CN106467571A - Conjugate of vitamin B1 and preparation method and application - Google Patents
Conjugate of vitamin B1 and preparation method and application Download PDFInfo
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- 239000011691 vitamin B1 Substances 0.000 title claims abstract description 54
- 238000002360 preparation method Methods 0.000 title claims abstract description 42
- 229960003495 thiamine Drugs 0.000 title claims abstract description 16
- 229930003451 Vitamin B1 Natural products 0.000 title claims description 13
- 235000010374 vitamin B1 Nutrition 0.000 title claims description 13
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 title claims 3
- 229930003270 Vitamin B Natural products 0.000 claims abstract description 29
- 235000019156 vitamin B Nutrition 0.000 claims abstract description 29
- 239000011720 vitamin B Substances 0.000 claims abstract description 29
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 16
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 15
- 230000002163 immunogen Effects 0.000 claims abstract description 15
- 241001465754 Metazoa Species 0.000 claims abstract description 7
- 230000006698 induction Effects 0.000 claims abstract description 3
- 108060003552 hemocyanin Proteins 0.000 claims abstract 2
- 239000000243 solution Substances 0.000 claims description 93
- 238000006243 chemical reaction Methods 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 238000000502 dialysis Methods 0.000 claims description 15
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 14
- 239000012153 distilled water Substances 0.000 claims description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- 239000008363 phosphate buffer Substances 0.000 claims description 9
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 7
- 244000061458 Solanum melongena Species 0.000 claims description 7
- 235000010288 sodium nitrite Nutrition 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 6
- 150000001718 carbodiimides Chemical class 0.000 claims description 6
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 6
- 229940088594 vitamin Drugs 0.000 claims description 6
- 229930003231 vitamin Natural products 0.000 claims description 6
- 235000013343 vitamin Nutrition 0.000 claims description 6
- 239000011782 vitamin Substances 0.000 claims description 6
- 238000000862 absorption spectrum Methods 0.000 claims description 5
- 239000000385 dialysis solution Substances 0.000 claims description 5
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 4
- 102000014914 Carrier Proteins Human genes 0.000 claims description 3
- 108010078791 Carrier Proteins Proteins 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 230000004071 biological effect Effects 0.000 claims description 2
- 229940127121 immunoconjugate Drugs 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- QYSGYZVSCZSLHT-UHFFFAOYSA-N octafluoropropane Chemical compound FC(F)(F)C(F)(F)C(F)(F)F QYSGYZVSCZSLHT-UHFFFAOYSA-N 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 11
- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 abstract description 9
- 235000019157 thiamine Nutrition 0.000 abstract description 3
- 239000011721 thiamine Substances 0.000 abstract description 3
- 230000001900 immune effect Effects 0.000 abstract 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 13
- 239000000047 product Substances 0.000 description 7
- 238000010521 absorption reaction Methods 0.000 description 6
- 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
- 239000007788 liquid Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 239000003547 immunosorbent Substances 0.000 description 3
- 239000008055 phosphate buffer solution Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 108010058846 Ovalbumin Proteins 0.000 description 2
- 206010047601 Vitamin B1 deficiency Diseases 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 208000002894 beriberi Diseases 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 210000005036 nerve Anatomy 0.000 description 2
- 238000011587 new zealand white rabbit Methods 0.000 description 2
- 229940092253 ovalbumin Drugs 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 230000035945 sensitivity Effects 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
- 208000014644 Brain disease Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 208000032274 Encephalopathy Diseases 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 208000035154 Hyperesthesia Diseases 0.000 description 1
- 208000006264 Korsakoff syndrome Diseases 0.000 description 1
- 206010029240 Neuritis Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 208000028017 Psychotic disease Diseases 0.000 description 1
- 206010040026 Sensory disturbance Diseases 0.000 description 1
- 208000032023 Signs and Symptoms Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 208000001871 Tachycardia Diseases 0.000 description 1
- 201000008485 Wernicke-Korsakoff syndrome Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 230000023852 carbohydrate metabolic process Effects 0.000 description 1
- 235000021256 carbohydrate metabolism Nutrition 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000001875 compounds Chemical class 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
- 230000029087 digestion Effects 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 201000002670 dry beriberi Diseases 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 201000001119 neuropathy Diseases 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000010408 sweeping Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000006794 tachycardia Effects 0.000 description 1
- 210000001519 tissue Anatomy 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
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- 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
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses a kind of formula（I）Vitamin B1（Thiamine）Conjugate, by vitamin B1It is coupled with the generation preferred bovine serum albumin of immunogenic carrier mass or hemocyanin and constitutes, wherein n is the vitamin B being combined with a bovine serum albumin molecule1Molecular number, n be integer 1 ~ 20, BSA be bovine serum albumin, molecular weight ranges be 6.6KDa ~ 6.9KDa.The invention also discloses the preparation method of described conjugate, will vitamin B1Couple together with producing immunogenic carrier mass, be combined into the conjugate with induction animal immuning system generated antibody.It is prepared for potency and reach 1:3200 antiserum, its lowest detection is limited to 0.18ppm.The present invention has simplicity, and fast, specifically, accurate feature, for preparing vitamin B1Enzyme-linked immunologic detecting kit provide the foundation.（I）.
Description
Technical field
The present invention designs a kind of conjugate of water soluble vitamins and preparation method and application, more particularly, to a kind of vitamin B1Conjugate and preparation method and application.Belong to vitamin field of immunodetection.
Background technology
Following denotations according to the present invention are applied to entire disclosure and claims：
BSA：Bovine serum albumin（Bovine Serum Albumin）, Sigma Products.
Vitamin B1：Thiamine, Sigma Products.
EDC：Ethyl [3- (dimethylamino) propyl group] carbodiimide, abbreviation EDC, Sigma Products.
PBS：Phosphate buffer（Phosphate buffer saline）（0.01M, pH 7.4）.
It is coated liquid：coating buffer（0.01M, pH 9.5）.
Dialyzer：Beijing Suo Laibao scientific ＆ technical corporation product.
Vitamin B1 belongs to water soluble vitamins, and vitimin supplement B1 can promote child growth, helps the digestion of carbohydrate, maintain nervous tissue, muscle, cardiomotility normal etc..Vitamin B1 is human energy metabolism, and particularly necessary to carbohydrate metabolism, old friend's body is generally relevant with the heat of picked-up to the requirement of thiamine.When the energy of human body is mainly derived from saccharide, the requirement of vitamin B1 is maximum.Vitamin B1 still maintains heart, necessary to nerve and digestive system normal function.When Vitamin B1 deficiency, by its degree, may occur in which following reaction successively：Nervous system reacts（Dry beriberi）, cardiovascular system reaction（Beriberi humida）Wernicke（Wei Buddhist nun's kirschner）Encephalopathy and Korsakoff syndrome（Korakaff's psychosis）.
Many nerve Signs and symptom are peripheral neuritiss, with acra sensory disturbance, including regional area hyperesthesia or stolidity.Cardiovascular symptom includes dyspnea during work, cardiopalmus, tachycardia and the abnormal situation of other electrocardiograms, and highoutput type heart failure.This exhaustion is referred to as " moist pedopathy ".It is with extensive edema.Therefore, study content in food and medicine for the vitamin B1 and detection method is very important.
In vitamin content measures, instrumental method such as high performance liquid chromatography, liquid chromatography mass spectrometric are used in conjunction and microbial method is most widely used method.These methods are accurately, stable, reliable, can be used as standard method.But instrumental method is expensive, time-consuming longer, and need professional and technical personnel, need large-scale instrument and equipment, and be likely to cause organic solvent pollution, so being difficult to for execute-in-place.Enzyme-Linked Immunospot（ELISA）Provide a kind of fabulous scanning means.This method has quickly, accurately, sensitive, and simply it is not necessary to the advantages of professional operates, this makes ELISA become a kind of preferable, can be used for the detection method of thing immunity conventional sweep.The core of Enzyme-Linked Immunospot is to need high-quality antibody.It is all small molecular organic compounds that most of vitamin include vitamin B1, does not have immunogenicity, referred to as hapten.Therefore it is necessary to these compounds are transformed into the immunogen that can cause animal immuning system generated antibody（Also referred to as complete antigen）.Through retrieval, not yet it is related to the immunogenic synthesis of vitamin B1 and the report of Antibody preparation in the world now, and the method for domestic detection vitamin B1 is mainly limited to liquid chromatography at present, the therefore immunogenic synthesis of research vitamin B1 and Antibody preparation just seems very necessary.
Content of the invention
For above-mentioned the deficiencies in the prior art, the problem to be solved in the present invention is：There is provided one kind that animal immune system can be caused to be directed to vitamin B1There is the immunogen of the antibody of specific reaction, i.e. vitamin B1Conjugate and prepare this immunogenic method.
The vitamin B of the present invention1Conjugate, by vitamin B1Hapten is constituted with producing antigenic carrier mass and being coupled, and described carrier mass is preferred protein, protein fragments, synthesis polypeptide or semi-synthetic polypeptide.
Above-mentioned vitamin B1Conjugate, wherein said protein is preferably bovine serum albumin.
Vitamin B of the present invention1Conjugate, its general structure is such as（I）
（I）
Wherein：n：It is the vitamin B being combined with a bovine serum albumin molecule1Molecular number, n be integer 1 ~ 20
BSA is bovine serum albumin（Bovine
Serum Albumin）, molecular weight ranges are 6.6KDa ~ 6.9KDa
Above-mentioned conjugate shows following physical chemical characteristicses：
（1）Outward appearance：Aubergine pulverulent solids
（2）Ultra-violet absorption spectrum：265nm.
Above-mentioned vitamin B1Conjugate, wherein preferably n is integer 1 ~ 10, and BSA molecular weight ranges are 6.7KDa ~ 6.8KDa.
Above-mentioned vitamin B1The preparation method of conjugate be：By vitamin B1With produce immunogenic carrier mass couple together, be combined into have induction animal immuning system generated antibody conjugate, and keep this conjugate biological activity constant.
Above-mentioned vitamin B1The preparation method of conjugate comprise the following steps that：
（1）The preparation of solution A：By vitamin B1With sodium nitrite（NaNO2）With mol ratio for 1：5 ~ 6 are dissolved in 300 μ L water, adjust pH in 1 ~ 2, ice bath lucifuge reaction 1h with concentrated hydrochloric acid, obtain solution A, standby；
（2）The preparation of solution B：In the aqueous solution of para-amino benzoic acid solution A being added dropwise to 0.05 ~ 0.1 M, adjust pH 9 ~ 10 with NaOH, under the conditions of 0 ~ 4 DEG C, react 4 hours, obtain solution B；
（3）The preparation of solution C：By vitamin B1, N-Hydroxysuccinimide（NHS）, ethyl [3- (dimethylamino）Propyl group] carbodiimide（EDC）, with mol ratio 1:2~8:5 ~ 15 ratio, NHS and EDC is dissolved in and is coated in buffer, and solution B is mixed with above-mentioned solution, room temperature reaction 2 hours, obtains solution C；
（4）The preparation of solution D：It is 7.38 ~ 7.56 that BSA is dissolved in pH, and concentration is the phosphate buffer of 0.01 M（PBS）In, it is made into the solution of 10.0 ± 5.0 mg/ml, standby；
（5）By vitamin B1Mol ratio with BSA is 15 ~ 50：Take solution C and solution D respectively, at ambient temperature, in the solution D under solution C is added dropwise to be stirred continuously, react 8 ~ 10h, obtain solution E；
（6）Solution E above-mentioned phosphate buffer stirring dialysis 70 ~ 80 hours, replacing afterwards is dialysed 70 ~ 80 hours with distilled water, changes a dialysis solution within every 6 ~ 8 hours, dialysis procedure is carried out at 0 ~ 4 DEG C.
（7）Solution E after lyophilizing dialysis, obtains aubergine vitamin B1Conjugate.
In above-mentioned preparation, vitamin B1It is preferably 1 with sodium nitrite mol ratio：6.
In above-mentioned preparation, the concentration of para-amino benzoic acid is preferably 0.07 M.
In above-mentioned preparation, vitamin B1It is preferably 1 with N-Hydroxysuccinimide, EDC mol ratio：5：10.
In above-mentioned preparation, vitamin B1It is preferably 50 with the mol ratio of BSA:1.
Can be successfully hapten vitamin B using technical scheme1It is coupled with carrier protein particularly bovine serum albumin BSA, thus having synthesized, immunoreation can be caused in animal body, produce the immunogen of antibody.Can be obtained to hapten vitamin B using this immunogen immune new zealand white rabbit1There is the antibody of specific reaction, through ELISA identification, using vitamin B of the present invention1The vitamin B prepared as immunogen of conjugate1Specific reaction antibody, its antiserum titre reaches 1：3200, its lowest detection is limited to 0.18 ppm.This is used for preparing vitamin B for this conjugate1Enzyme immunoassay detectable provide wide application space.In actual applications, the vitamin B of preparation1Specific antibody be plated in microwell plate it is possible to for checking vitamin B in actual sample1Content.Because the method has the characteristics that sensitivity height, high specificity, detection speed are fast, sample pre-treatments are simple, testing cost is low it is possible to the preliminary surface sweeping detection for actual sample is used.So not only can save substantial amounts of Check-Out Time, can be also used for execute-in-place, thus it is expensive together to compensate for instrumental method, complex pretreatment, need special technical staff's operation it is difficult to deficiency for scene.So, hapten vitamin B1Lay a good foundation with this Rapid examination method that synthesizes of carrier protein particularly bovine serum albumin BSA conjugate.
Specific embodiment
Embodiment
1
（1）The preparation of solution A：In 5 ml circle reaction bulbs, in stirring（120 revs/min）, add vitamin B1
17.358mg（0.05 mmol）, sodium nitrite 20.00
Mg, is dissolved in 300 μ L distilled water.Add 200 μ L concentrated hydrochloric acid, adjust pH between 1 ~ 2.Under condition of ice bath, lucifuge is reacted 1 hour, obtains yellow solution；
（2）The preparation of solution B：In 10 ml circle reaction bulbs, in stirring（120 revs/min）, add para-amino benzoic acid 10.00mg（0.07 mmol）, it is dissolved in 1000 μ L distilled water, solution A is added dropwise to wherein, and adjust pH between 9 ~ 10 with the NaOH of 1M, under the conditions of 0 ~ 4 DEG C, react 4 hours, obtain purplish red solution；
（3）The preparation of solution C：In 10 ml circle reaction bulbs, in stirring（120 revs/min）, add N-Hydroxysuccinimide（NHS）29.62mg（0.25 mmol）, ethyl [3- (dimethylamino）Propyl group] carbodiimide（EDC）98.66mg（0.50mmol）, it is dissolved in 500 μ L and be coated in buffer（0.01M, pH 9.5）, solution B is mixed with above-mentioned solution, room temperature reaction 2 hours, obtains solution C；
（4）The preparation of solution D：It is 7.40 that BSA is dissolved in pH, and concentration is in the phosphate buffer solution of 0.01M, is made into the solution of 10.0 mg/ml, standby；
（5）By vitamin B1Mol ratio with BSA is 50：Take solution C and solution D respectively, at ambient temperature, in the solution D under solution C is added dropwise to be stirred continuously, react 8 ~ 10h, obtain solution E;
(6) solution E above-mentioned phosphate buffer stirring dialysis 70 ~ 80 hours, replacing afterwards is dialysed 70 ~ 80 hours with distilled water, changes a dialysis solution within every 6 ~ 8 hours, dialysis procedure is carried out at 0 ~ 4 DEG C；
（7）Solution E after lyophilizing dialysis, obtains aubergine vitamin B1Conjugate.By ultra-violet absorption spectrum（Absorption value）Determine the coupling situation of product, the vitamin B of the present invention1Conjugate have feature ultraviolet absorption peak at 265 nm.
Embodiment
2
（1）The preparation of solution A：In 5 ml circle reaction bulbs, in stirring（120 revs/min）, add vitamin B1
17.358mg（0.05 mmol）, sodium nitrite 20.00
Mg, is dissolved in 300 μ L distilled water.Add 200 μ L concentrated hydrochloric acid, adjust pH between 1 ~ 2.Under condition of ice bath, lucifuge is reacted 1 hour, obtains yellow solution；
（2）The preparation of solution B：In 10 ml circle reaction bulbs, in stirring（120 revs/min）, add para-amino benzoic acid 6.86mg（0.05 mmol）, it is dissolved in 1000 μ L distilled water, solution A is added dropwise to wherein, and adjust pH between 9 ~ 10 with the NaOH of 1M, under the conditions of 0 ~ 4 DEG C, react 4 hours, obtain purplish red solution；
（3）The preparation of solution C：In 10 ml circle reaction bulbs, in stirring（120 revs/min）, add N-Hydroxysuccinimide（NHS）23,02mg（0.20 mmol）, ethyl [3- (dimethylamino）Propyl group] carbodiimide（EDC）115.02mg（0.60 mmol）, it is dissolved in 500 μ L and be coated in buffer（0.01M, pH 9.5）, solution B is mixed with above-mentioned solution, room temperature reaction 2 hours, obtains solution C；
（4）The preparation of solution D：It is 7.40 that BSA is dissolved in pH, and concentration is in the phosphate buffer solution of 0.01M, is made into the solution of 10.0 mg/ml, standby；
（5）By vitamin B1Mol ratio with BSA is 40：Take solution C and solution D respectively, at ambient temperature, in the solution D under solution C is added dropwise to be stirred continuously, react 8 ~ 10h, obtain solution E;
(6) solution E above-mentioned phosphate buffer stirring dialysis 70 ~ 80 hours, replacing afterwards is dialysed 70 ~ 80 hours with distilled water, changes a dialysis solution within every 6 ~ 8 hours, dialysis procedure is carried out at 0 ~ 4 DEG C；
（7）Solution E after lyophilizing dialysis, obtains aubergine vitamin B1Conjugate.By ultra-violet absorption spectrum（Absorption value）Determine the coupling situation of product, the vitamin B of the present invention1Conjugate have feature ultraviolet absorption peak at 265 nm.
Embodiment
3
（1）The preparation of solution A：In 5 ml circle reaction bulbs, in stirring（120 revs/min）, add vitamin B1
17.358mg（0.05 mmol）, sodium nitrite 20.00
Mg, is dissolved in 300 μ L distilled water.Add 200 μ L concentrated hydrochloric acid, adjust pH between 1 ~ 2.Under condition of ice bath, lucifuge is reacted 1 hour, obtains yellow solution；
（2）The preparation of solution B：In 10 ml circle reaction bulbs, in stirring（120 revs/min）, add para-amino benzoic acid 10.00mg（0.07 mmol）, it is dissolved in 1000 μ L distilled water, solution A is added dropwise to wherein, and adjust pH between 9 ~ 10 with the NaOH of 1M, under the conditions of 0 ~ 4 DEG C, react 4 hours, obtain purplish red solution；
（3）The preparation of solution C：In 10 ml circle reaction bulbs, in stirring（120 revs/min）, add N-Hydroxysuccinimide（NHS）29.62mg（0.25 mmol）, ethyl [3- (dimethylamino）Propyl group] carbodiimide（EDC）98.66mg（0.50mmol）, it is dissolved in 500 μ L and be coated in buffer（0.01M, pH 9.5）, solution B is mixed with above-mentioned solution, room temperature reaction 2 hours, obtains solution C；
（4）The preparation of solution D：It is 7.40 that BSA is dissolved in pH, and concentration is in the phosphate buffer solution of 0.01M, is made into the solution of 10.0 mg/ml, standby；
（5）By vitamin B1Mol ratio with BSA is 20：Take solution C and solution D respectively, at ambient temperature, in the solution D under solution C is added dropwise to be stirred continuously, react 8 ~ 10h, obtain solution E;
(6) solution E above-mentioned phosphate buffer stirring dialysis 70 ~ 80 hours, replacing afterwards is dialysed 70 ~ 80 hours with distilled water, changes a dialysis solution within every 6 ~ 8 hours, dialysis procedure is carried out at 0 ~ 4 DEG C；
（7）Solution E after lyophilizing dialysis, obtains aubergine vitamin B1Conjugate.By ultra-violet absorption spectrum（Absorption value）Determine the coupling situation of product, the vitamin B of the present invention1Conjugate have feature ultraviolet absorption peak at 265 nm.
Embodiment
4
The preparation of antibody and enzyme linked immunosorbent detection
1st, the preparation of antibody
Select the vitamin B prepared by above-described embodiment 11Conjugate as immunogen carry out animal immune test to prepare antibody
Take the vitamin B of 1mg/ml1Conjugate solution 1ml, add isopyknic Freund's complete adjuvant, fully emulsified after, give the male and healthy new zealand white rabbit of 6 body weight 2kg through subcutaneous multi-point injection, 1ml/ only, after 21 days, with the vitamin B of 0.5mg/ml1Conjugate solution 1ml fully emulsified with isopyknic incomplete Freund's adjuvant after carry out two and exempt from, after two exempt from, every 15 days booster immunizations once, immunity 5 times altogether.After immunity for the last time 7 days, heart extracting blood, room temperature stands 1 hour, and 0 ~ 4 DEG C is overnight, and 10000 revs/min are centrifuged 15 minutes, collects serum, and -20 DEG C preserve, standby.
2nd, the enzyme linked immunosorbent detection of antibody
（1）Titration
In the ELISA Plate in 96 holes, every hole adds the vitamin B of 100 μ L1Conjugate with ovalbumin（2μg/ml）, it is incubated 2 hours at 37 DEG C.Using washing liquid PBST after taking-up（1000ml pH 7.4, the PBS+ percent by volume of concentration 0.01M are 0.05% Tween 20）Washing 3 times；Add confining liquid（1000ml PBS+ mass percent by volume is 1% ovalbumin）250 μ L/ hole closings, 37 DEG C are incubated 2 hours, repeat to wash plate process after taking-up, add the diluent in proportion of antibody and negative serum（1:200, 1:400, 1:800,1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200,
1:102400）, every hole 100 μ L, 37 DEG C are incubated 0.5 hour, wash plate；After washing away antibody and negative serum, every hole adds 1:The goat anti-rabbit igg of 10000 horseradish peroxidase-labeled
100 μ L, 37 DEG C are incubated 0.5 hour, add the colour developing of substrate tetramethyl benzidine, every hole 100 μ L, room temperature places colour developing 10min, adds terminate liquid 100 μ L/ hole, uses microplate reader reading at 450nm after washing plate.
After measured：Vitamin B of the present invention1Conjugate antibody titer reach 1:3200.
The judgement of potency is more than 2 with P/N:1 serum highest extension rate is the enzyme linked immunosorbent detection potency of this antibody.
Wherein：The absorbance that above-mentioned P measures in a certain extension rate for test serum, the absorbance that above-mentioned N measures in corresponding extension rate for negative control.
（2）Specific assay：
Determination step is similar with titration, under the conditions of above-mentioned optimal envelope antigen with antibody concentration, plus adds vitamin B while antibody1Gradient solution（10-2-106ng/ml）, the antibody limited with envelope antigen competition binding, vitamin B1Concentration higher, antibody is just combined fewer with envelope antigen, thus colour developing more shallow, absorbance is lower.Again with blank（Only plus antibody, not plus vitamin B1Absorbance）Compare, to determine antibody specificity.
By measuring antibody specificity preferably, its lowest detectable limit can reach 0.18 ppm, and detection sensitivity is higher.
Claims (10)
1. a kind of vitamin B1Conjugate, by vitamin B1Hapten is coupled with the immunogenic carrier proteins Bovine albumin of generation or hemocyanin and constitutes.
2. vitamin B as claimed in claim 11Conjugate, the immunogenic carrier mass of wherein said generation is bovine serum albumin.
3. vitamin B as claimed in claim 21Conjugate, its general structure is such as（I）
（I）
Wherein：n：It is the Molecules of the vitamin B1 being combined with a bovine serum albumin molecule, described n is integer 1 ~ 20, BSA is bovine serum albumin（Bovine Serum Albumin）, molecular weight ranges are 6.6 KDa ~ 6.9 KDa；
Above-mentioned conjugate shows following physical chemical characteristicses：
Outward appearance：Aubergine pulverulent solids
Ultra-violet absorption spectrum：265nm.
4. vitamin B as claimed in claim 31Conjugate, it is characterized in that：Described n is integer 1 ~ 10, and BSA molecular weight ranges are 6.7 KDa ~ 6.8 KDa.
5. the vitamin B as any one of claim 1 ~ 41Conjugate preparation method, it is characterized in that：By vitamin with produce immunogenic carrier mass couple together, be combined into have induction animal immuning system generated antibody conjugate, and keep this conjugate biological activity constant.
6. vitamin B as claimed in claim 51Conjugate preparation method, completed by following steps：
（1）The preparation of solution A：By vitamin B1With sodium nitrite（NaNO2）With mol ratio for 1：5 ~ 6 are dissolved in 300 μ L water, adjust pH in 1 ~ 2, ice bath lucifuge reaction 1h with concentrated hydrochloric acid, obtain solution A, standby；
（2）The preparation of solution B：In the aqueous solution of para-amino benzoic acid solution A being added dropwise to 0.05 ~ 0.1 M, adjust pH 9 ~ 10 with NaOH, under the conditions of 0 ~ 4 DEG C, react 4 hours, obtain solution B；
（3）The preparation of solution C：By vitamin B1, N-Hydroxysuccinimide（NHS）, ethyl [3- (dimethylamino）Propyl group] carbodiimide（EDC）, with mol ratio 1:2~8:5 ~ 15 ratio, NHS and EDC is dissolved in and is coated in buffer, and solution B is mixed with above-mentioned solution, room temperature reaction 2 hours, obtains solution C；
（4）The preparation of solution D：It is 7.38 ~ 7.56 that BSA is dissolved in pH, and concentration is the phosphate buffer of 0.01 M（PBS）In, it is made into the solution of 10.0 ± 5.0 mg/ml, standby；
（5）By vitamin B1Mol ratio with BSA is 15 ~ 50：1 amount takes solution C and solution D respectively, at ambient temperature, in the solution D under solution C is added dropwise to be stirred continuously, reacts 8 ~ 10h, obtains solution E；
（6）Solution E above-mentioned phosphate buffer stirring dialysis 70 ~ 80 hours, replacing afterwards is dialysed 70 ~ 80 hours with distilled water, changes a dialysis solution within every 6 ~ 8 hours, dialysis procedure is carried out at 0 ~ 4 DEG C；
（7）Solution E after lyophilizing dialysis, obtains aubergine vitamin B1Conjugate.
7. vitamin B as claimed in claim 61Conjugate preparation method, it is characterized in that：Step（1）Described vitamin B1It is 1 with sodium nitrite mol ratio：6.
8. vitamin B as claimed in claim 61Preparation method, it is characterized in that：Step（2）The concentration of described para-amino benzoic acid is 0.07
M.
9. vitamin B as claimed in claim 61Preparation method, it is characterized in that：Step（3）Described vitamin B1It is 1 with N-Hydroxysuccinimide, EDC mol ratio：5:10.
10. vitamin B as claimed in claim 61Preparation method, it is characterized in that：Step（5）Described vitamin B1Mol ratio with BSA is 50:1.
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| CN110713986A (en) * | 2019-11-12 | 2020-01-21 | 江南大学 | Vitamin B1Monoclonal antibody hybridoma cell strain CBDD and application thereof |
| CN115232119A (en) * | 2021-04-25 | 2022-10-25 | 深圳泰乐德医疗有限公司 | Preparation method of vitamin B1 hapten, immunogen, coating antigen and monoclonal antibody |
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| CN102809658A (en) * | 2012-09-03 | 2012-12-05 | 南开大学 | Enzyme-linked immunosorbent assay kit of vitamin B2 |
| CN104076155A (en) * | 2014-07-10 | 2014-10-01 | 深圳市新产业生物医学工程股份有限公司 | Detection reagent for detecting 25-hydroxy vitamin D, as well as preparation method and application of detection reagent |
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| CN1827642A (en) * | 2006-03-14 | 2006-09-06 | 山东大学 | Ofloxacin couple and its preparing method and use |
| CN102809658A (en) * | 2012-09-03 | 2012-12-05 | 南开大学 | Enzyme-linked immunosorbent assay kit of vitamin B2 |
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| CN110713986A (en) * | 2019-11-12 | 2020-01-21 | 江南大学 | Vitamin B1Monoclonal antibody hybridoma cell strain CBDD and application thereof |
| CN110713986B (en) * | 2019-11-12 | 2023-05-12 | 江南大学 | Vitamin B strain 1 Monoclonal antibody hybridoma cell strain CBDD and application thereof |
| CN115232119A (en) * | 2021-04-25 | 2022-10-25 | 深圳泰乐德医疗有限公司 | Preparation method of vitamin B1 hapten, immunogen, coating antigen and monoclonal antibody |
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