CN105504047A - Cabazitaxel immunogen, specific antibody and detection reagent and preparation method thereof - Google Patents
Cabazitaxel immunogen, specific antibody and detection reagent and preparation method thereof Download PDFInfo
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- CN105504047A CN105504047A CN201610087472.4A CN201610087472A CN105504047A CN 105504047 A CN105504047 A CN 105504047A CN 201610087472 A CN201610087472 A CN 201610087472A CN 105504047 A CN105504047 A CN 105504047A
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- cabazitaxel
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- 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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- C07D—HETEROCYCLIC COMPOUNDS
- C07D305/00—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms
- C07D305/14—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- 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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Abstract
The invention discloses a cabazitaxel immunogen, a specific antibody and a detection reagent and a preparation method thereof and belongs to the field of biological detection. The cabazitaxel immunogen is synthesized by a brand new cabazitaxel derivative and has the advantage of high immunogenicity, can be induced to obtain the specific cabazitaxel antibody with high specificity and binding capacity and does not produce any cross reaction with 62 types of common medicines. The cabazitaxel detection reagent prepared by the antibody can conveniently and accurately determine the cabazitaxel content in a sample and facilitates clinical individualized treatment. Compared with the prior art, the detection reagent can achieve high-flux and quick detection on a full-automatic biochemical analyzer, has the advantages of being simple and convenient to operate, high in sensitivity and specificity, accurate in result and the like, can further effectively reduce the cabazitaxel detection cost and is suitable for clinical large-scale popularization and usage.
Description
Technical field
The invention belongs to field of biological detection, relate to Cabazitaxel immunogen, specific antibody and detection reagent and preparation method thereof.
Background technology
Cabazitaxel (Cabazitaxel) structural formula is as shown in formula III:
Cabazitaxel; another name: 7 β; 10 β ?dimethoxy docetaxel; chemical name: (2 α; 5 β; 7 β; 10 β; 13 α) ?4 ?Yi Xian Yang Ji ?13 ?[[(2R; 3S) ?3 ?[(tertbutyloxycarbonyl) amino] ?2 ?Qiang Ji ?3 ?hydrocinnamoyl] oxygen base] ?1 ?Qiang Ji ?7,10 ?Er Jia Yang Ji ?9 ?oxo Zi Shan ?5,20 ?Huan Yang Ji ?11 ?Xi ?2 ?benzoic ether; a kind of molecular design taxanes small molecule, anti-tumor drug, semi-synthetic by the precursor compound extracted from Ramulus et folium taxi cuspidatae.Cabazitaxel is the dimethoxy derivatives of Docetaxel, by the methoxylation to the latter's two hydroxyls, eliminate its to the P of multidrug resistance gene MDRl coding ?the avidity of glycoprotein (P ?gp), and this protein mediated multidrug resistance is considered to the classics mechanism that tumour cell resists traditional taxane chemotherapy medicine.As a kind of microtubule inhibitors, Cabazitaxel by with tubulin binding, what while promoting microtubule dimer to be assembled into microtubule, stop microtubule removes polymerisation process, thus suppress microtubule to decompose, increase the stability of microtubule, and then the mitotic division of inhibition tumor cell, tumour cell is blocked in the G2/M phase, and promote its apoptosis, the propagation of final Tumor suppression tissue.This product usually and prednisone coupling clinically, for the chemotherapy of advanced metastatic patients with prostate cancer.Close relation between the Plasma Concentration of Cabazitaxel and result for the treatment of, does not reach result for the treatment of lower than effective blood drug concentration, can cause serious toxic side effect higher than effective blood drug concentration.Therefore, fast, accurately, the Plasma Concentration of mensuration Cabazitaxel in tumour patient body of high-throughput, low cost, for reduction toxic side effect with to improve survival all significant.
At present, the main method of domestic and international monitoring Cabazitaxel 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 phase look spectrum ?tandem mass spectrometry (LC ?MS/MS), but these methods all can not meet the clinical demand of high-throughput and low cost.The Cabazitaxel 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 Cabazitaxel 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, adopts Cabazitaxel derivative to prepare the strong Cabazitaxel immunogen of immunogenicity and antibody thereof, and provides a kind of detection reagent of easy and simple to handle, highly sensitive, high specificity.Application homogeneous enzyme immunoassay detection technique realizes on automatic clinical chemistry analyzer the mensuration of Cabazitaxel, can high-throughput, rapid, accurately determine in sample Cabazitaxel content, and there is the advantages such as easy and simple to handle, highly sensitive, high specificity, result are accurate, effective reduction Cabazitaxel testing cost, be conducive to clinical individualized treatment, be applicable to clinically extensively promoting the use of.
One object of the present invention is the synthetic method providing a kind of Cabazitaxel derivative.
Another object of the present invention is the Cabazitaxel immunogen providing a kind of immunogenicity strong.
Another object of the present invention is to provide a kind of Cabazitaxel immunogenic preparation method.
Another object of the present invention is to provide the anti-Cabazitaxel specific antibody of the high specificity using Cabazitaxel immunogen of the present invention to prepare.
Another object of the present invention is to provide a kind of Cabazitaxel detection reagent.
Another object of the present invention is to provide the preparation method of Cabazitaxel detection reagent.
Immunogenicity is relevant with synthesized Cabazitaxel derivative molecular structure and selected kind of carrier, the immunogenic less immunogenic of Cabazitaxel in prior art, obtain antibody specificity, with the bonding force of Cabazitaxel, susceptibility is all not so good as the present invention.Cabazitaxel immunogen of the present invention, immunogenicity is high, can induce the anti-Cabazitaxel specific antibody obtaining high-titer.This antibodies specific is high, strong with the bonding force of Cabazitaxel.The Cabazitaxel detection reagent prepared by this antibody, can determine the Cabazitaxel content in sample quickly and accurately.
The technical solution used in the present invention is as follows:
A kind of Cabazitaxel immunogen, its structural formula is as shown in formula I:
In formula, R for connect Ji Tuan ?CO ?(CH
2)
n?COO ?, n is the integer between 1 to 20.Preferred R Wei ?CO ?(CH
2)
2?COO ?.
Carrier, for having immunogenic protein or polypeptide, is preferably serum protein, hemocyanin and thyroglobulin.Be more preferably bovine serum albumin.
The immunogenic route of synthesis of Cabazitaxel and method as follows:
1. the preparation method of Cabazitaxel derivative:
A kind of Cabazitaxel derivative, its structural formula is as shown in formula II:
In formula, R for connect Ji Tuan ?CO ?(CH
2)
n?COO ?, n is the integer between 1 to 20.
Synthetic route and the preparation process of above-mentioned Cabazitaxel derivative are as follows:
(1) take 0.5 ~ 2.0g Cabazitaxel, 1.0 ~ 4.0g compd A adds in reaction system, then hatch in the glass flask of continuous backflow with the benzene of 10 ~ 40ml pyridine or drying.
(2) by this reaction mixture 70 ?continuous heating 3 ~ 12 hours under the reflux temperature of 80 DEG C, then reaction mixture is slowly cooled to room temperature (can it be allowed at ambient temperature to cool voluntarily), remove unnecessary pyridine or the benzene on mixture upper strata 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 Cabazitaxel derivative.Remaining organic composition refers to the composition after reaction mixture removal pyridine or benzene.
(4) by 60% alcohol flushing more than 5 ~ 20 times of product distilled water preparation obtained above, to obtain the recrystallization of Cabazitaxel hemisuccinic acid fat (Cabazitaxel derivative).60% refers to volume parts, and namely ethanol contend is 60 parts, and distilled water volume is 40 parts.
(5) by tlc (TLC) this standard method of analysis, quantitative analysis is carried out to the productive rate of final product, the Cabazitaxel hemisuccinic acid ester leftover that final synthesis obtains by TLC experiment display its relative migration coefficient (Rf) be approximately 0.1 ?0.15, but Cabazitaxel in contrast demonstrates larger Rf value, be approximately 0.3 ?0.4.In standard building-up reactions, the average yield of end product Cabazitaxel derivative is approximately 97%.Above step (1) ?(5) method easy, the Cabazitaxel derivative purity obtained is high.
In the present invention, the compd A in the step (1) of the preparation process of Cabazitaxel derivative has selected Succinic anhydried to be synthesis material, thus the link radicals R of the final product Cabazitaxel derivative of gained Wei ?CO ?(CH
2)
2?COO ?.When n gets other values, select other contain 3 ?the docosandioic acid of 22 carbon atoms or corresponding acid anhydrides when testing, synthetic method is completely the same.When wherein selecting Succinic anhydried, n value is 2, and carbon atom number is 4.
2. the immunogenic synthesis of Cabazitaxel:
(1) carrier proteins 100 ~ 300mg is dissolved in 25 ~ 75ml0.2M, in the phosphoric acid buffer of pH8.5;
(2) following chemical is joined stirring and dissolving in small beaker: Cabazitaxel derivative, 1.5 ~ 6ml dimethyl formamide, 1.5 ~ 6ml ethanol, the 3.5 ~ 10.0ml10mM of 100 ~ 300mg the present invention synthesis, the potassium phosphate buffer of pH5.0,100 ~ 300mg1 ?Yi Ji ?3 ?(?3 ?dimethylaminopropyl) carbodiimide, 25 ~ 75mgN ?hydroxy thiosuccinimide, by these chemical at room temperature stirring and dissolving reaction 20 ~ 60min;
(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 Cabazitaxel immunogen.
When getting other integers in 1 ~ 20 scope in the present invention as n, the Cabazitaxel 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.
The linking group R Wei provided in the present invention ?CO ?(CH2) n ?COO ?, the Cabazitaxel derivative molecular structure that immunogenicity is strong and weak and synthesized, structure and the selected kind of carrier of linking group are relevant, choosing of linking group of the present invention also has the immunogenic effect of increase, when but n gets the arbitrary integer between 1 to 20, the Cabazitaxel immunogen using the Cabazitaxel derivative of different n value to prepare all possesses strong immunogenicity, can prepare the specific antibody of high-titer.
Anti-Cabazitaxel specific antibody of the present invention, obtain by producing after above-mentioned Cabazitaxel immunogen immune laboratory animal, the concrete steps of preparation are as follows:
(1) with PBS, the Cabazitaxel immunogen of above-mentioned synthesis is diluted to 1.0mg/ml, obtains antigenic solution, then mix with Freund's complete adjuvant with 0.5 ~ 2.0ml antigenic solution, laboratory animal is injected.
After (2) 2 ~ 3 weeks, then with the identical antigenic solution of 0.5 ~ 2.0ml and Freund's incomplete adjuvant, above-mentioned laboratory animal is injected once, afterwards every surrounding injection once, amount to injection 3 ~ 6 times.
(3) blood is got to above-mentioned laboratory animal, separation and purification obtain tiring be 1: 30000 ~ 1: 50000 anti-Cabazitaxel specific antibody.
Anti-Cabazitaxel specific antibody of the present invention is complete antibody molecule, also comprises and retaining and the antibody fragment of Cabazitaxel specific binding capacity or antibody derivatives.Antibody of the present invention is polyclonal antibody also can be monoclonal antibody, is preferably polyclonal antibody.
The polyclonal antibody that antibody of the present invention obtains animal booster immunization for Cabazitaxel 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 Cabazitaxel detection reagent, containing above-mentioned anti-Cabazitaxel 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 Cabazitaxel enzyme mark conjugate and enzyme substrates.
Above-mentioned enzyme mark conjugate be Pu Tao Tang ?6 ?Lin Suan Tuo Qing Mei ?haptens enzyme mark conjugate; Above-mentioned enzyme substrates be Pu Tao Tang ?6 ?phosphoric acid.Described haptens is Cabazitaxel derivative.
Cabazitaxel 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-Cabazitaxel specific antibody are mixed.Therefore, Cabazitaxel homogeneous enzyme immunoassay detection reagent comprises two class reagent:
(1) reagent A is mixed by anti-Cabazitaxel specific antibody and homogeneous phase enzyme substrates, and concrete preparation process is as follows:
1) by the Reduced nicotinamide-adenine dinucleotide (NAD of 2.0 ~ 6.0g oxidation state
+), 1.0 ~ 3.0g Pu Tao Tang ?6 ?phosphoric acid (G ?6 ?P) make homogeneous phase enzyme substrates with the Tris buffer solution of 0.5 ~ 2L55mM, pH=8.0;
2) be added in above-mentioned homogeneous phase enzyme substrates by the anti-Cabazitaxel specific antibody of preparation, the volume ratio of antibody and homogeneous phase enzyme substrates is 1:100 ~ 1:10000;
(2) reagent B by Pu Tao Tang ?6 ?Lin Suan Tuo Qing Mei ?hapten conjugation thing and Tris damping fluid mix, preparation method is as follows:
1) Pu Tao Tang ?6 ?the preparation of phosphate dehydrogenase (G6PDH) solution:
A. take the G6PDH that 5 ~ 25mg specification is 100KU, room-temperature dissolution contains 72.6mg (0.05M) Tris, 8mgMgCl in 6 ~ 20mL
2(3.3mM) with in the solution of 100mgNaCl, these pH value of solution=9.0;
B. add the Reduced nicotinamide-adenine dinucleotide (NADH) of 150 ~ 500mg reduction-state, 75 ~ 250mg Pu Tao Tang ?6 ?phosphoric acid (G ?6 ?P) and 0.25 ~ 1.50mL Trivalin SF;
C. 1 ~ 4mL dimethyl sulfoxide (DMSO) is dropwise added;
2) activation of Cabazitaxel derivative:
A. take 5 ~ 20mg Cabazitaxel derivative under anhydrous conditions, be dissolved in 300 ~ 1000 μ LDMF;
B. make above-mentioned solution temperature Jiang Dao ?2～?8 DEG C;
C. 1.5 ~ 6 μ L Tributylamines are added;
D. 0.5 ~ 3 μ L isobutyl chlorocarbonate is added;
E. ?2～?8 DEG C stir 15 ~ 60 minutes;
3) connection of G6PDH and Cabazitaxel derivative:
A. the Cabazitaxel derivative solution of above-mentioned activation is dropwise joined in the G6PDH solution of above-mentioned dissolving;
B.2 ?8 DEG C of stirrings are spent the night;
4) purified product: connect product by G ?25 gel chromatography column purification, the final product of acquisition is the de-hydrogen enzyme ?hapten conjugation thing of Portugal's grape sugar ?6 ?phosphorus acid, stores at 2 ?8 DEG C.
5) by the Pu Tao Tang of preparation ?6 ?Lin Suan Tuo Qing Mei ?hapten conjugation thing be added in the Tris damping fluid of 0.5 ~ 2L120mM, pH=8.2, the volume ratio of above-mentioned conjugate and Tris damping fluid is 1:100 ~ 1:10000.
Described anti-Cabazitaxel specific antibody and the volume ratio of homogeneous phase enzyme substrates are preferably 1: 550;
Described Cabazitaxel enzyme mark conjugate and the volume ratio of Tris damping fluid are preferably 1: 2500.
Cabazitaxel immunogens of the present invention is strong, immunogenicity is high, the anti-Cabazitaxel 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-Cabazitaxel specific antibody can determine the Cabazitaxel content 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 Cabazitaxel, accuracy is high, high specificity, tolerance range is all enhanced before comparing with detection efficiency, achieve 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 Cabazitaxel ELISA detection reaction curve;
Fig. 2 is Cabazitaxel homogeneous enzyme immunoassay response curve.
Embodiment
Embodiment one: the synthesis of Cabazitaxel derivative and detection by quantitative thereof
As n=2, the chemical structure of Cabazitaxel derivative is as shown in formula IV:
Synthetic route and the preparation process of above-mentioned Cabazitaxel derivative are as follows:
1. take 1g Cabazitaxel, 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 70 ?continuous heating 6 hours under the reflux temperature of 80 DEG C, then reaction mixture is slowly cooled to room temperature, removes 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 Cabazitaxel derivative.
4. by 60% alcohol flushing more than 10 times of product distilled water preparation obtained above, to obtain the recrystallization of Cabazitaxel hemisuccinic acid fat (Cabazitaxel derivative).
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 Cabazitaxel hemisuccinic acid ester leftover that final synthesis obtains by TLC experiment display its relative migration coefficient (Rf) be 0.1 ?0.15, but Cabazitaxel in contrast demonstrate larger Rf value be 0.3 ?0.4.In the standard building-up reactions of this experiment, the average yield of end product Cabazitaxel derivative reaches 97%.
Embodiment two: the immunogenic synthesis of Cabazitaxel
Cabazitaxel immunogen by the Cabazitaxel derivative shown in bovine serum albumin (BovineSerumAlbumin, BSA) Yu formula II ?CO ?(CH
2)
n?COO ?group be formed by connecting, in the present embodiment, describe this immunogenic synthetic method in detail for n=2, concrete steps are as follows:
1. BSA200mg is dissolved in 50ml0.2M, in the phosphoric acid buffer of pH8.5;
2. following chemical is joined the potassium phosphate buffer of stirred at ambient temperature solubilizing reaction 30min:200mg Cabazitaxel derivative, 3.5ml dimethyl formamide, 3.5ml ethanol, 7.0ml10mM, pH5.0 in small beaker, 200mg1 ?Yi Ji ?3 ?(?3 ?dimethylaminopropyl) carbodiimide, 50mgN ?hydroxy thiosuccinimide;
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 Cabazitaxel immunogen.
Embodiment three: the preparation of anti-Cabazitaxel specific antibody
Cabazitaxel 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 Cabazitaxel immunogen of 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, separation and purification obtain tiring be 1: 30000 ~ 1: 50000 anti-Cabazitaxel specific antibody.
Embodiment four: Cabazitaxel ELISA checks
Adopt obtained antibody to carry out the ELISA inspection of Cabazitaxel, this inspection utilizes competitive immunization analytical method to measure the Cabazitaxel content in liquid sample.
1. the foundation of Cabazitaxel ELISA examination criteria curve
(1) preparation of standard substance
Cabazitaxel 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 640.00ng/mL, 160.00ng/mL, 40.00ng/mL, 10.00ng/mL, 2.50ng/mL and 0.00ng/mL.Wherein, ELISA damping fluid contains 50.0mMTris, the BSA of 145mMNaCl and 0.25%.
(2) the ELISA method of inspection preparation standard curve of Cabazitaxel is utilized
With PBS, anti-Cabazitaxel antibody dilution prepared in embodiment three is become the final concentration solution of 1: 10000,100 μ L/ holes are coated on 96 hole elisa plates, 4 DEG C place 12 ?24h; After the above-mentioned 96 hole elisa plates being coated with anti-Cabazitaxel antibody being washed 3 times with PBS, add the BSA solution of 0.5% of 200 μ L/ holes, 4 DEG C of closed placements 8 ?16h.Then wash 3 times with PBS, add the standard substance in 20 μ L/ holes.Add again 100 μ L/ hole working concentrations HRP ?Cabazitaxel conjugate; After incubated at room temperature 30min, PBS washes plate 5 times; Then every hole adds 100 μ LTMB 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 Cabazitaxel content in testing sample
(1) testing sample is made
Preparation method: Cabazitaxel 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, be respectively 0.00ng/mL, 3.00ng/mL, 75.00ng/mL, 500.00ng/mL to final concentration, form whole blood sample that is blank, basic, normal, high concentration.This blank whole blood is not containing the healthy human blood of Cabazitaxel.
(2) testing method
Utilize the ELISA method of inspection of above-mentioned Cabazitaxel, 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 Cabazitaxel ELISA of contrast shown in Fig. 1 checks, calculates Cabazitaxel content in each sample, and carries out 3 multiple holes mensuration to each sample, and the actual content according to Cabazitaxel in above-mentioned sample calculates the rate of recovery, and result is as shown in table 1.
The ELISA of table 1 Cabazitaxel detects recovery experiment
From result in table 1: the Cabazitaxel rate of recovery adopting Cabazitaxel ELISA detection reagent of the present invention to measure in different concns sample is all higher, equal > 90%, illustrate that anti-Cabazitaxel specific antibody of the present invention may be used for the detection of Cabazitaxel in sample, and result precision is high.
Embodiment five: Pu Tao Tang ?6 ?Lin Suan Tuo Qing Mei ?the preparation of hapten conjugation thing
1. Pu Tao Tang ?6 ?the preparation of phosphate dehydrogenase (G6PDH) solution:
(1) accurately take the G6PDH that 15mg specification is 100KU, room-temperature dissolution contains 72.6mg (0.05M) Tris, 8mgMgCl in 12mL
2(3.3mM) with in the solution of 100mgNaCl, these pH value of solution=9.0, this step is carried out in beaker C.
(2) in above-mentioned beaker C, add the Reduced nicotinamide-adenine dinucleotide (NADH) of 225mg reduction-state, 135mg Pu Tao Tang ?6 ?phosphoric acid and 0.75mL Trivalin SF.
(3) in above-mentioned beaker C, 2mL dimethyl sulfoxide (DMSO) is dropwise added again.
2. the activation of Cabazitaxel derivative:
(1) take the above-mentioned Cabazitaxel derivative of 10mg under anhydrous conditions, be dissolved in 600 μ LDMF.
(2) make above-mentioned solution temperature Jiang Dao ?2～?8 DEG C.
(3) 3 μ L Tributylamines are added.
(4) 1.5 μ L isobutyl chlorocarbonates are added.
(5) ?2～?8 DEG C stir 30 minutes.
The connection of 3.G6PDH and Cabazitaxel derivative:
(1) the Cabazitaxel derivative solution of above-mentioned activation is dropwise joined in the G6PDH solution of above-mentioned dissolving.
(2) 2 ?8 DEG C stirrings are spent the night.
4. purified product:
By the solution in G ?25 gel chromatography column purification step 3, the final product of acquisition is the de-hydrogen enzyme ?hapten conjugation thing of Portugal's grape sugar ?6 ?phosphorus acid, stores at 2 ?8 DEG C.
Embodiment six: the preparation of Cabazitaxel homogeneous enzyme immunoassay detection reagent
1. the preparation of reagent A: by the Reduced nicotinamide-adenine dinucleotide (NAD of 4.036g (11.25mM) oxidation state
+), 1.711g (11.25mM) Pu Tao Tang ?6 ?phosphoric acid (G ?6 ?P) be placed in beaker D, make homogeneous phase enzyme substrates with the Tris buffer solution of 1L55mM, pH=8.0; Be added in above-mentioned homogeneous phase enzyme substrates by the anti-Cabazitaxel specific antibody of above-mentioned preparation, the volume ratio of antibody and homogeneous phase enzyme substrates is 1: 550.
2. the preparation of reagent B: Pu Tao Tang prepared by embodiment five ?6 ?Lin Suan Tuo Qing Mei ?hapten conjugation thing be added in the Tris damping fluid of 1L120mM, pH=8.2, the volume ratio of above-mentioned conjugate and Tris damping fluid is 1: 2500.
Embodiment seven: the inspection of Cabazitaxel 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) 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 steps auspicious BS480 automatic clinical chemistry analyzer reaction parameter
2. pattern detection
By the 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 Cabazitaxel standard substance, be respectively 50.00 to concentration, 250.00,1000.00ng/ml.Detection data and data analysis are in table 3.
Table 3 sample determination and precision and rate of recovery assessment
| Blood sample | Low | In | High |
| Sample concentration (ng/ml) | 50.00 | 250.00 | 1000.00 |
| 1 | 51.29 | 241.55 | 1094.00 |
| 2 | 50.18 | 245.42 | 1015.41 |
| 3 | 48.75 | 251.99 | 975.16 |
| 4 | 53.30 | 246.87 | 984.33 |
| 5 | 52.00 | 242.52 | 1055.56 |
| 6 | 49.09 | 253.00 | 1024.80 |
| 7 | 50.35 | 242.59 | 996.25 |
| 8 | 47.93 | 255.73 | 984.15 |
| 9 | 50.03 | 242.58 | 1000.03 |
| 10 | 48.33 | 245.01 | 1025.61 |
| Mean value (ng/ml) | 50.13 | 246.73 | 1015.53 |
| Standard deviation (SD) | 1.70 | 5.07 | 36.74 |
| Precision (CV%) | 3.39 | 2.05 | 3.62 |
| Rate of recovery % | 100.3 | 98.7 | 101.6 |
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%, 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 1000.00ng/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
The detected result display of table 4, the concentration that 62 kinds of Common drugs are equivalent to Cabazitaxel is all less than 0.1ng/ml.Visible, antibody of the present invention is the specific antibody of anti-Cabazitaxel, with other medicines no cross reaction.It should be noted that; the foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize specification sheets of the present invention and accompanying drawing content to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other correlative technology fields, be all in like manner included in scope of patent protection of the present invention.
Claims (10)
1. a Cabazitaxel 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 carrier, for having immunogenic protein or polypeptide, is selected from the one in serum protein, hemocyanin or thyroglobulin.
2. the immunogenic synthetic method of Cabazitaxel according to claim 1, is characterized in that, comprise following steps:
(1) carrier proteins 100 ~ 300mg is dissolved in 25 ~ 75ml0.2M, in the phosphoric acid buffer of pH8.5;
(2) following chemical is joined stirred at ambient temperature solubilizing reaction 20 ~ 60min:100 in small beaker ~ 300mg Cabazitaxel derivative, 1.5 ~ 6ml dimethyl formamide, 1.5 ~ 6ml ethanol, the potassium phosphate buffer of 3.5 ~ 10.0ml10mM, pH5.0,100 ~ 300mg1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 25 ~ 75mgN-hydroxy thiosuccinimide;
(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 Cabazitaxel immunogen;
In step (2), Cabazitaxel derivant structure 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.
3. the synthetic method of Cabazitaxel derivative, is characterized in that, concrete steps are:
(1) take 0.5 ~ 2.0g Cabazitaxel, 1.0 ~ 4.0g compd A adds in reaction system, then hatch in the glass flask of continuous backflow with the benzene of 10 ~ 40ml pyridine or drying; Described compd A is have the docosandioic acid of 3-22 carbon atom or corresponding acid anhydrides;
(2) by this reaction mixture continuous heating 3 ~ 12 hours under the reflux temperature of 70-80 DEG C, then reaction mixture is slowly cooled to room temperature, remove unnecessary pyridine or benzene;
(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 Cabazitaxel derivative;
(4) by 60% alcohol flushing of Cabazitaxel derivative distilled water preparation obtained above repeatedly, to obtain the recrystallization of Cabazitaxel hemisuccinic acid fat.
4. the synthetic method of Cabazitaxel derivative according to claim 3, is characterized in that, as n=2, synthetic route is:
5. an anti-Cabazitaxel specific antibody, it is characterized in that, for producing by after Cabazitaxel immunogen immune laboratory animal according to claim 1 the complete antibody molecule obtained, or for retaining and the antibody fragment of the ability of Cabazitaxel specific binding or antibody derivatives.
6. the anti-Cabazitaxel 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 Cabazitaxel immunogen to obtain animal booster immunization, or it is the monoclonal antibody obtained through somatic hybridization after immunity; Described laboratory animal is selected from the one of rabbit, goat, mouse, sheep, cavy or Malaysia and China.
7. a preparation method for anti-Cabazitaxel specific antibody, is characterized in that, comprise following steps:
(1) with PBS, Cabazitaxel immunogen according to claim 1 is diluted to 1.0mg/ml, obtains antigenic solution, then mix with Freund's complete adjuvant with 0.5 ~ 2.0ml antigenic solution, laboratory animal is injected;
After (2) 2 ~ 3 weeks, then with the identical antigenic solution of 0.5 ~ 2.0ml and Freund's incomplete adjuvant, above-mentioned laboratory animal is injected once, afterwards every surrounding injection once, amount to injection 3 ~ 6 times;
(3) blood is got to the laboratory animal of step (2), separation and purification obtain tiring be 1: 30000 ~ 1: 50000 anti-Cabazitaxel specific antibody.
8. a Cabazitaxel detection reagent, containing the anti-Cabazitaxel specific antibody described in claim 5 or 6 and indicator, described indicator is selected from the one in enzyme reagent, radio isotope reagent, fluorescent reagent or luminescence reagent; Described enzyme reagent is made up of Cabazitaxel enzyme mark conjugate and enzyme substrates, and Cabazitaxel enzyme mark conjugate is glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate, and enzyme substrates is G-6-P; Described haptens is Cabazitaxel derivative.
9. the preparation method of Cabazitaxel detection reagent according to claim 8, is characterized in that comprising following steps:
(1) reagent A: the Tris buffer solution of the Reduced nicotinamide-adenine dinucleotide of 2.0 ~ 6.0g oxidation state and 1.0 ~ 3.0g G-6-P, 0.5 ~ 2L55mM, pH=8.0 is made homogeneous phase enzyme substrates; Be added in above-mentioned homogeneous phase enzyme substrates by the anti-Cabazitaxel specific antibody described in claim 5 or 6, the volume ratio of anti-Cabazitaxel specific antibody and homogeneous phase enzyme substrates is 1:100 ~ 1:10000;
(2) reagent B: be added in the Tris damping fluid of 0.5 ~ 2L120mM, pH=8.2 by Cabazitaxel enzyme mark conjugate, the volume ratio of Cabazitaxel enzyme mark conjugate and Tris damping fluid is 1:100 ~ 1:10000.
10. the preparation method of Cabazitaxel detection reagent according to claim 9, is characterized in that the preparation method of described Cabazitaxel enzyme mark conjugate comprises following steps:
(1) preparation of glucose-6-phosphate dehydrogenase (G6PD) solution: take the glucose-6-phosphate dehydrogenase (G6PD) that 5 ~ 25mg specification is 100KU, room-temperature dissolution contains 72.6mg0.05MTris, 8mg3.3mMMgCl in 6 ~ 20mL
2with in the solution of 100mgNaCl, pH=9.0; Add the Reduced nicotinamide-adenine dinucleotide of 150 ~ 500mg reduction-state, 75 ~ 250mg G-6-P and 0.25 ~ 1.50mL Trivalin SF in the solution; Dropwise add 1 ~ 4mL dimethyl sulfoxide (DMSO) again;
(2) activation of Cabazitaxel derivative: take 5 ~ 20mg Cabazitaxel derivative under anhydrous conditions, is dissolved in 300 ~ 1000 μ L dimethyl formamides; Above-mentioned solution temperature is made to drop to-2 ~-8 DEG C; Add 1.5 ~ 6 μ L Tributylamines; Add 0.5 ~ 3 μ L isobutyl chlorocarbonate;-2 ~-8 DEG C are stirred 15 ~ 60 minutes;
(3) connection of glucose-6-phosphate dehydrogenase (G6PD) and Cabazitaxel derivative: the Cabazitaxel derivative solution that step (2) activates dropwise 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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| CN105753969A (en) * | 2016-04-06 | 2016-07-13 | 苏州博源医疗科技有限公司 | Asymmetric dimethylarginine immunogen, antibody, detecting reagent and preparation method |
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