CN103483444A - 用于检测和测定小檗碱的抗体、方法和试剂盒 - Google Patents
用于检测和测定小檗碱的抗体、方法和试剂盒 Download PDFInfo
- Publication number
- CN103483444A CN103483444A CN201210197148.XA CN201210197148A CN103483444A CN 103483444 A CN103483444 A CN 103483444A CN 201210197148 A CN201210197148 A CN 201210197148A CN 103483444 A CN103483444 A CN 103483444A
- Authority
- CN
- China
- Prior art keywords
- antibody
- immunogen
- berberine
- synthetic
- protein
- 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
- 238000000034 method Methods 0.000 title claims abstract description 38
- 229940093265 berberine Drugs 0.000 title abstract description 54
- QISXPYZVZJBNDM-UHFFFAOYSA-N berberine Natural products COc1ccc2C=C3N(Cc2c1OC)C=Cc4cc5OCOc5cc34 QISXPYZVZJBNDM-UHFFFAOYSA-N 0.000 title abstract description 52
- YBHILYKTIRIUTE-UHFFFAOYSA-N berberine Chemical compound C1=C2CC[N+]3=CC4=C(OC)C(OC)=CC=C4C=C3C2=CC2=C1OCO2 YBHILYKTIRIUTE-UHFFFAOYSA-N 0.000 title abstract description 51
- 230000002163 immunogen Effects 0.000 claims abstract description 34
- 239000000126 substance Substances 0.000 claims abstract description 19
- 238000001514 detection method Methods 0.000 claims abstract description 10
- 230000000890 antigenic effect Effects 0.000 claims abstract description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 6
- 239000000203 mixture Substances 0.000 claims description 23
- 238000002360 preparation method Methods 0.000 claims description 16
- 239000000463 material Substances 0.000 claims description 15
- 102000004169 proteins and genes Human genes 0.000 claims description 15
- 108090000623 proteins and genes Proteins 0.000 claims description 15
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 13
- 229940098773 bovine serum albumin Drugs 0.000 claims description 13
- 241001465754 Metazoa Species 0.000 claims description 12
- 210000002966 serum Anatomy 0.000 claims description 12
- 238000012360 testing method Methods 0.000 claims description 12
- 229910052799 carbon Inorganic materials 0.000 claims description 8
- 229920001184 polypeptide Polymers 0.000 claims description 8
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 8
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 claims description 5
- 125000001118 alkylidene group Chemical group 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 4
- 239000004793 Polystyrene Substances 0.000 claims description 3
- 229920005615 natural polymer Polymers 0.000 claims description 3
- 229920002223 polystyrene Polymers 0.000 claims description 3
- 229920001059 synthetic polymer Polymers 0.000 claims description 3
- 241000283690 Bos taurus Species 0.000 claims description 2
- 108010044091 Globulins Proteins 0.000 claims description 2
- 102000006395 Globulins Human genes 0.000 claims description 2
- 102000004895 Lipoproteins Human genes 0.000 claims description 2
- 108090001030 Lipoproteins Proteins 0.000 claims description 2
- 108010058846 Ovalbumin Proteins 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 108010071390 Serum Albumin Proteins 0.000 claims description 2
- 102000007562 Serum Albumin Human genes 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- 125000000217 alkyl group Chemical group 0.000 claims description 2
- 150000001720 carbohydrates Chemical group 0.000 claims description 2
- 150000001721 carbon Chemical group 0.000 claims description 2
- 108010074605 gamma-Globulins Proteins 0.000 claims description 2
- 150000004676 glycans Chemical class 0.000 claims description 2
- 229940092253 ovalbumin Drugs 0.000 claims description 2
- 229920001282 polysaccharide Polymers 0.000 claims description 2
- 239000005017 polysaccharide Substances 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 229920006395 saturated elastomer Polymers 0.000 claims description 2
- 210000001685 thyroid gland Anatomy 0.000 claims description 2
- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 claims 5
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims 2
- 230000000676 anti-immunogenic effect Effects 0.000 claims 1
- 102000013415 peroxidase activity proteins Human genes 0.000 claims 1
- 108040007629 peroxidase activity proteins Proteins 0.000 claims 1
- 229920000915 polyvinyl chloride Polymers 0.000 claims 1
- 239000004800 polyvinyl chloride Substances 0.000 claims 1
- 239000000941 radioactive substance Substances 0.000 claims 1
- 238000001179 sorption measurement Methods 0.000 claims 1
- 239000000427 antigen Substances 0.000 abstract description 16
- 102000036639 antigens Human genes 0.000 abstract description 16
- 108091007433 antigens Proteins 0.000 abstract description 16
- 239000011248 coating agent Substances 0.000 abstract description 14
- 238000000576 coating method Methods 0.000 abstract description 14
- 238000011002 quantification Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 26
- 239000000243 solution Substances 0.000 description 19
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 16
- 241000699666 Mus <mouse, genus> Species 0.000 description 14
- 239000000523 sample Substances 0.000 description 11
- 206010035226 Plasma cell myeloma Diseases 0.000 description 9
- 239000012980 RPMI-1640 medium Substances 0.000 description 9
- 201000000050 myeloid neoplasm Diseases 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 9
- 210000004408 hybridoma Anatomy 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- YWAGIZOVUVIWGD-UHFFFAOYSA-O 2-[(17-methoxy-5,7-dioxa-13-azoniapentacyclo[11.8.0.02,10.04,8.015,20]henicosa-1(13),2,4(8),9,14,16,18,20-octaen-16-yl)oxy]acetic acid Chemical compound C1=C2CC[N+]3=CC4=C(OCC(O)=O)C(OC)=CC=C4C=C3C2=CC2=C1OCO2 YWAGIZOVUVIWGD-UHFFFAOYSA-O 0.000 description 7
- 206010003445 Ascites Diseases 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 230000036039 immunity Effects 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 6
- GYFSYEVKFOOLFZ-UHFFFAOYSA-N Berberrubine Chemical compound [Cl-].C1=C2CC[N+]3=CC4=C(O)C(OC)=CC=C4C=C3C2=CC2=C1OCO2 GYFSYEVKFOOLFZ-UHFFFAOYSA-N 0.000 description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 6
- GLYPKDKODVRYGP-UHFFFAOYSA-O berberrubine Natural products C1=C2CC[N+]3=CC4=C(O)C(OC)=CC=C4C=C3C2=CC2=C1OCO2 GLYPKDKODVRYGP-UHFFFAOYSA-O 0.000 description 6
- GLYPKDKODVRYGP-UHFFFAOYSA-N burberrubine Natural products C12=CC=3OCOC=3C=C2CCN2C1=CC1=CC=C(OC)C(=O)C1=C2 GLYPKDKODVRYGP-UHFFFAOYSA-N 0.000 description 6
- 238000000502 dialysis Methods 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 238000001556 precipitation Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 239000002671 adjuvant Substances 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000004587 chromatography analysis Methods 0.000 description 5
- 239000002504 physiological saline solution Substances 0.000 description 5
- 210000000952 spleen Anatomy 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 150000003836 berberines Chemical group 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 238000012856 packing Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- FOCAUTSVDIKZOP-UHFFFAOYSA-N chloroacetic acid Chemical compound OC(=O)CCl FOCAUTSVDIKZOP-UHFFFAOYSA-N 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 238000004945 emulsification Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000012047 saturated solution Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 210000004988 splenocyte Anatomy 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 101150000595 CLMP gene Proteins 0.000 description 2
- 244000247747 Coptis groenlandica Species 0.000 description 2
- 235000002991 Coptis groenlandica Nutrition 0.000 description 2
- 101100382322 Drosophila melanogaster Acam gene Proteins 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 206010070834 Sensitisation Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000194017 Streptococcus Species 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 230000003187 abdominal effect Effects 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002949 hemolytic effect Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 230000000452 restraining effect Effects 0.000 description 2
- 230000008313 sensitization Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 241001480043 Arthrodermataceae Species 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 208000004429 Bacillary Dysentery Diseases 0.000 description 1
- 241001083847 Berberis Species 0.000 description 1
- 241001407408 Berberis fortunei Species 0.000 description 1
- 241001407382 Berberis julianae Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- 241000186227 Corynebacterium diphtheriae Species 0.000 description 1
- 241000218691 Cupressaceae Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 206010017915 Gastroenteritis shigella Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 241000589902 Leptospira Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 229920002534 Polyethylene Glycol 1450 Polymers 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 206010039587 Scarlet Fever Diseases 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical class OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 241000607626 Vibrio cholerae Species 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 206010001093 acute tonsillitis Diseases 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 238000003453 ammonium sulfate precipitation method Methods 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000024835 cytogamy Effects 0.000 description 1
- 230000037304 dermatophytes Effects 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 206010013023 diphtheria Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 208000001848 dysentery Diseases 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000001254 matrix assisted laser desorption--ionisation time-of-flight mass spectrum Methods 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000003032 molecular docking Methods 0.000 description 1
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229940005654 nitrite ion Drugs 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000009965 odorless effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000000242 pagocytic effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000012716 precipitator Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 208000008128 pulmonary tuberculosis Diseases 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 201000005113 shigellosis Diseases 0.000 description 1
- PCMORTLOPMLEFB-ONEGZZNKSA-N sinapic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC(OC)=C1O PCMORTLOPMLEFB-ONEGZZNKSA-N 0.000 description 1
- PCMORTLOPMLEFB-UHFFFAOYSA-N sinapinic acid Natural products COC1=CC(C=CC(O)=O)=CC(OC)=C1O PCMORTLOPMLEFB-UHFFFAOYSA-N 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 description 1
- 230000004960 subcellular localization Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 229940118696 vibrio cholerae Drugs 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biomedical Technology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明提供包括小檗碱或其衍生物的半抗原,与赋予抗原性的载体物质结合的前述半抗原的免疫原,与标记试剂结合的前述半抗原的偶联物(包被原),以及抗前述免疫原的抗体,该抗体能与完整的小檗碱中的至少一种结构性抗原决定部位结合。本发明还提供用于检测或定量样品中小檗碱的方法和试剂盒,以及前述偶联物和前述抗体在检测或定量小檗碱中的用途。本发明对小檗碱具有特异性,可用于样品中检测小檗碱的存在和测定小檗碱的含量。
Description
技术领域
本发明涉及小分子半抗原的抗体及其制备方法与应用,特别是涉及小檗碱及其衍生物的抗体及其制备方法与应用。
背景技术
本发明涉及用于检测和定量小檗碱的方法和试剂盒,以及其中使用的半抗原、免疫原、包被原和抗体。
其中“检测”是指定性分析物质是否存在。
其中“测定”是指对物质进行定量分析。
小檗碱是一种季铵生物碱,分子式[C20H18NO4]+Cl-,分子量371.8。又称黄连素。存在于黄连以及其它小檗属植物中。黄色针状结晶。熔点145℃。可溶于热水、乙醇、难溶于***、苯。能与氯仿和丙酮等生成复合物。又称黄连素。常用其盐酸盐。黄色结晶性粉末。无臭,味极苦。微溶于水,不溶于醚、氯仿、醇等。
药理作用
现代药理学研究表明其抗菌谱广,体外对多种革兰阳性及阴性菌均具抑菌作用,其中对溶血性链球菌、金葡菌、霍乱弧菌、脑膜炎球菌、志贺痢疾杆菌、伤寒杆菌、白喉杆菌等有较强的抑制作用,低浓度时抑菌,高浓度时杀菌。对流感病毒、阿米巴原虫、钩端螺旋体、某些皮肤真菌也有一定抑制作用。体外实验证实黄连素能增强白细胞及肝网状内皮***的吞噬能力。痢疾杆菌、溶血性链球菌、金葡菌等极易对本品产生耐药性。本品与青霉素、链霉素等并无交叉耐药性。小檗碱的盐酸盐(俗称盐酸黄连素)已广泛用于治疗胃肠炎、细菌性痢疾等,对肺结核、猩红热、急性扁桃腺炎和呼吸道感染也有一定疗效。中医常用黄连、黄柏、三颗针及十大功劳等作清热解毒药物,其中主要有效成分即小檗碱。
小檗碱化学结构如下:
目前建立了多种小檗碱的定性与定量检测和测定分析方法,如薄层层析法、HPLC法,HPLC-MS法等。其中最常用的方法是高效液相色谱法。但含小檗碱的生物样品(包括血浆、尿、唾液等)含有大量的影响含量测定的蛋白质及内源性物质;同时小檗碱可能呈结合状态,必须经过处理,排除内源性杂质和代谢物的干扰,使结合状态的小檗碱游离后才能测定;同时还需要浓缩以满足仪器检测灵敏度的要求,处理程序复杂,费时费力;同时由于小檗碱进入体内后,组织中的分布是极其微量的,即使经过富集,进行含量测定仍是极其困难的。另外,对于小檗碱在靶器官和组织中分布,以及细胞和亚细胞定位的研究,需要通过免疫组织化学和westblot等方法,但由于缺乏小檗碱的抗体,阻碍了此方面的研究。因此,有必要开发出一种用于检侧和测定生物样品中小檗碱的方法,对阐明相关药材或复方的作用机理,以及其体内分布和代谢研究,将具有特别的价值。
发明内容
本发明的半抗原小檗碱可提供确定的结构性抗原决定部位,但是它本身并不具备免疫原性,因此必须要偶联到适宜的赋予抗原性的载体物质上,这样所形成的免疫原才会在注射到宿主动物体内后诱发免疫应答。因此,本发明提供一种如下结构的免疫原:
其中R是0至6个碳的烷基桥(连接半抗原与载体物质的桥),优选R=0,即P1直接与小檗碱羰基碳原子结合,或R是C1-C6取代或未取代的、直链或支链的、饱和或不饱和的亚烷基,更优选R是C1-C4未取代的、直链的、饱和的亚烷基。
P是赋予抗原性的载体物质。载体物质选自蛋白质、蛋白质片合成的多肽或半合成的多肽。其中蛋白质、蛋白质片段选自白蛋白、血清蛋白、球蛋白、目镜蛋白和脂蛋白,优选为牛血清白蛋白、卵清蛋白、牛丙种球蛋白、甲状腺素结合球蛋白、锁眼形血蓝蛋白(keyholelimpet haemocyanin,KLH),更优选自锁眼形血蓝蛋白或牛血清蛋白(BSA)。合成多肽或半合成多肽是具有足够数量的可利用氨基的合成聚氨基酸,优选多聚赖氨酸。合成或天然聚合物是带有反应官能基的聚合物材料,特别是能够结合到半抗原产生免疫原的碳水化合物、酵母或多糖。
半抗原的制备本发明描述了将小檗碱进行结构改造后,得到9-O-羧甲基-小檗红碱,并用碳二亚胺法将半抗原与赋予抗原性的载体物质发生的偶联作用,产生免疫原。以小檗碱为起始原料,特定温度加热产生的混合物溶解于水,用三倍量的氯仿提取。氯仿被蒸发,残渣经Sephadex LH20和ODS凝胶柱色谱纯化得到小檗红碱。之后,在室温条件下向小檗红碱的氯仿溶液添加氯乙酸搅拌反应三小时,溶剂被蒸发后,残渣经层析得到9-O-羧甲基-小檗红碱,产物纯化经核磁共振氢谱和碳谱(1HNMR和13CNMR)确证。
免疫原的合成
9-O-羧甲基-小檗红碱和1-乙基-3-(3′-二甲基氨丙基)碳二亚胺盐酸盐(EDC)添加到吡啶溶液中。向混合物中滴加BSA或HSA溶液,然后在室温下搅拌3小时。随后,混合物在4度用水透析两天,换液五次,分装保存于-20℃的冰箱中。
合成免疫原的分析方法为了确认载体物质上结合有适当的半抗原,在免疫前,使用紫外分光度法或矩阵辅助紫外激光解析/电离飞行时间质谱(MALDI-TOF MS)对每个免疫原进行评估。小檗碱免疫原反应物和产物的吸收峰相比(200nm-400nm),可判断是否偶合,并根据反应物和产物的摩尔吸光系数计算偶联物与半抗原的比例。
使用voyager STR生物分光度测量方法研究站激光解析质谱与延迟萃取结合进行MALDI-TOF质谱。将每个要分析的等分试样在0.1%的三氟乙酸水溶液中稀释制成1mg/ml的试样溶液。使用芥子酸基质和牛血清白蛋白作为外标分析等分试样(1uL)。
对于优选的载体物质,牛血清白蛋白或KLH而言,优选半抗原与蛋白质的结合比为6-15∶1。
第二方面,免疫检测时需要有吸附在固相载体上的含有半抗原结构的包被原,因此,本发明提供一种如下结构的偶合物;
其中R是0至6个碳的烷基,P’是不同于免疫原载体的一种载体物质。
偶合物的合成
1.半抗原的制备 本发明描述了将小檗碱进行结构改造后,得到9-O-羧甲基-小檗红碱,并用碳二亚胺法将半抗原与赋予抗原性的载体物质发生的偶联作用,产生免疫原。以小檗碱为起始原料,特定温度加热产生的混合物溶解于水,用三倍量的氯仿提取。氯仿被蒸发,残渣经Sephadex LH20和ODS凝胶柱色谱纯化得到小檗红碱。之后,在室温条件下向小檗红碱的氯仿溶液添加氯乙酸搅拌反应三小时,溶剂被蒸发后,残渣经层析得到9-O-羧甲基-小檗红碱,产物纯化经核磁共振氢谱和碳谱(1HNMR和13CNMR)确证。
2.免疫原的合成
9-O-羧甲基-小檗红碱和1-乙基-3-(3′-二甲基氨丙基)碳二亚胺盐酸盐(EDC)添加到吡啶溶液中。向混合物中滴加BSA或HSA溶液,然后在室温下搅拌3小时。随后,混合物在4度用水透析两天,换液五次,分装保存于-20℃的冰箱中。
免疫检测时要将该复合物固定在支撑底物上。优选地,该方法进一步包括将所述复合物固定到支撑底物上,优选固体支持物,最优选聚苯乙烯固体支持物。
另一方面,本发明涉及对于本发明第一方面的免疫原产生的抗体,这些抗体能够至少与一个小檗碱结构上的表位结合,优选与完整的小檗碱结构表位相结合,该抗体是多克隆的,或是单克隆的,优选为单克隆抗体,且具有对小檗碱的特异性。
本发明进一步提供一种制备抗体的方法,该方法包含通过重复给予根据本发明的小檗碱的免疫原免疫动物,优选脊稚动物,最优选哺乳动物,并收集从免疫动物得到的血清的步骤。
另一方面,本发明包括在试样中检测或测定小檗碱的方法,该方法包括将供试样品和本发明的抗体一同与固定在固相支持物的偶联物接触,再与标记二抗接触,检测或测定标记信号,从校准曲线中推出样品中小檗碱的存在或含量。
另一方面,本发明还描述了如何将针对这种免疫原产生的抗体用于开发可用来检测和测定小檗碱的存在的通用分析方法和相应的试剂盒。本试剂盒包括用本发明的固定包被原或其混合物和本发明的抗体或其混合物。该试剂盒可以任意地包括应用所述缀合物和所述抗体在试样中检测或测定小檗碱的指导。优选地,试样是溶液,如生物流体。更优选地,试样是血清或尿。在本发明的方法和试剂盒中,首选各自不同的交联剂(免疫原的和包被原的)。
另一方面,本发明包括使用本发明的固定包被原或其混合物和本发明的抗体或其混合物在试验试样如生物流体中检测或测定小檗碱。
为了产生多克隆抗血清,将免疫原与Freund佐剂完全乳化,并且将混合物注射入宿主动物体内,如兔、羊、鼠、天竺鼠或马。进一步注射(加强)并取血清试样评价抗体效价。达到最佳效价时,随后将宿主动物放血得到适当体积的特异性的抗血清。要求的抗体纯化水平视计划的应用而定。对于许多目的,根本不需要纯化,但是,在其它情况下,例如抗体固定在固体载体上,纯化步骤能够除去不想要的物质和除去非特异性的结合。本发明特异性抗体作为试剂在免疫试验中用于测定或检测生物流体中的小檗碱的试剂是有用的。
单克隆抗体的制备本发明所述单克隆抗体是指获自基本上同源的抗体群的抗体,即组成该群体的抗体个体都相同,除了可能存在少量可能的自发突变。
本发明的抗体可以通过本领域内技术人员已知的各种技术进行制备。例如,本发明完全抗原,可被施用于动物以诱导单克隆抗体的产生。对于单克隆抗体,可利用杂交瘤技术来制备或可用重组DNA法制备。骨髓瘤细胞可选鼠类的骨髓瘤细胞系,包括衍生自MOPC-21和MPC-11小鼠肿瘤的骨髓瘤细胞系以及SP-2/0、NZO或X63-Ag8-653细胞,以及人骨髓瘤和小鼠-人杂合骨髓瘤细胞系。
对杂交瘤细胞生长于其中的培养基进行分析以检测具有所需特异性的单克隆抗体的产生,如通过体外结合分析,例如酶联免疫吸附分析(ELISA)或放射免疫分析(RIA)。表达抗体的细胞的位置可用FACS进行检测。然后,可将杂交瘤克隆通过有限稀释步骤形成亚克隆,并通过标准方法生长。为了达到这一目的而使用的适合的培养基包括,例如,DMEM或RPMI-1640培养基。此外,杂交瘤细胞可在动物体内作为腹水瘤生长。
由亚克隆分泌的单克隆抗体从培养基、腹水或血清中,通过常规的免疫球蛋白纯化工艺适当地得到分离,这些纯化工艺为例如,蛋白A-琼脂糖法(protein A-Sepharose)、羟基磷灰石层析、凝胶电泳、透析或亲和层析。
本发明的一个优选的方案中,抗小檗碱单克隆抗体采用Balb/C小鼠腹水生产单克隆抗体的方法制备。将约1-2×106杂交瘤细胞接种到致敏的小鼠腹腔内,2周内可见腹部明显胀大。抽取腹水,经饱和硫酸按沉淀法粗提出IgG,再将粗提的抗体经亲和层析柱(ProteinG-Sephrose)纯化。
具体实施方式
实施例1
免疫原和包被原的制备
免疫原和包被原采用同样的方法制备合成。小檗碱(3克),在190度加热15分钟。由此产生的混合物溶解于水,用三倍量的氯仿提取。氯仿被蒸发,残渣经Sephadex LH20和ODS凝胶柱色谱纯化得到小檗红碱2g。之后,在室温条件下向小檗红碱的氯仿溶液(100mg,0.31mol)添加氯乙酸300mg(3.17mol)搅拌反应三小时,溶剂被蒸发后,残渣经层析得到9-O-羧甲基-小檗红碱,将其与标准品进行核磁共振比对。
8毫克9-O-羧甲基-小檗红碱(0.021毫摩尔)和8毫克1-乙基-3-(3’-二甲基氨丙基)碳二亚胺盐酸盐(EDC,0.042毫摩尔)添加到1毫升吡啶溶液中。向混合物中滴加1毫升BSA或HSA溶液(8毫克),然后在室温下搅拌3小时。随后,混合物在4度用水透析两天,换液五次,冻干。
实施例2
免疫原和包被原的鉴定
精确配制适宜浓度的小檗碱与BSA、PLL标准溶液,将待检样品稀释到适当浓度后,用紫外分光光度计测出小檗碱、BSA、PLL、小檗碱-BSA、小檗碱-PLL的全波长图谱及最大吸收波长处的吸光值。将(A)和BSA(B)分别用PBS配置成0.05mg/ml和1mg/ml,用PBS将小檗碱-BSA(C)按1∶4稀释,在200-400nm紫外光谱范围内每1nm扫描一个点。测得这三种溶液在A物质和B物质最大吸收波长(am、bm)处的吸收值,即AAam、AAbm、ABam、ABbm、ACam、ACbm。根据公式A=εcl(c为摩尔浓度mol/l,l为比色杯厚度(cm))。计算A物质在A、B物质吸收峰波长处的摩尔分子消光值,即εAam、εAbm;以及B物质在A、B物质吸收峰波长处的摩尔分子消光值,即εBam、εBbm。
按以下公式计算A物质和B物质的偶联比:
GA/BSA=(Acam×εBbm-Acbm×εBam)/(Acbm×εAam-Acam×εAbm)。
实施例3
包被原固定
用碳酸盐缓冲溶液将包被原稀释到适当浓度,加到96孔酶标板中,每孔100μl,4℃过夜,0.05%PBS-T清洗三次,每次五分钟,再每孔加入100μl 10%血清,4℃过夜封闭未结合包被原的位点,0.05%PBS-T清洗三次后-20℃保存备用。
实施例4
用制备的免疫原免疫小鼠
饲养7周龄雄性BALB/c小鼠8只,用免疫原免疫其中7只,另外一只不免疫,以作阴性对照。免疫程序:第一次基础免疫,免疫剂量为50μg/只,用生理盐水稀释到适当体积,再加等体积弗式完全佐剂(FCA),采用注射器推拉法乳化成油包水(W/O)状态,可将一小滴混合液滴在清水的液面上,若混合液凝聚不散,则可认为混合液已乳化完全。将乳化好的抗原进行背部皮下多点注射(0.3ml/只);2周后以相同剂量的免疫原与弗式不完全佐剂(FIA)混合乳化,进行加强免疫,腹腔注射(0.3ml/只);此后每隔2-3周加强1次,方法同第二次。从每四次免疫开始,每次免疫后1周,断尾法采少量血,分离血清,间接ELISA法测效价。
采用棋盘滴定法测定血清的效价。将采得的血清从1∶100开始倍比稀释成8个梯度,以100μl/孔加入酶标板,37℃孵育1h后洗板。阴性对照用未免疫过的小鼠血清,空白对为PBS。加二抗:100μl/孔,37℃孵育1h,洗板。加显色液TMB:100μl/孔,于37℃孵育15min,在波长450nm处用酶标仪读出各孔的A值。以血清样品(P)与阴性血清(N)OD450比值大于2.1时定义为阳性,抗血清为阳性的最大稀释倍数的倒数定义为抗血清的效价。当效价大于64000时即可用于融合。
实施例5
单克隆抗体的制备
1、小鼠骨髓瘤细胞的准备
融合前7天复苏骨髓瘤细胞SP2/O,使细胞密度在2×105-6个/毫升,融合前一天换液,使细胞处于对数生长期。融合前用吸管将细胞轻轻从培养瓶中吹打下来,转移至50ml离心管,用RPMI-1640培养液洗1次(1000rpm,5min)。用RPMI-1640培养液重悬细胞,计数。
2、免疫脾细胞的制备
取待融合的Balb/C小鼠,融合前4天冲击免疫,50μg/只,不加佐剂,直接用生理盐水稀释到0.3ml,腹腔注射。融合当天断颈处死小鼠,浸泡于75%乙醇溶液5min,随后放入超净台里解剖。用无菌手术打开腹腔取出脾脏,放入合适大小的玻璃平皿中,按以下方法制成单个脾细胞悬液:用10ml注射器往脾内注入约0.2-0.5ml无血清1640培养液使其胀大,再用弯曲的针头多点刺破脾膜,然后用注射器吸取无血清1640培养液从脾的一端向另一端吹打,直至脾脏完全变白,用无血清1640培养液将平皿加满,静置1min,使非单细胞组织块沉淀,将单细胞悬液移入50mL塑料离心管中,1200rpm离心5min,弃上清,用20ml无血清1640培养液重悬细胞,计数。
3、细胞融合及培养
融合前先将1mL的PEG-1450和20mL的RPMI-1640基础培养基于37℃预温。将骨髓瘤细胞与脾细胞混合于50mL离心管内(脾细胞:1×108,骨髓瘤细胞:2.5×107),离心(1200rpm×5min),弃上清。在37℃水浴中沿管壁加入PEG 1ml,60秒内加完,边加边转动离心管,不宜吹打,加完后在37℃水浴中静止反应1min,再加入RPMI-1640基础培养基以终止PEG的作用,在1min内加完1ml,再在1min内加完2ml,然后逐渐加快速度,在五分钟内加完15ml。融合细胞终止后于900rpm×8min离心弃上清,加入预温的HAT培养基至所需的细胞浓度,并轻轻将细胞混匀,按100ul/孔加到96孔细胞培养板中。待融合细胞在HAT中培养7天左右后,以HAT培养基半量换液一次,第9-10天间接ELISA检测。
4、杂交瘤细胞的克隆化培养
采用的细胞克隆化培养方法是有限稀释法。将要克隆的细胞轻轻吹起,计数,然后稀释成80个/ml,种两排,100μl/孔。再稀释一倍,种两排,以此类推,最后得到四个浓度,使各孔的细胞分别为8个、4个、2个、1个。10天左右后观察胞,将只有一个杂交瘤集落的孔挑出来检测,若为阳性,再用同样的有限稀释法重复克隆2次,100%阳性后即得到稳定分泌单克隆株细胞株。得到的单克隆抗体细胞株在液氮中冻存。
实施例6
单克隆抗体的大量制备
采用腹水法大量制备单克隆抗体。
给6-8周龄的雌性Balb/C小鼠腹腔注射0.2mL/只福氏不完全佐剂致敏,3天后,接种杂交瘤细胞。将对数生长期的杂交瘤细胞以1000rpm离心5min,弃上清液,用生理盐水悬浮细胞沉淀,并将细胞数调至(1-2)×106个/mL,每只小鼠腹腔注射0.5mL细胞悬液。接种7-10天后,注意观察小鼠状态,可见小鼠腹部明显膨大,断颈处死小鼠,用镊子提起小鼠腹部皮肤,剪一小口,然后从两边向小鼠背部方向剪开,充分暴露腹部,然后用5ml注射器吸出腹腔内全部腹水。将取得的腹水于3000rpm离心20min。离心后收集上清,分装,于-20℃冻存。
实施例6
抗体的纯化
采用硫酸铵沉淀法对抗体进行纯化。
取20ml腹水,加生理盐水20ml,再逐滴加入(NH4)2SO4饱和溶液10ml,使成20%(NH4)2SO4溶液,边加边搅拌,充分混合后,静置30min。3000r/min离心20min,弃去沉淀。在上清液中再加(NH4)2SO4饱和溶液30ml,使成50%(NH4)2SO4溶液,充分混合,静置30min。3000r/min离心20min,弃上清。于沉淀中加20ml生理盐水,使之溶解,再加(NH4)2SO4饱和溶液10ml,使成33%(NH4)2SO4溶液,充分混合后,静置30min。3000r/min离心20min,弃上清,以除去白蛋白。重复步骤5,2~3次。用10ml生理盐水溶解沉淀,装入透析袋。在常水中透析过夜,再在生理盐水中于4℃透析24h,中间换液数次。离心去沉淀(去除杂蛋白),上清液即为粗提IgG。最后过SephadexG150层析柱。以0.01Mol/L pH7.4PBS(0.03Mol/LNaCl)洗脱,收集洗脱液。即可得到纯化的抗小檗碱抗体。
实施例7
建立间接ELISA法,测定及检测含小檗碱试样。
1、建立标准曲线
用PBS(pH 7.4)配制系列浓度(16、8、4、2、1、0.5、0.25、0μg·ml-1)的小檗碱标准溶液。用间接竞争ELISA方法测定小檗碱对包被抗原-抗体结合反应的抑制率。取50μl标准溶液与50μl按一定比例稀释的抗体混匀,加到连接有包被原的96孔酶标板中,37℃孵育一小时,洗板,加二抗100μl,37℃孵育一小时,洗板后显色,检测OD450值。以小檗碱浓度为0对应的孔A450值为AO,以其他各种浓度小檗碱对应的孔A450值为A,以抑制率(1-A/AO)×100%为纵坐标,以小檗碱浓度对数(Log2C)为横坐标,进行线性拟合,绘制标准曲线,建立直线回归方程。
2、小檗碱的检测
取50μl试样与50μl按一定比例稀释的抗体混匀,加到连接有包被原的96孔酶标板中,37℃孵育一小时,洗板,加二抗100μl,37℃孵育一小时,洗板后显色,检测OD450值。将OD450带到标准曲线中,计算试样中的小檗碱浓度。
Claims (21)
2.根据权利要求1的免疫原,R=0,即P1直接与甘草酸羰基碳原子结合。
3.根据权利要求1的免疫原,R是C1-C6取代或未取代的、直链或支链的、饱和或不饱和的亚烷基。
4.根据权利要求1或3的免疫原,R是C1-C4未取代的、直链的、饱和的亚烷基。
5.根据权利要求1的免疫原,其中载体物质为蛋白质、蛋白质片段、合成或天然多肽或半合成多肽、合成或天然聚合物。
6.根据权利要求1或5的免疫原,其中蛋白质、蛋白质片段选自白蛋白、血清蛋白、球蛋白、和脂蛋白。
7.根据权利要求1或5的免疫原,其中蛋白质、蛋白质片段选自牛血清白蛋白、卵清蛋白、牛丙种球蛋白、甲状腺素结合球蛋白、锁眼形血蓝蛋白(keyhole limpet haemocyanin,KLH)等。
8.根据权利要求1或5的免疫原,其中合成或天然多肽或半合成多肽是具有足够数量的可利用氨基的合成聚氨基酸。
9.根据权利要求1或5的免疫原,其中合成或天然聚合物选自碳水化合物、酵母或多糖。
10.抗如权利要求1至9的免疫原的抗体,其中抗体能与完整的甘草酸中的至少一种结构性抗原决定部位结合。
11.制备如权利要求10中的抗体的方法,该方法包括将如权利要求1至9的免疫原重复给予动物而使动物免疫,以及从免疫动物中收集所得到的血清抗体;以及利用免疫学上可接受的细胞系与产生抗体的免疫动物单克隆细胞系融合后产生的细胞系,所制备的单克隆抗体。
13.如权利要求12偶合物,其偶合物固定在固相支持物上。
14.根据权利要求13的固相支持物可为聚苯乙烯或聚氯乙烯等具有一定吸附作用的材料。
15.根据权利要求13或14的固相支持物,优选化学处理高吸附表面的聚苯乙烯酶标板。
16.用于检测和测定样品中甘草酸的方法,该方法包括将供试样品和如权利要求10和11中的抗体一同与如权利要求12至15中的固定在固相支持物的偶联物接触,再与标记二抗接触,检测或测定标记信号,从校准曲线中推出样品中甘草酸的存在或含量。
17.根据权利要求16的标记二抗,其标记试剂选自酶、发光物质、放射性物质或它们的混合物。
18.根据权利要求16的二抗,标记试剂是过氧化物酶。
19.根据权利要求16的二抗,标记试剂是辣根过氧化物酶(HRP)。
20.根据权利要求14或15的偶合物,发光物质是生物发光物质、化学发光物质或荧光物质。
21.用于检测和测定甘草酸的试剂盒,该试剂盒包括如权利要求12至15中任一项的偶联物或其混合物,以及如权利要求10或11的抗体或其混合物。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201210197148.XA CN103483444A (zh) | 2012-06-15 | 2012-06-15 | 用于检测和测定小檗碱的抗体、方法和试剂盒 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201210197148.XA CN103483444A (zh) | 2012-06-15 | 2012-06-15 | 用于检测和测定小檗碱的抗体、方法和试剂盒 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN103483444A true CN103483444A (zh) | 2014-01-01 |
Family
ID=49824079
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201210197148.XA Pending CN103483444A (zh) | 2012-06-15 | 2012-06-15 | 用于检测和测定小檗碱的抗体、方法和试剂盒 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN103483444A (zh) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1297153A (zh) * | 2000-09-27 | 2001-05-30 | 刘文泰 | 用特异性抗体进行中药成分测定的方法 |
CN1629160A (zh) * | 2003-12-15 | 2005-06-22 | 李耐三 | 13-已基小檗碱盐的制备及其抗病毒和抗菌作用 |
CN101422178A (zh) * | 2008-11-14 | 2009-05-06 | 宁夏农林科学院 | 小檗碱吡虫啉水剂 |
CN101486762A (zh) * | 2008-01-18 | 2009-07-22 | 北京中医药大学 | 用于检测和测定绿原酸的抗体、方法和试剂盒 |
CN101497663A (zh) * | 2008-01-29 | 2009-08-05 | 北京中医药大学 | 用于检测和测定黄芩苷的抗体、方法和试剂盒 |
-
2012
- 2012-06-15 CN CN201210197148.XA patent/CN103483444A/zh active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1297153A (zh) * | 2000-09-27 | 2001-05-30 | 刘文泰 | 用特异性抗体进行中药成分测定的方法 |
CN1629160A (zh) * | 2003-12-15 | 2005-06-22 | 李耐三 | 13-已基小檗碱盐的制备及其抗病毒和抗菌作用 |
CN101486762A (zh) * | 2008-01-18 | 2009-07-22 | 北京中医药大学 | 用于检测和测定绿原酸的抗体、方法和试剂盒 |
CN101497663A (zh) * | 2008-01-29 | 2009-08-05 | 北京中医药大学 | 用于检测和测定黄芩苷的抗体、方法和试剂盒 |
CN101422178A (zh) * | 2008-11-14 | 2009-05-06 | 宁夏农林科学院 | 小檗碱吡虫啉水剂 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2602849C (en) | Doxorubicin immunoassay | |
CA2602790C (en) | Docetaxel immunoassay | |
CN103739703B (zh) | 甘胆酸免疫原、抗甘胆酸特异性抗体及检测试剂 | |
CN102917592A (zh) | 利培酮免疫测定 | |
CN102621326B (zh) | 一种检测食品中呋喃它酮代谢物含量的方法 | |
CN108196054A (zh) | 一种检测甘草酸的试纸条及其制备方法和应用 | |
Yi et al. | Simultaneous detection of plant growth regulators jasmonic acid and methyl jasmonate in plant samples by a monoclonal antibody-based ELISA | |
CN109824673B (zh) | 一种佐匹克隆人工半抗原、人工抗原及其制备方法和应用 | |
CN114315722A (zh) | 唑虫酰胺人工半抗原及其抗体的制备与应用 | |
CN103159648A (zh) | 苄嘧磺隆通用半抗原、人工抗原及其制备方法与应用 | |
US6946547B2 (en) | Ecstasy-class analogs and use of same in detection of ecstasy-class compounds | |
CN101486762A (zh) | 用于检测和测定绿原酸的抗体、方法和试剂盒 | |
CN105884881A (zh) | 用于检测和测定木犀草苷的抗体、方法和试剂盒 | |
CN111763658A (zh) | 一株分泌抗二硝托胺单克隆抗体的杂交瘤细胞株及其应用 | |
CN111154000A (zh) | 一种抗西马特罗单克隆抗体及其应用 | |
CN103804491B (zh) | 1,5-脱水山梨醇免疫原及其特异性抗体及检测试剂 | |
CN106929479B (zh) | 一株维生素b2单克隆抗体杂交瘤细胞株gz-4及其应用 | |
CN101497663A (zh) | 用于检测和测定黄芩苷的抗体、方法和试剂盒 | |
CN103483444A (zh) | 用于检测和测定小檗碱的抗体、方法和试剂盒 | |
CN114014774A (zh) | 一种氟胺酮人工半抗原、人工抗原及其制备方法和应用 | |
CN103387611A (zh) | 用于检测和测定甘草酸的抗体、方法和试剂盒 | |
US8426143B2 (en) | Gemcitabine immunoassay | |
CN105237633A (zh) | 一种绿原酸完全抗原及其制备方法和应用 | |
CN114644572B (zh) | 一种溴己新半抗原、人工抗原、抗体及其制备方法和应用 | |
CN105440128A (zh) | 用于检测和测定连翘苷的抗体、方法和试剂盒 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
EXSB | Decision made by sipo to initiate substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
WD01 | Invention patent application deemed withdrawn after publication | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20140101 |