CN104788560B - Ciclosporin A immunogene, anti-Ciclosporin A specific antibody and Ciclosporin A detection reagent - Google Patents
Ciclosporin A immunogene, anti-Ciclosporin A specific antibody and Ciclosporin A detection reagent Download PDFInfo
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Abstract
A kind of obtained the invention discloses Ciclosporin A immunogene and its synthetic method and by the immunogene anti-Ciclosporin A antibody, detection reagent and preparation method thereof.Ciclosporin A immunogene prepared by the present invention, immunogenicity is high, can induce to obtain the anti-Ciclosporin A specific antibody of high-titer, and with 62 kinds of common drugs without any cross reaction；The Ciclosporin A detection reagent obtained by the Antibody preparation can accurately and quickly determine the Ciclosporin A content in sample.Compared with existing detection reagent in the market, detection reagent of the present invention has many advantages, such as that easy to operate, high sensitivity, high specificity, result are accurate, moreover it is possible to effectively reduce Ciclosporin A testing cost, be conducive to clinical large-scale promotion and use.
Description
Technical field
The invention belongs to biotechnologies, are related to Ciclosporin A immunogene, anti-Ciclosporin A specific antibody and ring
P0-357 A detection reagents.
Background technology
Ciclosporin A (Cyclosporin A), shown in structural formula such as formula (III)：
Ciclosporin A is a kind of fat-solubility cyclic peptide small-molecule drug of fungal source, it, which is that one kind is strong, exempts from
Epidemic disease inhibitor passes through the effect to too many levels during immune response, the proliferation of selective depression helper lymphocyte T (Th)
And function, immunoregulation effect is generated, is to play one of key agents of immunosuppressive action after organ transplant.Use cyclosporin
A treats the survival rate that can significantly improve the organ transplants such as skin, heart, kidney, liver, pancreas, marrow, lung, small intestine.This
Outside, Ciclosporin A can be additionally used in the treatment of various autoimmune disease.Therapeutic effect, toxic reaction and the blood medicine of Ciclosporin A
Concentration is closely related, and safe range is narrow, and medication individuation difference is big, and can not play treatment less than its effective blood drug concentration makees
With being excessively used, can cause serious toxic side effect, mainly renal toxicity and hepatotoxicity wind agitation, other invertibitys reactions further include
Diarrhea, Nausea and vomiting, hirsutism, is trembled with hypertension etc. at gingival hyperplasia.Since Ciclosporin A is mostly for Long term prophylatic
Medicine, and kidney, hepatotoxicity wind agitation are difficult to distinguish with rejection in kidney, liver transfer operation.Therefore, the monitoring of Ciclosporin A blood concentration
It has great significance for the clinical safety rational use of medicines for instructing organ transplant and autoimmune disorders.
The method of detection Ciclosporin A mainly includes both at home and abroad at present：High performance liquid chromatography, is immunized chemoluminescence method
Method, fluorescence polarization method, Electrochemiluminescince, Heterogeneus immunoassay etc., but these methods all have centainly in clinical practice
Defective.Deficient in stability is good currently on the market, high sensitivity, the Ciclosporin A detection reagent of high specificity, especially quality
Good Automated inspection reagent.Therefore, development ＆ production quality reaches clinical requirement, highly practical, cost-effective, can be applied to complete
The Ciclosporin A measure reagent of automatic biochemistry analyzer has become the hot spot of domestic and international external diagnosis reagent industry.
Invention content
The present invention using completely new cyclosporin A derivative in order to overcome the shortcomings of the prior art, prepare immunogene
The strong Ciclosporin A immunogene of property and its antibody, can be real with the Ciclosporin A homogeneous enzyme immunoassay detection reagent of the Antibody preparation
High-throughput, the rapid detection to Ciclosporin A on present automatic clinical chemistry analyzer.The detection reagent have it is easy to operate,
The advantages that high sensitivity, high specificity, accurate result, moreover it is possible to effectively reduce Ciclosporin A testing cost, be conducive to clinical expansion
It uses.
It is an object of the present invention to provide a kind of cyclosporin A derivatives.
It is another object of the present invention to provide a kind of strong Ciclosporin A immunogenes of immunogenicity.
It is another object of the present invention to provide a kind of preparation methods of Ciclosporin A immunogene.
A further object of the present invention is to provide the high specificity being prepared using Ciclosporin A immunogene of the present invention
Anti- Ciclosporin A specific antibody.
It is yet a further object of the present invention to provide a kind of Ciclosporin A detection reagents.
Immunogenicity is related with synthesized cyclosporin A derivative molecular structure and selected carrier, the prior art
The less immunogenic of middle Ciclosporin A immunogene, the specificity of acquired antibody, the binding force with Ciclosporin A, susceptibility
All not as good as the present invention.The Ciclosporin A immunogene of the present invention, immunogenicity is high, and the anti-ring spore that can induce to obtain high-titer is mould
Plain A specific antibodies.The antibody specificity is high, strong with the binding force of Ciclosporin A.The cyclosporin obtained by the Antibody preparation
A detection reagents can quickly and accurately determine the Ciclosporin A content in sample.The present invention is real by the following technical programs
Existing：
A kind of Ciclosporin A immunogene, shown in structural formula such as formula (I)：
In formula, R is linking groupN is the integer between 1 to 20, and preferably R is
Carrier is protein or polypeptide with immunogenicity, preferably haemocyanin, hemocyanin and thyroid gland ball egg
In vain, more preferably seralbumin, further preferably bovine serum albumin(BSA).
When R isWhen, the route of synthesis and method of the Ciclosporin A immunogene are as follows：
1. the preparation method of cyclosporin A derivative：
(1) weigh 0.08~0.32mmol Ciclosporin As and 0.08~0.32mmol compound A, co-dissolve in 3~
In 6mL DCM；28~112mg benzyltriethylammoinium chlorides are weighed, are dissolved in the KOH aqueous solutions of 3~6mL40%；It will be above-mentioned
Two kinds of solution are mixed into reaction mixture.
(2) above-mentioned reaction mixture is stirred at room temperature overnight；Solution after reaction HCl is adjusted into pH value to pH
=5.0~7.0, then by organic layer separation.
(3) organic phase is concentrated, is then purified with the rich layer chromatography of preparation, obtain 4.5~18mg white solids
Final product, yield 50%.
(4) obtained final product is analyzed and identified using nuclear magnetic resonance technique and Chromatography/Mass Spectrometry technology, it can be true
The fixed final gained compound is cyclosporin A derivative, shown in structural formula such as formula (II)：
In formula, R is linking groupN is the integer between 1 to 20.
In the present invention, the compound A the step of preparation process of cyclosporin A derivative in (1) has selected 2- (bromine first
Base) methyl acrylate is synthesis material, therefore the link group R of the final product cyclosporin A derivative of gained isWhen n takes other numerical value, other 2- (bromomethyl) methyl acrylate analogs are selected, such as 4- bromo-
2- methylene butyric acids methyl esters (n 2), the bromo- 2- methylene methyl valerates (n 3) of 5-, (n is the bromo- 2- methlenehexanoic acid methyl esters of 6-
Etc. 4) tested, synthetic method is completely the same.
2. the preparation process of Ciclosporin A immunogene：
(1) 100~300mg of carrier protein is dissolved in 25~75ml0.2M, in the phosphate buffer of pH8.5；
(2) following chemicals is added to stirring and dissolving in small beaker：The Ciclosporin A that 100~300mg present invention synthesizes
Derivative, 1.75~5.25ml dimethylformamides, 1.75~5.25ml ethyl alcohol, 3.5~10.5ml10mM, pH5.0 phosphoric acid
Potassium buffer solution, 100~300mg1- ethyls -3- (- 3- dimethylaminopropyls) carbodiimide, 25~75mg N- hydroxy succinyls
30~60min of dissolving reaction is stirred at room temperature in these chemicals by imines；
(3) solution dissolved is added dropwise in carrier protein solution, and is stirred overnight at 2~8 DEG C, obtain antigen；
Synthetic antigen by dialysis is purified, obtains Ciclosporin A immunogene.
In the present invention when n takes other integers in the range of 1~20, it can prepare in aforementioned manners as shown in formula (I)
Ciclosporin A immunogene.Carrier is still the protein with immunogenicity, can be haemocyanin, hemocyanin and first shape
Gland globulin.Preferably, carrier is haemocyanin.It is furthermore preferred that carrier is bovine serum albumin(BSA).
Since linking group mainly plays the connection function of small molecule derivative and carrier, immunogenicity power with it is synthesized
Cyclosporin A derivative molecular structure and selected carrier are related, therefore when theoretically n takes the arbitrary integer between 1 to 20,
Ciclosporin A immunogene prepared by cyclosporin A derivative is provided with strongly immunogenic without significant difference, can prepare efficiently
The specific antibody of valency.
A kind of anti-Ciclosporin A specific antibody is obtained by being produced after above-mentioned Ciclosporin A immunogen immune animal.
The anti-Ciclosporin A specific antibody is connect by Ciclosporin A immunogene obtained above using conventional method
Experimental animal is planted, antiserum is taken after booster immunization, is as follows：
(1) the BSA- Ciclosporin A immunogenes of above-mentioned synthesis are diluted to 1.0mg/ml with PBS, obtain antigenic solution, so
It is mixed afterwards with 1.0ml antigenic solutions with Freund's complete adjuvant, experimental animal is injected；
After (2) 2~3 weeks, then with antigenic solution identical 1.0ml and incomplete Freund's adjuvant above-mentioned experimental animal is injected
Once, it is primary every surrounding injection later, it injects 4 times altogether；
(3) blood is taken to above-mentioned experimental animal, it is 1 to isolate and purify to obtain potency:30000-1:50000 anti-Ciclosporin A
Specific antibody.
The anti-Ciclosporin A specific antibody of the present invention is complete antibody molecule, also includes retaining special with Ciclosporin A
The antibody fragment or antibody derivatives of different in nature binding ability.
The Anti-TNF-α that the antibody of the present invention obtains animal booster immunization for the single Ciclosporin A immunogene of use
Body or the monoclonal antibody to be obtained after being immunized through somatic hybridization；The experimental animal is rabbit, goat, mouse, silk floss
One kind of sheep, cavy or horse, preferably rabbit.
The present invention provides a kind of Ciclosporin A detection reagent, is tried containing above-mentioned anti-Ciclosporin A specific antibody and instruction
Agent.
Indicator of the present invention is selected from enzymatic reagent, radioactive isotope reagent, fluorescent reagent, luminescence reagent.Preferably, refer to
Show that reagent for enzymatic reagent, is made of the substrate of Ciclosporin A enzyme mark conjugate and enzyme.
Above-mentioned enzyme mark conjugate is glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate；The substrate of above-mentioned enzyme is Portugal
Grape sugar -6- phosphoric acid.
Ciclosporin A homogeneous enzyme immunoassay detection reagent before the use, in order to avoid the enzyme mark conjugate in indicator
React with the substrate of enzyme, the substrate of enzyme mark conjugate and enzyme is unmixed and separated, so by the substrate of enzyme with
Above-mentioned anti-Ciclosporin A specific antibody mixes.Therefore, Ciclosporin A homogeneous enzyme immunoassay detection reagent includes two classes
Reagent：
(1) reagent A is mixed by anti-Ciclosporin A specific antibody and homogeneous zymolyte, and specific preparation process is as follows：
1) by the nicotinamide adenine dinucleotide (NAD) of 2.018~8.072g (5.625~22.50mM) oxidation state,
0.856~3.422g (5.625~22.50mM) G-6-P (G-6-P), 0.5~2L 55mM, the Tris of pH=8.0
Homogeneous zymolyte is made in buffer solution；
2) the anti-Ciclosporin A specific antibody of preparation is added in above-mentioned homogeneous zymolyte, antibody and homogeneous zymolyte
Volume ratio be 1:100~1:10000, preferably 1：400；
(2) reagent B is mixed by glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation object with Tris buffer solutions, preparation side
Method is as follows：
1) preparation of glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) solution：
A. the G6PDH that 7.5~22.5mg specifications are 100KU is weighed, room-temperature dissolution contains 72.6mg in 6~18mL
(0.05M)Tris、8mg MgCl2In the solution of (3.3mM) and 100mg NaCl, pH value of solution=9.0；
B. the nicotinamide adenine dinucleotide (NADH) of 112.5~337.5mg reduction-states, 67.5~202.5mg are added in
G-6-P (G-6-P) and 0.375~1.125mL carbitols；
C. 1~3mL dimethyl sulfoxide (DMSO)s are added dropwise；
2) activation of cyclosporin A derivative：
A. 5~15mg cyclosporin A derivatives are weighed under anhydrous conditions, are dissolved in 300~900 μ L DMF；
B. above-mentioned solution temperature is made to drop to -2~-8 DEG C；
C. 1.5~4.5 μ L tri-n-butylamines are added in；
D. 0.75~2.25 μ L isobutyl chlorocarbonates are added in；
It stirs 30~60 minutes e.-2~-8 DEG C；
3) connection of G6PDH and cyclosporin A derivative：
A. the cyclosporin A derivative solution of above-mentioned activation is added dropwise in the G6PDH solution of above-mentioned dissolving；
B.2-8 it DEG C is stirred overnight；
4) purified product：
By G-25 gel chromatography column purification connection products, the final product of acquisition is glucose-6-phosphate dehydrogenase (G6PD)-half
Antigen conjugates store at 2-8 DEG C.
5) Tris that the glucose-6-phosphate dehydrogenase (G6PD) of preparation-hapten conjugation object is added to 120mM, pH=8.2 is buffered
In liquid, the volume ratio of above-mentioned conjugate and Tris buffer solutions is 1:100~1:10000, preferably 1：1500.
Ciclosporin A immunogens of the invention are strong, immunogenicity is high, the anti-Ciclosporin A specificity prepared
Antibody specificity is strong, potency is high, and with 62 kinds of common drugs without any cross reaction；It is special containing above-mentioned anti-Ciclosporin A
The homogeneous enzyme immunoassay detection reagent of heterogenetic antibody can easily and fast, accurately determine Ciclosporin A content in sample, and
And multiple samples can be measured simultaneously on automatic clinical chemistry analyzer, and realize the rapid measure of high throughput of Ciclosporin A, it is accurate
Exactness is high, high specificity, and accuracy is all enhanced, while realize detection process before being compared with detection efficiency
Full-automation, to the of less demanding of testing staff, it is easy to accomplish and promote the use of.
Description of the drawings
Fig. 1 cyclosporin A derivatives analyze and identify spectrogram；
Fig. 2 is the ELISA detection response curves of Ciclosporin A；
Fig. 3 is the homogeneous enzyme immunoassay response curve of Ciclosporin A.
Specific embodiment
It the synthesis of one cyclosporin A derivative of embodiment and its analyzes and identifies
Shown in the chemical constitution of cyclosporin A derivative such as formula (IV)：
The synthetic route and preparation process of above-mentioned cyclosporin A derivative are as follows：
1. 200mg (0.16mmol) Ciclosporin As and 29.2mg (0.16mmol) 2- (bromomethyl) methyl acrylate are weighed,
Co-dissolve is in 3mL DCM；56mg (0.24mmol) benzyltriethylammoinium chloride is weighed, the KOH for being dissolved in 3mL40% is water-soluble
In liquid；Above two solution is mixed into reaction mixture.
2. above-mentioned reaction mixture is stirred at room temperature overnight；Solution after reaction HCl is adjusted into pH value to PH
=6, then by organic layer separation.
3. organic phase is concentrated, then purified with the rich layer chromatography (prep-TLC) of preparation, obtain 9mg white solids
The final product of shape, yield 50%.
4. obtained final product is analyzed and identified using high performance liquid chromatography (HPLC) and mass spectrum (MSD) technology,
As a result as shown in Figure 1, it may be determined that the final gained compound is the cyclosporin A derivative shown in formula (IV).
In the present embodiment, 2- (bromomethyl) acrylic acid first has been selected in the step 1 of the preparation process of cyclosporin A derivative
Ester is synthesis material, therefore the link group R of the final product cyclosporin A derivative of gained isIt is elected
With other 2- (bromomethyl) methyl acrylate analogs, such as the bromo- 2- methylene butyric acids methyl esters of 4-, the bromo- 2- methylene valeric acid first of 5-
Bromo- 2- methlenehexanoic acid methyl esters of ester, 6- etc. are tested, and synthetic method is completely the same, and n is appointing in 2-20 in linking group R
Meaning integer.
The synthesis of two Ciclosporin A immunogene of embodiment
Ciclosporin A immunogene is shown in bovine serum albumin(BSA) (Bovine Serum Albumin, BSA) and formula (II)
Cyclosporin A derivativeGroup is formed by connecting, in the present embodiment, by taking n=1 as an example specifically
The synthetic method of the bright immunogene, is as follows：
1. bovine serum albumin(BSA) 200mg is dissolved in 50ml0.2M, in the phosphate buffer of pH8.5；
2. following chemicals is added to stirring and dissolving in small beaker：Cyclosporin A derivative, the 3.5ml of 200mg synthesis
Dimethylformamide, 3.5ml ethyl alcohol, the kaliumphosphate buffer of 7.0ml10mM, pH5.0,200mg1- ethyls -3- (- 3- diformazan ammonia
Propyl) carbodiimide, 50mg N- hydroxy thiosuccinimides, by these chemicals be stirred at room temperature dissolving reaction
30min；
3. the solution dissolved is added dropwise in BSA solution, and it is stirred overnight at 2~8 DEG C, obtains antigen；It will synthesis
Good antigen is purified by dialysis, obtains Ciclosporin A immunogene.
Embodiment three：The preparation of anti-Ciclosporin A specific antibody
The Ciclosporin A immunogene that embodiment two is prepared uses conventional method inoculation experiments animal rabbit, strengthens exempting from
Antiserum is taken after epidemic disease, is as follows：
1. the Ciclosporin A immunogene of above-mentioned synthesis is diluted to 1.0mg/ml with PBS, antigenic solution, Ran Houyong are obtained
1.0ml antigenic solutions are mixed with Freund's complete adjuvant, and experimental animal rabbit is injected.
After 2.2~3 weeks, then with antigenic solution identical 1.0ml and incomplete Freund's adjuvant above-mentioned experimental animal rabbit is noted
Penetrate primary, primary every surrounding injection later, injection 4 times altogether.
3. the experimental animal rabbit of pair step 2 takes blood, it is 1 to isolate and purify to obtain potency:30000-1:50000 anti-ring spore is mould
Plain A specific antibodies.
Example IV：The ELISA of Ciclosporin A is examined
1. the foundation of the ELISA examination criteria curves of Ciclosporin A
(1) preparation of standard items
Ciclosporin A powder (being purchased from Sigma companies) is dissolved in methanol solution, is prepared into the storing liquid of 1mg/ml.With
ELISA buffer solutions storing liquid is diluted to successively 400.00ng/mL, 150.00ng/mL, 50.00ng/mL, 15.00ng/mL,
The standard solution of 5.00ng/mL and 0.00ng/mL.Wherein, ELISA buffer solutions contain 50.0mM Tris, 145mM NaCl and
0.25% BSA.
(2) standard curve is prepared using the ELISA methods of inspection of Ciclosporin A
Anti- Ciclosporin A antibody prepared in embodiment three is diluted to 1 with PBS:8000 final concentration solution, 100 μ
L/ holes are coated on 96 hole elisa plates, 4 DEG C of placement 12-24h；With PBS by the above-mentioned 96 hole enzymes for being coated with anti-Ciclosporin A antibody
After yoke plate washs 3 times, the 0.5% BSA solution in 200 μ L/ holes is added in, 8-16h is placed in 4 DEG C of closings.Then it is washed 3 times with PBS,
Add in the standard items in 20 μ L/ holes.Add the HRP- Ciclosporin A conjugates of 100 μ L/ holes working concentrations；It is incubated at room temperature
PBS board-washings 5 times after 30min；Then 100 μ L tmb substrates are added in per hole, are incubated at room temperature 30min.Again 100 μ L are added in per hole to terminate
Liquid (2M sulfuric acid).Measure the light absorption value of 450nm.The light absorption value calibration of 450nm according to corresponding to each standard items, it is bent to make standard
Line, as a result as shown in Figure 2.
2. the detection of Ciclosporin A content in sample to be tested
(1) sample to be tested is made
Preparation method：Ciclosporin A powder (being purchased from Sigma companies) is dissolved in the storage that 1 μ g/mL are made in methanol solution
Liquid, and this storing liquid is diluted in blank whole blood, until final concentration is respectively 0.00,5.00,50.00,400.00ng/mL, shape
Into the whole blood sample of blank, basic, normal, high concentration.The blank whole blood is the healthy human blood without Ciclosporin A.
(2) test method
Using the ELISA methods of inspection of above-mentioned Ciclosporin A, by the whole blood sample generation of above-mentioned blank, basic, normal, high concentration
For standard items, test above-mentioned blank, basic, normal, high concentration whole blood sample 450nm light absorption value.
(3) test result
The standard curve that the ELISA of Ciclosporin A shown in compares figure 1 is examined, calculates Ciclosporin A in each sample
Content, and 3 multiple holes are carried out to each sample and are measured, the rate of recovery is calculated according to the actual content of Ciclosporin A in above-mentioned sample,
The results are shown in Table 1.
The ELISA detection recovery experiments of 1 Ciclosporin A of table
From result in table 1：It is measured in various concentration sample using the ELISA detection reagents of Ciclosporin A of the present invention
The Ciclosporin A rate of recovery it is all higher, equal ＞ 90% illustrates that anti-Ciclosporin A specific antibody of the present invention can be used
The detection of Ciclosporin A in sample, and result precision is high.
Embodiment five：The preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation object
The preparation of glucose-6-phosphate dehydrogenase (G6PD) 1. (G6PDH) solution：
(1) accurate to weigh the G6PDH that 15mg specifications are 100KU, room-temperature dissolution contains 72.6mg (0.05M) in 12mL
Tris、8mg MgCl2In the solution of (3.3mM) and 100mg NaCl, pH value of solution=9.0, this step carries out in beaker C.
(2) nicotinamide adenine dinucleotide (NADH) of 225mg reduction-states, 135mg grapes are added in above-mentioned beaker C
Sugar -6- phosphoric acid (G-6-P) and 0.75mL carbitols (Carbitol).
(3) 2mL dimethyl sulfoxide (DMSO)s (dimethy sulfoxide, DMSO) are added dropwise again in above-mentioned beaker C.
2. the activation of cyclosporin A derivative：
(1) the above-mentioned cyclosporin A derivatives of 10mg are weighed under anhydrous conditions, are dissolved in 600 μ L DMF.
(2) above-mentioned solution temperature is made to drop to -2~-8 DEG C.
(3) 3 μ L tri-n-butylamines (tributylamine) are added in.
(4) 1.5 μ L isobutyl chlorocarbonates (isobutylchloroformate) are added in.
(5) -2~-8 DEG C are stirred 30 minutes.
The connection of 3.G6PDH and cyclosporin A derivative：
(1) the cyclosporin A derivative solution of above-mentioned activation is added dropwise in the G6PDH solution of above-mentioned dissolving.
(2) it is stirred overnight for 2-8 DEG C.
4. purified product：
By the solution in G-25 gel chromatographies column purification step 3, the final product of acquisition is G-6-P dehydrogenation
Enzyme-hapten conjugation object, stores at 2-8 DEG C.
Embodiment six：The preparation of Ciclosporin A homogeneous enzyme immunoassay detection reagent
1. the preparation of reagent A：By the nicotinamide adenine dinucleotide (NAD) of 4.036g (11.25mM) oxidation state,
1.711g (11.25mM) G-6-P (G-6-P) is placed in beaker D, with 1L 55mM, the Tris buffer solutions of pH=8.0
Homogeneous zymolyte is made in dissolving；The anti-Ciclosporin A specific antibody of above-mentioned preparation is added in above-mentioned homogeneous zymolyte, antibody
Volume ratio with homogeneous zymolyte is 1:400.
2. the preparation of reagent B：Glucose-6-phosphate dehydrogenase (G6PD) prepared by embodiment five-hapten conjugation object is added to
In the Tris buffer solutions of 120mM, pH=8.2, the volume ratio of above-mentioned conjugate and Tris buffer solutions is 1:1500.
Embodiment seven：Ciclosporin A homogeneous enzyme immunoassay is examined and result
1. obtain standard curve：
(1) it sets and steps auspicious BS-480 automatic clinical chemistry analyzers response parameter (being shown in Table 2).
(2) operating procedure is：First reagent adding A, adds standard items, is eventually adding reagent B.After adding in reagent B, measure not
With the OD at time point340Light absorption value calculates reaction rate during various criterion product concentration, needs constantly to adjust in actual mechanical process
The volume ratio of reagent A and reagent B, while survey luminous point is adjusted, comparatively ideal reaction normal curve graph is finally obtained, such as Fig. 3 institutes
Show.
Table 2 steps auspicious BS-480 automatic clinical chemistry analyzers response parameter
2. pattern detection：By the obtained standard curve of homogeneous enzyme immunoassay detection reagent of the present invention, replication is low,
Middle and high concentration Quality Control sample 10 times, above-mentioned Quality Control sample are：Ciclosporin A standard items are dissolved in people's whole blood, until concentration point
It Wei 20.0,200.0,800.0ng/ml.Detection data and data analysis are shown in Table 3.
3 sample of table measure and precision and rate of recovery assessment
| Blood sample | It is low | In | It is high |
| Sample concentration (ng/ml) | 20.0 | 200.0 | 800.0 |
| 1 | 20.2 | 201.4 | 805.2 |
| 2 | 19.3 | 203.5 | 793.1 |
| 3 | 20.7 | 198.0 | 811.9 |
| 4 | 20.9 | 203.6 | 795.2 |
| 5 | 19.8 | 202.7 | 805.7 |
| 6 | 20.7 | 199.4 | 808.5 |
| 7 | 20.3 | 197.5 | 792.0 |
| 8 | 19.9 | 204.2 | 810.1 |
| 9 | 19.0 | 201.8 | 789.9 |
| 10 | 20.5 | 203.6 | 800.6 |
| Average value (ng/ml) | 20.13 | 201.57 | 802.48 |
| Standard deviation (SD) | 0.6273 | 2.4554 | 8.1482 |
| Precision (CV%) | 3.12 | 1.22 | 1.02 |
| Rate of recovery % | 100.7 | 100.8 | 100.3 |
Testing result：The accuracy that the 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 below 5%.
Embodiment eight：Interfering effects of drug is tested
It chooses 62 kinds of Common drugs and carries out Interference Detection, adjustment concentration is to 1.00 μ g/ml, using the homogeneous enzyme of embodiment seven
Immunization method is measured：
1. reagent A haptoreaction prepared by interference medicament to be measured and embodiment six, adds reagent B；
2. the OD of the above-mentioned mixed solution of detection340Light absorption value obtains the dense of respective substance according to the standard curve of embodiment seven
Degree.
62 kinds of common medicine names and measurement result are referring specifically to table 4.
4 common interference drug monitoring result of table
Measurement result is shown：The concentration that above-mentioned 62 kinds of Common drugs are equivalent to Ciclosporin A is respectively less than 0.01 μ g/ml.By
This is as it can be seen that the antibody of the present invention is the specific antibody of anti-Ciclosporin A, with other medicines no cross reaction.
Claims (3)
1. a kind of preparation method of Ciclosporin A detection reagent, which is characterized in that preparation process is：
(1) reagent A：By 2.018~8.072g, 5.625~22.50mM oxidation state nicotinamide adenine dinucleotide and
0.856~3.422g, 5.625~22.50mM G-6-Ps, 0.5~2L 55mM, the Tris buffer solutions of pH=8.0 are molten
Homogeneous zymolyte is made in solution；Anti- Ciclosporin A specific antibody is added in above-mentioned homogeneous zymolyte, anti-Ciclosporin A is special
Property antibody and homogeneous zymolyte volume ratio be 1:400；
(2) reagent B：Ciclosporin A enzyme mark conjugate is added in the Tris buffer solutions of 120mM, pH=8.2, Ciclosporin A enzyme
The volume ratio for marking conjugate and Tris buffer solutions is 1:1500；
The preparation method of the anti-Ciclosporin A specific antibody comprises the steps of：
1. Ciclosporin A immunogene is diluted to 1.0mg/ml with PBS, obtain antigenic solution, then with 1.0ml antigenic solutions with
Freund's complete adjuvant mixes, and experimental animal is injected；
2. after 2~3 weeks, then it is primary to the injection of above-mentioned experimental animal with antigenic solution identical 1.0ml and incomplete Freund's adjuvant,
It is primary every surrounding injection later, it injects 4 times altogether；
3. taking blood to the experimental animal of step 2., it is 1 to isolate and purify to obtain potency:30000-1:50000 anti-Ciclosporin A is special
Heterogenetic antibody, the experimental animal are rabbit；
The Ciclosporin A immunogene, shown in structural formula such as formula (I)：
In formula, R isCarrier is the bovine serum albumin(BSA) with immunogenicity；The enzyme mark conjugate
For glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate.
2. the preparation method of Ciclosporin A detection reagent according to claim 1, which is characterized in that the ring spore is mould
The preparation method of plain A enzymes mark conjugate comprises the steps of：
(a) preparation of glucose-6-phosphate dehydrogenase (G6PD) solution：Weigh the glucose -6- phosphorus that 7.5~22.5mg specifications are 100KU
Acidohydrogenase, room-temperature dissolution contain 72.6mg 0.05M Tris, 8mg 3.3mM MgCl in 6~18mL2With 100mg NaCl's
In solution, pH=9.0；In the solution add in 112.5~337.5mg reduction-states nicotinamide adenine dinucleotide, 67.5~
202.5mg G-6-Ps and 0.375~1.125mL carbitols；1~3mL dimethyl sulfoxide (DMSO)s are added dropwise again；
(b) activation of cyclosporin A derivative：5~15mg cyclosporin A derivatives are weighed under anhydrous conditions, are dissolved in 300
In~900 μ L dimethylformamides；Above-mentioned solution temperature is made to drop to -2~-8 DEG C；Add in 1.5~4.5 μ L tri-n-butylamines；It adds in
0.75~2.25 μ L isobutyl chlorocarbonates；- 2~-8 DEG C are stirred 30~60 minutes；
(c) connection of glucose-6-phosphate dehydrogenase (G6PD) and cyclosporin A derivative：The Ciclosporin A that step (b) activates is spread out
Biological solution is added dropwise in the glucose-6-phosphate dehydrogenase (G6PD) solution of step (a) dissolving；2-8 DEG C is stirred overnight；
(d) purified product：By G-25 gel chromatography column purification connection products, the final product of acquisition is G-6-P
Dehydrogenase-hapten conjugation object, stores at 2-8 DEG C；
The cyclosporin A derivative, shown in structural formula such as formula (II)：
In formula, R is
3. the preparation method of Ciclosporin A detection reagent according to claim 1, which is characterized in that the ring spore is mould
The preparation method of plain A immunogenes, comprises the steps of：
(I) 100~300mg of bovine serum albumin(BSA) is dissolved in 25~75ml 0.2M, in the phosphate buffer of pH 8.5；
(II) following chemicals is added to stirring and dissolving in small beaker：Ring spore described in 100~300mg claims 2 is mould
Plain A derivatives, 1.75~5.25ml dimethylformamides, 1.75~5.25ml ethyl alcohol, 3.5~10.5ml 10mM, pH 5.0
Kaliumphosphate buffer, 100~300mg 1- ethyls -3- (3- dimethylaminopropyls) carbodiimide, 25~75mg N- hydroxy ambers
30~60min of dissolving reaction is stirred at room temperature in above-mentioned chemicals by amber acid imide；
(III) solution dissolved is added dropwise in bovine serum albumin solution, and is stirred overnight at 2~8 DEG C, obtain antigen；
Synthetic antigen by dialysis is purified, obtains Ciclosporin A immunogene.
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| CN107782889A (en) * | 2017-12-22 | 2018-03-09 | 太原瑞盛生物科技有限公司 | A kind of Ciclosporin A immunologic function test reagent and its preparation and detection method |
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| CN102295698A (en) * | 2011-05-19 | 2011-12-28 | 福州金域医学检验所有限公司 | Cyclosporine A immunogen, cyclosporine A specific antibody, detection reagent, and detection kit |
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| CN102295698A (en) * | 2011-05-19 | 2011-12-28 | 福州金域医学检验所有限公司 | Cyclosporine A immunogen, cyclosporine A specific antibody, detection reagent, and detection kit |
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