CN102295698B - Cyclosporine A immunogen, cyclosporine A specific antibody, detection reagent, and detection kit - Google Patents
Cyclosporine A immunogen, cyclosporine A specific antibody, detection reagent, and detection kit Download PDFInfo
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- CN102295698B CN102295698B CN201110130344.0A CN201110130344A CN102295698B CN 102295698 B CN102295698 B CN 102295698B CN 201110130344 A CN201110130344 A CN 201110130344A CN 102295698 B CN102295698 B CN 102295698B
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Abstract
The invention discloses a cyclosporine A immunogen, and a cyclosporine A specific antibody, a detection reagent and a detection kit obtained therefrom. A specific cyclosporine A derivative is used to prepare a cyclosporine A enzyme-labeled conjugate and the cyclosporine A immunogen through a chemosynthesis method, the cyclosporine A specific antibody is prepared from the cyclosporine A immunogen, and the homogeneous enzyme immune detection reagent of cyclosporine A is prepared from above substances. The detection reagent of the invention which has the advantages of high sensitivity and strong specificity can realize the high flux, and rapid and automatic detection of cyclosporine A by means of a fully-automatic biochemical analyzer.
Description
Technical field
The present invention relates to Ciclosporin A immunogen, anti-Ciclosporin A specific antibody, detectable and detection kit.
Background technology
Structural formula is such as Ciclosporin A (Cyclosporine A)（II）It is shown：
Ciclosporin A is a kind of ring-type undecapeptide, is mainly used in suppressing kidney, heart and liver migration process
In immunological rejection, it can also be used to treat diabetes, malaria and autoimmune disease.But due to its effectively treatment concentration
Scope is narrower, the immunological rejection of transplant organ can not be suppressed when concentration is relatively low and can be caused after overdose a series of tight
The side effect of weight is such as：Renal dysfunction, hypertension, cardiovascular spasm, headache, diarrhoea, lymphoma etc..Therefore, in therapeutic process
In carry out monitor drug concentration and be very important.
At present, high performance liquid chromatography is mainly to the method that Ciclosporin A carries out monitor drug concentration, puts the method for exempting from and glimmering
Light polarization method.High performance liquid chromatography elapsed time is long, and sample pre-treatments and operating process are complicated, high to personnel requirement；Put and exempt from
The radioactive ray of method generates greatly harm to the health of operator；Fluorescence polarization method need reagent rely primarily on into
Mouthful and expense is extremely expensive.
Homogeneous enzyme immunoassay inspection is a kind of competitive reaction, and the Ciclosporin A dissociated in reaction system can be mould with ring spore
Plain A enzymes mark conjugate competitive binding Ciclosporin A specific antibody, whole reaction is betided in a liquid phase homogeneous system,
Without the need for being separated by solid phase.The Ciclosporin A dissociated in sample is more, and the antibody of competition binding is more, the ring that antibody is discharged
P0-357 A enzyme mark conjugates are also more, and the signal obtained by enzyme mark conjugate and substrate reactions is also stronger.Continuous mode
In, after the Ciclosporin A and antibody response in sample, enzyme mark conjugate is further added, by the change of OD340nm light absorption values
Changing can calculate the content of sample.
Homogeneous enzyme immunoassay detection method speed is fast, simple to operate, sensitivity is high, high specificity and can be in full-automatic biochemical point
The rapid detection of high flux to monitoring medicine is realized in analyzer.It has been widely used in the small molecules such as illegal drug in the world
High throughput testing, because Ciclosporin A is bigger than general small molecule, prepared by the enzyme mark conjugate of its specific antibody and correlation difficult
Degree is larger, determines the Ciclosporin A content in human sample using homogeneous enzyme immunoassay detectable and has great importance.
The content of the invention
It is an object of the present invention to provide a kind of Ciclosporin A immunogen.
Further object is that offer is mould using the anti-ring spore that Ciclosporin A immunogen of the present invention is prepared
Plain A specific antibodies.
It is yet a further object of the present invention to provide a kind of detectable containing anti-Ciclosporin A antibody of the invention.
A further object of the present invention is to provide a kind of Ciclosporin A detection kit.
The technical solution used in the present invention is：
Ciclosporin A immunogen, its structural formula such as formula（I）It is shown：
In formula, R is linking group, and carrier has immunogenicity.
Preferably, carrier is with immunogenic protein.
R is preferably-O- (CH2)n- COO-, n are the integers between 1-20, and particularly, R is-O- (CH2)4-COO- 。
A kind of anti-Ciclosporin A specific antibody, is obtained by producing after above-mentioned Ciclosporin A immunogen immune animal.
A kind of Ciclosporin A detectable, is coupled containing above-mentioned anti-Ciclosporin A specific antibody, Ciclosporin A enzyme mark
The substrate of thing and enzyme.
Ciclosporin A detection kit, containing above-mentioned anti-Ciclosporin A specific antibody and the anti-Ciclosporin A of detection
The indicator of specific antibody and Ciclosporin A complex.
The Ciclosporin A immunogen of the present invention, immunogenicity is high, can prepare the Ciclosporin A antibody of specificity.
The anti-Ciclosporin A specific antibody high specificity of the present invention, with Common drugs without any cross reaction, sensitivity
Height, can reach 12.5 ng/ml, far below ng/ml~450 ng/ml of clinical application scope 100 of Ciclosporin A.
The present invention Ciclosporin A detectable and detection kit can easily and fast, accurately determine in sample
Ciclosporin A content.
The Ciclosporin A detectable and detection kit of the present invention can simultaneously be determined on automatic clinical chemistry analyzer
Multiple samples, realize the rapid measure of high flux of Ciclosporin A, sensitivity height, high specificity, degree of accuracy and detection efficiency phase
Than being all enhanced before, while realizing the full-automation of detection process.
Description of the drawings
Fig. 1 is Ciclosporin A homogeneous enzyme immunoassay reaction normal curve chart.
Specific embodiment
The technical solution used in the present invention is：
Ciclosporin A immunogen, its structural formula such as formula（I）It is shown：
In formula, R is linking group, and carrier has immunogenicity.Preferably, carrier is with immunogenic protein.
Although other sufficiently large immunogenic materials that possess can also be as carrier, under normal circumstances from protein as load
Body.The most frequently used immunogenic carrier includes serum albumin, hemocyanin（KLH）And Elityran.The selection of carrier is
The basic general knowledge of those skilled in the art.
R is preferably-O- (CH2)n- COO-, n are the integers between 1-20, and particularly, R is-O- (CH2)4-COO- 。
A kind of anti-Ciclosporin A specific antibody, is obtained by above-mentioned Ciclosporin A immunogen immune animal.
" antibody " of indication refers not only to complete antibody molecule in the present invention, also including reservation complete antibody specificity knot
The antibody fragment or derivant of conjunction ability.It can also be monoclonal antibody that the antibody of the present invention can be polyclonal antibody, excellent
Elect polyclonal antibody as.
The antibody of the present invention can be prepared by prior art.The typical method for obtaining polyclonal antibody is using single
One immunogen, after adding or being not added with adjuvant, at one or more position of animal immunity is carried out, and host animal includes：
Rabbit, goat, mice, sheep, Cavia porcelluss or horse.Persistent immunological is carried out always, until antibody titer reaches highest.Animal timing is taken a blood sample
Appropriate specific corrosioning anteserum is obtained, antiserum can be with purification.Monoclonal antibody can be made by somatocyte hybriding technology.
A kind of Ciclosporin A detectable, is coupled containing above-mentioned anti-Ciclosporin A specific antibody, Ciclosporin A enzyme mark
The substrate of thing and enzyme.Wherein, Ciclosporin A enzyme mark conjugate can be obtained by chemical synthesis process.
Ciclosporin A detection kit, it is special containing above-mentioned anti-Ciclosporin A specific antibody and detection Ciclosporin A
The indicator of heterogenetic antibody and Ciclosporin A complex.
Indicator is selected from enzymatic reagent, radiosiotope reagent, fluorometric reagent, luminescence reagent and chemical illuminating reagent.
Preferably, indicator is made up of the substrate of Ciclosporin A enzyme mark conjugate and enzyme.
Sample to be tested is various physiology samples, and such as blood sample needs the pre-treatment for carrying out blood sample before measure.
With reference to embodiment, the present invention is further illustrated.
Cyclosporin A derivative chemical constitution such as formula used in following examples（III）It is shown.
N in this formula and subsequent chemistry formula is the integer between 1-20.
The synthetic route of the cyclosporin derivative is as follows：
Specific synthesis step is as follows：
The synthesis of compound 1
（1）Accurately weigh 1.0 g 4- penetenoic acids（10.0 mmol, 1.0 eq）With 1.6 g N-hydroxy-succinamides
（14.0 mmol, 1.4 eq）It is added in the mL round-bottomed flasks of drying 100 containing 40 mL tetrahydrofurans, is placed in magnetic force
Stir on agitator.
（2）Accurately weigh 2.5 g 1,3- dicyclohexylcarbodiimides（12.0 mmol, 1.2 eq）, it is added to 20 mL
In tetrahydrofuran, it is placed in magnetic stirring apparatuss and fully mixes.
（3）At 23 DEG C, the mixture in step 2 is added dropwise in the mixture of step 1, and is stirred continuously, institute
Some solid particles quickly dissolve, and produce a kind of white precipitate rapidly.
（4）The mixture was stirred overnight, filters, concentrated in vacuo and using flash chromatography on silica gel purification column purification (eluent：
Petrol ether/ethyl acetate=1/1), obtain 1.5 g compound as white solid 1（Yield：75%）.
The synthesis of compound 2
（1）Accurately weigh following compound：400 mg Ciclosporin As（0.332 mmol, 1.0 eq）, 600 mg compounds
1,3- diisopropylideneacetone -4 of 1 (3.0 mmol, 9.0 eq) and 56 mg, 5- glyoxalidine -2- methene base-cyclohexyl phosphines -
Benzylidene dichloro ruthenium（0.0066 mmol, 0.2 mmol）It is dissolved in 10 ml CH2Cl2In
（2）Above-mentioned mixed solution is added in the flame-dried Schlenk pipes of 100 ml Jing, Jing is stirred vigorously and returns
Stream condensation completes reaction for 22 hours, and after cooling, the mixed liquor obtains a kind of solid crude extract Jing after concentrated in vacuo, by the crude extract
Jing purification by flash chromatography（Eluent：15% acetonitrile-ethyl acetate）Obtain 400 mg yellow compounds 2（Yield：85%）.
The synthesis of cyclosporin derivative
（1）Weigh 1 g compounds 2（0.739 mmol, 1 eq）With 166 mg Lithium hydrates（LiOH·H2O）
（3.695 mmol, 5eq）, in being dissolved in 5 ml distilled water and 20 ml tetrahydrofurans, after being stirred overnight under room temperature,
（2）By the concentration of compound Jing negative pressure and dilute with water, with the 1N hydrochloric acid accurate adjustment pH value of solution to 5, and second is used
Acetoacetic ester is extracted.
（3）After extracting and demixing, organic layer is separated and Jing Na2SO4Absorbent drying, Jing is filtered, concentrated in vacuo and efficient liquid phase
300 mg cyclosporin A derivative white solids are obtained after chromatogram purification（Yield：32%）.
NMR (Nuclear Magnetic Resonance) spectrum is carried out using the MHz of Bruker Avance III plus 400 to cyclosporin derivative to sweep
Retouch, internal standard adopts TMS.As a result it is as follows：1H NMR (CDCl3, 400MHz): 0.69-2.40 (72H, m), 2.48-2.60
(5H, m), 2.67-3.10 (12H, m), 3.25-3.48 (5H, m), 3.79-3.81 (1H, m), 4.43-4.46
(1H, m), 4.70-5.40 (13H, m), 5.58-5.67 (1H, m), 7.29-7.41 (1H, m), 7.80-7.82
(1H, m);It is characterized as formula（Ⅲ）Shown cyclosporin derivative.
Using Chromatography/Mass Spectrometry technology（LCMS）Derivant to obtaining is analyzed identification, and instrument is Agilent company
QQ-TOF mass spectrometry instrument LC/MSD1200 is serial, and ion source is using cation or negative ionization mode.Chromatographic column specification is：
Welchrom XB-C18 (50 × 4.6 mm, 5 μm), column temperature is 30 DEG C, and flow velocity is 1.5 mL/min, and mobile phase is second
Nitrile-water ratio is 30%-95%.
LCMS results show：Purity 99.7%；The min of retention time 2.713；Molecular weight 1259；Molecule from for： 1260
(M+1)。
The immunogenic synthesis of BSA- Ciclosporin As
Ciclosporin A immunogen passes through-O- (CH by bovine serum albumin and cyclosporin A derivative2)4- COO- groups connect
Connect and form, specific synthetic method is as follows：
1) by BSA（200 mg）It is dissolved in the M of 50 ml 0.2, the phosphate buffer of pH 8.5（Phosphate buffer
Solution, PBS）In；
2) following chemicals are added to into stirring and dissolving in small beaker：200 mg cyclosporin A derivatives, 3.5 ml bis-
Methyl nitrosourea（Dimethylformamide, DMF）, 3.5 ml ethanol, the mM of 7.0 ml 10, pH 5.0 potassium phosphate buffering
Liquid, 400 mg 1- ethyl -3- (- 3- dimethylaminopropyls) carbodiimides, 50 mg N- hydroxy thiosuccinimides（N-
hydroxysuccinimide, Sulfo-NHS）, stirring and dissolving reaction 30 minutes at room temperature；
3) solution for having dissolved is dropped in BSA solution, and is stirred overnight at 2~8 DEG C, obtain antigen；Will synthesis
Good antigen is dialysed through neutral phosphate buffer liquid（4×4L）Purification, obtains Ciclosporin A immunogen, is stored in -20 DEG C.
The preparation of anti-Ciclosporin A specific antibody
Resulting Ciclosporin A immunogen is adopted into conventional method inoculation experiments animal rabbit, anti-blood is taken after booster immunization
Clearly, comprise the following steps that：
The Ciclosporin A immunogen of synthesis is diluted to into 1.0 mg/ml with PBS, then with the antigenic solution of 1.0 ml with
Freund's complete adjuvant mixes, and rabbit is injected；
After 2~3 weeks, then rabbit is injected once with 1.0 ml identicals antigenic solutions and incomplete Freund's adjuvant, afterwards
Every surrounding once, altogether twice, the antibody titer of acquisition is about 1：30000.
The preparation of glucose-6-phosphate dehydrogenase (G6PD)-Ciclosporin A conjugate
1. glucose-6-phosphate dehydrogenase (G6PD)（G6PDH）The preparation of solution：
A) 37.5 mg G6PDH are weighed（100KU）.Room-temperature dissolution is in the M tirs of 6 mL 0.05,3.3 mM MgCl2,
0.85% NaCl, pH=9.0.G6PDH（100KU）:~0.04 µM；
B) 112.5 mg NADH, 67.5 mg G-6-P and 0.375 mL carbitols are added in enzymatic solution
（Carbitol）；
C) 1.125 mL dimethyl sulfoxide (dimethy sulfoxide, DMSO) are added dropwise over.
2. the activation of cyclosporin A derivative
A) weigh 10 mg cyclosporin A derivatives under anhydrous conditions to be dissolved in 210 L DMSO and 90 L DMF；
B) whole solution temperature is made to drop to 2-8 DEG C；
C) 6 L tri-n-butylamines are added（tributylamine）；
D) 3.5 L isobutyl chlorocarbonates are added（isobutylchloroformate）；
E) 2-8 DEG C is stirred 30 minutes.
3. the connection of enzyme and Ciclosporin A
During a) the cyclosporin A derivative solution of activation to be added dropwise to the enzymatic solution for dissolving；
B) 2-8 DEG C is stirred overnight；
4. enzyme mark conjugate is obtained by G-25 gel chromatography column purifications
5. BSA is added in enzyme mark conjugate solution to final concentration 0.5%, NaN3To 0.1%.It is stored in 2-8 DEG C.
The preparation of Ciclosporin A homogeneous enzyme immunoassay reagent
1. the preparation of reagent A：The Ciclosporin A specific antibody of preparation is added in homogeneous zymolyte（11.25 mM
NAD is dissolved in pH8.55 mM Tris buffer）, the ratio of antibody and substrate is 1:100-1:10000.
2. the preparation of reagent B：The enzyme mark conjugate of preparation is added in the buffer of enzyme（120 mM Tris, pH=
8.2）, the ratio of enzyme mark conjugate is 1:100-1:10000.
Ciclosporin A homogeneous enzyme immunoassay is checked
By automatic clinical chemistry analyzer, first add sample, then reagent adding A（Antibody and Substrate cocktail）, it is eventually adding examination
Agent B（Enzymatic solution）, determine the OD of different time points340Light absorption value, calculates reaction rate during different sample concentrations, practical operation
During need constantly to adjust the ratio of reagent A and reagent B, optimize sample pretreatment process, show that comparatively ideal reaction normal is bent
Line chart, as shown in Figure 1.
The standard curve obtained by homogeneous enzyme immunoassay detectable, the basic, normal, high concentration blood sample of replication（Will
Ciclosporin A standard substance are dissolved in blank whole blood substrate, and to concentration 60.0,300.0,600.0 ng/ml are respectively）5 times, send out
The response rate of existing sample is high（＞ 90%）.
Using test sample（Ciclosporin A standard substance are dissolved in 50.0 mM Tris buffer）Carry out ciclosporin
The sensitivity test of A homogeneous enzyme immunoassay reagents, the sensitivity of reagent has reached 12.5 ng/mL（Confidence level is 99.73%, 12.5
Ng/ml samples are in the range of 3 times of standard deviations with 0 ng/ml samples without intersecting）.
Interfering effects of drug is tested
32 kinds of common compounds and medicine are chosen, it is 10.0 μ g/ml to adjust its concentration, carries out interference test measure, is tested
As a result it is as shown in the table：
The method checked by Ciclosporin A homogeneous enzyme immunoassay is measured to above-claimed cpd, is as a result feminine gender.Can
See, the antibody of the present invention is the specific antibody of anti-Ciclosporin A.
Using the anti-Ciclosporin A specific antibody of the present invention, carry out with the substrate of Ciclosporin A enzyme mark conjugate and enzyme
Combination, you can obtain the Ciclosporin A detectable or test kit of the present invention.
Claims (9)
1. Ciclosporin A immunogen, its structural formula such as formula（I）It is shown：
In formula, R is linking group, and carrier has immunogenicity, and R is-O- (CH2)4- COO-, carrier is bovine serum albumin, its
Preparation method comprises the steps：
The synthesis of compound 1
Accurately weigh 1.0 g 4- penetenoic acids and 1.6 g N-hydroxy-succinamides are added to containing 40 mL tetrahydrofurans
In being dried 100 mL round-bottomed flasks, it is placed in being stirred on magnetic stirring apparatuss；
2.5 g 1 are accurately weighed, 3- dicyclohexylcarbodiimides in being added to 20 mL tetrahydrofurans, are placed in magnetic stirring apparatuss
In fully mix；
At 23 DEG C, the mixture in step 2 is added dropwise in the mixture of step 1, and is stirred continuously, all of solid
Granule quickly dissolves, and produces a kind of white precipitate rapidly；
The mixture was stirred overnight, filters, concentrated in vacuo and using flash chromatography on silica gel purification column purification, eluent：Petroleum ether/
Ethyl acetate=1/1, obtains 1.5 g compound as white solid 1；
The synthesis of compound 2
Accurately weigh following compound：1, the 3- diisopropylidenes third of 400 mg Ciclosporin As, 600 mg compounds 1 and 56 mg
Ketone -4,5- glyoxalidine -2- methene bases-cyclohexyl phosphine-benzylidene dichloro rutheniums, are dissolved in 10 ml CH2Cl2In；
Above-mentioned mixed solution is added in the flame-dried Schlenk pipes of 100 ml Jing, Jing is stirred vigorously and reflux condensation mode
Reaction is completed within 22 hours, after cooling, the mixed liquor obtains a kind of solid crude extract Jing after concentrated in vacuo, and crude extract Jing is quick
Chromatogram purification, eluent：15% acetonitrile-ethyl acetate, obtains 400 mg yellow compounds 2；
The synthesis of cyclosporin A derivative
Weigh 1 g compounds 2, and 166 mg Lithium hydrate LiOHH2O, is dissolved in 5 ml distilled water and 20 ml tetra-
In hydrogen furan, it is stirred overnight under room temperature；
By the concentration of compound Jing negative pressure and dilute with water, with the 1N hydrochloric acid accurate adjustment pH value of solution to 5, and ethyl acetate is used
Extraction；
After extracting and demixing, organic layer is separated and Jing Na2SO4Absorbent drying, Jing is filtered, concentrated in vacuo and high performance liquid chromatography is pure
Cyclosporin A derivative white solid is obtained after change；
The immunogenic synthesis of BSA- Ciclosporin As
Ciclosporin A immunogen passes through-O- (CH by bovine serum albumin and cyclosporin A derivative2)4- COO- groups connect and
Into specific synthetic method is as follows：
200 mg BSA are dissolved in into the M of 50 ml 0.2, in the phosphate buffer of pH 8.5；
Following chemicals are added to into stirring and dissolving in small beaker：200 mg cyclosporin A derivatives, 3.5 ml dimethyl acyls
Amine, 3.5 ml ethanol, the mM of 7.0 ml 10, the kaliumphosphate buffer of pH 5.0,400 mg 1- ethyl -3- (- 3- diformazan ammonia
Propyl group) carbodiimide, 50 mg N- hydroxy thiosuccinimides, at room temperature stirring and dissolving react 30 minutes；
The solution for having dissolved is dropped in BSA solution, and is stirred overnight at 2~8 DEG C, obtain antigen；Synthetic is resisted
Original obtains Ciclosporin A immunogen through neutral phosphate buffer liquid dialysis 4 × 4L purification.
2. a kind of anti-Ciclosporin A specific antibody, the Ciclosporin A immunogen immune by described in claim 1 any one
Production after animal is obtained.
3. a kind of Ciclosporin A detectable, containing anti-Ciclosporin A specific antibody, ciclosporin described in claim 2
The substrate of A enzyme mark conjugates and enzyme.
4. Ciclosporin A detectable according to claim 3, it is characterised in that：Ciclosporin A enzyme mark conjugate includes
Glucose-6-phosphate dehydrogenase (G6PD)-Ciclosporin A enzyme mark conjugate.
5. Ciclosporin A detection kit, anti-containing the anti-Ciclosporin A specific antibody described in claim 2 and detection
The indicator of Ciclosporin A specific antibody and Ciclosporin A complex.
6. Ciclosporin A detection kit according to claim 5, it is characterised in that：Indicator is selected from enzymatic reagent, puts
Injectivity isotope reagent and luminescence reagent.
7. Ciclosporin A detection kit according to claim 5, it is characterised in that：Indicator is by Ciclosporin A enzyme
The substrate of mark conjugate and enzyme is constituted.
8. Ciclosporin A detection kit according to claim 7, it is characterised in that：Ciclosporin A enzyme mark conjugate bag
Include glucose-6-phosphate dehydrogenase (G6PD)-Ciclosporin A enzyme mark conjugate.
9. Ciclosporin A detection kit according to claim 5, it is characterised in that：Anti- Ciclosporin A specific antibody
With reference on stable surface, its indicator is made up of the substrate of Ciclosporin A enzyme mark conjugate and enzyme.
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| CN104764883A (en) * | 2015-03-31 | 2015-07-08 | 上海云泽生物科技有限公司 | Immunoassay method and kit for detecting concentration of cyclosporine A |
| CN104788560B (en) * | 2015-05-16 | 2018-06-19 | 苏州博源医疗科技有限公司 | Ciclosporin A immunogene, anti-Ciclosporin A specific antibody and Ciclosporin A detection reagent |
| CN108794621A (en) * | 2017-05-04 | 2018-11-13 | 南开大学 | Conjugate of cyclosporin and the preparation method and application thereof |
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| DK0487301T3 (en) * | 1990-11-20 | 2000-09-18 | Dade Behring Marburg Gmbh | Method for Stabilizing Enzyme Conjugates |
| CN101929998A (en) * | 2009-06-22 | 2010-12-29 | 常州赛德瑞尔生物科技有限公司 | Fast food safety homogeneous immunological detection reagent |
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