CN104530222A - Paclitaxel immunogen, anti-paclitaxel specific antibody and paclitaxel detection reagent - Google Patents
Paclitaxel immunogen, anti-paclitaxel specific antibody and paclitaxel detection reagent Download PDFInfo
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- CN104530222A CN104530222A CN201410799415.XA CN201410799415A CN104530222A CN 104530222 A CN104530222 A CN 104530222A CN 201410799415 A CN201410799415 A CN 201410799415A CN 104530222 A CN104530222 A CN 104530222A
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- taxol
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- 229930012538 Paclitaxel Natural products 0.000 title claims abstract description 97
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 51
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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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
- C07K14/46—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
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4717—Plasma globulins, lactoglobulin
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- 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
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- 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
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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Abstract
The invention discloses a paclitaxel immunogen and a synthetic method of the paclitaxel immunogen as well as an anti-paclitaxel specific antibody and a detection reagent obtained by the paclitaxel immunogen and a preparation method thereof. The paclitaxel immunogen prepared by the invention is high in immunogenicity, can be induced to obtain a high titer anti-paclitaxel specific antibody and is free of any cross reactions with 62 common medicines. The paclitaxel detection reagent prepared by the antibody can be used for precisely and quickly determining the content of paclitaxel in a sample. Compared with existing detection reagents in the market, the detection reagent has the advantages of being simple and convenient to operate, high in sensitivity, strong in specificity, accurate in result and the like and can be used for effectively lowering the paclitaxel detection cost, thereby facilitating clinical large-scaled popularization and use.
Description
Technical field
The invention belongs to biological technical field, relate to Taxol Immunogen, anti-taxol specific antibody and taxol detection reagent.
Background technology
Taxol (Paclitaxel), its structural formula is as shown in formula III:
Taxol is from yew (having another name called Japanese yew), extract the tricyclic diterpene alkaloids medicament obtained, having unique induction tubulin polymerization thus the antitumor action of T suppression cell division, is a clinical line medication for the treatment of the cancers such as mammary cancer, uterus carcinoma, ovarian cancer and nonsmall-cell lung cancer at present.But taxol has the side effects such as bone marrow density reduction, anaphylaxis, neutrophilic granulocyte minimizing, ypotension, bradyrhythmia, nausea and vomiting, result for the treatment of and its Plasma Concentration have the dependency of height.Therefore, by the concentration level of taxol in monitoring patient body, effectively can control and alleviate these side effects, reaching optimal result for the treatment of, significant for clinical rational drug use.
At present, the main method of domestic and international monitoring taxol Plasma Concentration is the traditional methods such as enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC), reversed-phased high performace liquid chromatographic (RP-HPLC), Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), but these methods are all not suitable for clinical large-scale application.Although the taxol of domestic existing application latex enhancing immune turbidimetry measures test kit listing, growing clinical detection demand far can not be met.The taxol 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 taxol 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
The defect that the present invention exists to overcome prior art, adopt brand-new D51-7059 to prepare the strong Taxol Immunogen of immunogenicity and antibody thereof, the taxol homogeneous enzyme immunoassay detection reagent prepared with this antibody can be implemented on automatic clinical chemistry analyzer taxol high-throughput, rapid detection.This detection reagent has the advantages such as easy and simple to handle, highly sensitive, high specificity, result are accurate, effectively can also reduce taxol testing cost, is conducive to clinical expansion and uses.
One object of the present invention is to provide a kind of D51-7059.
Another object of the present invention is the Taxol Immunogen providing a kind of immunogenicity strong.
Another object of the present invention is the preparation method providing a kind of Taxol Immunogen.
Another object of the present invention is to provide the anti-taxol specific antibody of the high specificity using Taxol Immunogen of the present invention to prepare.
Another object of the present invention is to provide a kind of taxol detection reagent.
Immunogenicity is relevant with synthesized D51-7059 molecular structure and selected kind of carrier, the less immunogenic of Taxol Immunogen in prior art, obtain antibody specificity, with the bonding force of taxol, susceptibility is all not so good as the present invention.Taxol Immunogen of the present invention, immunogenicity is high, can induce the anti-taxol specific antibody obtaining high-titer.This antibodies specific is high, strong with the bonding force of taxol.The taxol detection reagent prepared by this antibody, can determine the content of taxol in sample quickly and accurately.The present invention is achieved by the following technical solutions:
A kind of Taxol Immunogen, its structural formula is as shown in formula I:
In formula, R is linking group-CO-(CH
2)
n-COO-, n are the integers between 1 to 20, and preferred R is-CO-(CH
2)
2-COO-.
Carrier, for having immunogenic protein or polypeptide, is preferably serum protein, hemocyanin and thyroglobulin, is more preferably serum albumin, more preferably bovine serum albumin.
When R is-CO-(CH
2)
nduring-COO-, route of synthesis and the method for this Taxol Immunogen are as follows:
1. the preparation method of D51-7059:
A kind of D51-7059, its structural formula is as shown in formula II:
In formula, R is linking group-CO-(CH
2)
n-COO-, n are the integers between 1 to 20.
(1) take 1g taxol, 2g compd A adds in reaction system, then hatch in the glass flask of continuous backflow with the benzene of 20ml pyridine or drying.
(2) by this reaction mixture continuous heating 6 hours under the reflux temperature of 70-80 DEG C, then reaction mixture is slowly cooled to room temperature, remove unnecessary pyridine or benzene gently.
(3) remaining organic composition is continued under condition of negative pressure pass into nitrogen gas stream and make its evaporation drying, the dry bottom product obtained is D51-7059.
(4) by 60% alcohol flushing more than 10 times of product distilled water preparation obtained above, to obtain the recrystallization of taxol half amber pearl acid fat (D51-7059).
(5) by tlc (TLC) this standard method of analysis, quantitative analysis is carried out to the productive rate of final product, in this experiment, the taxol hemisuccinate residue that final synthesis obtains is approximately 0.1-0.15 by its relative migration coefficient (Rf) of TLC experiment display, but taxol in contrast demonstrates larger Rf value, is approximately 0.3-0.4.In the standard building-up reactions of this experiment, the average yield of end product D51-7059 is approximately 95% or higher.
In the present invention, the compd A in the step (1) of the preparation process of D51-7059 has selected Succinic anhydried to be synthesis material, therefore the link radicals R of the final product D51-7059 of gained is-CO-(CH
2)
2-COO-.When n gets other numerical value, select other=docosandioic acid containing 3-22 carbon atom or corresponding acid anhydrides be when testing, and synthetic method is completely the same.
2. the preparation process of Taxol Immunogen:
(1) carrier proteins 200mg is dissolved in 50ml 0.2M, in the phosphoric acid buffer of pH 8.5;
(2) following chemical is joined stirring and dissolving in small beaker: D51-7059,3.5ml dimethyl formamide, 3.5ml ethanol, the 7.0ml 10mM of 200mg the present invention synthesis, the potassium phosphate buffer of pH 5.0,200mg 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 50mg N-hydroxy thiosuccinimide, by these chemical at room temperature stirring and dissolving reaction 30min;
(3) solution dissolved is dropped in carrier protein solution, and stir at 2 ~ 8 DEG C and spend the night, obtain antigen; Synthetic antigen is carried out purifying through dialysis, obtains Taxol Immunogen.
When getting other integers in 1 ~ 20 scope in the present invention as n, the Taxol Immunogen as shown in formula I can be prepared with aforesaid method.Carrier, still for having immunogenic protein, can be serum protein, hemocyanin and thyroglobulin.Preferably, carrier is serum protein.Preferred, carrier is bovine serum albumin.
Because linking group mainly plays the ligation of small molecule derivative and carrier, immunogenicity is strong and weak relevant with synthesized D51-7059 molecular structure and selected kind of carrier, therefore when n gets the arbitrary integer between 1 to 20 in theory, Taxol Immunogen prepared by D51-7059 is without significant difference, all possess strong immunogenicity, the specific antibody of high-titer can be prepared.
A kind of anti-taxol specific antibody, obtains by producing after above-mentioned Taxol Immunogen immune animal.
Described anti-taxol specific antibody adopts ordinary method inoculation experiments animal by above-mentioned obtained Taxol Immunogen, and get antiserum(antisera) after booster immunization, concrete steps are as follows:
(1) with PBS, the BSA-Taxol Immunogen of above-mentioned synthesis is diluted to 1.0mg/ml, obtains antigenic solution, then mix with Freund's complete adjuvant with 1.0ml antigenic solution, laboratory animal is injected;
After (2) 2 ~ 3 weeks, then with the identical antigenic solution of 1.0ml and Freund's incomplete adjuvant, above-mentioned laboratory animal is injected once, afterwards every surrounding injection once, amount to injection 4 times;
(3) get blood to above-mentioned laboratory animal, separation and purification obtains the anti-taxol specific antibody of tiring as 1:30000-1:50000.
Anti-taxol specific antibody of the present invention is complete antibody molecule, also comprises and retaining and the antibody fragment of taxol specific binding capacity or antibody derivatives.
The polyclonal antibody that antibody of the present invention obtains animal booster immunization for Taxol Immunogen that employing is single, or for after immunity through monoclonal antibody that somatic hybridization obtains; Described laboratory animal is the one of rabbit, goat, mouse, sheep, cavy or horse, is preferably rabbit.
The invention provides a kind of taxol detection reagent, containing above-mentioned anti-taxol specific antibody and indicator.
Indicator of the present invention is selected from enzyme reagent, radio isotope reagent, fluorescent reagent, luminescence reagent.Preferably, indicator is enzyme reagent, is made up of Japanese yew alcoholase mark conjugate and enzyme substrates.
Above-mentioned enzyme mark conjugate is glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate; Above-mentioned enzyme substrates is G-6-P.
Taxol homogeneous enzyme immunoassay detection reagent before the use, in order to avoid the substrate of the enzyme mark conjugate in indicator and enzyme reacts, the substrate of enzyme mark conjugate and enzyme is unmixed and separated, so the substrate of enzyme and above-mentioned anti-taxol specific antibody are mixed.Therefore, taxol homogeneous enzyme immunoassay detection reagent comprises two class reagent:
(1) reagent A is mixed by anti-taxol specific antibody and homogeneous phase enzyme substrates, and concrete preparation process is as follows:
1) Reduced nicotinamide-adenine dinucleotide NAD, 1.711g (11.25mM) G-6-P (G-6-P) of 4.036g (11.25mM) oxidation state is made homogeneous phase enzyme substrates with the Tris buffer solution of 1L 55mM, pH=8.0;
2) be added in above-mentioned homogeneous phase enzyme substrates by the anti-taxol specific antibody of preparation, the volume ratio of antibody and homogeneous phase enzyme substrates is 1:100 ~ 1:10000;
(2) reagent B is mixed by glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing and Tris damping fluid, and preparation method is as follows:
1) preparation of glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) solution:
A. take the G6PDH that 15mg specification is 100KU, room-temperature dissolution contains 72.6mg (0.05M) Tris, 8mg MgCl in 12mL
2(3.3mM) with in the solution of 100mg NaCl, these pH value of solution=9.0;
B. the Reduced nicotinamide-adenine dinucleotide (NADH) of 225mg reduction-state is added, 135mg G-6-P (G-6-P) and 0.75mL Trivalin SF;
C. 2mL dimethyl sulfoxide (DMSO) is dropwise added;
2) activation of D51-7059:
A. take 10mg D51-7059 under anhydrous conditions, be dissolved in 600 μ L DMF;
B. above-mentioned solution temperature is made to drop to-2 ~-8 DEG C;
C. 3 μ L Tributylamines are added;
D. 1.5 μ L isobutyl chlorocarbonates are added;
E.-2 ~-8 DEG C are stirred 30 minutes;
3) connection of G6PDH and D51-7059:
A. the D51-7059 dropwise of above-mentioned activation is joined in the G6PDH solution of above-mentioned dissolving;
B.2-8 a DEG C stirring is spent the night;
4) purified product:
Connect product by G-25 gel chromatography column purification, the final product of acquisition is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, stores at 2-8 DEG C.
5) be added in the Tris damping fluid of 120mM, pH=8.2 by the glucose-6-phosphate dehydrogenase (G6PD) of preparation-hapten conjugation thing, the volume ratio of above-mentioned conjugate and Tris damping fluid is 1:100 ~ 1:10000.
Taxol Immunogen high specificity of the present invention, immunogenicity are high, the anti-taxol specific antibody high specificity prepared, height of tiring, and with common 62 kinds of medicines without any cross reaction; Homogeneous enzyme immunoassay detection reagent containing above-mentioned anti-taxol specific antibody can determine the content of taxol in sample easily and fast, exactly, and can on automatic clinical chemistry analyzer the multiple sample of Simultaneously test, realize the rapid mensuration of high-throughput of taxol, accuracy is high, high specificity, tolerance range is all enhanced before comparing with detection efficiency, achieves the full-automation of testing process simultaneously, less demanding to testing staff, is easy to realize and promote the use of.
Accompanying drawing explanation
Fig. 1 is taxol ELISA detection reaction curve;
Fig. 2 is taxol homogeneous enzyme immunoassay response curve.
Embodiment
The synthesis of embodiment one D51-7059 and detection by quantitative thereof
The chemical structure of D51-7059 is as shown in formula IV:
Synthetic route and the preparation process of above-mentioned D51-7059 are as follows:
1. take 1g taxol, 2g Succinic anhydried adds in reaction system, then hatch in the glass flask of continuous backflow with the benzene of 20ml pyridine or drying;
2., by this reaction mixture continuous heating 6 hours under the reflux temperature of 70-80 DEG C, then reaction mixture is slowly cooled to room temperature, remove unnecessary pyridine or benzene gently;
3. remaining organic composition is continued under condition of negative pressure to pass into nitrogen gas stream and make its evaporation drying, the dry bottom product obtained is D51-7059;
4. by 60% alcohol flushing more than 10 times of product distilled water preparation obtained above, to obtain the recrystallization of taxol half amber pearl acid fat (D51-7059);
5. by standard method of analysis tlc (TLC), quantitative analysis is carried out to the productive rate of final product, the taxol hemisuccinate residue that final synthesis obtains is 0.1-0.15 by its relative migration coefficient (Rf) of TLC experiment display, but taxol in contrast demonstrates larger Rf value for 0.3-0.4, the average yield of end product D51-7059 is greater than 95%.
The synthesis of embodiment two Taxol Immunogen
Taxol Immunogen is by-CO-(the CH of the D51-7059 shown in bovine serum albumin (Bovine Serum Albumin, BSA) Yu formula II
2)
n-COO-group is formed by connecting, and in the present embodiment, describe this immunogenic synthetic method in detail for n=2, concrete steps are as follows:
1. bovine serum albumin 200mg is dissolved in 50ml 0.2M, in the phosphoric acid buffer of pH 8.5;
2. following chemical is joined stirring and dissolving in small beaker: D51-7059,3.5ml dimethyl formamide, 3.5ml ethanol, the 7.0ml 10mM of 200mg synthesis, the potassium phosphate buffer of pH 5.0,200mg 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 50mg N-hydroxy thiosuccinimide, by these chemical at room temperature stirring and dissolving reaction 30min;
3. the solution dissolved is dropped in BSA solution, and stir at 2 ~ 8 DEG C and spend the night, obtain antigen; Synthetic antigen is carried out purifying through dialysis, obtains Taxol Immunogen.
Embodiment three: the preparation of anti-taxol specific antibody
Taxol Immunogen embodiment two prepared adopts ordinary method inoculation experiments animal rabbit, and get antiserum(antisera) after booster immunization, concrete steps are as follows:
1. with PBS, the Taxol Immunogen of above-mentioned synthesis is diluted to 1.0mg/ml, obtains antigenic solution, then mix with Freund's complete adjuvant with 1.0ml antigenic solution, experimental animal rabbit is injected.
After 2.2 ~ 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.
3. the experimental animal rabbit of pair step 2 gets blood, and separation and purification obtains the anti-taxol specific antibody of tiring as 1:30000-1:50000.
Embodiment four: taxol ELISA checks
1. the foundation of taxol ELISA examination criteria curve
(1) preparation of standard substance
Taxol 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 400.00ng/mL, 150.00ng/mL, 50.00ng/mL, 15.00ng/mL, 5.00ng/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 taxol is utilized
With PBS, antitaxol antibody prepared in embodiment three is diluted to 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 antitaxol 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-taxol 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 content of taxol in testing sample
(1) testing sample is made
Preparation method: taxol 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, 5.00,50.00,300.00ng/mL, forms whole blood sample that is blank, basic, normal, high concentration.This blank whole blood is not containing the healthy human blood of taxol.
(2) testing method
Utilize the ELISA method of inspection of above-mentioned taxol, 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 taxol ELISA of contrast shown in Fig. 1 checks, calculates content of taxol in each sample, and carries out 3 multiple holes mensuration to each sample, and the actual content according to taxol in above-mentioned sample calculates the rate of recovery, and result is as shown in table 1.
The ELISA of table 1 taxol detects recovery experiment
From result in table 1: the taxol rate of recovery adopting taxol ELISA detection reagent of the present invention to measure in different concns sample is all higher, equal > 90%, illustrate that anti-taxol specific antibody of the present invention may be used for the detection of taxol in sample, and result precision is high.
Embodiment five: the preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing
1. the preparation of glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) solution:
(1) accurately take the G6PDH that 15mg specification is 100KU, room-temperature dissolution contains 72.6mg (0.05M) Tris, 8mg MgCl in 12mL
2(3.3mM) with in the solution of 100mg NaCl, these pH value of solution=9.0, this step is carried out in beaker C.
(2) in above-mentioned beaker C, the Reduced nicotinamide-adenine dinucleotide NADH of 225mg reduction-state is added, 135mg G-6-P (G-6-P) and 0.75mL Trivalin SF (Carbitol).
(3) in above-mentioned beaker C, 2mL dimethyl sulfoxide (DMSO) (dimethy sulfoxide, DMSO) is dropwise added again.
2. the activation of D51-7059:
(1) take the above-mentioned D51-7059 of 10mg under anhydrous conditions, be dissolved in 600 μ L DMF.
(2) above-mentioned solution temperature is made to drop to-2 ~-8 DEG C.
(3) 3 μ L Tributylamines (tributylamine) are added.
(4) 1.5 μ L isobutyl chlorocarbonates (isobutylchloroformate) are added.
(5)-2 ~-8 DEG C are stirred 30 minutes.
The connection of 3.G6PDH and D51-7059:
(1) the D51-7059 dropwise of above-mentioned activation is joined in the G6PDH solution of above-mentioned dissolving.
(2) 2-8 DEG C of stirring is spent the night.
4. purified product:
By the solution in G-25 gel chromatography column purification step 3, the final product of acquisition is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, stores at 2-8 DEG C.
Embodiment six: the preparation of taxol homogeneous enzyme immunoassay detection reagent
1. the preparation of reagent A: Reduced nicotinamide-adenine dinucleotide NAD, 1.711g (11.25mM) G-6-P (G-6-P) of 4.036g (11.25mM) oxidation state is placed in beaker D, makes homogeneous phase enzyme substrates with the Tris buffer solution of 1L 55mM, pH=8.0; Be added in above-mentioned homogeneous phase enzyme substrates by the anti-taxol specific antibody of above-mentioned preparation, the volume ratio of antibody and homogeneous phase enzyme substrates can be 1:100 ~ 1:10000, and ratio is in the present embodiment 1:400.
2. the preparation of reagent B: glucose-6-phosphate dehydrogenase (G6PD) embodiment five prepared-hapten conjugation thing is added in the Tris damping fluid of 120mM, pH=8.2, the volume ratio of above-mentioned conjugate and Tris damping fluid can be 1:100 ~ 1:10000, and ratio is in the present embodiment 1:1500.
Embodiment seven: the inspection of taxol homogeneous enzyme immunoassay and result
1. obtain typical curve:
(1) auspicious BS200 automatic clinical chemistry analyzer reaction parameter (see table 2) advanced in years is set.
(2) operation steps 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 steps auspicious BS200 automatic clinical chemistry analyzer reaction parameter
2. pattern detection: the typical curve obtained by homogeneous enzyme immunoassay detection reagent of the present invention, replication basic, normal, high concentration Quality Control sample 10 times, above-mentioned Quality Control sample is: be dissolved in by Taxol Standard in human serum, be respectively 0.30 to concentration, 1.50,5.00 μ g/ml.Detection data and data analysis are in table 3.
Table 3 sample determination and precision and rate of recovery assessment
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 drugs and carry out Interference Detection, adjustment concentration to 10.0 μ g/ml, adopts the homogeneous enzyme immunoassay method of embodiment seven to measure:
1. by reagent A contact reacts prepared by interference medicament to be measured and embodiment six, then add reagent B;
2. detect the OD of above-mentioned mixing solutions
340light absorption value, obtains the concentration of respective substance according to the typical curve of embodiment seven.
62 kinds of common medicine names and measurement result are specifically see table 4.
Table 4 common interference drug determination result
Measurement result shows: the concentration that above-mentioned 62 kinds of Common drugs are equivalent to taxol is all less than 0.1 μ g/ml.As can be seen here, antibody of the present invention is the specific antibody of anti-taxol, with other medicines no cross reaction.
Claims (10)
1. a Taxol Immunogen, its structural formula is as shown in formula I:
In formula, R is linking group-CO-(CH
2)
n-COO-, n are the integers between 1 to 20, and preferred R is-CO-(CH
2)
2-COO-; Carrier, for having immunogenic protein or polypeptide, is selected from the one in serum protein, hemocyanin or thyroglobulin, is preferably serum protein, is more preferably bovine serum albumin.
2. a preparation method for Taxol Immunogen as claimed in claim 1, is characterized in that comprising following steps:
(1) carrier proteins 200mg is dissolved in 50ml 0.2M, in the phosphoric acid buffer of pH 8.5;
(2) following chemical is joined stirring and dissolving in small beaker: 200mg D51-7059,3.5ml dimethyl formamide, 3.5ml ethanol, 7.0ml 10mM, the potassium phosphate buffer of pH 5.0,200mg 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 50mg N-hydroxy thiosuccinimide, by these chemical at room temperature stirring and dissolving reaction 30min;
(3) solution dissolved is dropped in carrier protein solution, and stir at 2 ~ 8 DEG C and spend the night, obtain antigen; Synthetic antigen is carried out purifying through dialysis, obtains Taxol Immunogen.
3. Taxol Immunogen preparation method according to claim 2, is characterized in that described D51-7059 structural formula is as shown in formula II:
Above-mentioned R is linking group-CO-(CH
2)
n-COO-, n are the integer between 1 to 20, preferred n=2.
4. the D51-7059 according to any one of claim 2-3, is characterized in that being synthesized by following steps:
(1) take 1g taxol, 2g compd A adds in reaction system, then hatch in the glass flask of continuous backflow with the benzene of 20ml pyridine or drying;
(2) by this reaction mixture continuous heating 6 hours under the reflux temperature of 70-80 DEG C, then reaction mixture is slowly cooled to room temperature, remove unnecessary pyridine or benzene gently;
(3) remaining organic composition is continued under condition of negative pressure pass into nitrogen gas stream and make its evaporation drying, obtain dry bottom product;
(4) 60% alcohol flushing more than 10 times of product distilled water preparation step (3) obtained, obtains recrystallized material and is D51-7059.
Described compd A is docosandioic acid containing 3-22 carbon atom or corresponding acid anhydrides, is preferably Succinic anhydried.
5. an anti-taxol specific antibody, by the complete antibody molecule produced after the Taxol Immunogen immunization experiment animal described in claim 1-2 any one, or for retaining and the antibody fragment of taxol specific binding capacity or antibody derivatives.
6. the anti-taxol specific antibody of one according to claim 5, it is characterized in that described complete antibody molecule, antibody fragment or antibody derivatives, for the polyclonal antibody adopting single Taxol Immunogen to obtain animal booster immunization, or it is the monoclonal antibody obtained through somatic hybridization after immunity; Described laboratory animal is the one of rabbit, goat, mouse, sheep, cavy or horse, is preferably rabbit.
7. a preparation method for the anti-taxol specific antibody according to any one of claim 5-6, is characterized in that comprising following steps:
(1) with PBS, bovine serum albumin-Taxol Immunogen is diluted to 1.0mg/ml, obtains antigenic solution, then mix with Freund's complete adjuvant with 1.0ml antigenic solution, laboratory animal is injected;
After (2) 2 ~ 3 weeks, then with the identical antigenic solution of 1.0ml and Freund's incomplete adjuvant, above-mentioned laboratory animal is injected once, afterwards every surrounding injection once, amount to injection 4 times;
(3) get blood to the laboratory animal of step (2), separation and purification obtains the anti-taxol specific antibody of tiring as 1:30000-1:50000.
8. a taxol detection reagent, containing the anti-taxol specific antibody described in claim 5-7 and indicator, described indicator is selected from the one in enzyme reagent, radio isotope reagent, fluorescent reagent or luminescence reagent, is preferably enzyme reagent; Described enzyme reagent is made up of Japanese yew alcoholase mark conjugate and enzyme substrates, and enzyme mark conjugate is preferably glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate, and enzyme substrates is preferably G-6-P.
9. a preparation method for taxol detection reagent as claimed in claim 8, is characterized in that comprising following steps:
(1) reagent A: the Tris buffer solution of the Reduced nicotinamide-adenine dinucleotide of 4.036g, 11.25mM oxidation state and 1.711g, 11.25mM G-6-P 1L 55mM, pH=8.0 is made homogeneous phase enzyme substrates; Be added in above-mentioned homogeneous phase enzyme substrates by the anti-taxol specific antibody according to any one of claim 5-7, the volume ratio of anti-taxol specific antibody and homogeneous phase enzyme substrates is 1:100 ~ 1:10000;
(2) reagent B: Japanese yew alcoholase mark conjugate is added in the Tris damping fluid of 120mM, pH=8.2, the volume ratio of Japanese yew alcoholase mark conjugate and Tris damping fluid is 1:100 ~ 1:10000;
Described anti-taxol specific antibody and the volume ratio of homogeneous phase enzyme substrates are preferably 1:400;
Described Japanese yew alcoholase mark conjugate and the volume ratio of Tris damping fluid are preferably 1:1500.
10. the taxol detection reagent according to Claim 8 described in-9 any one, is characterized in that the preparation method of described Japanese yew alcoholase mark conjugate comprises following steps:
(1) preparation of glucose-6-phosphate dehydrogenase (G6PD) solution: take the glucose-6-phosphate dehydrogenase (G6PD) that 15mg specification is 100KU, room-temperature dissolution contains 72.6mg 0.05M Tris, 8mg 3.3mM MgCl in 12mL
2with in the solution of 100mg NaCl, pH=9.0; Add the Reduced nicotinamide-adenine dinucleotide of 225mg reduction-state, 135mg G-6-P and 0.75mL Trivalin SF in the solution; Dropwise add 2mL dimethyl sulfoxide (DMSO) again;
(2) activation of D51-7059: take 10mg D51-7059 under anhydrous conditions, is dissolved in 600 μ L dimethyl formamides; Above-mentioned solution temperature is made to drop to-2 ~-8 DEG C; Add 3 μ L Tributylamines; Add 1.5 μ L isobutyl chlorocarbonates;-2 ~-8 DEG C are stirred 30 minutes;
(3) connection of glucose-6-phosphate dehydrogenase (G6PD) and D51-7059: the D51-7059 dropwise that step (2) activates is joined in the glucose-6-phosphate dehydrogenase (G6PD) solution that step (1) dissolves; 2-8 DEG C of stirring is spent the night;
(4) purified product: connect product by G-25 gel chromatography column purification, the final product of acquisition is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, stores at 2-8 DEG C.
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Application publication date: 20150422 |