CN106680485A - Enzyme-linked immunoassay kit for spectinomycin medicament and application thereof - Google Patents
Enzyme-linked immunoassay kit for spectinomycin medicament and application thereof Download PDFInfo
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- CN106680485A CN106680485A CN201611046359.8A CN201611046359A CN106680485A CN 106680485 A CN106680485 A CN 106680485A CN 201611046359 A CN201611046359 A CN 201611046359A CN 106680485 A CN106680485 A CN 106680485A
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- spectinomycin
- enzyme
- detecting kit
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- immunologic detecting
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- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 title claims abstract description 91
- 229960000268 spectinomycin Drugs 0.000 title claims abstract description 89
- 239000003814 drug Substances 0.000 title claims abstract description 21
- 238000002965 ELISA Methods 0.000 title claims abstract description 15
- 102000004190 Enzymes Human genes 0.000 claims abstract description 47
- 108090000790 Enzymes Proteins 0.000 claims abstract description 47
- 239000000243 solution Substances 0.000 claims abstract description 24
- 238000001514 detection method Methods 0.000 claims abstract description 19
- 235000013336 milk Nutrition 0.000 claims abstract description 19
- 239000008267 milk Substances 0.000 claims abstract description 19
- 210000004080 milk Anatomy 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 15
- 229940092253 ovalbumin Drugs 0.000 claims abstract description 11
- 239000000758 substrate Substances 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims description 25
- 230000001900 immune effect Effects 0.000 claims description 21
- 239000003593 chromogenic compound Substances 0.000 claims description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 9
- 238000004140 cleaning Methods 0.000 claims description 9
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 8
- 239000012086 standard solution Substances 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 7
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims description 6
- 229940005654 nitrite ion Drugs 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- 230000008878 coupling Effects 0.000 claims description 5
- 238000010168 coupling process Methods 0.000 claims description 5
- 238000005859 coupling reaction Methods 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 claims description 4
- NPWGWQRXHVJJRD-UHFFFAOYSA-N N-hydroxyglycine Chemical compound ONCC(O)=O NPWGWQRXHVJJRD-UHFFFAOYSA-N 0.000 claims description 4
- 108010058846 Ovalbumin Proteins 0.000 claims description 4
- 239000007983 Tris buffer Substances 0.000 claims description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 4
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 108010088751 Albumins Proteins 0.000 claims description 2
- 102000009027 Albumins Human genes 0.000 claims description 2
- 102000014914 Carrier Proteins Human genes 0.000 claims description 2
- 108010078791 Carrier Proteins Proteins 0.000 claims description 2
- 108060003552 hemocyanin Proteins 0.000 claims description 2
- 239000003755 preservative agent Substances 0.000 claims description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims 1
- 108010000912 Egg Proteins Proteins 0.000 claims 1
- 102000002322 Egg Proteins Human genes 0.000 claims 1
- 235000014103 egg white Nutrition 0.000 claims 1
- 210000000969 egg white Anatomy 0.000 claims 1
- 235000013601 eggs Nutrition 0.000 claims 1
- 210000001685 thyroid gland Anatomy 0.000 claims 1
- 238000005406 washing Methods 0.000 abstract description 2
- 239000012089 stop solution Substances 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 23
- 241001494479 Pecora Species 0.000 description 11
- 230000015572 biosynthetic process Effects 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000003547 immunosorbent Substances 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 206010035226 Plasma cell myeloma Diseases 0.000 description 4
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- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene chloride Substances ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 4
- 201000000050 myeloid neoplasm Diseases 0.000 description 4
- 102100033620 Calponin-1 Human genes 0.000 description 3
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 description 3
- 101000652736 Homo sapiens Transgelin Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 238000005138 cryopreservation Methods 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- XOJVVFBFDXDTEG-UHFFFAOYSA-N pristane Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 210000004988 splenocyte Anatomy 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- -1 2- iminothiolanes hydrochloride Chemical class 0.000 description 1
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical class CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 235000011330 Armoracia rusticana Nutrition 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 206010018612 Gonorrhoea Diseases 0.000 description 1
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 206010068676 Pneumoretroperitoneum Diseases 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 208000005727 Retropneumoperitoneum Diseases 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 241001655322 Streptomycetales Species 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 239000004411 aluminium Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 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
- 230000007910 cell fusion Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- KZNICNPSHKQLFF-UHFFFAOYSA-N dihydromaleimide Natural products O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229960003276 erythromycin Drugs 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 125000004836 hexamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012473 microbial detection method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 150000003222 pyridines Chemical class 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 238000007142 ring opening reaction Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- DSLBDAPZIGYINM-UHFFFAOYSA-N sulfanium;chloride Chemical compound S.Cl DSLBDAPZIGYINM-UHFFFAOYSA-N 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- ATGUDZODTABURZ-UHFFFAOYSA-N thiolan-2-ylideneazanium;chloride Chemical class Cl.N=C1CCCS1 ATGUDZODTABURZ-UHFFFAOYSA-N 0.000 description 1
- IMFACGCPASFAPR-UHFFFAOYSA-N tributylamine Chemical class CCCCN(CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-N 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
Classifications
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- 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
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides an enzyme-linked immunoassay kit for spectinomycin medicaments, which comprises an enzyme label plate, an enzyme-labeled anti-antibody, a spectinomycin standard, a substrate developing solution, a stop solution, a washing solution and a spectinomycin antibody, wherein the enzyme label plate is coated with a spectinomycin hapten-ovalbumin conjugate. The invention also discloses a method for detecting spectinomycin in milk by using the enzyme-linked immunoassay kit, which comprises the steps of firstly treating a milk sample to be detected, then detecting by using the enzyme-linked immunoassay kit, and finally analyzing a detection result. The enzyme-linked immunoassay kit provided by the invention has the advantages of simple structure, convenient use and carrying, and the detection method is efficient, accurate, simple and convenient, and is suitable for qualitative and quantitative detection of large-batch samples.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technology, and in particular to a kind of enzyme linked immunosorbent detection reagent of spectinomycin medicine
Box, its detection for being particularly suitable for spectinomycin medicament residue in milk.
Technical background
Spectinomycin is a kind of aminocyclitol antibiotic produced by streptomycete, is that a new special efficacy specially controls the anti-of gonorrhoea
Raw element.Spectinomycin can prevent the synthesis of bacterial cell wall proteins and can hinder the release of synthetic protein, extensively should
For veterinary field.Because spectinomycin has powerful restraining and sterilizing bacteria effect and growth of animal etc. can be promoted to act on, some
Producer illegally adds in feed, while cultivating user does not observe the off-drug period again, causes the residual of spectinomycin in milk exceeded
Phenomenon it is more and more, the health to the people causes extremely serious influence.In view of this kind of situation, national governments and international group
Knit and all start to implement more and more strict surveillance and control measure to residue of veterinary drug.The traditional detection method of spectinomycin has microbial detection
Method, thin-layered chromatography, high performance liquid chromatography, gas chromatography, the series connection mass-spectrometric technique of Ultra Performance Liquid Chromatography one etc., these sides
The spectinomycin detection that method is used in milk, the cumbersome sequence of pre-treatment is complicated, and the measure cycle is long, or even needs expensive instrument, into
This is higher, it is impossible to meet the real-time detection of spectinomycin in milk sample.
The content of the invention
For above-mentioned the deficiencies in the prior art, the invention provides a kind of enzyme linked immunosorbent detection reagent of spectinomycin medicine
Box, it includes ELISA Plate, enzyme mark antiantibody, spectinomycin standard items, substrate nitrite ion, terminate liquid, cleaning solution, spectinomycin
Antibody, is coated with spectinomycin haptens-ovalbumin conjugate on the ELISA Plate.
Spectinomycin haptens-ovalbumin the conjugate is obtained by spectinomycin haptens with ovalbumin coupling.
The spectinomycin haptens is obtained by spectinomycin with carboxymethyl azanol reaction.
The spectinomycin antibody is spectinomycin monoclonal antibody or spectinomycin polyclonal antibody.
The spectinomycin antibody is prepared by spectinomycin hapten-carrier protein conjugate as immunogene, institute
Carrier protein is stated for bovine serum albumin(BSA), thyroprotein, human serum albumins or hemocyanin, the spectinomycin haptens
It is spectinomycin-carboxymethyl azanol.
The concentration of the spectinomycin standard solution is respectively 0 μ g/L, 0.05 μ g/L, 0.15 μ g/L, 0.45 μ g/L,
1.5 μ g/L, 4.5 μ g/L.
The marker enzyme of the enzyme mark antiantibody is horseradish peroxidase, and the assay chromogenic substrate solution is chromogenic substrate A liquid
With chromogenic substrate B liquid, the chromogenic substrate A liquid is urea peroxide, and chromogenic substrate B liquid is tetramethyl benzidine, and terminate liquid is
2mol/L hydrochloric acid.
The cleaning solution is the Tris buffer solutions containing 1.0-1.5% polysorbas20s and 0.5 ‰ Proclin300 preservatives, institute
State percentage, permillage and be volume ratio.
Another object of the present invention additionally provides a kind of enzyme linked immunosorbent detection reagent of the above-mentioned spectinomycin medicine of application
The method of spectinomycin residual in box detection milk sample, it mainly includes：Milk sample to be measured is processed first；Then with above-mentioned
The enzyme-linked immunologic detecting kit of spectinomycin medicine is detected；Ultimate analysis testing result.
The enzyme-linked immunologic detecting kit of spectinomycin medicine of the present invention mainly using indirect competitive ELISA method it is qualitative or
The residual quantity of spectinomycin medicine in quantitative determination sample；Pre-treatment to milk sample is less demanding, sample pretreatment process
It is fairly simple, can be while quick detection high-volume milk sample.Enzyme-linked immunologic detecting kit of the invention, the letter of this body structure
Single, easy to use, carrying convenience, detection method are efficient, accurate, easy, be suitable to qualitative, the quantitative detection of batch samples.This
The enzyme-linked immunologic detecting kit of invention can play a significant role in spectinomycin residue detection.
Brief description of the drawings
Fig. 1：Spectinomycin hapten synthesis technology path；
Fig. 2：Spectinomycin standard items suppression curve.
Specific embodiment
The present invention is expanded on further with reference to specific embodiment.These embodiments are merely to illustrate the present invention, and
It is not limited to the scope of the present invention.
The enzyme-linked immunologic detecting kit of the spectinomycin medicine of embodiment 1
The enzyme-linked immunologic detecting kit of spectinomycin medicine, it includes：
1) 96 holes or 48 hole elisa Plates, are coated with spectinomycin haptens-ovalbumin conjugate thereon；
2) enzyme mark antiantibody, is with the sheep anti mouse antiantibody of horseradish peroxidase-labeled；
3) 6 bottles of spectinomycin standard solution, concentration is respectively 0 μ g/L, 0.05 μ g/L, 0.15 μ g/L, 0.45 μ g/L,
1.5 μ g/L, 4.5 μ g/L；
4) substrate nitrite ion is made up of substrate colour developing A liquid and substrate colour developing B liquid, and substrate nitrite ion A liquid is urea peroxide, bottom
Thing nitrite ion B liquid tetramethyl benzidines；
5) terminate liquid is 2mol/L hydrochloric acid；
6) cleaning solution is to contain 1.0%~1.5% polysorbas20 (percentage is volume ratio) and 0.5 ‰ Proclin300
Tris buffer solutions (permillage is volume ratio)；
7) spectinomycin antibody, is spectinomycin monoclonal antibody.
The preparation of the enzyme-linked immunologic detecting kit main component of embodiment 2
1st, immunogene and coating antigen synthesize
Immunogene synthesis includes two steps, and the first step is anti-by spectinomycin and carboxymethyl azanol reaction synthesis spectinomycin half
Spectinomycin haptens and bovine serum albumin(BSA) coupling are obtained immunogene by original, second step.
1) synthesis of spectinomycin haptens, route map is shown in Fig. 1
Weigh 100mg spectinomycins and 140mg carboxymethyl azanols be dissolved in 3mL pyridines, be stirred at room temperature 24 hours, dry up,
Added water 10mL, and pH 9 or so is adjusted to 0.2mol/L NaOH, uses CH2Cl2Extract 3 times, each 20mL abandons CH2Cl2, water layer use
HCl is adjusted to pH 3, then uses CH2Cl2Extract 3 times, each 20mL, with anhydrous sodium sulfate dehydration, filtrate is blown in filtering at 45 DEG C
Dry, 60 DEG C dry 3 hours, obtain white crystal, as spectinomycin haptens.
2) immunogene synthesis
Weigh 50mg spectinomycin haptens, plus 2mL dioxane, 30 μ L tri-n-butylamines, 30 μ L isobutyl chlorocarbonates,
10 DEG C of stirring 1h, reaction solution is poured into bovine serum albumin(BSA), and (160mg bovine serum albumin(BSA)s are dissolved in 5mL H2O and 5mL dioxies six
Ring), pH 8.5 is adjusted to NaOH, then 6h is stirred at 10 DEG C.Dialysed at 4 DEG C 24h with the distilled water for flowing, dialyzate is through cold
It is lyophilized dry, obtain immunogene.
3) coating antigen synthesis
It is coupled with ovalbumin with spectinomycin haptens and is obtained, method synthesizes with immunogene.
2nd, the preparation of monoclonal antibody
1) animal immune
The Balb/c mouse of 8 week old are selected, with immunogene and the spectinomycin haptens-ox blood containing Fu Shi Freund's complete adjuvants
Pure protein conjugate carries out hypodermic injection, immunizing dose be 150 μ g/ only, every 2 weeks booster immunizations once, the 4th booster immunization
One week after, it is 1 to obtain potency:1000 antibody serum.
2) preparation of myeloma cell and splenocyte
SP2/0 myeloma cell makes cell be in exponential phase, system using conventional RPMI-1640 nutrient solution cultures
Into SP2/0 myeloma cell's cell suspension；Learn from else's experience immune mouse spleen, crush, the stainless steel filtered through gauze for crossing 400 mesh is made
Splenocyte suspension, it is standby after counting.
3) cell fusion and monoclonal
By 1 × 108SP2/0 myeloma cell and 5 × 108Splenocyte is merged under PEG effects, for secretory antibody
Culture hole, uses indirect competitive ELISA to determine to screen positive hole.Monoclonal is carried out to positive hole using limiting dilution assay,
Hybridoma cell strain until obtaining secrete monoclonal antibody.The antibody specificity is directed to spectinomycin, and sensitivity can reach
To 0.05 μ g/L.
4) cell cryopreservation and recovery
The monoclonal hybridoma strain of spectinomycin is made (1-2) × 10 of frozen stock solution6The cell suspension of individual/mL,
Each cryopreservation tube 1mL, is stored in liquid nitrogen using gradually falling temperature method.Cryopreservation tube is taken out during recovery, is immediately placed in 37 DEG C of water-baths
Speed is melted, and then moves to 5min on ice, then is transferred in conical flask, is added in the complete culture solution of 10mL warms, moves into CO2Incubator
Middle culture.
5) a large amount of productions of monoclonal antibody and purifying
The Balb/c raettins of health are taken, only, pneumoretroperitoneum injects the Dan Ke of spectinomycin to Intraperitoneal injection norphytane 0.5mL/ within 7 days
Grand hybridoma cell strain 3 × 105Individual/only, and observe every other day, ascites is gathered after 10-20 days.First entered with octanoic acid-saturated ammonium sulfate method
Row ascites preliminary purification, is purified, after purification using the AKTA purifier100 purifying instrument equipped with Protein-A pillars afterwards
- 20 DEG C of preservations of antibody.
3rd, prepared by sheep anti mouse antiantibody：It, using sheep as immune animal, is immunogene to disease-free thalline sheep with mouse source antibody to be
It is immunized, is obtained sheep anti mouse antiantibody.
4th, enzyme marks the preparation of sheep anti mouse antiantibody
Sheep anti mouse antiantibody and horseradish peroxidase thing enzyme (HRP) are carried out into idol using maleimide base class active ester method
Connection.
1) using 4- (N- maleimidomethyls) hexamethylene -1- carboxylic acids succinimide ester (SMCC) activation horseradish mistakes
Oxide thing enzyme, takes the HRP solution of 100 μ L about 10mg/mL, by SMCC：ALP molar feed ratios (3~5):1 adds SMCC, 25
DEG C reaction 30min after, purified with G25 desalting columns and obtain solution I.
2) Traut ' s reagents (2- iminothiolanes hydrochloride) are used to activate sheep anti mouse antiantibody antiantibody, and with ammonia
The material of base introduces terminal sulfhydryl group by ring-opening reaction.The sheep anti mouse antiantibody solution of 100 μ L about 10mg/mL is taken, by 2- imido
Base sulfane hydrochloride：Sheep anti mouse antiantibody is (5~10):1 adds 2- iminothiolane hydrochlorides, after 25 DEG C of reaction 30min, uses
The purifying of G25 desalting columns obtains solution II.
3) by above-mentioned solution I and II mix, by 25 DEG C react 1h, plus 10 μ L 1mol/L 2 mercapto ethanol, 25 DEG C reaction
After 30min, separated on AKTA purifier 100 with Superdex 200, elution buffer is phosphate buffer,
Flow velocity is that 1mL/min collects eluent.
Traditional sodium periodate oxidizing process coupling, horseradish peroxidase can produce many and resist in the presence of oxidation
The site that body is combined, reduces the enzymatic activity of enzyme marker, and many condensates, the inventive method are mixed with the conjugate for making preparation
Be that the orientation of antibody and enzyme is coupled, do not reduce the activity of horseradish peroxidase and polymer is less, the difference between batch of conjugate compared with
Small, real-time stability preferably, extends the shelf life of product, it is ensured that the quality of product.The method cannot be only used for antiantibody
With horseradish peroxidase, it may also be used for the coupling of other general antibody, enzyme labelled antibody is prepared, for Salmonella.
5th, the preparation of ELISA Plate
With 0.05mol/L, pH8.0 phosphate coating buffer or 0.05mol/L pH9.6 carbonate coating buffers are by spectinomycin
Haptens-ovalbumin is diluted to 0.05~0.15 μ g/ml, and 100 μ L are added per hole, and 37 DEG C incubate 2h or 4 DEG C overnight, with washing
Liquid is washed 2 times, is patted dry, and then adds 3% skimmed milk power of 200 μ L to be closed in every hole again, and 37 DEG C of incubation 2h incline in hole
Liquid, is preserved after drying with aluminium film vacuum sealing.
The detection of spectinomycin in the milk sample of embodiment 3
1st, enzyme-linked immunologic detecting kit Cleaning Principle：
After sample solution or standard solution is added in ELISA Plate micropore, enzyme mark antiantibody, spectinomycin are added
Antibody working solution, the spectinomycin medicine remained in sample and coated spectinomycin haptens-ovalbumin idol on ELISA Plate
Connection thing competition spectinomycin antibody, enzyme mark antiantibody be amplified effect, with substrate nitrite ion colour developing, sample light absorption value with it is big
The content of miromycin medicine is negatively correlated, and the residual quantity of spectinomycin in sample is drawn by comparing with standard curve.According to enzyme
The depth of color on target, comparing with the spectinomycin standard solution color of series concentration can grand sight roughly in judgement sample
The concentration range of mycin residual quantity.
2nd, enzyme-linked immunologic detecting kit detecting step：
1) taking out needs the ELISA Plate for being coated with spectinomycin haptens-ovalbumin conjugate of quantity, each sample
It is parallel 2 holes to be done with standard items.
2) the μ L of standard items/sample 50 are added in corresponding micropore, being subsequently adding the μ L/ of enzyme mark sheep anti mouse antiantibody 50
Hole, adds the μ L/ holes of spectinomycin monoclonal antibody working solution 50, and vibration is mixed, and is reacted in the rearmounted 25 DEG C of light protected environments of cover plate
20min。
3) cover plate film is opened, liquid in hole is dried, the μ L/ holes of cleaning solution 250 are added, is washed 4-5 times, outwell cleaning solution, used
Blotting paper is patted dry.Cleaning solution is to contain 1.0%~1.5% polysorbas20 (percentage is volume ratio) and 0.5 ‰ Proclin300
Tris buffer solutions (permillage is volume ratio).
4) the μ L/ holes of substrate colour developing A liquid urea peroxide 50 are added, the μ L/ of substrate colour developing B liquid tetramethyl benzidine 50 are added
Hole, mixes, and is placed in after cover plate in 25 DEG C of light protected environments and reacts 10min.
5) the μ L/ holes of terminate liquid 2mol/L hydrochloric acid 50 are added, is mixed, setting ELIASA determines OD values at 450nm.
3rd, Analysis of test results
With the absorbance values (B) of the standard solution of each concentration for being obtained divided by first standard solution (0
Standard) absorbance (B0) multiplied by with 100%, obtaining percentage absorbance.With spectinomycin standard concentration (μ g/L)
Logarithm value is X-axis, and percentage absorbance is Y-axis, draws canonical plotting.The percentage for calculating sample solution with same method is inhaled
Shading value, the concentration of corresponding each sample can then read the residual quantity of spectinomycin from standard curve.
The standard curve of experimental example 1 is tested
Standard curve constitutes (0 μ g/L, 0.05 μ g/L, 0.15 μ g/L, 0.45 μ g/L, 1.5 μ g/L, 4.5 μ g/ by 6 concentration
L), number of times is respectively determined 5 times to 6 standard points, to determine working concentration scope and IC50.Taken the logarithm with normal concentration point, inhibiting rate
(B/B0) it is ordinate drafting standard curve, IC50It is 0.2955 μ g/L, sees Fig. 2, regression equation is：Y=B/B0=-
0.3621log(X)+0.3083；The equation for releasing concentration value X is：
Concentration value X=10^(-(B/B0-0.3083)÷0.3621)。
The sample preci-sion and accuracy of experimental example 2 is tested
1st, sample precision test:
Precision is represented with the coefficient of variation.Measure is added to milk sample with the spectinomycin of 0.45 μ g/L concentration,
Three kits of different batches are taken respectively, and each concentration is repeated 3 times, and the coefficient of variation is calculated respectively, the results are shown in Table 1.
The milk sample coefficient of variation of table 1 is determined
Result shows that the coefficient of variation of sample is 3.77-7.03% in milk.
2. sample accuracy test
The degree of accuracy is represented with the rate of recovery.The spectinomycin standard solution that concentration is 0.1 μ g/L is taken, respectively to 5 milk
Sample is added recovery test, each concentration do 3 it is parallel, the rate of recovery is calculated respectively, result of calculation is shown in Table 2.
The TIANZHU XINGNAO Capsul of table 2 is determined
Result shows milk sample TIANZHU XINGNAO Capsul between 86.0%-112%, shows enzyme linked immunosorbent detection examination of the present invention
The accuracy rate that agent box determines milk is higher.
The cross reacting rate of experimental example 3 is tested
Selection has 4 kinds of drug monitoring cross reacting rates of similar structures and similar functions with spectinomycin, by various medicines
The standard curve of thing respectively obtains its 50% inhibition concentration (i.e. IC50).Kit of the present invention is calculated to other medicines with following formula
Cross reacting rate.Analog cross reacting rate is smaller, then this kit is better to the specificity of the detection of spectinomycin.Hand over
Fork reactivity (%)=(spectinomycin IC50/ spectinomycin analog IC50) × 100%, the results are shown in Table 3.
The cross reacting rate of table 3 is determined
| Medicine name | Cross reacting rate (%) |
| Spectinomycin | 100% |
| Neomycin | ＜ 0.1% |
| Erythromycin | ＜ 0.5% |
| Streptomysin | ＜ 0.1% |
| Kanamycins | ＜ 1% |
The kit keeping quality of experimental example 4 is tested
Kit preservation condition is 2~8 DEG C, by the measure of 12 months, the maximum absorbance value (zero standard) of kit,
50% inhibition concentration, spectinomycin add actual measured value within normal range (NR).
The enzyme-linked immunologic detecting kit of the spectinomycin medicine of embodiment 4
It is antibody work liquid level spectinomycin polyclonal antibody working solution, enzyme mark antiantibody with the difference of embodiment 1
It is the goat-anti rabbit-anti antibody of horseradish peroxidase-labeled.
Claims (9)
1. a kind of enzyme-linked immunologic detecting kit of spectinomycin medicine, including ELISA Plate, enzyme mark antiantibody, spectinomycin mark
Quasi- product, substrate nitrite ion, terminate liquid, cleaning solution, spectinomycin antibody, be coated with the ELISA Plate spectinomycin haptens-
Ovalbumin conjugate.
2. enzyme-linked immunologic detecting kit as claimed in claim 1, it is characterised in that the spectinomycin haptens-egg white egg
White conjugate is obtained by spectinomycin haptens with ovalbumin coupling.
3. enzyme-linked immunologic detecting kit as claimed in claim 1 or 2, it is characterised in that the spectinomycin haptens is by big
Miromycin is obtained with carboxymethyl azanol reaction.
4. enzyme-linked immunologic detecting kit as claimed in claim 3, it is characterised in that the spectinomycin antibody is that grand sight is mould
Plain monoclonal antibody or spectinomycin polyclonal antibody.
5. enzyme-linked immunologic detecting kit as claimed in claim 4, it is characterised in that the spectinomycin antibody is mould by grand sight
Plain hapten-carrier protein conjugate is prepared as immunogene, and the carrier protein is bovine serum albumin(BSA), thyroid gland egg
In vain, human serum albumins or hemocyanin, the spectinomycin haptens are spectinomycin-carboxymethyl azanol.
6. enzyme-linked immunologic detecting kit as claimed in claim 1 or 2, it is characterised in that the spectinomycin standard solution
Concentration be respectively 0 μ g/L, 0.05 μ g/L, 0.15 μ g/L, 0.45 μ g/L, 1.5 μ g/L, 4.5 μ g/L.
7. enzyme-linked immunologic detecting kit as claimed in claim 1 or 2, it is characterised in that the enzyme marks the mark of antiantibody
Enzyme is horseradish peroxidase, and the assay chromogenic substrate solution is chromogenic substrate A liquid and chromogenic substrate B liquid, and the chromogenic substrate A liquid is
Urea peroxide, chromogenic substrate B liquid is tetramethyl benzidine, and terminate liquid is 2mol/L hydrochloric acid.
8. enzyme-linked immunologic detecting kit as claimed in claim 1 or 2, it is characterised in that the cleaning solution is to contain 1.0-
The Tris buffer solutions of 1.5% polysorbas20 and 0.5 ‰ Proclin300 preservatives, the percentage, permillage are volume ratio.
9. spectinomycin is residual in the enzyme-linked immunologic detecting kit detection milk sample described in a kind of use claim any one of 1-8
The method stayed, mainly includes the following steps that：Milk sample to be measured is processed first；Then with described in claim any one of 1-8
Enzyme-linked immunologic detecting kit is detected；Ultimate analysis testing result.
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Cited By (1)
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