CN108164596A - Conjugate of Dioscin and preparation method and application - Google Patents
Conjugate of Dioscin and preparation method and application Download PDFInfo
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- CN108164596A CN108164596A CN201611135732.7A CN201611135732A CN108164596A CN 108164596 A CN108164596 A CN 108164596A CN 201611135732 A CN201611135732 A CN 201611135732A CN 108164596 A CN108164596 A CN 108164596A
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- dioscin
- conjugate
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- solution
- azurin
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- CJNUQCDDINHHHD-APRUHSSNSA-N dioscin Natural products C[C@@H]1CC[C@@]2(OC1)O[C@H]3C[C@H]4[C@@H]5CC=C6C[C@H](CC[C@@H]6[C@H]5CC[C@]4(C)[C@H]3[C@@H]2C)O[C@@H]7O[C@H](CO)[C@@H](O[C@@H]8O[C@@H](C)[C@H](O)[C@@H](O)[C@H]8O)[C@H](O)[C@H]7O[C@@H]9O[C@@H](C)[C@H](O)[C@@H](O)[C@H]9O CJNUQCDDINHHHD-APRUHSSNSA-N 0.000 title claims abstract description 74
- HDXIQHTUNGFJIC-UHFFFAOYSA-N (25R)-spirost-5-en-3beta-ol 3-O-<O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranoside> Natural products O1C2(OCC(C)CC2)C(C)C(C2(CCC3C4(C)CC5)C)C1CC2C3CC=C4CC5OC1OC(CO)C(O)C(O)C1OC1OC(C)C(O)C(O)C1O HDXIQHTUNGFJIC-UHFFFAOYSA-N 0.000 title claims abstract description 70
- VNONINPVFQTJOC-RXEYMUOJSA-N Collettiside III Natural products O([C@@H]1[C@@H](O)[C@H](O[C@H]2[C@H](O)[C@H](O)[C@@H](O)[C@H](C)O2)[C@H](CO)O[C@@H]1O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]5[C@H](C)[C@@]6(O[C@H]5C4)OC[C@H](C)CC6)CC3)CC=2)CC1)[C@H]1[C@H](O)[C@H](O)[C@@H](O)[C@H](C)O1 VNONINPVFQTJOC-RXEYMUOJSA-N 0.000 title claims abstract description 70
- VNONINPVFQTJOC-ZGXDEBHDSA-N dioscin Chemical compound O([C@@H]1[C@@H](CO)O[C@H]([C@@H]([C@H]1O)O[C@H]1[C@@H]([C@H](O)[C@@H](O)[C@H](C)O1)O)O[C@@H]1CC2=CC[C@H]3[C@@H]4C[C@H]5[C@@H]([C@]4(CC[C@@H]3[C@@]2(C)CC1)C)[C@@H]([C@]1(OC[C@H](C)CC1)O5)C)[C@@H]1O[C@@H](C)[C@H](O)[C@@H](O)[C@H]1O VNONINPVFQTJOC-ZGXDEBHDSA-N 0.000 title claims abstract description 70
- VNONINPVFQTJOC-UHFFFAOYSA-N polyphyllin III Natural products O1C2(OCC(C)CC2)C(C)C(C2(CCC3C4(C)CC5)C)C1CC2C3CC=C4CC5OC(C(C1O)OC2C(C(O)C(O)C(C)O2)O)OC(CO)C1OC1OC(C)C(O)C(O)C1O VNONINPVFQTJOC-UHFFFAOYSA-N 0.000 title claims abstract description 70
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 108010007337 Azurin Proteins 0.000 claims abstract description 12
- 230000005847 immunogenicity Effects 0.000 claims abstract description 9
- 241001465754 Metazoa Species 0.000 claims abstract description 7
- 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
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 12
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 claims description 12
- 239000008363 phosphate buffer Substances 0.000 claims description 11
- 238000000502 dialysis Methods 0.000 claims description 9
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 5
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 4
- 229920001184 polypeptide Polymers 0.000 claims description 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 238000003786 synthesis reaction Methods 0.000 claims description 4
- 238000000862 absorption spectrum Methods 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000012634 fragment Substances 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 235000002722 Dioscorea batatas Nutrition 0.000 claims 3
- 235000006536 Dioscorea esculenta Nutrition 0.000 claims 3
- 240000001811 Dioscorea oppositifolia Species 0.000 claims 3
- 235000003416 Dioscorea oppositifolia Nutrition 0.000 claims 3
- 239000001397 quillaja saponaria molina bark Substances 0.000 claims 3
- 229930182490 saponin Natural products 0.000 claims 3
- 150000007949 saponins Chemical class 0.000 claims 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 1
- 238000001514 detection method Methods 0.000 abstract description 12
- 102000004190 Enzymes Human genes 0.000 abstract description 6
- 108090000790 Enzymes Proteins 0.000 abstract description 6
- 230000001900 immune effect Effects 0.000 abstract description 3
- 238000011587 new zealand white rabbit Methods 0.000 abstract description 3
- 210000002966 serum Anatomy 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 238000003556 assay Methods 0.000 description 3
- 239000003547 immunosorbent Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- 241000982119 Antipalpus Species 0.000 description 1
- 235000011330 Armoracia rusticana Nutrition 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 241000908494 Dioscorea nipponica Species 0.000 description 1
- 235000017008 Dioscorea nipponica Nutrition 0.000 description 1
- 241000234272 Dioscoreaceae Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241000130764 Tinea Species 0.000 description 1
- 208000002474 Tinea Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 229940126678 chinese medicines Drugs 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 239000003433 contraceptive agent Substances 0.000 description 1
- 230000002254 contraceptive effect Effects 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000000586 desensitisation Methods 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
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 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
- 230000006698 induction Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- PEFNSGRTCBGNAN-QNDFHXLGSA-N luteolin 7-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=C2C(=O)C=C(C=3C=C(O)C(O)=CC=3)OC2=C1 PEFNSGRTCBGNAN-QNDFHXLGSA-N 0.000 description 1
- KBGKQZVCLWKUDQ-UHFFFAOYSA-N luteolin-glucoside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC(O)=CC2=C1C(=O)C=C(C=1C=C(O)C(O)=CC=1)O2 KBGKQZVCLWKUDQ-UHFFFAOYSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- -1 small molecule Organic compound Chemical class 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 150000005856 steroid saponins Chemical class 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 239000009792 yinqiao Substances 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
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
Abstract
Conjugate of Dioscin and preparation method and application.The invention discloses a kind of conjugates of the Dioscin of logical formula (I), it is made of Dioscin with generating carrier mass keyhole azurin (KLH) coupling of immunogenicity, wherein n is the molecular number of Dioscin combined with a keyhole azurin molecule, KLH is keyhole azurin, and molecular weight ranges are 450KDa~13000KDa.The invention also discloses the preparation methods of the conjugate, i.e., Dioscin are connected with generating the carrier mass of immunogenicity, be combined into the conjugate for inducing animal immuning system generated antibody.The conjugate of the Dioscin of the present invention is prepared for the antiserum that potency reaches 1: 16000, lowest detection is limited to 0.53ng/ml by the way that new zealand white rabbit is immunized.The present invention have simplicity, fast, specifically, it is accurate the characteristics of, the enzyme-linked immunologic detecting kit to prepare Dioscin provides the foundation.
Description
Technical field
The present invention relates to a kind of conjugate of small haptens and preparation method and application more particularly to Dioscins
Conjugate and preparation method and application.
Background technology
Following denotations of the present invention are suitable for the whole instruction and claims：
Dioscin：Chinese Industrial Standards (CIS) quality inspection institute product
KLH：Keyhole azurin (Keyhole Limpet Hemocyanin), Sigma Products
DMF：Dimethylformamide, Beijing lark prestige (J＆K) Products
Sodium metaperiodate：Beijing lark prestige (J＆K) Products
Bag filter：Beijing Suo Laibao (Solarbio) Products
PBS：Phosphate buffer (Phosphate Buffer Saline) (0.01M, pH=7.40)
CBS：Carbonate buffer solution (Carbonate Buffer Saline) (0.05M, pH=9.6).
Dioscin (Dioscin) is Dioscoreaceae plant rhizoma dioscoreae nipponicae (Dioscorea nipponica Makino) medicinal material
Main steroid saponin chemical composition.Dioscin has the function of cough-relieving, eliminating the phlegm, desensitization, anti-inflammatory, also there is insecticidal and anti-palpus
The effect of the fungies such as tinea trichobacteria etc., and can be as synthesizing steroid hormone and the raw material of contraceptive.In recent years, Dioscin exists
The effect of antitumor, hypoglycemic, immunological regulation, prevention cardiovascular and cerebrovascular disease etc. increasingly causes global concern, contains
The drug (such as Wei C Yinqiao Granules) of Dioscin has listed, and is widely used in the prevention and treatment of coronary heart disease.Many Chinese medicines, Chinese medicine
All contain Dioscin in medicine materical crude slice and Chinese patent drug.Therefore, correlated quality detection and mirror are carried out by index ingredient of Dioscin
Setting analysis has great significance.
At present, the common assay method of Dioscin has gravimetric method, colorimetric method, coulomb method and thin layer chromatography scanning etc., these
Quantitative analysis method trivial operations, specificity is not strong, and accuracy is not high.Some are highly sensitive and highly selective for developed recently
Quantitative approach, such as gas chromatography, high performance liquid chromatography and chromatography spectrum joint technology.Although these methods are accurate, surely
It is fixed, it is reliably, but since instrumental method is expensive, bothersome longer, and organic solvent pollution is caused, large-scale instrument and equipment is needed,
Special technical staff is needed, so being difficult to apply to execute-in-place.Enzyme-Linked Immunospot (ELISA) provides a kind of fabulous
Detection means.This method has quickly, accurately, simply, does not need to the advantages that special messenger operates, this causes ELISA method to become one
The ideal detection means of kind.The core of Enzyme-Linked Immunospot is to need the antibody of high quality.Dioscin is a kind of small molecule
Organic compound, without immunogenicity, referred to as haptens.Therefore it is necessary to Dioscin, which is converted into, can cause animal immune
System generates the immunogene (also referred to as comlete antigen) of antibody.Through retrieval, there has been no about the immune of Dioscin in the world
Original synthesis and the report of antibody preparation process, it is impossible to meet detection needs, therefore the synthesis for studying the immunogene of Dioscin is shown
It obtains increasingly important.
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 Dioscin, i.e. conjugate of Dioscin and preparation method thereof.Together
When, the present invention also provides the Dioscin conjugate as immunogene in Dioscin idiosyncrasy antibody is prepared
Application.
The conjugate of the Dioscin of the present invention is coupled by Dioscin haptens with generating the carrier mass of immunogenicity
It forms, the carrier mass is preferred protein, protein fragments, synthesis polypeptide or semi-synthetic polypeptide.
The conjugate of above-mentioned Dioscin, wherein the carrier mass for generating immunogenicity is keyhole azurin.
The conjugate of Dioscin of the present invention, general structure such as (I)
Wherein, n is the Molecules of Dioscin combined with a keyhole azurin, and KLH is keyhole azurin
(Keyhole Limpet Hemocyanin), molecular weight ranges are 450KDa~13000KDa；
Above-mentioned conjugate shows following physical chemical characteristics：
(1) appearance：Light gray powder solid；
(2) ultra-violet absorption spectrum：270nm.
The preparation method of the conjugate of above-mentioned Dioscin is：By Dioscin and the carrier mass for generating immunogenicity
It connects, is combined into the conjugate with induction animal immuning system generated antibody, and keep the bioactivity of the conjugate
It is constant.
The preparation method of the conjugate of above-mentioned Dioscin is as follows：
(1) preparation of solution A：10.0mg micromolecular compound Dioscins are dissolved in dimethylformamide (DMF),
It adds in containing 2.5mg sodium periodate solution 0.5ml, is protected from light is stirred to react 1h at room temperature；
(2) preparation of solution B：30mg KLH are dissolved in 10ml 0.05M carbonate buffer solutions (CBS), it is spare；
(3) solution A is added dropwise in solution B, is protected from light is stirred to react 6h at room temperature, obtain solution C；
(4) solution C is transferred in bag filter, stirs dialysis 72h with a concentration of 0.01M phosphate buffers (PBS), later
Replacement distilled water 20~30h of dialysis, a dialyzate is replaced per 6h, dialysis procedure carries out at 4 DEG C；
(5) dialyzate is lyophilized, obtains the conjugate of light grey Dioscin.
In above-mentioned preparation, Dioscin is 1: 1 with sodium metaperiodate molar ratio.
In above-mentioned preparation, the mass number ratio of Dioscin and keyhole azurin is 1: 3.
The conjugate of Dioscin of the present invention is as immunogene in Dioscin idiosyncrasy antibody is prepared
Using.
It can be successfully haptens Dioscin and carrier protein particularly keyhole using technical scheme of the present invention
Azurin KLH couplings are got up, and can be triggered an immune response in animal body so as to synthesize, and generate the completely immune of antibody
It is former --- the conjugate of Dioscin.
By the use of Dioscin of the present invention conjugate as immunogen immune new zealand white rabbit, successfully obtain
There is to haptens Dioscin the antibody of idiosyncrasy.Through ELISA experimental identifications, Dioscin of the present invention is utilized
The galuteolin idiosyncrasy antibody that conjugate is prepared as immunogene, antiserum titre reach 1: 16000, lowest detection
It is limited to 0.53ng/ml.
The conjugate of above-mentioned Dioscin and the Dioscin idiosyncrasy antibody of high-titer are successfully prepared, for system
The enzyme-linked immunologic detecting kit of standby Dioscin provides the foundation.It is quickly, special since the method for the invention has simply
It is different, it is accurate the characteristics of, available for the assay of Dioscin, can execute-in-place, save a large amount of detection time, compensate for instrument
Device method is time-consuming longer, the deficiency that large-scale instrument and equipment is needed to support.
Specific embodiment：
Embodiment 1
(1) preparation of solution A：10.0mg micromolecular compound Dioscins are dissolved in dimethylformamide (DMF),
It adds in containing 2.5mg sodium periodate solution 0.5ml, is protected from light is stirred to react 1h at room temperature；
(2) preparation of solution B：30mg KLH are dissolved in 10ml 0.05M carbonate buffer solutions (CBS), it is spare；
(3) solution A is added dropwise in solution B, is protected from light is stirred to react 6h at room temperature, obtain solution C；
(4) solution C is transferred in bag filter, stirs dialysis 72h with a concentration of 0.01M phosphate buffers (PBS), later
Replacement distilled water 20~30h of dialysis, a dialyzate is replaced per 6h, dialysis procedure carries out at 4 DEG C；
(5) dialyzate is lyophilized, obtains the conjugate of light grey Dioscin.It is determined by ultra-violet absorption spectrum (absorption value)
The coupling situation of product, the conjugate of Dioscin of the invention have feature ultraviolet absorption peak at 270nm.
Embodiment 2
The preparation of antibody and enzyme linked immunosorbent detection
1. the preparation of antibody
The conjugate of the Dioscin prepared by above-described embodiment 1 is selected to carry out animal immune experiment as immunogene to make
Standby antibody.
The solution 1ml of the conjugate of the Dioscin of 1mg/ml is taken, adds in isometric Freund's complete adjuvant, it is fully emulsified
Afterwards, through subcutaneous multi-point injection to the male and healthy new zealand white rabbit of 6 weight 2kg, 1ml/ only, after 21 days, with 0.5mg/
Two are carried out after the conjugate solution 1ml of the Dioscin of ml and isometric incomplete Freund's adjuvant are fully emulsified to exempt from, two exempt from
Afterwards, it is primary every 15 days booster immunizations, it is immunized 5 times altogether.After immune 7 days of last time, heart extracting blood is stored at room temperature 1 hour, and 4
DEG C overnight, 10000 revs/min centrifuge 15 minutes, collect serum, -20 DEG C preservation, it is spare.
2. the enzyme linked immunosorbent detection of antibody
(1) titration
On the ELISA Plate in 96 holes, the Dioscin of 100ul and the conjugate (2ug/ of bovine serum albumin(BSA) are added in per hole
Ml), 2h is incubated at 37 DEG C.After taking-up using washing lotion PBST (1000ml pH 7.4, concentration 0.01M PBS+ percents by volume be
0.05%Tween 20) it washs 3 times；Add in confining liquid (1000ml PBS+ mass percents by volume are 5% skimmed milk powers)
250ul/ holes are closed, and 37 DEG C of incubation 2h repeat board-washing process, add in dilution (1 in proportion of antibody and negative serum after taking-up
: 200,1: 400,1: 800,1: 1600,1: 3200,1: 6400,1: 12800,1: 25600,1: 51200,1: 102400), per hole
100ul, 37 DEG C of incubation 0.5h, board-washing；After antibody and negative serum is washed away, 1: 10000 horseradish peroxidating is added in per hole
The goat anti-rabbit igg 100ul of object enzyme label, 37 DEG C are incubated 0.5h, the colour developing of substrate tetramethyl benzidine are added in after board-washing, per hole
100ul is placed at room temperature for colour developing 10min, terminate liquid 100ul/ holes is added in, with microplate reader reading at 450nm.
After measured：The conjugate antibody titer of Dioscin of the present invention is up to 1: 16000.
The judgement of potency using P/N more than 2: 1 serum highest extension rate as the antibody enzyme linked immunosorbent detection potency.
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 that extension rate is answered 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
Add in Dioscin gradient solution (10 simultaneously-2-106Ng/ml), the antibody limited with envelope antigen competitive binding, Dioscin
Concentration it is higher, antibody is just combined fewer with envelope antigen, more shallow so as to develop the color, and absorbance value is lower.Again with blank control
(only adding antibody, do not add the absorbance value of Dioscin) compares, to determine antibody specificity.
Preferable by measuring antibody specificity, minimum detection limit can reach 0.53ng/ml, and detection sensitivity is higher.
Claims (8)
1. a kind of conjugate of Dioscin is coupled by Dioscin haptens with generating the carrier mass of immunogenicity,
The carrier mass is preferred protein, protein fragments, synthesis polypeptide or semi-synthetic polypeptide.
2. the conjugate of Dioscin as described in claim 1, wherein the carrier mass for generating immunogenicity is key
Hole azurin.
3. the conjugate of Dioscin as claimed in claim 2, general structure such as (I)
Wherein, n is the Molecules of Dioscin combined with a keyhole azurin, and KLH is keyhole azurin
(Keyhole Limpet Hemocyanin), molecular weight ranges are 450KDa~13000KDa；
Above-mentioned conjugate shows following physical chemical characteristics：
(1) appearance：Light gray powder solid；
(2) ultra-violet absorption spectrum：270nm.
4. the preparation method of the conjugate of Dioscin according to any one of claims 1 to 3, it is characterized in that：By Chinese yam
Saponin(e is connected with generating the carrier mass of immunogenicity, is combined into the coupling for inducing animal immuning system generated antibody
Object, and keep the bioactivity of the conjugate constant.
5. the preparation method of the conjugate of Dioscin as claimed in claim 4, is completed by following steps：
(1) preparation of solution A：10.0mg micromolecular compound Dioscins are dissolved in dimethylformamide (DMF), are added in
Containing 2.5mg sodium periodate solution 0.5ml, it is protected from light is stirred to react 1h at room temperature；
(2) preparation of solution B：30mg KLH are dissolved in 10ml 0.05M carbonate buffer solutions (CBS), it is spare；
(3) solution A is added dropwise in solution B, is protected from light is stirred to react 6h at room temperature, obtain solution C；
(4) solution C is transferred in bag filter, is stirred dialysis 72h with a concentration of 0.01M phosphate buffers (PBS), is replaced later
With distilled water 20~30h of dialysis, a dialyzate is replaced per 6h, dialysis procedure carries out at 4 DEG C；
(5) dialyzate is lyophilized, obtains the conjugate of light grey Dioscin.
6. the preparation method of the conjugate of Dioscin as claimed in claim 5, it is characterized in that：Chinese yam described in step (1)
Saponin(e is 1: 1 with sodium metaperiodate molar ratio.
7. the preparation method of the conjugate of Dioscin as claimed in claim 5, it is characterized in that：Chinese yam described in step (2)
The mass number ratio of saponin(e and keyhole azurin is 1: 3.
8. the conjugate of Dioscin according to any one of claims 1 to 3 is as immunogene to prepare Dioscin special
Application in reagin.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102786594A (en) * | 2012-05-09 | 2012-11-21 | 中国药科大学 | Sarsasapogenin monoclonal antibody and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102786594A (en) * | 2012-05-09 | 2012-11-21 | 中国药科大学 | Sarsasapogenin monoclonal antibody and application |
Non-Patent Citations (3)
| Title |
|---|
| DRIEDGER AND SPORNS: "Development of an Antibody against Diosgenin and Spiroaminoketal Alkaloids.", 《FOOD AND AGRICULTURAL IMMUNOLOGY》 * |
| W PHROMPITTAYARAT ET AL.: "Production of polyclonal antibodies against dioscin.", 《PLANTA MED》 * |
| 李祥: "薯蓣皂素分析方法综述", 《陕西科技大学学报》 * |
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