CN104031142B - The folic acid complete antigen synthetic method of a kind of high specific and application thereof - Google Patents
The folic acid complete antigen synthetic method of a kind of high specific and application thereof Download PDFInfo
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- CN104031142B CN104031142B CN201410254563.3A CN201410254563A CN104031142B CN 104031142 B CN104031142 B CN 104031142B CN 201410254563 A CN201410254563 A CN 201410254563A CN 104031142 B CN104031142 B CN 104031142B
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- folic acid
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- carrier protein
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- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 title claims abstract description 145
- 239000011724 folic acid Substances 0.000 title claims abstract description 73
- 235000019152 folic acid Nutrition 0.000 title claims abstract description 73
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 title claims abstract description 72
- 229960000304 folic acid Drugs 0.000 title claims abstract description 72
- 239000000427 antigen Substances 0.000 title claims abstract description 45
- 102000036639 antigens Human genes 0.000 title claims abstract description 45
- 108091007433 antigens Proteins 0.000 title claims abstract description 45
- 238000010189 synthetic method Methods 0.000 title claims abstract description 11
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 26
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 26
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000006243 chemical reaction Methods 0.000 claims abstract description 22
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims abstract description 8
- 241001465754 Metazoa Species 0.000 claims abstract description 8
- 238000000502 dialysis Methods 0.000 claims abstract description 7
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims abstract description 6
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 claims abstract description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 5
- 125000000250 methylamino group Chemical group [H]N(*)C([H])([H])[H] 0.000 claims abstract description 4
- 230000008878 coupling Effects 0.000 claims abstract description 3
- 238000010168 coupling process Methods 0.000 claims abstract description 3
- 238000005859 coupling reaction Methods 0.000 claims abstract description 3
- 239000012460 protein solution Substances 0.000 claims abstract description 3
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims abstract 2
- 239000002253 acid Substances 0.000 claims description 10
- 230000033228 biological regulation Effects 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- 150000001875 compounds Chemical class 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 101000609762 Gallus gallus Ovalbumin Proteins 0.000 claims description 2
- 239000000376 reactant Substances 0.000 claims description 2
- 108010042653 IgA receptor Proteins 0.000 claims 1
- 102100034014 Prolyl 3-hydroxylase 3 Human genes 0.000 claims 1
- 238000002965 ELISA Methods 0.000 abstract description 10
- 238000001514 detection method Methods 0.000 abstract description 10
- 238000004458 analytical method Methods 0.000 abstract description 6
- 229960002989 glutamic acid Drugs 0.000 abstract description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 abstract description 4
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-Glutamic acid Natural products OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 abstract description 2
- 150000002224 folic acids Chemical class 0.000 abstract description 2
- 238000001179 sorption measurement Methods 0.000 abstract description 2
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 abstract 1
- LRQKBLKVPFOOQJ-UHFFFAOYSA-N 2-aminohexanoic acid Chemical class CCCCC(N)C(O)=O LRQKBLKVPFOOQJ-UHFFFAOYSA-N 0.000 abstract 1
- CPNGPNLZQNNVQM-UHFFFAOYSA-N pteridine Chemical compound N1=CN=CC2=NC=CN=C21 CPNGPNLZQNNVQM-UHFFFAOYSA-N 0.000 abstract 1
- 238000000034 method Methods 0.000 description 11
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 5
- 239000011248 coating agent Substances 0.000 description 5
- 238000000576 coating method Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 2
- HNXQXTQTPAJEJL-UHFFFAOYSA-N 2-aminopteridin-4-ol Chemical compound C1=CN=C2NC(N)=NC(=O)C2=N1 HNXQXTQTPAJEJL-UHFFFAOYSA-N 0.000 description 2
- JOAQINSXLLMRCV-UHFFFAOYSA-N 4-{[(2-amino-4-hydroxypteridin-6-yl)methyl]amino}benzoic acid Chemical compound C1=NC2=NC(N)=NC(O)=C2N=C1CNC1=CC=C(C(O)=O)C=C1 JOAQINSXLLMRCV-UHFFFAOYSA-N 0.000 description 2
- MSTNYGQPCMXVAQ-KIYNQFGBSA-N 5,6,7,8-tetrahydrofolic acid Chemical compound N1C=2C(=O)NC(N)=NC=2NCC1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 MSTNYGQPCMXVAQ-KIYNQFGBSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- -1 acyl glutamic acid Chemical compound 0.000 description 2
- 239000005515 coenzyme Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229940005654 nitrite ion Drugs 0.000 description 2
- BRBAEHHXGZRCBK-UHFFFAOYSA-N pentrinitrol Chemical compound [O-][N+](=O)OCC(CO)(CO[N+]([O-])=O)CO[N+]([O-])=O BRBAEHHXGZRCBK-UHFFFAOYSA-N 0.000 description 2
- 229950006286 pentrinitrol Drugs 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000011931 Nucleoproteins Human genes 0.000 description 1
- 108010061100 Nucleoproteins Proteins 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 150000008065 acid anhydrides Chemical class 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229960004050 aminobenzoic acid Drugs 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- GYOZYWVXFNDGLU-XLPZGREQSA-N dTMP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)C1 GYOZYWVXFNDGLU-XLPZGREQSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229940014144 folate Drugs 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 150000003949 imides Chemical class 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000000366 juvenile effect Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 201000010193 neural tube defect Diseases 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 229940055726 pantothenic acid Drugs 0.000 description 1
- 235000019161 pantothenic acid Nutrition 0.000 description 1
- 239000011713 pantothenic acid Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000004144 purine metabolism Effects 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 239000012048 reactive intermediate Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 208000035581 susceptibility to neural tube defects Diseases 0.000 description 1
- 150000003512 tertiary amines Chemical class 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 229960001295 tocopherol Drugs 0.000 description 1
- 239000011732 tocopherol Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- IMFACGCPASFAPR-UHFFFAOYSA-N tributylamine Chemical compound CCCCN(CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-N 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
Classifications
-
- 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
-
- 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
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The folic acid complete antigen synthetic method of a kind of high specific and application thereof, belong to technical field of biochemical industry.The present invention be in carrier protein solution, add 6 aminocaproic acids and water-soluble carbodiimide after react under room temperature, obtain having modified the carrier protein B of linking arm after dialysis.Folic acid (N { 4 [(2 amino 1; 4 dihydro 4 oxo 6 pteridine) methylamino] benzoyl } L glutamic acid) it is dissolved in dimethyl sulfoxide; add dicyclohexylcarbodiimide and N-Hydroxysuccinimide; reaction generates folic acid derivatives; carry out coupling with the amino on carrier protein B linking arm again, obtain complete antigen.Result shows, the antiserum titre obtained with the antigen-immunized animal of the present invention reaches 81000, detection limit 0.042ng/mL, 503nhibiting concentration 0.3ng/mL.The antibody specificity produced is high, highly sensitive.The antigen of the present invention or antibody are used for the preparation of immune affinity column and set up ELISA adsorption analysis method and Colloidal gold stripes, are used for detecting folic acid residual.
Description
Technical field
The present invention relates to folic acid complete antigen synthetic method and the application thereof of a kind of high specific, belong to biochemical technology
Field.
Background technology
Folic acid (folic acid or folate) has another name called dish acyl glutamic acid, one of vitamin B group, be aminoacid, pyrimidine and
The carrier of single carbon transfer in purine metabolism, the synthesis to nucleoprotein plays coenzyme effect；Meanwhile, folic acid is also that many desmoenzymes are anti-
The coenzyme answered；The shortage of folic acid, can cause the synthesis of dTMP to be restricted, make DNA biosynthesis block, and it is lean to produce huge juvenile cell
Blood.Current research it turned out, women pregnant before or First Trimester lack folic acid be neural tube defects occur Important cause of disease it
One.Folic acid is in close relations with homocysteine metabolism, has important meaning biology to prevention cardiovascular and cerebrovascular disease
Justice.And, also there are some researches show, the generation of folic acid and many cancers, prevent and treat relevant.In China, folic acid is as one
Planting food additive can be according in the legal interpolation of national food safety standard to food.
Folic acid, chemical name N-{4-[(2-amino-Isosorbide-5-Nitrae-dihydro-4-oxo-6-pteridine) methylamino] benzoyl }-L-
Glutamic acid, molecular formula C19H19N7O6, molecular weight 441.40(presses international relative atomic mass in 2007), by pterin pyridine, to amino
Benzoic acid and Pidolidone combine synthesis.
At present, according to the difference of detection object, mainly there are colorimetry thin layer chromatography, gas in China to the detection method of folic acid
Phase chromatography mass spectrometry (GC/MS), high performance liquid chromatography (HPLC), microbial method, isotope radioimmunology, enzyme linked immunological
Absorption method (Enzyme-linked immunosorbent assay, ELISA), colloidal gold strip method etc..
Instrument analytical method exist sample must dilute through multistep, filter, the pretreatment process such as affinity column enrichment, there is preparation
Complicated, loaded down with trivial details shortcoming, its testing cost is high, and the cycle is long, it is impossible to meet high-volume sample rapid screening, and on-the-spot quickly inspection
The requirement surveyed.ELISA and colloidal gold strip are owned by France in immuno analytical method, have higher sensitivity and specificity, detection
Time the highest and easy and simple to handle to the purity requirement of sample, it is adaptable to the field quick detection of great amount of samples.Regardless of whether be instrument
Affinity column in analysis method pretreatment process is enriched with or during the detection of immune analysis method, its vital impact
Factor or the antigen of high specific and antibody.
Traditional folic acid artificial antigen typically uses mixed anhydride method, and the amino in little molecule can be in the effect of tertiary amine
React with isobutyl chlorocarbonate down, generate reactive intermediate mixed acid anhydride, then react with the primary amino radical on carrier protein, formed
Amide cross-bond.And folic acid small molecule carrier albumen directly couples, due to the sterically hindered pass between carrier protein and little molecule
System, the antibody obtained eventually through immune animal often for the part-structure of little molecule, the specificity of antibody and sensitive
Degree can be greatly affected, especially the friendship to materials such as petrin, pteroic acid, tetrahydrofolic acid, dihydrofoilic acid and para-amino benzoic acid
Fork phenomenon is inevitable.
The generation of animal immune antibody is relevant with the structure of carrier molecule.Equally, carrier molecule not only affects generation
The quantity of antibody, has an effect on type and the affinity of antibody.There are some researches show, the polyatom between hapten and carrier structure
The existence of clearance space is more prone to the antibody with high specific and high-affinity.
The present invention is that modification has the 6-aminocaprolc acid of certain space length as carrier linking arm on carrier protein, will
Hapten is coupled on the linking arm of carrier protein, forms the complete antigen between carrier and hapten with certain space interval.
Summary of the invention
It is an object of the invention to for existing folic acid antigen synthetic technology and the deficiency of corresponding antibodies and defect, it is provided that one
Plant novel folic acid complete antigen synthetic method so that the folic acid monoclonal antibody preparing high specific is possibly realized, the present invention
Another object be to provide the application of folic acid complete antigen.
Technical scheme, the folic acid complete antigen synthetic method of a kind of high specific, step is: at carrier protein
After solution adds 6-aminocaprolc acid (ε-ACA) and water-soluble carbodiimide (EDC), react under room temperature, modified after dialysis
Shown in the carrier protein B(formula 1 of linking arm)；Folic acid (N-{4-[(2-amino-1,4-dihydro-4-oxo-6-pteridine) first ammonia
Base] benzoyl }-Pidolidone) it is dissolved in dimethyl sulfoxide (DMSO), add dicyclohexylcarbodiimide (DCC) and N-hydroxyl
Base succimide (NHS), reaction generates folic acid derivatives, adds carrier protein B, regulates pH value in reaction 8.5, with carrier egg
Amino on white B carries out coupling, and dialysis obtains the folic acid complete antigen (formula 2) of high specific.Complete antigen is dialysed, then
Carry out ultraviolet qualification (Fig. 1).
Carrier protein modifies route:
Formula 1
Folic acid artificial antigen's synthetic route is:
Formula 2
The folic acid complete antigen synthetic method of described high specific, specifically comprises the following steps that
(1) carrier protein is modified: take carrier protein 50mg and be dissolved in 5mL pH9.6 carbonate buffer solution, add
100mg 6-aminocaprolc acid, 30mg water-soluble carbodiimide；Regulation system pH value, in 6 ~ 8, reacts 4h under room temperature；Buffer with PBS
Liquid is dialysed 2 days, and period changes water 4 times, i.e. obtains having modified the carrier protein B of linking arm；
(2) preparation of folic acid complete antigen:
Taking 25mg folic acid, add 2.5mL DMSO and dissolve, regulation pH value is 7.4, then is separately added into 10mg N-hydroxyl fourth two
Acid imide NHS, 23mg DCC, lucifuge mixing under room temperature, stirring reaction 4h, then carrier protein B 100mg prepared by step (1)
Adding in reactant liquor, regulation pH value is 8.5, lucifuge reaction 2h at room temperature 25 DEG C；Dialysing 2 days with PBS, period changes water
4 times, i.e. obtain folic acid complete antigen；
The application of the folic acid complete antigen of described high specific, it is characterised in that: exempted from by folic acid complete antigen compound
Epidemic disease animal obtains polyclonal antibody or monoclonal antibody, and is applied to detect in folic acid.
Described N-{-[(2-amino-Isosorbide-5-Nitrae-dihydro-4-oxo-6-pteridine) methylamino] benzoyl }-Pidolidone,
6-aminocaprolc acid purity is all higher than 95%.
Described carrier protein is: bovine serum albumin BSA, chicken ovalbumin OVA.
The application in preparing folic acid antibody of the above-mentioned folic acid complete antigen compound falls within protection scope of the present invention.
The antibody that above-mentioned folic acid complete antigen compound immune animal obtains falls within protection scope of the present invention, described anti-
Body is polyclonal antibody or monoclonal antibody.
Above-mentioned folic acid complete antigen compound or antibody application in detection folic acid fall within the scope of protection of the invention.
Beneficial effects of the present invention: the present invention is novel folic acid complete antigen synthetic method, and folic acid complete antigen presents
The specific folic acid entirety antigenic structure gone out so that the folic acid monoclonal antibody filtering out high specific is possibly realized.
Test result indicate that, the antiserum titre obtained with the antigen-immunized animal of the present invention is up to 81000, and detection is limited to
0.042ng/mL, 503nhibiting concentration is 0.3ng/mL.The antibody specificity produced is high, highly sensitive.The antigen or anti-of the present invention
Body can be used for the preparation of immune affinity column and sets up ELISA adsorption analysis method and Colloidal gold stripes.
Accompanying drawing explanation
Fig. 1, folic acid antigen ultraviolet spectrogram.
Detailed description of the invention
Experimental technique used in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Embodiment 1 modifies the preparation of carrier protein
Take bovine serum albumin 50mg and be dissolved in 5mL pH9.6 carbonate buffer solution, add 100mg 6-amino own
Acid, 30mg water-soluble carbodiimide.Regulation system pH value, in 6 ~ 8, reacts 4h under room temperature.Dialyse 2 days with PBS, period
Change water 4 times, i.e. obtain having modified the bovine serum albumin carrier protein B of linking arm.
The preparation of embodiment 2 folic acid complete antigen
Taking 25mg folic acid, add 2.5mL DMSO and dissolve, regulation pH value is 7.4, then is separately added into 10mg NHS, 23mg
DCC, lucifuge mixing under room temperature, stirring reaction 4h, add carrier protein B 100mg, regulation pH value is 8.5, room temperature 25 DEG C
Lower lucifuge reaction 2h.Dialysing 2 days with PBS, period changes water 4 times, i.e. obtains folic acid complete antigen.
The preparation of embodiment 3 folic acid coating antigen
Take 25mg folic acid, add 2.5mL DMSO and dissolve, zero degree pre-cooling 30 minutes.Under zero degree, it is separately added into three positive fourths
Amine, isobutyl chlorocarbonate (hapten folic acid, tri-n-butylamine, the mol ratio of isobutyl chlorocarbonate are 1:1.2:1.2), 0 DEG C anti-
Answer 1 hour.Taking 100mg oralbumin/bovine serum albumin, add 10mL 0.1M pH9.6 carbonate buffer solution, 0 DEG C pre-
Cold 30 minutes.Under the conditions of 0 DEG C, the hapten solution of activation is added drop-wise at a slow speed in protein solution, under the conditions of 0 DEG C, reacts 1h,
Then 24h is reacted under room temperature.Dialysing 2 days with PBS, period changes water 4 times, i.e. obtains folic acid coating antigen.
The sero-fast preparation of embodiment 4 folic acid
With the prepared antigen of embodiment 2 as immunogen, selecting 6-8 week old, female BAl BIc/C mice is immune animal, uses
Freund adjuvant carries out immunity, immune mouse 5.
Freund adjuvant immunization method is: head exempts to take appropriate immunogen and mixes with equal-volume Freund's complete adjuvant, after emulsifying is good
Through the immunity of neck dorsal sc multi-point injection, at interval of 3 weeks booster immunizations once.
The sero-fast mensuration of embodiment 5 folic acid
One, using indirect ELISA method detection serum titer, concrete operation step is as follows:
It is coated: the coating antigen 0.05M pH9.6 carbonate buffer solution in embodiment 3 is started multiple proportions from 1 g/mL dilute
Release, 100 L/ holes, 37 DEG C of reaction 2h.
Washing: inclined by solution in plate, dries, and washs 3 times with cleaning mixture, each 3min.
Close: after patting dry, add 200 L/hole confining liquid, 37 DEG C of reaction 2h.Washing post-drying is standby.
Sample-adding: antiserum is started doubling dilution from 1:1000, and join each dilution be coated in hole, 100 L/
Hole, 37 DEG C of reaction 1h；Fully after washing, add the HRP-sheep anti-mouse igg of 1:3000 dilution, 100 L/hole, 37 DEG C of reaction 1h.
After colour developing: ELISA Plate taken out, fully washing, every hole adds the TMB nitrite ion of 100 L, 37 DEG C of lucifuge reactions
15min。
Terminate and measure: every hole adds 50 L stop buffers to terminate reaction, then with the OD in each hole of microplate reader mensuration450Value.
Result interpretation: with OD450Value is more than or equal to the serum corresponding to 2.1 times (i.e. P/N >=2.1) of negative control hole
Highly diluted multiple is the ELISA titer of serum.
Two, lowest detectable limit, half suppression and specific detection
Concrete operation step is as follows:
The working concentration of coating antigen and antibody is determined, with OD by above-mentioned indirect ELISA square formation titrimetry450It is worth 1.5 left
Antigen corresponding time right and antibody concentration are the suitableeest working concentration.
It is coated: coating antigen is diluted to the suitableeest working concentration, 100 L/hole, 37 DEG C of reaction 2h with being coated buffer.
Washing and closing: method operates with above-mentioned indirect elisa method.
Preparation folic acid standard solution: folic acid standard substance DMSO is configured to the mother solution of 0.5mg/mL, then, at sample-adding
Before, then become to need concentration with the PBS solution doubling dilution of 0.01mol/L, the pH7.4 containing 10% methanol.
Sample-adding: every hole adds each concentration standards of folic acid of 50 L doubling dilutions, then adds the 50 the suitableeest dilutions in L/ hole
The antiserum of multiple, 37 DEG C of reaction 1h.Fully after washing, the HRP-sheep anti-mouse igg of addition 1:3000 dilution, 100 L/hole, 37
DEG C reaction 1h.
After chromogenic reaction: ELISA Plate taken out, fully washing, every hole adds the TMB nitrite ion of 100 L, and 37 DEG C of lucifuges are anti-
Answer 15min.
7) terminate and measure: every hole adds 50 L stop buffers to terminate reaction, then with the OD in each hole of microplate reader mensuration450
Value.
8) data process: with the logarithm of each concentration of folic acid as abscissa, with the OD that each concentration of folic acid is corresponding450Value is sat for vertical
Mark, draws standard curve, calculation of half inhibitory concentration (IC50, i.e. OD450Value is when the A0 that zero standard solution is corresponding drops to 50%
Corresponding standard concentration), thus judge whether antiserum has specificity to folic acid.
9) standard substance of folic acid are changed into para-amino benzoic acid, pantothenic acid, nicotinic acid, glutamic acid, petrin, pteroic acid, VB1, VB12,
Methotrexate, biotin, D-tocopherol, tetrahydrofolic acid, measure IC as stated above50Value, and calculate cross reacting rate.
Cross reacting rate (%)=IC50(folic acid)/IC50(intersection thing)
Experiment sets 3 repetitions, results averaged.
Result shows, after four exempt from, mouse resisting anteserum titer is up to 81000, and detection is limited to 0.042ng/mL, folic acid IC50For
0.3ng/mL, the cross reacting rate of each analog is respectively less than 0.1%.
Claims (4)
1. the folic acid complete antigen synthetic method of a high specific, it is characterised in that step is: add in carrier protein solution
Enter 6-aminocaprolc acid ε-ACA and water-soluble carbodiimide EDC, at room temperature react, after dialysis, obtain having modified the load of linking arm
Body protein B；By folic acid N-{4-[(2-amino-1,4-dihydro-4-oxo-6-pteridine) methylamino] benzoyl }-Pidolidone
Being dissolved in dimethyl sulfoxide DMSO, add dicyclohexylcarbodiimide DCC and N-hydroxysuccinimide NHS, reaction generates leaf
Acid derivative, adds the carrier protein B having modified linking arm, and the amino in regulation pH value in reaction 8.5, with carrier protein B enters
Row coupling, dialysis, obtain the folic acid complete antigen of high specific.
The folic acid complete antigen synthetic method of high specific the most according to claim 1, it is characterised in that: described carrier protein
For bovine serum albumin BSA or chicken ovalbumin OVA.
The folic acid complete antigen synthetic method of high specific the most according to claim 1, it is characterised in that specifically comprise the following steps that
(1) carrier protein is modified: take carrier protein 50mg and be dissolved in 5mL pH9.6 carbonate buffer solution, add 100mg
6-aminocaprolc acid, 30mg water-soluble carbodiimide；Regulation system pH value, in 6 ~ 8, reacts 4h under room temperature；With PBS dialysis 2
My god, period changes water 4 times, i.e. obtains having modified the carrier protein B of linking arm；
(2) preparation of folic acid complete antigen:
Taking 25mg folic acid, add 2.5mL DMSO and dissolve, regulation pH value is 7.4, then it is sub-to be separately added into 10mg N-maloyl
Amine NHS, 23mg DCC, lucifuge mixing under room temperature, stirring reaction 4h, the carrier egg having modified linking arm prepared by step (1)
White B 100mg adds in reactant liquor, and regulation pH value is 8.5, lucifuge reaction 2h at room temperature 25 DEG C；With PBS dialysis 2
My god, period changes water 4 times, i.e. obtains folic acid complete antigen.
4. the application of the folic acid complete antigen of high specific described in claim 1, it is characterised in that: by folic acid complete antigen
Compound immune animal obtains polyclonal antibody or monoclonal antibody, and is applied to detect in folic acid.
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| CN105622745A (en) * | 2016-01-22 | 2016-06-01 | 深圳市新产业生物医学工程股份有限公司 | Derivative of amino acid fragment of angiotensin II, angiotensin II antigen and preparation method and application of angiotensin II antigen |
| CN106290903A (en) * | 2016-08-03 | 2017-01-04 | 北京德奥平生物技术有限公司 | A kind of ADMA immunization measures reagent and detection method |
| CN114181102B (en) * | 2021-11-10 | 2023-06-23 | 深圳市检验检疫科学研究院 | Hexaresorcinol hapten, artificial antigen, antibody and synthesis method and application thereof |
| CN116120430A (en) * | 2023-02-27 | 2023-05-16 | 浙江准策生物技术有限公司 | Folic acid complete antigen and antibody, and preparation method and application thereof |
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