CN107353200A - A kind of vanillylmandelic acid (VMA) derivative, its synthetic method and a kind of vanillylmandelic acid (VMA) immunogene, its preparation method and its application - Google Patents
A kind of vanillylmandelic acid (VMA) derivative, its synthetic method and a kind of vanillylmandelic acid (VMA) immunogene, its preparation method and its application Download PDFInfo
- Publication number
- CN107353200A CN107353200A CN201710499551.0A CN201710499551A CN107353200A CN 107353200 A CN107353200 A CN 107353200A CN 201710499551 A CN201710499551 A CN 201710499551A CN 107353200 A CN107353200 A CN 107353200A
- Authority
- CN
- China
- Prior art keywords
- vma
- vanillylmandelic acid
- acid
- vanillylmandelic
- immunogene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- CGQCWMIAEPEHNQ-UHFFFAOYSA-N Vanillylmandelic acid Chemical compound COC1=CC(C(O)C(O)=O)=CC=C1O CGQCWMIAEPEHNQ-UHFFFAOYSA-N 0.000 title claims abstract description 356
- 238000002360 preparation method Methods 0.000 title claims abstract description 33
- 238000010189 synthetic method Methods 0.000 title claims abstract description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 53
- 238000001514 detection method Methods 0.000 claims abstract description 37
- 239000000243 solution Substances 0.000 claims description 76
- 102000004190 Enzymes Human genes 0.000 claims description 42
- 108090000790 Enzymes Proteins 0.000 claims description 42
- 102100031126 6-phosphogluconolactonase Human genes 0.000 claims description 35
- 108010029731 6-phosphogluconolactonase Proteins 0.000 claims description 35
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 claims description 35
- 150000001875 compounds Chemical class 0.000 claims description 28
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 27
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 24
- 238000006243 chemical reaction Methods 0.000 claims description 24
- 239000007983 Tris buffer Substances 0.000 claims description 18
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 18
- 238000002347 injection Methods 0.000 claims description 16
- 239000007924 injection Substances 0.000 claims description 16
- 239000000758 substrate Substances 0.000 claims description 16
- 102000014914 Carrier Proteins Human genes 0.000 claims description 14
- 108010078791 Carrier Proteins Proteins 0.000 claims description 14
- 230000000890 antigenic effect Effects 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 239000002671 adjuvant Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 238000010171 animal model Methods 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 10
- 239000000427 antigen Substances 0.000 claims description 9
- 102000036639 antigens Human genes 0.000 claims description 9
- 108091007433 antigens Proteins 0.000 claims description 9
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 9
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 9
- 239000012460 protein solution Substances 0.000 claims description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- 241001465754 Metazoa Species 0.000 claims description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 6
- 235000009499 Vanilla fragrans Nutrition 0.000 claims description 6
- 235000012036 Vanilla tahitensis Nutrition 0.000 claims description 6
- 229940098773 bovine serum albumin Drugs 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 230000003053 immunization Effects 0.000 claims description 6
- 238000002649 immunization Methods 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims description 5
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims description 5
- 230000021615 conjugation Effects 0.000 claims description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- IMFACGCPASFAPR-UHFFFAOYSA-N tributylamine Chemical class CCCCN(CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-N 0.000 claims description 5
- QBYIENPQHBMVBV-HFEGYEGKSA-N (2R)-2-hydroxy-2-phenylacetic acid Chemical compound O[C@@H](C(O)=O)c1ccccc1.O[C@@H](C(O)=O)c1ccccc1 QBYIENPQHBMVBV-HFEGYEGKSA-N 0.000 claims description 4
- CCMKPCBRNXKTKV-UHFFFAOYSA-N 1-hydroxy-5-sulfanylidenepyrrolidin-2-one Chemical class ON1C(=O)CCC1=S CCMKPCBRNXKTKV-UHFFFAOYSA-N 0.000 claims description 4
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical class CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 claims description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 4
- 210000004369 blood Anatomy 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 4
- 150000001718 carbodiimides Chemical class 0.000 claims description 4
- 238000000502 dialysis Methods 0.000 claims description 4
- NLFBCYMMUAKCPC-KQQUZDAGSA-N ethyl (e)-3-[3-amino-2-cyano-1-[(e)-3-ethoxy-3-oxoprop-1-enyl]sulfanyl-3-oxoprop-1-enyl]sulfanylprop-2-enoate Chemical compound CCOC(=O)\C=C\SC(=C(C#N)C(N)=O)S\C=C\C(=O)OCC NLFBCYMMUAKCPC-KQQUZDAGSA-N 0.000 claims description 4
- 239000012467 final product Substances 0.000 claims description 4
- 238000005227 gel permeation chromatography Methods 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 4
- 230000003647 oxidation Effects 0.000 claims description 4
- 238000007254 oxidation reaction Methods 0.000 claims description 4
- 229910052698 phosphorus Inorganic materials 0.000 claims description 4
- 239000011574 phosphorus Substances 0.000 claims description 4
- 239000012264 purified product Substances 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 241000283707 Capra Species 0.000 claims description 3
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 3
- 241001494479 Pecora Species 0.000 claims description 3
- IWYDHOAUDWTVEP-UHFFFAOYSA-N R-2-phenyl-2-hydroxyacetic acid Natural products OC(=O)C(O)C1=CC=CC=C1 IWYDHOAUDWTVEP-UHFFFAOYSA-N 0.000 claims description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 238000002474 experimental method Methods 0.000 claims description 3
- 238000009396 hybridization Methods 0.000 claims description 3
- 125000005647 linker group Chemical group 0.000 claims description 3
- 229960002510 mandelic acid Drugs 0.000 claims description 3
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 3
- 230000000392 somatic effect Effects 0.000 claims description 3
- 230000009870 specific binding Effects 0.000 claims description 3
- 102000009843 Thyroglobulin Human genes 0.000 claims description 2
- 108010034949 Thyroglobulin Proteins 0.000 claims description 2
- 230000008878 coupling Effects 0.000 claims description 2
- 238000010168 coupling process Methods 0.000 claims description 2
- 238000005859 coupling reaction Methods 0.000 claims description 2
- 108060003552 hemocyanin Proteins 0.000 claims description 2
- 230000005847 immunogenicity Effects 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 229920001184 polypeptide Polymers 0.000 claims description 2
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 claims description 2
- 210000002966 serum Anatomy 0.000 claims description 2
- 229960002175 thyroglobulin Drugs 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 2
- 101710088194 Dehydrogenase Proteins 0.000 claims 1
- 244000263375 Vanilla tahitensis Species 0.000 claims 1
- 239000012634 fragment Substances 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 230000004907 flux Effects 0.000 abstract description 3
- 239000007788 liquid Substances 0.000 description 14
- 238000012360 testing method Methods 0.000 description 11
- 238000002965 ELISA Methods 0.000 description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 9
- 229940088597 hormone Drugs 0.000 description 8
- 239000005556 hormone Substances 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 210000002700 urine Anatomy 0.000 description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 244000290333 Vanilla fragrans Species 0.000 description 5
- 150000003943 catecholamines Chemical class 0.000 description 4
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 238000007689 inspection Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 244000144725 Amygdalus communis Species 0.000 description 3
- 235000011437 Amygdalus communis Nutrition 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- UCTWMZQNUQWSLP-UHFFFAOYSA-N adrenaline Chemical compound CNCC(O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-UHFFFAOYSA-N 0.000 description 3
- 235000020224 almond Nutrition 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- CGQCWMIAEPEHNQ-QMMMGPOBSA-N (2s)-2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)acetic acid Chemical compound COC1=CC([C@H](O)C(O)=O)=CC=C1O CGQCWMIAEPEHNQ-QMMMGPOBSA-N 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- NBSCHQHZLSJFNQ-GASJEMHNSA-N D-Glucose 6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@H]1O NBSCHQHZLSJFNQ-GASJEMHNSA-N 0.000 description 2
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical class ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 2
- VFRROHXSMXFLSN-UHFFFAOYSA-N Glc6P Natural products OP(=O)(O)OCC(O)C(O)C(O)C(O)C=O VFRROHXSMXFLSN-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000012937 correction Methods 0.000 description 2
- 230000001934 delay Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 229940045189 glucose-6-phosphate Drugs 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 2
- 229910052753 mercury Inorganic materials 0.000 description 2
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000013062 quality control Sample Substances 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- ICBPURKUPVLVCM-UHFFFAOYSA-N 1,5-dimethyl-2-phenylpyrazol-3-one;2-hydroxy-2-phenylacetic acid Chemical compound OC(=O)C(O)C1=CC=CC=C1.CN1C(C)=CC(=O)N1C1=CC=CC=C1 ICBPURKUPVLVCM-UHFFFAOYSA-N 0.000 description 1
- AVMSWPWPYJVYKY-UHFFFAOYSA-N 2-Methylpropyl formate Chemical compound CC(C)COC=O AVMSWPWPYJVYKY-UHFFFAOYSA-N 0.000 description 1
- OLSDAJRAVOVKLG-UHFFFAOYSA-N 3-hydroxymandelic acid Chemical compound OC(=O)C(O)C1=CC=CC(O)=C1 OLSDAJRAVOVKLG-UHFFFAOYSA-N 0.000 description 1
- 206010001367 Adrenal insufficiency Diseases 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 208000007530 Essential hypertension Diseases 0.000 description 1
- 241000628997 Flos Species 0.000 description 1
- 206010020850 Hyperthyroidism Diseases 0.000 description 1
- 206010061216 Infarction Diseases 0.000 description 1
- 102000010909 Monoamine Oxidase Human genes 0.000 description 1
- 108010062431 Monoamine oxidase Proteins 0.000 description 1
- 239000007832 Na2SO4 Substances 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 235000006041 Prunus persica f compressa Nutrition 0.000 description 1
- 240000006522 Prunus persica f. compressa Species 0.000 description 1
- 230000001919 adrenal effect Effects 0.000 description 1
- 208000017515 adrenocortical insufficiency Diseases 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003913 calcium metabolism Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000007574 infarction Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 230000005226 mechanical processes and functions Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- -1 methoxy adrenaline Chemical compound 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 208000028591 pheochromocytoma Diseases 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002889 sympathetic effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C59/40—Unsaturated compounds
- C07C59/58—Unsaturated compounds containing ether groups, groups, groups, or groups
- C07C59/64—Unsaturated compounds containing ether groups, groups, groups, or groups containing six-membered aromatic rings
- C07C59/66—Unsaturated compounds containing ether groups, groups, groups, or groups containing six-membered aromatic rings the non-carboxylic part of the ether containing six-membered aromatic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/09—Preparation of carboxylic acids or their salts, halides or anhydrides from carboxylic acid esters or lactones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/093—Preparation of carboxylic acids or their salts, halides or anhydrides by hydrolysis of —CX3 groups, X being halogen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/30—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group
- C07C67/31—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by introduction of functional groups containing oxygen only in singly bound form
-
- 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
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of vanillylmandelic acid (VMA) derivative, its synthetic method and a kind of vanillylmandelic acid (VMA) immunogene, its preparation method and its application；It is high with anti-vanillylmandelic acid (VMA) specific antibody high specificity, potency made from vanillylmandelic acid (VMA) derivative, and with 92 kinds of common chaff interferences without any cross reaction, therefore higher accuracy, precision, sensitivity and specificity can be embodied；The application of vanillylmandelic acid (VMA) immunogene is to be used to vanillylmandelic acid (VMA) immunogene prepare anti-vanillylmandelic acid (VMA) specific antibody, with by anti-vanillylmandelic acid (VMA) specific antibody be used for prepare vanillylmandelic acid (VMA) detection reagent, the detection reagent can be realized on automatic clinical chemistry analyzer to vanillylmandelic acid (VMA) high flux, rapid detection.
Description
Technical field
The present invention relates to a kind of vanillylmandelic acid (VMA) derivative, its synthetic method and a kind of vanillylmandelic acid (VMA) immunogene, its system
Preparation Method and its application, belong to biological technical field.
Background technology
Vanillylmandelic acid (VMA) (Vanillymandelic Acid, VMA), its structural formula is as shown in formula VI：
Vanillylmandelic acid (VMA) is also known as 3-methoxy-4-hydroxy mandelic acid (DL-3-Methoxy-4-hydroxyman
Delic acid, MHMA) or vanilla mandelic acid, it is that the whole last reign of a dynasty of adrenal medullary hormone catecholamine (CA) class material thanks to production
Thing, CA almost all are metabolized in vivo, and least a portion of CA acts on through monoamine oxidase, generate p- dihydroxy tussol, most of CA
It is changed into 3- methoxies adrenaline under the effect of CA-O- transmethylases or removes methoxy adrenaline, is finally metabolized as 3- methoxies
Base -4- hydroxy mandelic acids.Internal CA metabolism is very fast, and its metabolite is discharged by urine more.Clinically in 24h urine
VMA content rise sees pheochromocytoma, neuroblastoma, essential hypertension, miocardial infarction, chronic kidney not
Entirely, hyperthyroidism or decline, adrenal insufficiency, sympathetic nerve physiological function exception etc..Therefore, determine
Clinical diagnosis of the VMA discharge rate for above-mentioned disease is significant in 24h urine.
Classical VMA detection methods mainly have：Visible spectrophotometry, the method operating process is cumbersome, poor accuracy；
Gas chromatography, this method is higher to instrument requirements, complex operation；High phase liquid phase-electrochemical process, the method is easily by instrument quality
With the interference of working environment, the sensitivity of measure and selectivity are both needed to further be lifted；Heavy nitrogen, the method is cumbersome, can
Low by property, the rate of recovery, repeatability, stability need to be improved.Above assay method does not adapt to the need of VMA clinical examinations
Will, deficient in stability is good in the market, high sensitivity, the VMA detection reagents of high specificity, the especially measured automation of matter
Testing reagent.Therefore, research and develop a kind of quality and reach clinical examination requirement, practical, cost-effective, can be applied to full-automatic life
The VMA detection reagents for changing analyzer are imperative.
The content of the invention
For overcome the deficiencies in the prior art, of the invention first purpose is that providing a kind of vanillylmandelic acid (VMA) derives
Thing, the derivative are new synthetic, are not present in nature.
Realize that the purpose of the present invention can reach by adopting the following technical scheme that：A kind of vanillylmandelic acid (VMA) derivative, its
Structural formula is as shown in formula I：
Second object of the present invention is to provide a kind of synthetic method of vanillylmandelic acid (VMA) derivative, and the synthetic method has
Not in conventional synthesis process, and there is good synthetic effect, greatly improve the combined coefficient of vanillylmandelic acid (VMA) derivative.
Realize that the purpose of the present invention can reach by adopting the following technical scheme that：A kind of vanillylmandelic acid (VMA) as described above
The synthetic method of derivative, synthetic route are shown below：
Course of reaction includes following：
Synthesize compounds Ⅳ step：Compound ii, compound III are dissolved in DMF, and added
K2CO3After being reacted, through extracting, drying, concentrating, purifying and obtain compounds Ⅳ；
Synthesize chemical compounds I step：Compounds Ⅳ and benzyltriethylammoinium chloride are dissolved in chloroform, it is molten that reaction is made
Liquid；NaOH solution is added dropwise in reaction solution, pH value of solution=4 are adjusted after reaction, then through extracting, drying, concentrating, purifying,
Obtain chemical compounds I i.e. vanillylmandelic acid (VMA) derivative.
Third object of the present invention is to provide a kind of preparation method of vanillylmandelic acid (VMA) immunogene.
Realize that the purpose of the present invention can reach by adopting the following technical scheme that：A kind of system of vanillylmandelic acid (VMA) immunogene
Preparation Method, including：
Dissolve carrier protein step：Carrier protein 100-300g is dissolved in 25-75mL 0.2M, pH=8.5 phosphoric acid delays
In fliud flushing, carrier protein solution is obtained；
Mixed liquor preparation process：Following solution is mixed and stirred for dissolving reaction 30-60min, obtains mixed liquor；
100-300mg vanillylmandelic acid (VMA) derivatives；
1.75-5.25mL dimethylformamide；
1.75-5.25mL ethanol；
3.5-10.5mL 10mM, pH=5 kaliumphosphate buffer；
100-300mg 1- ethyls -3- (- 3- dimethylaminopropyls) carbodiimide,
25-75mg N- hydroxy thiosuccinimides；
Immunogene preparation process：Mixed liquor is added dropwise in carrier protein solution, and at least 8h is stirred at 2-8 DEG C, is obtained
To antigen；Antigen is purified by dialysis, obtains vanillylmandelic acid (VMA) immunogene.
Fourth object of the present invention is to provide a kind of vanillylmandelic acid (VMA) immunogene.
Realize that the purpose of the present invention can reach by adopting the following technical scheme that：A kind of vanillylmandelic acid (VMA) as described above
Immunogene, its structural formula is as shown in formula V：
Wherein ,-(CH2)3- CO- is linking group；R is carrier, is serum for protein or polypeptide with immunogenicity
One kind in albumen, hemocyanin and thyroglobulin.
Further, R is bovine serum albumin.
The 5th purpose of the present invention is to provide a kind of application of vanillylmandelic acid (VMA) immunogene, for vanillylmandelic acid (VMA) is exempted from
Epidemic focus is used to prepare anti-vanillylmandelic acid (VMA) specific antibody, and by anti-vanillylmandelic acid (VMA) specific antibody for preparing vanilla almond
Sour detection reagent, the detection reagent can be realized on automatic clinical chemistry analyzer to vanillylmandelic acid (VMA) high flux, rapid
Detection.
Realize that the purpose of the present invention can reach by adopting the following technical scheme that：A kind of vanillylmandelic acid (VMA) as described above
The application of immunogene, including vanillylmandelic acid (VMA) immunogene is used to prepare anti-vanillylmandelic acid (VMA) specific antibody, and by anti-vanilla
Mandelic acid specific antibody is used to prepare vanillylmandelic acid (VMA) detection reagent.
Further, anti-vanillylmandelic acid (VMA) specific antibody is caused after immunization experiment animal：Complete antibody molecule, tool
Have and one kind in the antibody fragment and antibody derivatives of vanillylmandelic acid (VMA) specific binding capacity；Complete antibody molecule, antibody
Fragment and antibody derivatives, to use single vanillylmandelic acid (VMA) immunogene to the Anti-TNF-α obtained by animal booster immunization
Body, or be through the monoclonal antibody obtained by somatic hybridization after being immunized；Described experimental animal is rabbit, goat, mouse, silk floss
One kind of sheep, cavy or Malaysia and China；
The preparation method of anti-vanillylmandelic acid (VMA) specific antibody is：
Immune step：Vanillylmandelic acid (VMA) immunogene is diluted to 0.1-3mg/mL with PBS, obtains antigenic solution, Ran Houyong
0.5-5mL antigenic solutions are mixed with equivalent Freund's complete adjuvant, and first time injection is carried out to experimental animal；After 2-3 weeks, 0.5- is used
5mL antigenic solutions mix second of injection of progress with equivalent incomplete Freund's adjuvant；It is molten with 0.5-5mL antigens every 4 weeks afterwards
With equivalent incomplete Freund's adjuvant hybrid injection once, co-injection three to six times in step are immunized in liquid；
Separation antibody step：To taking blood through immune experimental animal and isolating and purifying, potency is obtained as 1:30000-50000
Anti- vanillylmandelic acid (VMA) specific antibody.
Further, vanillylmandelic acid (VMA) detection reagent includes anti-vanillylmandelic acid (VMA) specific antibody, vanillylmandelic acid (VMA) enzyme mark
The substrate of conjugate and enzyme；Vanillylmandelic acid (VMA) enzyme mark conjugate is glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate；
The substrate of enzyme is G-6-P.
Further, vanillylmandelic acid (VMA) detection reagent includes reagent A and reagent B：
Reagent A is the Substrate cocktail of anti-vanillylmandelic acid (VMA) specific antibody and enzyme, is prepared by following steps：
By the NADH and 0.856- of 2.018-8.072g, 5.625-22.50mM oxidation state
3.422g, 5.625-22.50mM G-6-P are made homogeneously with 0.5-2L, 55mM, pH=8 Tris buffer solutions
Zymolyte；Anti- vanillylmandelic acid (VMA) specific antibody is added in homogeneous zymolyte, anti-vanillylmandelic acid (VMA) specific antibody with it is homogeneous
The volume ratio of zymolyte is 1:100-10000, obtain reagent A；
Reagent B is vanillylmandelic acid (VMA) enzyme mark conjugate solution, is prepared by following steps：
Vanillylmandelic acid (VMA) enzyme mark conjugate is added in 120mM, pH=8.2 Tris buffer solutions, vanillylmandelic acid (VMA) enzyme mark
The volume ratio of conjugate and Tris buffer solutions is 1:100-10000.
Further, vanillylmandelic acid (VMA) enzyme mark conjugate is prepared by the following method to obtain：
The preparation process of glucose-6-phosphate dehydrogenase (G6PD) solution：Weigh 7.5-22.5mg specifications be 100KU glucose-
6- phosphate dehydrogenases, it is dissolved in 6-18mL 0.05M containing 72.6mg Tris, 8mg 3.3mM MgCl2It is molten with 100mg NaCl
In liquid, pH=9 is adjusted；Then NADH, the 67.5-202.5mg of 112.5-337.5mg reduction-states are added
G-6-P and 0.375-1.125mL carbitols；1-3mL dimethyl sulfoxide (DMSO)s are added dropwise again, obtain glucose -6- phosphorus
Acidohydrogenase solution；
The activation step of vanillylmandelic acid (VMA) derivative：5-15mg vanillylmandelic acid (VMA) derivatives are weighed under anhydrous conditions, it is molten
Solution is in 300-900 μ L dimethylformamides；Solution temperature is set to add 1.5-4.5 μ L tri-n-butylamines, 0.75- after being down to -2~-8 DEG C
2.25 μ L isobutyl chlorocarbonates, 30-60min is stirred under the conditions of -2~-8 DEG C；
The Connection Step of glucose-6-phosphate dehydrogenase (G6PD) and vanillylmandelic acid (VMA) derivative：The vanillylmandelic acid (VMA) that will have been activated
Derivative solution is added dropwise in glucose-6-phosphate dehydrogenase (G6PD) solution；2-8 DEG C of stirring at least 8h, obtains connection product；
Purified product step：Connection product is purified by G-25 gel chromatography columns, the final product of acquisition is glucose -6-
Phosphate dehydrogenase-hapten conjugation thing, is stored at 2-8 DEG C.
Compared with prior art, the beneficial effects of the present invention are：
1st, vanillylmandelic acid (VMA) derivative and synthetic method of the invention are targetedly recent studies on and design, in existing skill
In art and it is not present；
2nd, the present invention is with vanillylmandelic acid (VMA) immunogene made from vanillylmandelic acid (VMA) derivative and antibody its sensitivity, specificity
Immunogene and antibody all than directly using the preparation of the vanillylmandelic acid (VMA) original is high, and the anti-vanillylmandelic acid (VMA) specific antibody prepared is special
It is different in nature strong, potency is high, and with 92 kinds of common chaff interferences without any cross reaction, thus can embody it is higher accurate
Property, precision, sensitivity and specificity；
3rd, immunologic function test reagent of the invention can realize on automatic clinical chemistry analyzer to vanillylmandelic acid (VMA) high flux,
Rapid detection, multiple samples can be determined simultaneously, have easy to operate, high sensitivity, high specificity, result accurate etc. excellent
Point, moreover it is possible to effectively reduce vanillylmandelic acid (VMA) testing cost, be advantageous to clinical expansion use.
Brief description of the drawings
Fig. 1 is the standard curve of embodiment 4ELISA detections；
Fig. 2 is the standard curve of the vanillylmandelic acid (VMA) immunologic detection method of embodiment 7；
Fig. 3 is dissipating for the vanillylmandelic acid (VMA) clinical samples immune detection of embodiment 9 and high performance liquid chromatography detection correction data
Point diagram.
Embodiment
Below, with reference to accompanying drawing and embodiment, the present invention is described further：
First, vanillylmandelic acid (VMA) derivative and its synthesis：
Vanillylmandelic acid (VMA) derivative, its structural formula is as shown in formula I：
The synthetic route for synthesizing the vanillylmandelic acid (VMA) derivative process is：
Course of reaction includes following：
Synthesize compounds Ⅳ step：Compound ii, compound III are dissolved in DMF, and added
K2CO3After being reacted, through extracting, drying, concentrating, purifying and obtain compounds Ⅳ；
Synthesize chemical compounds I step：Compounds Ⅳ and benzyltriethylammoinium chloride are dissolved in chloroform, it is molten that reaction is made
Liquid；NaOH solution is added dropwise in reaction solution, pH value of solution=4 are adjusted after reaction, then through extracting, drying, concentrating, purifying,
Obtain chemical compounds I i.e. vanillylmandelic acid (VMA) derivative.
2nd, the preparation method of vanillylmandelic acid (VMA) immunogene：
Dissolve carrier protein step：Carrier protein 100-300g is dissolved in 25-75mL 0.2M, pH=8.5 phosphoric acid delays
In fliud flushing, carrier protein solution is obtained；
Mixed liquor preparation process：Following solution is mixed and stirred for dissolving reaction 30-60min, obtains mixed liquor；
100-300mg vanillylmandelic acid (VMA) derivatives；
1.75-5.25mL dimethylformamide；
1.75-5.25mL ethanol；
3.5-10.5mL 10mM, pH=5 kaliumphosphate buffer；
100-300mg 1- ethyls -3- (- 3- dimethylaminopropyls) carbodiimide,
25-75mg N- hydroxy thiosuccinimides；
Immunogene preparation process：Mixed liquor is added dropwise in carrier protein solution, and at least 8h is stirred at 2-8 DEG C, is obtained
To antigen；Antigen is purified by dialysis, obtains vanillylmandelic acid (VMA) immunogene；
Obtained vanillylmandelic acid (VMA) immunogene, its structural formula is as shown in formula V：
Wherein ,-(CH2)3- CO- is linking group；R is carrier, is bovine serum albumin.
3rd, the application of vanillylmandelic acid (VMA) immunogene：
1st, anti-vanillylmandelic acid (VMA) specific antibody is prepared：
Anti- vanillylmandelic acid (VMA) specific antibody is caused after immunization experiment animal：Complete antibody molecule, have and vanilla
One kind in the antibody fragment and antibody derivatives of mandelic acid specific binding capacity；Complete antibody molecule, antibody fragment and anti-
Syntaxy thing, to use single vanillylmandelic acid (VMA) immunogene to the polyclonal antibody obtained by animal booster immunization, Huo Zhewei
Through the monoclonal antibody obtained by somatic hybridization after immune；Described experimental animal include but is not limited to rabbit, goat, mouse,
One kind of sheep, cavy or Malaysia and China, preferably rabbit；
The preparation method of anti-vanillylmandelic acid (VMA) specific antibody is：
Immune step：Vanillylmandelic acid (VMA) immunogene is diluted to 0.1-3mg/mL with PBS, obtains antigenic solution, Ran Houyong
0.5-5mL antigenic solutions are mixed with equivalent Freund's complete adjuvant, and first time injection is carried out to experimental animal；After 2-3 weeks, 0.5- is used
5mL antigenic solutions mix second of injection of progress with equivalent incomplete Freund's adjuvant；It is molten with 0.5-5mL antigens every 4 weeks afterwards
With equivalent incomplete Freund's adjuvant hybrid injection once, co-injection three to six times in step are immunized in liquid；
Separation antibody step：To taking blood through immune experimental animal and isolating and purifying, potency is obtained as 1:30000-50000
Anti- vanillylmandelic acid (VMA) specific antibody.
2nd, vanillylmandelic acid (VMA) detection reagent is prepared：
Vanillylmandelic acid (VMA) detection reagent includes anti-vanillylmandelic acid (VMA) specific antibody, vanillylmandelic acid (VMA) enzyme mark conjugate and enzyme
Substrate；Vanillylmandelic acid (VMA) enzyme mark conjugate is glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate；The substrate of enzyme is
G-6-P；
Specifically, vanillylmandelic acid (VMA) detection reagent before the use, in order to avoid the enzyme mark conjugate in indicator and
The substrate of enzyme reacts, and the substrate of enzyme mark conjugate and enzyme is not mix and separated, thus by the substrate of enzyme with it is above-mentioned
Anti- vanillylmandelic acid (VMA) specific antibody mixes.Therefore, vanillylmandelic acid (VMA) detection reagent includes two class reagents, reagent A and
Reagent B：
Reagent A is the Substrate cocktail of anti-vanillylmandelic acid (VMA) specific antibody and enzyme, is prepared by following steps：
By the NADH and 0.856- of 2.018-8.072g, 5.625-22.50mM oxidation state
3.422g, 5.625-22.50mM G-6-P are made homogeneously with 0.5-2L, 55mM, pH=8 Tris buffer solutions
Zymolyte；Anti- vanillylmandelic acid (VMA) specific antibody is added in homogeneous zymolyte, anti-vanillylmandelic acid (VMA) specific antibody with it is homogeneous
The volume ratio of zymolyte is 1:100-10000, obtain reagent A；
Reagent B is vanillylmandelic acid (VMA) enzyme mark conjugate solution, is prepared by following steps：
Vanillylmandelic acid (VMA) enzyme mark conjugate is added in 120mM, pH=8.2 Tris buffer solutions, vanillylmandelic acid (VMA) enzyme mark
The volume ratio of conjugate and Tris buffer solutions is 1:100-10000；
The volume of anti-vanillylmandelic acid (VMA) specific antibody and homogeneous zymolyte is preferably 1:1250；
The volume ratio of vanillylmandelic acid (VMA) enzyme mark conjugate and Tris buffer solutions is preferably 1:2500.
Wherein, vanillylmandelic acid (VMA) enzyme mark conjugate is prepared by following steps：
The preparation process of glucose-6-phosphate dehydrogenase (G6PD) solution：Weigh 7.5-22.5mg specifications be 100KU glucose-
6- phosphate dehydrogenases, it is dissolved in 6-18mL 0.05M containing 72.6mg Tris, 8mg 3.3mM MgCl2It is molten with 100mg NaCl
In liquid, pH=9 is adjusted；Then NADH, the 67.5-202.5mg of 112.5-337.5mg reduction-states are added
G-6-P and 0.375-1.125mL carbitols；1-3mL dimethyl sulfoxide (DMSO)s are added dropwise again, obtain glucose -6- phosphorus
Acidohydrogenase solution；
The activation step of vanillylmandelic acid (VMA) derivative：5-15mg vanillylmandelic acid (VMA) derivatives are weighed under anhydrous conditions, it is molten
Solution is in 300-900 μ L dimethylformamides；Solution temperature is set to add 1.5-4.5 μ L tri-n-butylamines, 0.75- after being down to -2~-8 DEG C
2.25 μ L isobutyl chlorocarbonates, 30-60min is stirred under the conditions of -2~-8 DEG C；
The Connection Step of glucose-6-phosphate dehydrogenase (G6PD) and vanillylmandelic acid (VMA) derivative：The vanillylmandelic acid (VMA) that will have been activated
Derivative solution is added dropwise in glucose-6-phosphate dehydrogenase (G6PD) solution；2-8 DEG C of stirring at least 8h, obtains connection product；
Purified product step：Connection product is purified by G-25 gel chromatography columns, the final product of acquisition is glucose -6-
Phosphate dehydrogenase-hapten conjugation thing, is stored at 2-8 DEG C.
The structural formula of vanillylmandelic acid (VMA) derivative is the same as formula I.
Embodiment 1：
The synthesis of vanillylmandelic acid (VMA) derivative and its analyze and identify
Route synthesis vanillylmandelic acid (VMA) derivative is synthesized by the following way：
Specific synthesis step is as follows：
1) synthesis of compounds Ⅳ：
20g (131.6mmol) compound ii, 33.2g (170mmol) compound III is weighed, co-dissolve is in 300mL
In DMF (DMF), 36.4g (263mmol) K is then added2CO3, reaction mixture is made；Reaction is mixed
Liquid is closed to be stirred at room temperature 12 hours；After reaction terminates, 200mL purified waters are added in reaction mixture, then with 300mL bis-
Chloromethanes (DCM) is extracted, and extraction step is repeated 3 times；The organic phase for the combination being obtained by extraction is passed through into Na2SO4It is dried
Processing, then concentrated；The residue obtained after concentration is passed through into silica dehydrator post (PE:EA=5:1) purified, obtained
28g compounds Ⅳs, yield 80%.
Examine：Using Bruker Avance III plus 400MHz and VARIAN MERCURY plus 300M to upper
State compounds Ⅳ and carry out NMR spectrum scanning, using TMS as internal standard；As a result it is as follows：1H-NMR(400MHz,CDCI3):δ
9.86(s,1H),7.46(m,2H),7.01(m,1H),4.19(m,4H),3.93(s,3H),2.58(m,2H),2.22(m,2H),
1.26(m,3H).It is characterized as the compounds Ⅳ shown in structural formula.
2) synthesis of chemical compounds I：
Weigh 3g (11.3mmol) compounds Ⅳ, 0.15g (0.6mmol) benzyltriethylammoinium chloride, co-dissolve in
30mL chloroforms (CHCl3) in reaction solution is made；It is that reaction is added dropwise in 15g/mL NaOH solutions under the conditions of 56 DEG C by concentration
In solution, and stirred 2.5 hours under the conditions of 56 DEG C；The residue that reaction obtains after terminating is adjusted to pH=4 with 1N HCI,
Acidification is carried out, is then extracted with 30mL ethyl acetate (EA), extraction step is repeated 3 times；The combination that will be obtained by extraction
Organic phase be dried and concentration；The residue obtained after concentration is purified by pre-HPLC, obtains 0.4g
The chemical compounds I of white solid, as vanillylmandelic acid (VMA) derivative (VMA derivatives), yield 12.4%.
Examine：Using Bruker Avance III plus 400MHz and VARIAN MERCURY plus 300M to upper
State compound as white solid and carry out NMR spectrum scanning, using TMS as internal standard.As a result it is as follows：1H-NMR(400MHz,
CD3OD):δ7.08(s,1H),6.93(m,2H),5.06(s,1H),4.04(m,2H),3.83(s,3H),2.50(m,2H),
2.03(m,2H).It is characterized as the chemical compounds I shown in structural formula.
Embodiment 2
The synthesis of vanillylmandelic acid (VMA) immunogene：
The structural formula of vanillylmandelic acid (VMA) immunogene is as shown in formula V：
Vanillylmandelic acid (VMA) immunogene by carrier and vanillylmandelic acid (VMA) derivative-(CH2)3- CO- groups are formed by connecting；R is
Bovine serum albumin(BSA) (Bovine Serum Albumin, BSA).
The preparation method of the immunogene comprises the following steps that：
Dissolve carrier protein step：Bovine serum albumin 200g is dissolved in 50mL 0.2M, pH=8.5 phosphate buffer
In, obtain carrier protein solution；
Mixed liquor preparation process：Following solution is mixed and stirred for dissolving reaction 30min, obtains mixed liquor；
200mg vanillylmandelic acid (VMA) derivatives；
3.5mL dimethylformamide；
3.5mL ethanol；
7mL 10mM, pH=5 kaliumphosphate buffer；
200mg 1- ethyls -3- (- 3- dimethylaminopropyls) carbodiimide,
50mg N- hydroxy thiosuccinimides；
Immunogene preparation process：Mixed liquor is added dropwise in BAS solution, and at least 8h is stirred at 2-8 DEG C, is resisted
It is former；Antigen is purified by dialysis, obtains vanillylmandelic acid (VMA) immunogene.
Embodiment 3
The preparation of anti-vanillylmandelic acid (VMA) specific antibody：
The preparation method of anti-vanillylmandelic acid (VMA) specific antibody is：
Immune step：Vanillylmandelic acid (VMA) immunogene is diluted to 1mg/mL with PBS, antigenic solution is obtained, is then resisted with 1mL
Original solution is mixed with equivalent Freund's complete adjuvant, and first time injection is carried out to experimental animal rabbit；After 2-3 weeks, with 1mL antigenic solutions
Second of injection of progress is mixed with equivalent incomplete Freund's adjuvant；Afterwards 0.5-5mL antigenic solutions and equivalent Freund were used every 4 weeks
Once, co-injection four times in step are immunized in Freund's incomplete adjuvant hybrid injection；
Separation antibody step：To taking blood through immune experimental animal rabbit and isolating and purifying, potency is obtained as 1:30000-
50000 anti-vanillylmandelic acid (VMA) specific antibody.
Embodiment 4
The ELISA of vanillylmandelic acid (VMA) is examined：
1st, the foundation of the ELISA examination criteria curves of vanillylmandelic acid (VMA)：
1) preparation of standard items：
Vanillylmandelic acid (VMA) powder (being purchased from Sigma companies) is dissolved in methanol solution, is prepared into 1mg/mL storing liquid.With
Storing liquid is diluted to 16mg/L, 8mg/L, 4mg/L, 2mg/L, 1mg/L and 0mg/L standard liquid by ELISA buffer solutions successively.
Wherein, ELISA buffer solutions contain 50mM Tris, 145mM NaCl and percent by volume 0.25% BSA.
2) standard curve is prepared using the ELISA methods of inspection of vanillylmandelic acid (VMA)：
Anti- vanillylmandelic acid (VMA) antibody prepared in embodiment 3 is diluted to 1 with PBS:10000 final concentration solution, 100
μ L/ holes are coated on 96 hole elisa plates, 4 DEG C of placement 12-24h；With PBS by above-mentioned 96 holes for being coated with anti-vanillylmandelic acid (VMA) antibody
After elisa plate washs 3 times, the BSA solution of the percent by volume 0.5% in 200 μ L/ holes is added, 8-16h is placed in 4 DEG C of closings.Then
Washed 3 times with PBS, add the standard items in 20 μ L/ holes.Add the HRP- vanillylmandelic acid (VMA)s coupling of 100 μ L/ holes working concentrations
Thing；PBS board-washings 5 times after incubation 30min at room temperature；Then 100 μ L tmb substrates are added per hole, are incubated at room temperature 30min.Again per hole
Add 100 μ L terminate liquids (2M sulfuric acid).Determine 450nm light absorption value.The light absorption value of 450nm according to corresponding to each standard items is determined
Mark, standard curve is made, as a result as shown in Figure 1.
2nd, in testing sample vanillylmandelic acid (VMA) content detection：
1) testing sample is made：
Preparation method：Vanillylmandelic acid (VMA) powder (being purchased from Sigma companies) is dissolved in the storage that 1mg/mL is made in methanol solution
Liquid storage, and this storing liquid is diluted in blank diaper, is respectively 0,2,8,16mg/L to final concentration, formed respectively blank, it is low,
The urine specimen of middle and high concentration.Blank diaper is the Healthy People urine without vanillylmandelic acid (VMA).
2) method of testing：
Using the ELISA methods of inspection of above-mentioned vanillylmandelic acid (VMA), by the urine specimen generation of above-mentioned blank, basic, normal, high concentration
For standard items, test above-mentioned blank, the urine specimen of basic, normal, high concentration 450nm light absorption value.
3) test result：
The standard curve that the ELISA of vanillylmandelic acid (VMA) shown in compares figure 1 is examined, calculates vanilla almond in each sample
Acid content, and 3 multiple holes measure are carried out to each sample, recovery is calculated according to the actual content of vanillylmandelic acid (VMA) in above-mentioned sample
Rate, as a result as shown in Table 1.
The ELISA testing results of the vanillylmandelic acid (VMA) of form 1
From result in form 1：Various concentrations sample is determined using the ELISA detection reagents of vanillylmandelic acid (VMA) of the present invention
In the vanillylmandelic acid (VMA) rate of recovery it is all higher, between 99%-101%, illustrate that anti-vanillylmandelic acid (VMA) of the present invention is special
Heterogenetic antibody can be used for the detection of vanillylmandelic acid (VMA) in sample, and result precision is high.
Embodiment 5
The preparation method of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing is：
The preparation process of glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) solution：
Accurate to weigh the G6PDH that 15mg specifications are 100KU, room-temperature dissolution is in 12mL Tris containing 72.6mg0.05M, 8mg
3.3mM MgCl2In 100mg NaCl solution, pH=9 is adjusted, this step is carried out in beaker.
NADH (NADH), the 135mg glucose -6- phosphorus of 225mg reduction-states are added in beaker
Sour (G-6-P) and 0.75mL carbitols (Carbitol).
2mL dimethyl sulfoxide (DMSO)s (dimethy sulfoxide, DMSO) are added dropwise again in beaker, obtain glucose -6-
Phosphate dehydrogenase enzyme solutions.
The activation step of vanillylmandelic acid (VMA) derivative：
The vanillylmandelic acid (VMA) derivative of the synthesis of 10mg embodiments 1 is weighed under anhydrous conditions, is dissolved in 600 μ L dimethyl methyls
In acid amides (DMF)；Above-mentioned solution temperature is set to add 3 μ L tri-n-butylamines (tributylamine), 1.5 μ L chlorine after dropping to -2~-8 DEG C
Iso-butyl formate (isobutylchloroformate), -2~-8 DEG C of stirring 30min.
G6PDH and vanillylmandelic acid (VMA) derivative Connection Step：
The vanillylmandelic acid (VMA) derivative solution activated is added dropwise in G6PDH solution, 2-8 DEG C is stirred overnight (extremely
Few 8h), obtain connection product.
Purified product step：
Connection product is purified by G-25 gel chromatography columns, the final product of acquisition is glucose-6-phosphate dehydrogenase (G6PD)-half
Antigen conjugates, stored at 2-8 DEG C.
Embodiment 6
The preparation of vanillylmandelic acid (VMA) detection reagent：
Vanillylmandelic acid (VMA) detection reagent before the use, in order to avoid the enzyme mark conjugate and the substrate of enzyme in indicator
React, the substrate of enzyme mark conjugate and enzyme is not mix and separated, so the substrate of enzyme and above-mentioned anti-vanilla is flat
Peach acid specific antibody mixes.Therefore, vanillylmandelic acid (VMA) detection reagent includes two class reagents, reagent A and reagent B：
The preparation of reagent A：
By the NADH (NAD of 4.036g (11.25mM) oxidation state+) and 1.711g (11.25mM)
G-6-P (G-6-P) is placed in beaker, and homogeneous zymolyte is made with 1L, 55mM, pH=8 Tris buffer solutions；
The anti-vanillylmandelic acid (VMA) specific antibody prepared in embodiment 3 is added in homogeneous zymolyte, the body of antibody and homogeneous zymolyte
Product is than being 1:1250, obtain reagent A.
Reagent B preparation：
Glucose-6-phosphate dehydrogenase (G6PD) prepared by embodiment 5-hapten conjugation thing is added to 120mM, pH=8.2
In Tris buffer solutions, the volume ratio of vanillylmandelic acid (VMA) enzyme mark conjugate and Tris buffer solutions is 1:2500.
Embodiment 7
Vanillylmandelic acid (VMA) immunity inspection and result
1st, standard curve is obtained：
1) set and step auspicious BS-480 automatic clinical chemistry analyzers response parameter, as shown in Table 2.
The Biochemical Analyzer parameter setting of form 2
2) operating procedure is：First reagent adding A, adds standard items, is eventually adding reagent B.After adding reagent B, measure is not
With the OD at time point340Light absorption value, reaction rate during various criterion product concentration is calculated, needs constantly to adjust in actual mechanical process
The volume ratio of reagent A and reagent B, while light-metering point is adjusted, comparatively ideal reaction normal curve map is finally drawn, such as Fig. 2 institutes
Show.
2nd, pattern detection：By the obtained standard curve of vanillylmandelic acid (VMA) detection reagent of the present invention, replication is low,
Middle and high concentration Quality Control sample 10 times, above-mentioned Quality Control sample are：Vanillylmandelic acid (VMA) standard items are dissolved in human urine, to concentration
Respectively 2,8,16mg/L.Detection data and data analysis are shown in Table lattice 3.
The testing result of the sample of form 3
Testing result：The degree of accuracy of the vanillylmandelic acid (VMA) detection reagent measure of the present invention is high, and the rate of recovery is in 99%-
Between 101%, precision is high, and CV is below 4%.
Embodiment 8
Medicine and hormone interference test：
Chaff interference chooses 62 kinds of Common drugs and 30 kinds of common hormones and hormone metabolism thing carries out Interference Detection, adjusts concentration
To 1mg/L, it is measured using the vanillylmandelic acid (VMA) immunity inspection of embodiment 7：
Reagent A haptoreaction prepared by chaff interference to be measured and embodiment 6, adds reagent B；
Detect the OD of above-mentioned mixed solution340Light absorption value, the concentration of respective substance is obtained according to Fig. 2.
62 kinds of Common drugs claim with 30 kinds of common hormones and hormone metabolism name and measurement result is referring specifically to form 4.
The common interference thing measurement result of form 4
Measurement result is shown：Above-mentioned 62 kinds of Common drugs and 30 kinds of common hormones and hormone metabolism thing are equivalent to vanilla almond
The concentration of acid is respectively less than 0.01mg/L.As can be seen here, antibody of the invention is the specific antibody of anti-vanillylmandelic acid (VMA), and common
Chaff interference no cross reaction.
Embodiment 9
100 clinical samples are entered using the vanillylmandelic acid (VMA) detection reagent of high performance liquid chromatography and embodiment 6 respectively
Row measure, and make correlation analysis, the data of measure are referring to form 5.
The clinical samples measurement result correction data of form 5
Above-mentioned data are mapped, referring to Fig. 3, obtained linear equation is：Y=1.003x-0.0442, coefficient R2
=0.999, show that the degree of accuracy of vanillylmandelic acid (VMA) detection reagent measure vanillylmandelic acid (VMA) clinical samples is high.
For those skilled in the art, technical scheme that can be as described above and design, make other each
Kind is corresponding to be changed and deforms, and all these change and deformed the protection model that should all belong to the claims in the present invention
Within enclosing.
Claims (10)
1. a kind of vanillylmandelic acid (VMA) derivative, it is characterised in that its structural formula is as shown in formula I：
A kind of 2. synthetic method of vanillylmandelic acid (VMA) derivative as claimed in claim 1, it is characterised in that the synthetic route
It is shown below：
Course of reaction includes following：
Synthesize compounds Ⅳ step：Compound ii, compound III are dissolved in DMF, and add K2CO3Enter
After row reaction, through extracting, drying, concentrating, purifying and obtain compounds Ⅳ；
Synthesize chemical compounds I step：Compounds Ⅳ and benzyltriethylammoinium chloride are dissolved in chloroform, reaction solution is made；Will
NaOH solution is added dropwise in reaction solution, and pH value of solution=4 are adjusted after reaction, then through extracting, drying, concentrating, purifying, is changed
Compound I is vanillylmandelic acid (VMA) derivative.
A kind of 3. preparation method of vanillylmandelic acid (VMA) immunogene, it is characterised in that including：
Dissolve carrier protein step：Carrier protein 100-300g is dissolved in 25-75mL 0.2M, pH=8.5 phosphate buffer
In, obtain carrier protein solution；
Mixed liquor preparation process：Following solution is mixed and stirred for dissolving reaction 30-60min, obtains mixed liquor；
100-300mg vanillylmandelic acid (VMA) derivatives as claimed in claim 1；
1.75-5.25mL dimethylformamide；
1.75-5.25mL ethanol；
3.5-10.5mL 10mM, pH=5 kaliumphosphate buffer；
100-300mg 1- ethyls -3- (- 3- dimethylaminopropyls) carbodiimide,
25-75mg N- hydroxy thiosuccinimides；
Immunogene preparation process：Mixed liquor is added dropwise in carrier protein solution, and at least 8h is stirred at 2-8 DEG C, is resisted
It is former；Antigen is purified by dialysis, obtains vanillylmandelic acid (VMA) immunogene.
4. a kind of vanillylmandelic acid (VMA) immunogene as claimed in claim 3, it is characterised in that its structural formula is as shown in formula V：
Wherein ,-(CH2)3- CO- is linking group；R is carrier, is serum egg for protein or polypeptide with immunogenicity
In vain, one kind in hemocyanin and thyroglobulin.
5. vanillylmandelic acid (VMA) immunogene as claimed in claim 4, it is characterised in that the R is bovine serum albumin.
6. the application of a kind of vanillylmandelic acid (VMA) immunogene as described in claim 3-5 is any, it is characterised in that including by vanilla
Mandelic acid immunogene is used to prepare anti-vanillylmandelic acid (VMA) specific antibody, and anti-vanillylmandelic acid (VMA) specific antibody is used to prepare
Vanillylmandelic acid (VMA) detection reagent.
7. the application of vanillylmandelic acid (VMA) immunogene as claimed in claim 6, it is characterised in that the anti-vanillylmandelic acid (VMA) is special
Property antibody be immunization experiment animal after caused by：Complete antibody molecule, have it is anti-with vanillylmandelic acid (VMA) specific binding capacity
One kind in body fragment and antibody derivatives；The complete antibody molecule, antibody fragment and antibody derivatives, for using single
Vanillylmandelic acid (VMA) immunogene is to the polyclonal antibody obtained by animal booster immunization, or is through obtained by somatic hybridization after being immunized
The monoclonal antibody arrived；Described experimental animal is one kind of rabbit, goat, mouse, sheep, cavy or Malaysia and China；
The preparation method of the anti-vanillylmandelic acid (VMA) specific antibody is：
Immune step：Vanillylmandelic acid (VMA) immunogene is diluted to 0.1-3mg/mL with PBS, antigenic solution is obtained, then uses 0.5-
5mL antigenic solutions are mixed with equivalent Freund's complete adjuvant, and first time injection is carried out to experimental animal；After 2-3 weeks, 0.5-5mL is used
Antigenic solution mixes second of injection of progress with equivalent incomplete Freund's adjuvant；Afterwards every 4 weeks with 0.5-5mL antigenic solutions with
Once, co-injection three to six times in step are immunized in equivalent incomplete Freund's adjuvant hybrid injection；
Separation antibody step：To taking blood through immune experimental animal and isolating and purifying, potency is obtained as 1:30000-50000's is anti-
Vanillylmandelic acid (VMA) specific antibody.
8. the application of vanillylmandelic acid (VMA) immunogene as claimed in claim 7, it is characterised in that the vanillylmandelic acid (VMA) detection examination
Agent includes the substrate of anti-vanillylmandelic acid (VMA) specific antibody, vanillylmandelic acid (VMA) enzyme mark conjugate and enzyme；The vanillylmandelic acid (VMA) enzyme
Mark conjugate is glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate；The substrate of the enzyme is G-6-P.
9. the application of vanillylmandelic acid (VMA) immunogene as claimed in claim 8, it is characterised in that the vanillylmandelic acid (VMA) detection examination
Agent includes reagent A and reagent B：
The reagent A is the Substrate cocktail of anti-vanillylmandelic acid (VMA) specific antibody and enzyme, is prepared by following steps：
By the NADH of 2.018-8.072g, 5.625-22.50mM oxidation state and 0.856-3.422g,
Homogeneous zymolyte is made with 0.5-2L, 55mM, pH=8 Tris buffer solutions in 5.625-22.50mM G-6-Ps；
Anti- vanillylmandelic acid (VMA) specific antibody is added in homogeneous zymolyte, anti-vanillylmandelic acid (VMA) specific antibody and homogeneous zymolyte
Volume ratio is 1:100-10000, obtain reagent A；
The reagent B is vanillylmandelic acid (VMA) enzyme mark conjugate solution, is prepared by following steps：
Vanillylmandelic acid (VMA) enzyme mark conjugate is added in 120mM, pH=8.2 Tris buffer solutions, the coupling of vanillylmandelic acid (VMA) enzyme mark
The volume ratio of thing and Tris buffer solutions is 1:100-10000.
10. the application of vanillylmandelic acid (VMA) immunogene as claimed in claim 9, it is characterised in that the vanillylmandelic acid (VMA) enzyme mark
Conjugate is prepared by the following method to obtain：
The preparation process of glucose-6-phosphate dehydrogenase (G6PD) solution：Weigh the glucose -6- phosphorus that 7.5-22.5mg specifications are 100KU
Acidohydrogenase, it is dissolved in 6-18mL 0.05M containing 72.6mg Tris, 8mg 3.3m M MgCl2With 100mg NaCl solution
In, adjust pH=9；Then NADH, the 67.5-202.5mg Portugals of 112.5-337.5mg reduction-states are added
Grape sugar -6- phosphoric acid and 0.375-1.125mL carbitols；1-3mL dimethyl sulfoxide (DMSO)s are added dropwise again, obtain G-6-P
Dehydrogenase solution；
The activation step of vanillylmandelic acid (VMA) derivative：5-15mg vanillylmandelic acid (VMA) derivatives are weighed under anhydrous conditions, are dissolved in
In 300-900 μ L dimethylformamides；Solution temperature is set to add 1.5-4.5 μ L tri-n-butylamines, 0.75-2.25 after being down to -2~-8 DEG C
μ L isobutyl chlorocarbonates, 30-60min is stirred under the conditions of -2~-8 DEG C；
The Connection Step of glucose-6-phosphate dehydrogenase (G6PD) and vanillylmandelic acid (VMA) derivative：The vanillylmandelic acid (VMA) activated is derived
Thing solution is added dropwise in glucose-6-phosphate dehydrogenase (G6PD) solution；2-8 DEG C of stirring at least 8h, obtains connection product；
Purified product step：Connection product is purified by G-25 gel chromatography columns, the final product of acquisition is G-6-P
Dehydrogenase-hapten conjugation thing, is stored at 2-8 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710499551.0A CN107353200A (en) | 2017-06-27 | 2017-06-27 | A kind of vanillylmandelic acid (VMA) derivative, its synthetic method and a kind of vanillylmandelic acid (VMA) immunogene, its preparation method and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710499551.0A CN107353200A (en) | 2017-06-27 | 2017-06-27 | A kind of vanillylmandelic acid (VMA) derivative, its synthetic method and a kind of vanillylmandelic acid (VMA) immunogene, its preparation method and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107353200A true CN107353200A (en) | 2017-11-17 |
Family
ID=60274080
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710499551.0A Pending CN107353200A (en) | 2017-06-27 | 2017-06-27 | A kind of vanillylmandelic acid (VMA) derivative, its synthetic method and a kind of vanillylmandelic acid (VMA) immunogene, its preparation method and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107353200A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109704954A (en) * | 2018-12-21 | 2019-05-03 | 苏州博源医疗科技有限公司 | 3-(3- hydroxy phenyl) derivative of -3- hydracrylic acid, homogeneous enzyme immunoassay detection reagent and preparation method thereof |
| CN112225672A (en) * | 2020-10-16 | 2021-01-15 | 湖南苏阳医疗科技有限公司 | Methoxy norepinephrine derivative, immunogen, specific antibody, preparation method and application thereof |
| CN112266330A (en) * | 2020-10-15 | 2021-01-26 | 湖南苏阳医疗科技有限公司 | Methoxy adrenaline derivative, immunogen, anti-methoxy adrenaline specific antibody, preparation method and application thereof |
| CN114085149A (en) * | 2021-08-09 | 2022-02-25 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Megastigmatrienone hapten, artificial antigen, preparation methods of megastigmatrienone hapten and artificial antigen, antibody and application of megastigmatrienone hapten and artificial antigen |
| CN114685477A (en) * | 2020-12-25 | 2022-07-01 | 苏州博源医疗科技有限公司 | Linezolid derivative, immunogen, anti-linezolid specific antibody, and preparation method and application thereof |
| CN114685470A (en) * | 2020-12-25 | 2022-07-01 | 苏州博源医疗科技有限公司 | Risperidone key group derivative, immunogen, anti-risperidone specific antibody, and preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0450095A1 (en) * | 1989-10-19 | 1991-10-09 | Yamasa Shoyu Kabushiki Kaisha (Yamasa Corporation) | Method of assaying d-vanillylmandelic acid, and reagent and kit therefor |
| EP0453504B1 (en) * | 1989-01-12 | 1995-04-12 | Cancer Research Campaign Technology Limited | Derivatives of hydroxymethoxy mandelic acid (hmma), homovanillic acid (hva), antibodies and labelled substances prepared therefrom, and immunoassays using these |
| CN105175530A (en) * | 2015-08-14 | 2015-12-23 | 苏州博源医疗科技有限公司 | Vanilmandelic acid immune detection reagent and preparation method thereof |
| CN105294427A (en) * | 2014-06-11 | 2016-02-03 | 上海予利化学科技有限公司 | Resolving synthetic method for paramethoxymandelic acid |
| CN106596917A (en) * | 2016-12-19 | 2017-04-26 | 苏州博源医疗科技有限公司 | Homovanillic acid homogeneous enzyme immuno-assay reagent, preparation method and detection method |
-
2017
- 2017-06-27 CN CN201710499551.0A patent/CN107353200A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0453504B1 (en) * | 1989-01-12 | 1995-04-12 | Cancer Research Campaign Technology Limited | Derivatives of hydroxymethoxy mandelic acid (hmma), homovanillic acid (hva), antibodies and labelled substances prepared therefrom, and immunoassays using these |
| EP0450095A1 (en) * | 1989-10-19 | 1991-10-09 | Yamasa Shoyu Kabushiki Kaisha (Yamasa Corporation) | Method of assaying d-vanillylmandelic acid, and reagent and kit therefor |
| CN105294427A (en) * | 2014-06-11 | 2016-02-03 | 上海予利化学科技有限公司 | Resolving synthetic method for paramethoxymandelic acid |
| CN105175530A (en) * | 2015-08-14 | 2015-12-23 | 苏州博源医疗科技有限公司 | Vanilmandelic acid immune detection reagent and preparation method thereof |
| CN106596917A (en) * | 2016-12-19 | 2017-04-26 | 苏州博源医疗科技有限公司 | Homovanillic acid homogeneous enzyme immuno-assay reagent, preparation method and detection method |
Non-Patent Citations (1)
| Title |
|---|
| G.W. MELLOR等: "Production and characterisation of antibodies to vanillylmandelic acid", 《JOURNAL OF IMMUNOLOGICAL METHODS》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109704954A (en) * | 2018-12-21 | 2019-05-03 | 苏州博源医疗科技有限公司 | 3-(3- hydroxy phenyl) derivative of -3- hydracrylic acid, homogeneous enzyme immunoassay detection reagent and preparation method thereof |
| CN109704954B (en) * | 2018-12-21 | 2021-05-04 | 苏州博源医疗科技有限公司 | 3- (3-hydroxyphenyl) -3-hydroxypropionic acid derivative, homogeneous enzyme immunoassay reagent and preparation method thereof |
| CN112266330A (en) * | 2020-10-15 | 2021-01-26 | 湖南苏阳医疗科技有限公司 | Methoxy adrenaline derivative, immunogen, anti-methoxy adrenaline specific antibody, preparation method and application thereof |
| CN112266330B (en) * | 2020-10-15 | 2023-02-24 | 长沙博源医疗科技有限公司 | Methoxyarenol derivative, immunogen, anti-methoxyadrol specific antibody, preparation method and application thereof |
| CN112225672A (en) * | 2020-10-16 | 2021-01-15 | 湖南苏阳医疗科技有限公司 | Methoxy norepinephrine derivative, immunogen, specific antibody, preparation method and application thereof |
| CN112225672B (en) * | 2020-10-16 | 2023-03-28 | 长沙博源医疗科技有限公司 | Methoxy norepinephrine derivative, immunogen, specific antibody, preparation method and application thereof |
| CN114685477A (en) * | 2020-12-25 | 2022-07-01 | 苏州博源医疗科技有限公司 | Linezolid derivative, immunogen, anti-linezolid specific antibody, and preparation method and application thereof |
| CN114685470A (en) * | 2020-12-25 | 2022-07-01 | 苏州博源医疗科技有限公司 | Risperidone key group derivative, immunogen, anti-risperidone specific antibody, and preparation method and application thereof |
| CN114685470B (en) * | 2020-12-25 | 2023-08-18 | 长沙博源医疗科技有限公司 | Risperidone key group derivative, immunogen, anti-risperidone specific antibody, and preparation methods and applications thereof |
| CN114685477B (en) * | 2020-12-25 | 2023-11-17 | 长沙博源医疗科技有限公司 | Linezolid derivative, immunogen, linezolid-resistant specific antibody, and preparation methods and applications thereof |
| CN114085149A (en) * | 2021-08-09 | 2022-02-25 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Megastigmatrienone hapten, artificial antigen, preparation methods of megastigmatrienone hapten and artificial antigen, antibody and application of megastigmatrienone hapten and artificial antigen |
| CN114085149B (en) * | 2021-08-09 | 2023-10-20 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Megastigmatrienone hapten and artificial antigen as well as preparation methods, antibodies and applications thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107353200A (en) | A kind of vanillylmandelic acid (VMA) derivative, its synthetic method and a kind of vanillylmandelic acid (VMA) immunogene, its preparation method and its application | |
| CN106645692B (en) | Estriol homogeneous enzyme immunoassay detection reagent, preparation method and detection method | |
| CN102768284B (en) | Preparation method of immunodetection reagent of carbamazepine homogeneous enzyme | |
| CN107365342B (en) | Aldosterone derivative, immunogene and synthetic method, specific antibody and detection reagent and preparation method, kit | |
| CN104530222A (en) | Paclitaxel immunogen, anti-paclitaxel specific antibody and paclitaxel detection reagent | |
| CN104447984B (en) | Docetaxel immunogene, anti-Docetaxel specific antibody and Docetaxel detection reagent | |
| CN104788560B (en) | Ciclosporin A immunogene, anti-Ciclosporin A specific antibody and Ciclosporin A detection reagent | |
| CN109111494A (en) | Derivatives of estradiol, immunogene, antibody, enzyme mark conjugate, detection reagent and preparation method thereof | |
| CN104804079B (en) | Imatinib immunogene, derivative and synthetic method, specific antibody and detection reagent and preparation method | |
| CN104569373A (en) | Methotrexate homogenous enzyme immunoassay reagent as well as preparation method and detection method thereof | |
| CN105175530A (en) | Vanilmandelic acid immune detection reagent and preparation method thereof | |
| CN105175531A (en) | Hydroxyproline immunogen, specific antibody and detection reagent, and preparation methods thereof | |
| CN104774256B (en) | Catecholamine immunogene, derivative and synthetic method, specific antibody and detection reagent and preparation method | |
| CN105131106A (en) | 5-hydroxyindoleacetic acid immunogen, antibody and detection reagent, and preparation methods thereof | |
| CN107973836B (en) | Aldosterone derivative, preparation method thereof and aldosterone homogeneous enzyme immunoassay reagent | |
| CN110003300A (en) | A kind of derivative of 17OHS, detection reagent and preparation method | |
| CN109928958A (en) | A kind of voriconazole derivate, its synthetic method and a kind of voriconazole immunogene, preparation method and its application | |
| CN106596917B (en) | Homovanillic acid homogeneous enzyme immunoassay detection reagent, preparation method and detection method | |
| CN105131105A (en) | Cortisol immunogen, derivative, antibody, detection reagent and preparation method | |
| CN102295698B (en) | Cyclosporine A immunogen, cyclosporine A specific antibody, detection reagent, and detection kit | |
| CN104987392A (en) | Dehydroepiandrosterone immunogen, derivative, antibody and detection reagent as well as preparation method | |
| CN107132349A (en) | A kind of homocysteine automatic detection reagent, preparation method and detection method | |
| CN108120838B (en) | Dipeptide derivative for detecting angiotensin II and preparation method and application thereof | |
| CN108586562B (en) | A kind of cortex 01 derivatives and the preparation method and application thereof | |
| CN108490195A (en) | A kind of vitamin B12 immunization assay method and its reagent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171117 |