized in that, has the structural formula shown in formula I:
Wherein, R is-(CH
2) n-COO-, n be integer between 1 to 20.
2. a 5 FU 5 fluorouracil immunogen, is characterized in that, has the structural formula shown in formula II:
Wherein, R is linking group-(CH
2) n-COO-, n be integer between 1 to 20, carrier is for having immunogenic protein or polypeptide; Preferred described carrier is any one in serum protein, hemocyanin or thyroglobulin.
3. the immunogenic preparation method of 5 FU 5 fluorouracil, is characterized in that, described preparation method comprises:
Prepare 5 FU 5 fluorouracil derivative according to claim 1: and
Described 5 FU 5 fluorouracil derivative is connected with carrier, obtains described 5 FU 5 fluorouracil immunogen;
Wherein, described carrier is for having immunogenic protein or polypeptide.
4. preparation method according to claim 3, is characterized in that, described 5 FU 5 fluorouracil derivative is by 5 FU 5 fluorouracil and Br-(CH
2) n-COOH is substituted reaction in the basic conditions and is prepared from; Preferably, as n=1, the preparation process of described 5 FU 5 fluorouracil derivative is as follows:
5. the preparation method according to claim 3 or 4, is characterized in that, the step that described 5 FU 5 fluorouracil derivative is connected with described carrier comprises:
Step S1, prepares carrier soln and 5 FU 5 fluorouracil derivative solution; Preferred described carrier is any one in serum protein, hemocyanin or thyroglobulin;
Step S2, drops to described 5 FU 5 fluorouracil derivative solution in described carrier soln, obtains 5 FU 5 fluorouracil immunogen crude product;
Step S3, carries out purifying to described immunogen crude product, obtains described 5 FU 5 fluorouracil immunogen; Preferred described purifying adopts the mode of dialysis to carry out.
6. preparation method according to claim 5, is characterized in that, described step S1 comprises:
Step S11, by described carrier solubilizes in the sodium phosphate buffer of 0.15 ~ 0.25M, pH 8.2 ~ 8.7, obtains described carrier soln; Wherein, the mass volume ratio of described carrier and described phosphoric acid buffer is 3:1 ~ 5:1, is preferably 4:1;
Step S12, mixes the potassium phosphate buffer of described 5 FU 5 fluorouracil derivative and 8 ~ 12mM, pH 4.8 ~ 5.3 and organic solvent, obtains mixture; Wherein, described organic solvent comprises dimethyl formamide, ethanol, 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide and N-hydroxy thiosuccinimide;
The mass ratio of preferred described 5 FU 5 fluorouracil derivative and described 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide and N-hydroxy thiosuccinimide is 1:0.8 ~ 1.1:0.20 ~ 0.30; The volume ratio of described dimethyl formamide and described ethanol and described potassium phosphate buffer is 1:0.8 ~ 1.1:1.8 ~ 2.2; More preferably the mass volume ratio of the cumulative volume of described 5 FU 5 fluorouracil derivative and described dimethyl formamide, described ethanol and described potassium phosphate buffer is 95 ~ 105:6 ~ 8;
Step S13, at room temperature carries out stirring 20 ~ 40min to described mixture, obtains described 5 FU 5 fluorouracil derivative solution.
7. preparation method according to claim 6, is characterized in that, described step S2 comprises:
Step S21, drops to described 5 FU 5 fluorouracil derivative solution in described carrier soln, obtains mixing solutions;
Step S22, carries out low temperature stirring to described mixing solutions, obtains described immunogen crude product;
Wherein, the mass ratio of described 5 FU 5 fluorouracil derivative and described carrier is 0.8 ~ 1.1:0.8 ~ 1.1; The temperature that described low temperature stirs is 2 ~ 8 DEG C, and the time that described low temperature stirs is more than or equal to 24h.
8. an anti-5 FU 5 fluorouracil antibody, described anti-5 FU 5 fluorouracil antibody obtains by after immunogen immune animal, it is characterized in that, described immunogen is 5 FU 5 fluorouracil immunogen according to claim 2.
9. antibody according to claim 8, is characterized in that, described antibody is polyclonal antibody or monoclonal antibody.
10. antibody according to claim 8, is characterized in that, the polypeptide fragment that described antibody is complete albumen or can be combined with described immunogens.
11. antibody according to claim 8, is characterized in that, the described animal of described immunogen immune comprise rabbit, goat, mouse, sheep, cavy or Malaysia and China any one.
12. 1 kinds of 5 FU 5 fluorouracil detection kit, is characterized in that, described test kit comprises the anti-5 FU 5 fluorouracil antibody according to any one of 5 FU 5 fluorouracil derivative according to claim 1 and/or claim 8 to 11.
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Cited By (5)
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| CN107132347A (en) * | 2016-11-25 | 2017-09-05 | 南方医科大学 | A kind of time-resolved fluorescence detection kit of 5 fluorouracil blood concentrations of real-time monitoring |
| US10286079B2 (en) | 2015-09-22 | 2019-05-14 | The Regents Of The University Of California | Modified cytotoxins and their therapeutic use |
| US10654864B2 (en) * | 2015-09-22 | 2020-05-19 | The Regents Of The University Of California | Modified cytotoxins and their therapeutic use |
| CN113501877A (en) * | 2021-08-23 | 2021-10-15 | 苏州科铭生物技术有限公司 | Preparation method of anti-5-fluorouracil monoclonal antibody and enzyme-linked immunoassay method of 5-fluorouracil |
| CN114907274A (en) * | 2022-05-11 | 2022-08-16 | 宁夏医科大学 | 5-fluorouracil-1-alkyl acid derivative, preparation method and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10286079B2 (en) | 2015-09-22 | 2019-05-14 | The Regents Of The University Of California | Modified cytotoxins and their therapeutic use |
| US10654864B2 (en) * | 2015-09-22 | 2020-05-19 | The Regents Of The University Of California | Modified cytotoxins and their therapeutic use |
| AU2016326392B2 (en) * | 2015-09-22 | 2021-02-11 | The Regents Of The University Of California | Modified cytotoxins and their therapeutic use |
| CN107132347A (en) * | 2016-11-25 | 2017-09-05 | 南方医科大学 | A kind of time-resolved fluorescence detection kit of 5 fluorouracil blood concentrations of real-time monitoring |
| CN113501877A (en) * | 2021-08-23 | 2021-10-15 | 苏州科铭生物技术有限公司 | Preparation method of anti-5-fluorouracil monoclonal antibody and enzyme-linked immunoassay method of 5-fluorouracil |
| CN114907274A (en) * | 2022-05-11 | 2022-08-16 | 宁夏医科大学 | 5-fluorouracil-1-alkyl acid derivative, preparation method and application thereof |
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