CN108794621A - Conjugate of cyclosporin and the preparation method and application thereof - Google Patents
Conjugate of cyclosporin and the preparation method and application thereof Download PDFInfo
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- CN108794621A CN108794621A CN201710319414.4A CN201710319414A CN108794621A CN 108794621 A CN108794621 A CN 108794621A CN 201710319414 A CN201710319414 A CN 201710319414A CN 108794621 A CN108794621 A CN 108794621A
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- Prior art keywords
- cyclosporin
- conjugate
- solution
- preparation
- serum albumin
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- 229930182912 cyclosporin Natural products 0.000 title claims abstract description 89
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 title claims abstract description 85
- 108010036949 Cyclosporine Proteins 0.000 title claims abstract description 84
- 229960001265 ciclosporin Drugs 0.000 title claims abstract description 84
- 229930105110 Cyclosporin A Natural products 0.000 title claims abstract description 80
- 238000002360 preparation method Methods 0.000 title claims abstract description 31
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 18
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 18
- 108010058846 Ovalbumin Proteins 0.000 claims abstract description 9
- 229940092253 ovalbumin Drugs 0.000 claims abstract description 8
- 230000005847 immunogenicity Effects 0.000 claims abstract description 7
- 241001465754 Metazoa Species 0.000 claims abstract description 6
- 230000008878 coupling Effects 0.000 claims abstract description 4
- 238000010168 coupling process Methods 0.000 claims abstract description 4
- 238000005859 coupling reaction Methods 0.000 claims abstract description 4
- 230000001939 inductive effect Effects 0.000 claims abstract 2
- 239000000243 solution Substances 0.000 claims description 76
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 22
- 238000000502 dialysis Methods 0.000 claims description 20
- 239000006228 supernatant Substances 0.000 claims description 20
- 239000008363 phosphate buffer Substances 0.000 claims description 19
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims description 16
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- 239000000047 product Substances 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 9
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 210000002966 serum Anatomy 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims description 5
- 108010036941 Cyclosporins Proteins 0.000 claims description 5
- 239000012071 phase Substances 0.000 claims description 5
- 239000012048 reactive intermediate Substances 0.000 claims description 5
- 229940014800 succinic anhydride Drugs 0.000 claims description 5
- ISCMYZGMRHODRP-UHFFFAOYSA-N 3-(iminomethylideneamino)-n,n-dimethylpropan-1-amine Chemical compound CN(C)CCCN=C=N ISCMYZGMRHODRP-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 150000007513 acids Chemical class 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000012074 organic phase Substances 0.000 claims description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 4
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 241000283690 Bos taurus Species 0.000 claims description 3
- RZZPDXZPRHQOCG-OJAKKHQRSA-O CDP-choline(1+) Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OCC[N+](C)(C)C)O[C@H]1N1C(=O)N=C(N)C=C1 RZZPDXZPRHQOCG-OJAKKHQRSA-O 0.000 claims description 2
- 238000000862 absorption spectrum Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 13
- 238000000034 method Methods 0.000 abstract description 10
- 102000004190 Enzymes Human genes 0.000 abstract description 4
- 108090000790 Enzymes Proteins 0.000 abstract description 4
- 230000001900 immune effect Effects 0.000 abstract description 3
- 238000011587 new zealand white rabbit Methods 0.000 abstract description 3
- 239000008280 blood Substances 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 239000000427 antigen Substances 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- -1 dichloromethanes Alkane Chemical class 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000003018 immunosuppressive agent Substances 0.000 description 3
- 239000013049 sediment Substances 0.000 description 3
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 108010078791 Carrier Proteins Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 239000003547 immunosorbent Substances 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 108010042653 IgA receptor Proteins 0.000 description 1
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical class CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 102100034014 Prolyl 3-hydroxylase 3 Human genes 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 241001149960 Tolypocladium inflatum Species 0.000 description 1
- 241000223259 Trichoderma Species 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000010322 bone marrow transplantation Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- PMATZTZNYRCHOR-UHFFFAOYSA-N cyclosporine a Chemical compound CCC1NC(=O)C(C(O)C(C)CC=CC)N(C)C(=O)C(C(C)C)N(C)C(=O)C(CC(C)C)N(C)C(=O)C(CC(C)C)N(C)C(=O)C(C)NC(=O)C(C)NC(=O)C(CC(C)C)N(C)C(=O)C(C(C)C)NC(=O)C(CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-UHFFFAOYSA-N 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 208000024908 graft versus host disease Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 150000002466 imines Chemical class 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
Abstract
The invention discloses a kind of conjugates of the cyclosporin of logical formula (I), are made of with carrier mass bovine serum albumin or the ovalbumin coupling for generating immunogenicity cyclosporin haptens.Wherein n is the molecular number of the cyclosporin combined with a bovine serum albumin molecule, and the n is integer 1~20, and BSA is bovine serum albumin, and molecular weight ranges are 6.6KDa~6.9KDa.The invention also discloses the preparation methods of the conjugate, i.e., connect cyclosporin with the carrier mass for generating immunogenicity, be combined into the conjugate for inducing animal immuning system generated antibody.The conjugate of the cyclosporin of the present invention is prepared for potency up to 50,000 antiserum, lowest detection is limited to 20ppb by the way that new zealand white rabbit is immunized.The present invention has method easy, and quickly, specifically, accurate feature, the enzyme-linked immunologic detecting kit to prepare cyclosporin provides the foundation.
Description
Technical field
The present invention relates to a kind of conjugate of cyclosporin and the preparation method and application thereof more particularly to a kind of cyclosporins
Conjugate and the preparation method and application thereof.Belong to immunosuppressant drug blood concentration immunologic surveillance field.
Background technology
Following denotations of the present invention are suitable for the whole instruction and claims：
BSA：Haemocyanin (Bovine Serum Albumin), Sigma Products
PBS：Phosphate buffer (Phosphate Buffered Saline) (0.01M, pH=7.40)
Sephadex-G75：Sephadex, Sigma Products
cBSA：Through ethylene diamine-modified bovine serum albumin
Dialysis membrane：Suo Laibao Science and Technology Ltd.s
Cyclosporin：Sigma Products
EDC：Ethyl [3- (dimethylamino) propyl] carbodiimide, abbreviation EDC, Sigma Products
Cyclosporin is a kind of Western medicine, and molecular formula is C62H111N11O12, it is that one kind being widely used in prevention of organ transplant row
The immunosuppressor of reprimand.It is by the active activity for reaching inhibition immune system with growth for inhibiting T cell.Cyclosporine in
Fungi by Sandoz drugmakers of Norway scientist in soil sample in 1969 --- porous trichoderma (Tolypocladium
Inflatum it is separated for the first time in).Occurred suitable for preventing the transplanting of the organ or tissues such as allogeneic kidney, liver, the heart, marrow
Rejection；Prevent and treat the graft-versus-host reaction occurred when bone-marrow transplantation.This product is often and cortex hormone of aadrenaline
Etc. immunosuppressor use in conjunction, to improve curative effect.
The blood concentration of cyclosporine is related to immunosuppressive intensity, also related to liver reaction of renal toxicity, the toxicity of drug
It reacts and the rejection clinical manifestation of organ transplant is not easy to differentiate sometimes, the effective concentration of cyclosporine connects very much again with poisoning concentration
Closely (less than 2 times), different patients have different pharmacokinetics to change in addition, therefore in the process of clinical application of cyclosporine
In, it is necessary to therapeutic drug monitoring is carried out, this is conducive to the survival rate for improving transplant organ, mitigates adverse reaction.
Instrument analytical method such as high performance liquid chromatography is widely used method.These methods are accurate, stablize, is reliable,
Standard method can be used as.But instrumental method is expensive, time-consuming longer, and causes organic solvent pollution, and large-scale instrument is needed to set
It is standby, need special technical staff.Enzyme-Linked Immunospot (ELISA) provides a kind of fabulous detection means.The method has
Quickly, accurately, simply, the advantages that not needing professional's operation, this makes ELISA method become a kind of ideal.It can be used for often
Advise detection method.The core of Enzyme-Linked Immunospot is to need the antibody of high quality.Therefore the immunogene of cyclosporin is studied
The Antibody preparation of synthesis and high quality just seems very necessary.
Invention content
In view of the above shortcomings of the prior art, the problem to be solved in the present invention is：There is provided a kind of can cause animal immune system
System generates the immunogene for the antibody for having idiosyncrasy for cyclosporin, the i.e. conjugate and preparation method thereof of cyclosporin.Together
When, the present invention also provides the conjugates of the cyclosporin as immunogene in preparing cyclosporin idiosyncrasy antibody
Using.
The conjugate of the cyclosporin of the present invention, by cyclosporin haptens and the antigenic carrier mass cow's serum of generation
Albumen or ovalbumin coupling are constituted.
Wherein：The carrier mass of above-mentioned generation immunogenicity is preferably bovine serum albumin.
The conjugate of cyclosporin of the present invention, general structure such as (I)
Wherein：N is the Molecules of the cyclosporin combined with a bovine serum albumin molecule, the n be integer 1~
20, BSA be bovine serum albumin (Bovine serum albumin), molecular weight ranges 6.6KDa~6.9Kda；
Above-mentioned conjugate shows following physical chemical characteristics：
(1) appearance：White powder solid；
(2) ultra-violet absorption spectrum：250nm, 277nm
The conjugate of above-mentioned cyclosporin, it is characterized in that：The n is integer 5~15, BSA molecular weight ranges 6.6KDa
~6.9Kda
The preparation method of the conjugate of above-mentioned cyclosporin is：By cyclosporin and the carrier mass for generating immunogenicity
It connects, is combined into and generates the conjugate of antibody with induction animal immune, and keep the bioactivity of the conjugate constant.
The preparation method of the conjugate of above-mentioned cyclosporin, is completed by following steps：
(1) preparation of solution A：50mg cyclosporins (0.042mmol) are added in 25mL round-bottomed flasks, with 10mL dichloromethanes
Alkane dissolves.14mg metachloroperbenzoic acids (MCPBA) are added thereto, room temperature is stirred overnight, and it is water-soluble that sodium bicarbonate is added later
Liquid is extracted with dichloromethane, merges organic phase, and vacuum rotary steam obtains the dissolving of product diluted hydrochloric acid aqueous solution and 5h is stirred at room temperature, it
PH to 7 is adjusted afterwards, and succinic anhydride, HOSu NHS, ethyl [3- (dimethylamino) propyl] carbodiimide (EDC) is added,
It is dissolved in DMF- water two-phase mixtures solvents with molar ratio 1: 2~8: 5~15, reaction generates cyclosporin similar to object and ethyl
The reactive intermediate of [3- (dimethylamino) propyl] carbodiimide is solution A；
(2) preparation of cBSA：Under the conditions of 0~4 DEG C, it is 7.38~7.56 that ethylenediamine, which is first dissolved in pH, a concentration of 0.01M
It is 7.38~7.56 with concentrated hydrochloric acid tune pH in~002M phosphate buffer solutions；With ethylenediamine: BSA: EDC molar ratio be 15~
25: 1: 15~25 amount weighs BSA and EDC, is then added in ethylenediamine solution, 2~4 are stirred to react under the conditions of 20 ± 5 DEG C
Hour；It is small with the stirring dialysis 70~80 of above-mentioned phosphate buffer by the reaction solution of ethylenediamine and BSA under the conditions of 0~4 DEG C
When, it then uses distilled water instead and dialyses 20~30 hours, replace a dialyzate within every 6 hours；At 0~4 DEG C, with 13000 revs/min
The solution 15 minutes after above-mentioned dialysis is centrifuged, supernatant is taken；Supernatant is lyophilized, obtains white powder solid cBSA, it is spare；
(3) configuration of solution B：It is 7.38~7.56 that cBSA, which is dissolved in pH, and a concentration of 0.01M~0.02M phosphoric acid buffers are molten
In liquid, it is made into the solution of 10.0 ± 5.0mg/ml, it is spare；
(4) it is that 15~50: 1 amount takes solution A and solution B respectively by the molar ratio of cyclosporin derivative and cBSA, 20
At a temperature of DEG C ± 5 DEG C, solution A is added dropwise in the solution B under the state of being stirred continuously, reacts 4~6 hours, obtain solution
C；
(5) solution C stirs dialysis 70~80 hours with above-mentioned phosphate buffer, then to use distilled water dialysis instead 20~30 small
When, replace a dialyzate within every 6 hours；Then at 0~4 DEG C, with the solution 15 after 13000 revs/min of above-mentioned dialysis of centrifugation
Minute, take supernatant；
(6) supernatant is lyophilized, obtains the conjugate of white cyclosporin.
In the preparation method of the conjugate of above-mentioned cyclosporin：Cyclosporin described in step (1), hydroxysuccinimidyl acyl
The molar ratio of imines, EDC is preferably 1: 5: 10.
In the preparation method of the conjugate of above-mentioned cyclosporin：Step (2) ethylenediamine and bovine serum albumin,
The molar ratio of EDC is 20: 1: 20.
In the preparation method of the conjugate of above-mentioned cyclosporin：Phosphate buffer pH described in step (2) (3) is preferred
It is 7.40, concentration is preferably 0.01M.
In the preparation method of the conjugate of above-mentioned cyclosporin：Step (4) cyclosporin and bovine serum albumin
Molar ratio be 30: 1.
The conjugate of cyclosporin of the present invention is as immunogene in preparing cyclosporin idiosyncrasy antibody
Using.
It can be successfully haptens cyclosporin and carrier protein especially cow's serum using technical scheme of the present invention
Protein B SA couplings are got up, and can be triggered an immune response in animal body to synthesize, be generated complete immunogene-ring of antibody
The conjugate of p0-357.
Using the conjugate of cyclosporin of the present invention as immunogen immune new zealand white rabbit, successfully obtain
There is to haptens cyclosporin the antibody of idiosyncrasy.Through ELISA experimental identifications, cyclosporin of the present invention is utilized
The cyclosporin idiosyncrasy antibody that conjugate is prepared as immunogene, antiserum titre reach 50,000, lowest detection
It is limited to 10ppb.
The conjugate of above-mentioned cyclosporin and the cyclosporin idiosyncrasy antibody of high-titer are successfully prepared, for system
The enzyme-linked immunologic detecting kit of standby cyclosporin provides the foundation.In practical applications, the cyclosporin of preparation spy
Different reagin is plated in micropore disk, so that it may quickly to detect the content of cyclosporin in blood sample.Due to the method for the invention
With simple, quickly, specifically, accurate feature, it is possible to the assay for cyclosporin in blood sample.Not only may be used in this way
To save a large amount of detection time, it is time-consuming longer to compensate for instrumental method, needs large-scale instrument and equipment to support, needs special technology
The deficiency of personnel's operation.So haptens cyclosporin and the carrier protein especially synthesis of bovine serum albumin BSA conjugate and
It is sero-fast to be successfully prepared as this fast detection method and lay a good foundation.
Specific implementation mode
Embodiment 1
(1) preparation of solution A：50mg cyclosporins (0.042mmol) are added in 25mL round-bottomed flasks, with 10mL dichloromethanes
Alkane dissolves.14mg metachloroperbenzoic acids (MCPBA) are added thereto, room temperature is stirred overnight, and it is water-soluble that sodium bicarbonate is added later
Liquid is extracted with dichloromethane, merges organic phase, and vacuum rotary steam obtains the dissolving of product diluted hydrochloric acid aqueous solution and 5h is stirred at room temperature, it
PH to 7 is adjusted afterwards, and succinic anhydride, HOSu NHS, ethyl [3- (dimethylamino) propyl] carbodiimide (EDC) is added,
It is dissolved in DMF- water two-phase mixtures solvents with molar ratio 1: 2~8: 5~15, reaction generates cyclosporin similar to object and ethyl
The reactive intermediate of [3- (dimethylamino) propyl] carbodiimide is solution A.
(2) preparation of cBSA：Under the conditions of 0~4 DEG C, it is 7.40 that ethylenediamine 18.0mg, which is first dissolved in 20ml pH, a concentration of
It is 7.40 with concentrated hydrochloric acid tune pH in 0.01M phosphate buffer solutions；Weigh respectively 1000.0mgBSA (molecular weight 68,000) and
Then 57.51mgEDC is added in ethylenediamine solution, is stirred to react under the conditions of 20 DEG C 2 hours；By reacting for ethylenediamine and BSA
Solution, with above-mentioned phosphate buffer stirring dialysis 70 hours, then uses distilled water instead and dialyses 24 hours, often under the conditions of 0~4 DEG C
Replace a dialyzate within 6 hours；At 0~4 DEG C, with the solution 15 minutes after 13000 revs/min of above-mentioned dialysis of centrifugation, supernatant is taken
Liquid；Supernatant is lyophilized, obtains white powder solid cBSA, it is spare；
(3) configuration of solution B：It is 7.40 that cBSA, which is dissolved in pH, in a concentration of 0.01M phosphate buffer solutions, is made into
The solution of 10.0mg/ml, it is spare；
(4) it is that 20: 1 amounts take solution A and solution B respectively by the molar ratio of cyclosporin and cBSA, at a temperature of 20 DEG C,
Solution A is added dropwise in the solution B under the state of being stirred continuously, and is reacted 6 hours, is obtained solution C；
(5) solution C stirs dialysis 70 hours with above-mentioned phosphate buffer, then uses distilled water instead and dialyse 24 hours, every 6 hours
Replace a dialyzate；Then at 0~4 DEG C, with the solution 15 minutes after 13000 revs/min of above-mentioned dialysis of centrifugation, supernatant is taken
Liquid；
(6) supernatant is lyophilized, obtains the conjugate of white cyclosporin.
Embodiment 2
(1) preparation of solution A：50mg cyclosporins (0.042mmol) are added in 25mL round-bottomed flasks, with 10mL dichloromethanes
Alkane dissolves.14mg metachloroperbenzoic acids (MCPBA) are added thereto, room temperature is stirred overnight, and it is water-soluble that sodium bicarbonate is added later
Liquid is extracted with dichloromethane, merges organic phase, and vacuum rotary steam obtains the dissolving of product diluted hydrochloric acid aqueous solution and 5h is stirred at room temperature, it
PH to 7 is adjusted afterwards, and succinic anhydride, HOSu NHS, ethyl [3- (dimethylamino) propyl] carbodiimide (EDC) is added,
It is dissolved in DMF- water two-phase mixtures solvents with molar ratio 1: 2~8: 5~15, reaction generates cyclosporin similar to object and ethyl
The reactive intermediate of [3- (dimethylamino) propyl] carbodiimide is solution A.
(2) preparation of cBSA：Under the conditions of 0~4 DEG C, it is 7.40 that ethylenediamine 18.0mg, which is first dissolved in 20ml pH, a concentration of
It is 7.40 with concentrated hydrochloric acid tune pH in 0.01M phosphate buffer solutions；Weigh respectively 1000.0mgBSA (molecular weight 68,000) and
Then 57.51mgEDC is added in ethylenediamine solution, is stirred to react under the conditions of 20 DEG C 2 hours；By reacting for ethylenediamine and BSA
Solution, with above-mentioned phosphate buffer stirring dialysis 70 hours, then uses distilled water instead and dialyses 30 hours, often under the conditions of 0~4 DEG C
Replace a dialyzate within 6 hours；At 0~4 DEG C, with the solution 15 minutes after 13000 revs/min of above-mentioned dialysis of centrifugation, supernatant is taken
Liquid；Supernatant is lyophilized, obtains white powder solid cBSA, it is spare；
(3) configuration of solution B：It is 7.40 that cBSA, which is dissolved in pH, in a concentration of 0.01M phosphate buffer solutions, is made into
The solution of 10.0mg/ml, it is spare；
(4) it is that 30: 1 amounts take solution A and solution B respectively by the molar ratio of cyclosporin and cBSA, at a temperature of 25 DEG C,
Solution A is added dropwise in the solution B under the state of being stirred continuously, and is reacted 4 hours, is obtained solution C；
(5) solution C stirs dialysis 72 hours with above-mentioned phosphate buffer, then uses distilled water instead and dialyse 30 hours, every 6 hours
Replace a dialyzate；Then at 0~4 DEG C, with the solution 15 minutes after 13000 revs/min of above-mentioned dialysis of centrifugation, supernatant is taken
Liquid；
(6) supernatant is lyophilized, obtains the conjugate of white cyclosporin.
Embodiment 3
The preparation of antibody is purified and detection
1. the preparation of antibody
It selects the conjugate of the cyclosporin prepared by above-described embodiment 2 to carry out animal immune as immunogene to test to make
Standby antibody.
Isometric Freund's complete adjuvant is added in the solution 1ml for taking the conjugate of the cyclosporin of 1mg/ml, fully emulsified
Afterwards, through subcutaneous multi-point injection give four weight 2kg male and healthy new zealand white rabbit, 1ml/ only, with same amount antigen after 15 days
Exempt from the fully emulsified progress two afterwards of incomplete Freund's adjuvant, after two exempt from, primary every 15 days booster immunizations, amount of antigen halves,
It is immunized 5 times altogether.After last time is 7 days immune, heart extracting blood is stored at room temperature 1 hour, and 0-4 DEG C overnight, 13000 revs/min of centrifugations 15
Minute, serum is collected, -20 DEG C of preservations are spare.
2. the purifying of antibody
Saturated ammonium sulfate is added to the final concentration of body of ammonium sulfate into the antiserum of above-mentioned preparation under stirring
Product percentage 50%, 0-4 DEG C stands overnight, and has sediment precipitation；It is centrifuged 15 minutes with 13000 revs/min, abandons supernatant, Xiang Chen
The PBS of 0.01M, pH 7.4 is added in starch to dissolving is precipitated, saturated ammonium sulfate is then added to the final concentration of ammonium sulfate
For percent by volume 33%, 0-4 DEG C stands overnight, and has sediment precipitation；It is centrifuged 15 minutes with 13000 revs/min, abandons supernatant,
Into sediment be added 0.01M, pH 7.4 PBS to precipitate dissolving.By the above-mentioned purified PBS of 0.01M, pH 7.4,0-4
DEG C dialysis, change dialyzate 3 times, then be added quality percent by volume be 0.02% sodium azide, -20 DEG C preservation, it is spare.
3. the enzyme linked immunosorbent detection of antibody
(1) titration：Method is using conventional Immunofluorescent antibody detection method；
On the ELISA Plate in 96 holes, it is coated with the cyclosporin in the holes 100ul/ and the conjugate (10ug/ml) of ovalbumin,
0-4 DEG C stands overnight, and then uses the PBST PBS+ percent by volume 0.05%Tween20 of concentration 0.01M (1000ml pH7.4)
Board-washing four times；It is closed with the holes 250ul/ confining liquid (1000mlPBST+ mass percents by volume are 1% ovalbumins), room temperature is put
3 hours are set, board-washing；After washing away confining liquid, add the antiserum in the holes 100ul/, is placed at room temperature for 2 hours, board-washing；Washing away anti-blood
After clear, the goat anti-rabbit igg 100ul of 1: 1000 horseradish peroxidase-labeled is added per hole, is placed at room temperature for 1 hour, washes
Plate；Substrate o-phenylene diamine colour developing is added, is placed at room temperature for 10min, adds 2M HCL and terminates.Microplate reader A492nm detections.
After measured：The conjugate antibody titer of cyclosporin of the present invention is up to 50,000.
Serum highest extension rate of the judgement of potency with P/N more than 2: 1 is the enzyme linked immunosorbent detection potency of the antibody.
Wherein：Above-mentioned P is the absorbance value that test serum is measured in a certain extension rate, and above-mentioned N is negative control in phase
The absorbance value for answering multiple to measure.
(2) specific assay：
Determination step is similar with titration, under the conditions of above-mentioned best envelope antigen and antibody concentration, adds antibody
Cyclosporin solution (from 100ppm-1ppt) is added simultaneously, and the limited antibody of envelope antigen competitive binding, cyclosporin it is dense
Degree it is higher, antibody just combined with envelope antigen it is fewer, to colour developing it is more shallow, absorbance value is lower.(only add with blank control again
Antibody does not add the absorbance value of cyclosporin) it compares, to determine antibody specificity.
Preferable by measuring antibody specificity, minimum detection limit can reach 10ng/ml, and detection sensitivity is higher, and
It is less with the cross reaction of other structures analog.
Embodiment 4
Prepare the conjugate of cyclosporin and ovalbumin
(1) preparation of solution A：By cyclosporin 19.4mg, HOSu NHS 35.0mg, EDC 60.00mg dissolvings
In 7ml dimethylformamides, reaction generates the reactive intermediate of cyclosporin and EDC, spare.
(2) configuration of solution B：It is 7.40 that ovalbumin, which is dissolved in pH, in a concentration of 0.01M phosphate buffer solutions, is made into
The solution of 10.0mg/ml, it is spare；
(3) it is that 30: 1 amounts take solution A and solution B respectively by the molar ratio of cyclosporin and ovalbumin, in 25 DEG C of temperature
Under, solution A is added dropwise in the solution B under the state of being stirred continuously, reacts 5 hours, obtain solution C；
(4) solution C stirs dialysis 72 hours with above-mentioned phosphate buffer, then uses distilled water instead and dialyse 24 hours, every 6 hours
Replace a dialyzate；Then at 0~4 DEG C, with the solution 15 minutes after 13000 revs/min of above-mentioned dialysis of centrifugation, supernatant is taken
Liquid；
(5) supernatant is lyophilized, obtains the conjugate of white cyclosporin and ovalbumin.
Claims (10)
1. a kind of conjugate of cyclosporin, by cyclosporin haptens and the carrier mass bovine serum albumin for generating immunogenicity
Or ovalbumin coupling is constituted.
2. the conjugate of cyclosporin as described in claim 1, wherein the carrier mass cow's serum for generating immunogenicity
Albumen.
3. the conjugate of cyclosporin as claimed in claim 2, general structure such as (I)
Wherein：N is the Molecules of the cyclosporin combined with a bovine serum albumin molecule, and the n is integer 1~20,
CBSA is bovine serum albumin (Bovine serum albumin), molecular weight ranges 6.6KDa~6.9Kda；
Above-mentioned conjugate shows following physical chemical characteristics：
(1) appearance：White powder solid；
(2) ultra-violet absorption spectrum：250nm, 277nm.
4. the conjugate of cyclosporin as claimed in claim 3, it is characterized in that：The n is integer 5~15, cBSA molecular weight
Range 6.6KDa~6.9Kda.
5. the preparation method of the conjugate of cyclosporin according to any one of claims 1 to 4, it is characterized in that：Ring spore is mould
Element is connected with the carrier mass for generating immunogenicity, is combined into the conjugate for inducing animal immune generation antibody, and
Keep the bioactivity of the conjugate constant.
6. the preparation method of the conjugate of cyclosporin as claimed in claim 5, is completed by following steps：
(1) preparation of solution A：50mg cyclosporins (0.042mmol) are added in 25mL round-bottomed flasks, with 10mL dichloromethane
Dissolving.14mg metachloroperbenzoic acids (MCPBA) are added thereto, room temperature is stirred overnight, and it is water-soluble that sodium bicarbonate is added later
Liquid is extracted with dichloromethane, merges organic phase, and vacuum rotary steam obtains the dissolving of product diluted hydrochloric acid aqueous solution and 5h is stirred at room temperature, it
PH to 7 is adjusted afterwards, and succinic anhydride, HOSu NHS, ethyl [3- (dimethylamino) propyl] carbodiimide (EDC) is added,
It is dissolved in DMF- water two-phase mixtures solvents with molar ratio 1: 2~8: 5~15, reaction generates cyclosporin similar to object and ethyl
The reactive intermediate of [3- (dimethylamino) propyl] carbodiimide is solution A
(2) preparation of cBSA：Under the conditions of 0~4 DEG C, it is 7.38~7.56 that ethylenediamine, which is first dissolved in pH, a concentration of 0.01M~
It is 7.38~7.56 with concentrated hydrochloric acid tune pH in 0.02M phosphate buffer solutions；With ethylenediamine: BSA: EDC molar ratio is 15~25
: 1: 15~25 amount weighs BSA and EDC, is then added in ethylenediamine solution, and it is small to be stirred to react 2~4 under the conditions of 20 ± 5 DEG C
When；By the reaction solution of ethylenediamine and BSA under the conditions of 0~4 DEG C, dialysed 70~80 hours with the stirring of above-mentioned phosphate buffer,
Then it uses distilled water instead to dialyse 20~30 hours, replaces a dialyzate within every 6 hours；At 0~4 DEG C, with 13000 revs/min from
The solution 15 minutes after dialysis is stated in the heart, takes supernatant；Supernatant is lyophilized, obtains white powder solid cBSA, it is spare；
(3) configuration of solution B：It is 7.38~7.56 that cBSA, which is dissolved in pH, in a concentration of 0.01M~0.02M phosphate buffer solutions,
It is made into the solution of 10.0 ± 5.0mg/ml, it is spare；
(4) it is that 15~50: 1 amount takes solution A and solution B respectively by the molar ratio of cyclosporin and cBSA, in 20 DEG C of ± 5 DEG C of temperature
Under, solution A is added dropwise in the solution B under the state of being stirred continuously, reacts 4~6 hours, obtain solution C；
(5) solution C stirs dialysis 70~80 hours with above-mentioned phosphate buffer, then uses distilled water instead and dialyse 20~30 hours, and every 6
Hour replaces a dialyzate；Then it at 0~4 DEG C, with the solution 15 minutes after 13000 revs/min of above-mentioned dialysis of centrifugation, takes
Supernatant；
(6) supernatant is lyophilized, obtains the conjugate of white cyclosporin.
7. the preparation method of the conjugate of cyclosporin as claimed in claim 6, it is characterized in that：Addition described in step (1)
Succinic anhydride, HOSu NHS, ethyl [3- (dimethylamino) propyl] carbodiimide (EDC), with molar ratio 1: 2~8: 5
~15.
8. the preparation method of the conjugate of cyclosporin as claimed in claim 6, it is characterized in that：Step (2) described ethylenediamine
Molar ratio with bovine serum albumin, EDC is 20: 1: 20.
9. the preparation method of the conjugate of cyclosporin as claimed in claim 6, it is characterized in that：Step (4) the ring spore is mould
The molar ratio of element and bovine serum albumin is 30: 1.
10. the conjugate of cyclosporin according to any one of claims 1 to 4 is as immunogene to prepare cyclosporin special
Application in reagin.
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