CN112798778A - Immunofluorescence chromatography detection card and method for synchronously detecting florfenicol and trimethoprim contents in poultry eggs - Google Patents
Immunofluorescence chromatography detection card and method for synchronously detecting florfenicol and trimethoprim contents in poultry eggs Download PDFInfo
- Publication number
- CN112798778A CN112798778A CN202110285602.6A CN202110285602A CN112798778A CN 112798778 A CN112798778 A CN 112798778A CN 202110285602 A CN202110285602 A CN 202110285602A CN 112798778 A CN112798778 A CN 112798778A
- Authority
- CN
- China
- Prior art keywords
- trimethoprim
- florfenicol
- detection
- solution
- microspheres
- 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.)
- Granted
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 99
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical compound COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 title claims abstract description 90
- 229960001082 trimethoprim Drugs 0.000 title claims abstract description 90
- AYIRNRDRBQJXIF-NXEZZACHSA-N (-)-Florfenicol Chemical compound CS(=O)(=O)C1=CC=C([C@@H](O)[C@@H](CF)NC(=O)C(Cl)Cl)C=C1 AYIRNRDRBQJXIF-NXEZZACHSA-N 0.000 title claims abstract description 89
- 229960003760 florfenicol Drugs 0.000 title claims abstract description 89
- 235000013601 eggs Nutrition 0.000 title claims abstract description 36
- 244000144977 poultry Species 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000004587 chromatography analysis Methods 0.000 title claims abstract description 19
- 238000010166 immunofluorescence Methods 0.000 title claims abstract description 19
- 239000004005 microsphere Substances 0.000 claims abstract description 89
- 239000012528 membrane Substances 0.000 claims abstract description 24
- 238000006243 chemical reaction Methods 0.000 claims abstract description 20
- 241000283707 Capra Species 0.000 claims abstract description 12
- 238000003908 quality control method Methods 0.000 claims abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000010521 absorption reaction Methods 0.000 claims abstract description 4
- 238000002156 mixing Methods 0.000 claims description 54
- 239000000243 solution Substances 0.000 claims description 41
- 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 claims description 34
- 239000006228 supernatant Substances 0.000 claims description 29
- 108010090804 Streptavidin Proteins 0.000 claims description 27
- 229960002685 biotin Drugs 0.000 claims description 23
- 239000011616 biotin Substances 0.000 claims description 23
- 239000002244 precipitate Substances 0.000 claims description 22
- 239000000523 sample Substances 0.000 claims description 20
- 235000020958 biotin Nutrition 0.000 claims description 17
- 239000007853 buffer solution Substances 0.000 claims description 14
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 14
- 239000008055 phosphate buffer solution Substances 0.000 claims description 12
- 239000003761 preservation solution Substances 0.000 claims description 11
- 238000009210 therapy by ultrasound Methods 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 230000006287 biotinylation Effects 0.000 claims description 10
- 238000007413 biotinylation Methods 0.000 claims description 10
- 238000007865 diluting Methods 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 229930006000 Sucrose Natural products 0.000 claims description 9
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 9
- 239000005018 casein Substances 0.000 claims description 9
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 9
- 235000021240 caseins Nutrition 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- 239000005720 sucrose Substances 0.000 claims description 9
- 239000012224 working solution Substances 0.000 claims description 9
- 239000000020 Nitrocellulose Substances 0.000 claims description 7
- 239000002202 Polyethylene glycol Substances 0.000 claims description 7
- 230000008878 coupling Effects 0.000 claims description 7
- 238000010168 coupling process Methods 0.000 claims description 7
- 238000005859 coupling reaction Methods 0.000 claims description 7
- 229920001220 nitrocellulos Polymers 0.000 claims description 7
- 229920001223 polyethylene glycol Polymers 0.000 claims description 7
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000007987 MES buffer Substances 0.000 claims description 6
- 239000000427 antigen Substances 0.000 claims description 6
- 102000036639 antigens Human genes 0.000 claims description 6
- 108091007433 antigens Proteins 0.000 claims description 6
- 230000000903 blocking effect Effects 0.000 claims description 6
- 239000003365 glass fiber Substances 0.000 claims description 6
- 238000007789 sealing Methods 0.000 claims description 6
- 238000005507 spraying Methods 0.000 claims description 6
- 238000000108 ultra-filtration Methods 0.000 claims description 6
- 239000002699 waste material Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 241000287828 Gallus gallus Species 0.000 claims description 4
- 238000003860 storage Methods 0.000 claims description 4
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 claims description 3
- 229920004890 Triton X-100 Polymers 0.000 claims description 3
- 239000013504 Triton X-100 Substances 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 239000012470 diluted sample Substances 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 3
- 239000010931 gold Substances 0.000 claims description 3
- 229910052737 gold Inorganic materials 0.000 claims description 3
- 238000007790 scraping Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 2
- 238000002203 pretreatment Methods 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 238000000861 blow drying Methods 0.000 abstract description 2
- 235000013594 poultry meat Nutrition 0.000 description 19
- 239000000126 substance Substances 0.000 description 12
- 238000011084 recovery Methods 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000003556 assay Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000003317 immunochromatography Methods 0.000 description 3
- 244000144972 livestock Species 0.000 description 3
- 241000272517 Anseriformes Species 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 241000272201 Columbiformes Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000286209 Phasianidae Species 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000000622 liquid--liquid extraction Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000017448 oviposition Effects 0.000 description 1
- 235000013613 poultry product Nutrition 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 229940124530 sulfonamide Drugs 0.000 description 1
- 150000003456 sulfonamides Chemical class 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000004506 ultrasonic cleaning Methods 0.000 description 1
Images
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
- G01N33/94—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
- G01N33/9446—Antibacterials
-
- 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
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses an immunofluorescence chromatography detection card and a method for synchronously detecting the content of florfenicol and trimethoprim in poultry eggs; the immunofluorescence chromatography detection card and the immunofluorescence chromatography detection method are simple and time-saving in sample pretreatment operation, free of blow-drying, high in detection method sensitivity, suitable for on-site instant detection, and capable of synchronously detecting florfenicol and trimethoprim contents in poultry eggs, and technically characterized by comprising a bottom plate, wherein a water absorption paper pad, a reaction membrane, a combination pad and a sample pad are sequentially connected to the bottom plate from left to right, the reaction membrane is marked with three lines of a florfenicol detection line T1, a trimethoprim detection line T2 and a quality control C line, and the combination pad is coated with florfenicol detection microspheres, trimethoprim detection microspheres and goat anti-chicken IgY polyclonal antibody labeled microspheres; belongs to the technical field of biological detection.
Description
Technical Field
The invention discloses an egg detection method, in particular to an immunofluorescence chromatography detection card and method for synchronously detecting the content of florfenicol and trimethoprim in eggs, belonging to the technical field of biological detection.
Background
Florfenicol is a novel broad-spectrum antibiotic of chloramphenicol, has obvious advantages in safety and effectiveness, and is widely used in livestock and poultry breeding industry. Trimethoprim is named trimethoprim, belongs to a broad-spectrum antibacterial synergist, has weak bactericidal capability, has obvious synergistic effect when being used together with sulfonamides and some antibiotics, and is widely used in livestock and poultry breeding industry.
However, the florfenicol and trimethoprim are used in a large amount, so that the detection rate of the florfenicol and trimethoprim in livestock and poultry products is high, and the public health is seriously threatened, so that the national standard of food safety sets a strict detection limit for the two antibiotics, and the egg laying period of the laying hens is forbidden.
At present, in the prior art, florfenicol and trimethoprim detection chromatograms, enzyme-linked methods, immunochromatography and the like are used, some methods depend on high-end equipment seriously, some methods have high requirements on samples to be detected, the pretreatment is complex, or only one of the methods can be detected, so that the working efficiency is reduced seriously.
Disclosure of Invention
In view of the above problems, a first object of the present invention is to provide an immunofluorescent layer assay card which has high assay sensitivity, is easy and time-saving to operate, does not need to be dried, has high sensitivity, is suitable for on-site real-time assay, and can synchronously assay the florfenicol and trimethoprim contents in poultry eggs.
The invention also aims to provide a method for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs, which is simple to operate, can effectively eliminate interference items, improves the detection sensitivity and is suitable for poultry egg detection in any place.
In order to achieve the above object, a first technical solution provided by the present invention is as follows:
an immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in poultry eggs comprises a bottom plate, wherein a water absorption paper pad, a reaction membrane, a combination pad and a sample pad are sequentially connected to the bottom plate from left to right, the reaction membrane is marked with a florfenicol detection line T1, a trimethoprim detection line T2 and a quality control C line, and the combination pad is coated with florfenicol detection microspheres, trimethoprim detection microspheres and goat anti-chicken IgY polyclonal antibody labeled microspheres;
the florfenicol detection microsphere is prepared by combining a long-chain biotinylated florfenicol monoclonal antibody connected with a polyethylene glycol long-chain lengthened arm (-dPEG 24-) and a streptavidin fluorescent microsphere through biotin and streptavidin;
the trimethoprim detection microsphere is prepared by combining a long-chain biotinylated trimethoprim monoclonal antibody connected with a polyethylene glycol long-chain lengthened arm (-dPEG 24-) and a streptavidin fluorescent microsphere through biotin and streptavidin.
Further, the immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs is prepared by the following steps:
(1) diluting chicken IgY to 0.1mg/mL by using 0.01M phosphate buffer solution with 3% sucrose sugar and pH7.4 to serve as quality control C-line working solution;
(2) diluting the florfenicol coupling antigen to 0.2mg/mL by using a phosphate buffer solution of 0.01M with pH7.4 and containing 3% sucrose sugar to be used as a working solution of a florfenicol detection line;
(3) diluting the trimethoprim coupling antigen to 0.2mg/mL by using a phosphate buffer solution of 0.01M and pH7.4 containing 3% sucrose sugar, and using the diluted sample as a working solution of a trimethoprim detection line;
(4) scribing three lines of a florfenicol detection line T1, a trimethoprim detection line T2 and a quality control line C on a nitrocellulose membrane by using a gold spraying membrane scribing instrument, wherein the distance between two adjacent lines is 5mm, and the scribing concentration is 1 mul/cm; after finishing the scraping, the paper is dried in a drying oven at 45 ℃ for 48 hours.
Further, the immunofluorescence chromatography detection card for synchronously detecting the content of the florfenicol and the trimethoprim in the poultry eggs is characterized in that the sample pad is prepared by the following steps:
(1) preparing 0.02M phosphate buffer solution of pH7.4 containing 1% casein, 0.15M NaCl, 1% PEG 8000 and 1.5% Triton X-100 as a treatment solution;
(2) treating fluid prepared in step (1) at a concentration of 50. mu.l/cm2Uniformly coating the mixture on glass fiber RB65, and drying for 16h at 45 ℃.
Further, the immunofluorescence chromatography detection card for synchronously detecting the content of the florfenicol and the trimethoprim in the poultry eggs is prepared by the following steps: mixing florfenicol detection microspheres, trimethoprim detection microspheres and goat anti-chicken IgY polyclonal antibody marking microspheres, uniformly spraying onto a glass fiber membrane 8964 pad with the width of 1.2cm, and drying at 45 ℃.
Further, the immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs is prepared by the following steps: :
(1) adding 1mL of 0.05M MES buffer solution into a 2mL centrifuge tube, adding 1mg of fluorescent microspheres, and uniformly mixing by vortex; adding 4. mu.l NHS (25 mg/ml) and 4. mu.l EDC (25 mg/ml) into the centrifuge tube, mixing uniformly by vortex, and reacting for 20min on a rotary incubator;
(2) centrifugally separating the activated fluorescent microspheres, removing supernatant, taking precipitate, adding 1000 mu l of 0.02M borate buffer solution with pH8.0, performing ultrasonic treatment for 2s, performing interval for 5s, and repeating the step for 5 times to break up the microspheres;
(3) adding 100 μ g goat anti-chicken IgY, mixing uniformly by vortex, placing on a rotary incubator, and reacting for 60 min;
(4) adding 100 μ l of 10% casein blocking solution into each tube, mixing uniformly by vortex, placing on a rotary incubator after completing ultrasonic treatment, and sealing for 60 min; and centrifuging again, sucking supernatant, retaining precipitate, adding 1mL of microsphere preservation solution, and mixing completely for later use.
Further, the preparation process of the immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in the poultry egg comprises three steps:
the first step is as follows: the preparation method of the long-chain biotinylation florfenicol monoclonal antibody and the long-chain biotinylation trimethoprim monoclonal antibody comprises the following steps:
(1) taking out long-chain Biotin (NHS-dPEG 24-Biotin), balancing for 30min at room temperature, and dissolving with DMSO to a final concentration of 10mM for use;
(2) respectively taking 1ml of florfenicol monoclonal antibody (2 mg/ml) and 1ml of trimethoprim monoclonal antibody (2 mg/ml) into a 2ml centrifuge tube, adding 27 mu l of Biotin NHS-dPEG24-Biotin prepared in the previous step into each tube, uniformly mixing by vortex, and reacting for 30min on a rotary incubator;
(3) transferring the mixture to an ultrafiltration tube after the reaction is finished, centrifuging the mixture for 15min at 12000g, and discarding waste liquid;
(4) adding 2ml of PBS buffer solution into the ultrafiltration tube, 12000g, centrifuging 15ml, and discarding waste liquid;
(5) repeating the step (4) for 5 times, re-dissolving 200 mu l of PBS, collecting the labeled antibody, and measuring the concentration by using a BCA kit.
The second step is that: the preparation method of the streptavidin fluorescent microsphere comprises the following steps:
(1) adding 1mL of marking buffer solution 0.05M MES buffer solution into a 2mL centrifuge tube, adding 1mg of fluorescent microspheres, and uniformly mixing by vortex; adding 4 μ l NHS (25 mg/ml) and 4 μ l EDC (25 mg/ml), mixing by vortex, and reacting for 20min on a rotary incubator;
(2) centrifugally separating the activated fluorescent microspheres, discarding supernatant, taking precipitate, adding 1000 μ l of borate buffer solution, performing ultrasonic treatment for 2s, performing intermittent treatment for 5s, and repeating the steps for 5 times to break up the microspheres;
(3) adding 100 mu g of streptavidin into the activated microspheres, uniformly mixing by vortex, and placing on a rotary incubator for reaction for 60 min;
(4) adding 100 μ l of 10% casein blocking solution into each tube, mixing uniformly by vortex, placing on a rotary incubator after ultrasonic treatment, sealing for 60min, centrifuging, removing supernatant, and collecting precipitate;
(5) adding 1mL of microsphere preservation solution into the precipitate, fully and uniformly mixing, carrying out centrifugal separation, sucking supernatant, and taking the precipitate;
(6) repeating the step (5) for 2-3 times to fully remove unreacted streptavidin; then adding 1mL of microsphere storage solution, and fully and uniformly mixing for later use.
The third step: the preparation process of the florfenicol detection microsphere and the trimethoprim detection microsphere comprises the following steps:
(1) respectively adding 5 mu g of each long-chain biotinylation florfenicol monoclonal antibody and long-chain biotinylation trimethoprim monoclonal antibody into 2 tubes of fluorescent microspheres containing 1ml of streptavidin, uniformly mixing in a vortex manner, and reacting for 30min on a rotary incubator;
(2) after the reaction is finished, centrifuging at 16500rpm for 20min, sucking the supernatant, and taking the precipitate;
(3) adding 1mL of microsphere preservation solution, mixing well, centrifuging at 16500rpm for 20min, and removing supernatant;
(4) repeating the step (3) for 3 times, and fully removing the unreacted biotin-labeled antibody;
(5) adding 1mL of microsphere storage solution, and mixing well for later use.
The second technical scheme provided by the invention is as follows:
a method for synchronously detecting the content of florfenicol and trimethoprim in poultry eggs comprises the following steps:
firstly), pretreating a sample to obtain a solution to be detected;
secondly), adding a solution to be detected into the detection card of claim 1, horizontally standing for 10min, and then inserting the solution into a fluorescence immunochromatographic instrument to read the result;
and thirdly), the result is brought into the standard curve, and the content of the florfenicol and the trimethoprim in the sample can be calculated.
Wherein, the pretreatment method in the step one) comprises the following steps:
(1) adding the extract into the fully homogenized egg to-be-detected liquid, manually shaking and uniformly mixing for extraction, centrifuging and taking supernatant;
(2) adding n-hexane and a redissolution into the supernatant, shaking, standing and layering to obtain a lower water phase as a solution to be detected;
in the method for synchronously detecting the content of the florfenicol and the trimethoprim in the poultry eggs, the extracting solution is ethyl acetate containing 0.5 to 1.5 weight percent of trichloroacetic acid.
In the method for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs, the complex solution is PBS buffer solution containing 0.15M NaCl and 0.05M PB, and the pH value is 6.6.
According to the method for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs, the volume ratio of the supernatant to the n-hexane to the complex solution is as follows: 1:5:0.5-1.
Compared with the prior art, the technical scheme provided by the invention has the following technical advantages:
1. the technical scheme provided by the invention is that a long-chain biotinylation monoclonal antibody and a streptavidin fluorescent microsphere are used for preparing a labeled microsphere through the affinity action of Biotin and streptavidin, the long-chain Biotin (NHS-dPEG 24-Biotin) is connected with a polyethylene glycol long-chain lengthened arm (-dPEG 24-), and the other end of the lengthened arm is connected with NHS for coupling and labeling the monoclonal antibody, so that the steric hindrance effect can be effectively reduced, the labeling efficiency is improved, and a Biotin-streptavidin system has the signal amplification effect and can further improve the detection sensitivity.
2. The technology provided by the invention can be used for directly loading the sample for detection after being extracted by a liquid-liquid extraction method, the pretreatment operation is simple, the processes of blow drying, column passing and the like are not needed, the content of the florfenicol and the trimethoprim can be synchronously detected, and the application range is wide.
3. The technology provided by the invention can synchronously detect the content of the florfenicol and the trimethoprim.
Drawings
FIG. 1 is a schematic diagram of a test card structure and test principle provided in the present application;
FIG. 2 is a schematic representation of the principle of the long chain biotinylated monoclonal antibody provided herein;
FIG. 3 is a schematic diagram of the preparation process of streptavidin fluorescent microspheres;
FIG. 4 is a schematic diagram of the preparation process of detection microspheres
Detailed Description
The following claims are hereby incorporated into the detailed description of the invention with further limitations, but not to be construed as limiting the invention in any way, so that any and all modifications that come within the scope of the claims are to be embraced by the invention.
Example 1
The immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in poultry eggs provided by the embodiment comprises a bottom plate, wherein a water absorption paper pad, a reaction membrane, a combination pad and a sample pad are sequentially connected to the bottom plate from left to right, the reaction membrane is marked with three lines of a florfenicol detection line T1, a trimethoprim detection line T2 and a quality control C line, and the combination pad is coated with florfenicol detection microspheres, trimethoprim detection microspheres and goat anti-chicken IgY polyclonal antibody labeled microspheres;
the florfenicol detection microsphere is prepared by combining a long-chain biotinylated florfenicol monoclonal antibody connected with a polyethylene glycol long-chain lengthened arm (-dPEG 24-) and a streptavidin fluorescent microsphere through biotin and streptavidin;
the trimethoprim detection microsphere is prepared by combining a long-chain biotinylated trimethoprim monoclonal antibody connected with a polyethylene glycol long-chain lengthened arm (-dPEG 24-) and a streptavidin fluorescent microsphere through biotin and streptavidin.
Wherein:
the reaction membrane is prepared by the following method:
(1) diluting chicken IgY to 0.1mg/mL by using 0.01M phosphate buffer solution with 3% sucrose sugar and pH7.4 to serve as quality control C-line working solution;
(2) diluting the florfenicol coupling antigen to 0.2mg/mL by using a phosphate buffer solution of 0.01M with pH7.4 and containing 3% sucrose sugar to be used as a working solution of a florfenicol detection line;
(3) diluting the trimethoprim coupling antigen to 0.2mg/mL by using a phosphate buffer solution of 0.01M and pH7.4 containing 3% sucrose sugar, and using the diluted sample as a working solution of a trimethoprim detection line;
(4) scribing three lines of a florfenicol detection line, a trimethoprim detection line and a quality control C line on a nitrocellulose membrane by using a gold spraying membrane scribing instrument, wherein the distance between two adjacent lines is 5mm, and the scribing concentration is 1 mul/cm; after finishing the scraping, the paper is dried in a drying oven at 45 ℃ for 48 hours.
The preparation method of the sample pad comprises the following steps:
(1) preparing 0.02M phosphate buffer solution of pH7.4 containing 1% casein, 0.15M NaCl, 1% PEG 8000 and 1.5% Triton X-100 as a treatment solution;
(2) treating fluid prepared in step (1) at a concentration of 50. mu.l/cm2Uniformly coating the mixture on glass fiber RB65, and drying for 16h at 45 ℃.
The goat anti-chicken IgY polyclonal antibody labeled microsphere is prepared by the following steps:
(1) adding 1mL of 0.05M MES buffer solution into a 2mL centrifuge tube, adding 1mg of fluorescent microspheres, and uniformly mixing by vortex; adding 4. mu.l NHS (25 mg/ml) and 4. mu.l EDC (25 mg/ml) into the centrifuge tube, mixing uniformly by vortex, and reacting for 20min on a rotary incubator;
(3) centrifugally separating the activated fluorescent microspheres, removing supernatant, taking precipitate, adding 1000 mu l of 0.02M borate buffer solution with pH8.0, performing ultrasonic treatment for 2s, performing interval for 5s, and repeating the step for 5 times to break up the microspheres;
(4) adding 100 μ g goat anti-chicken IgY, mixing uniformly by vortex, placing on a rotary incubator, and reacting for 60 min;
(5) adding 100 μ l of 10% casein blocking solution into each tube, mixing uniformly by vortex, placing on a rotary incubator after completing ultrasonic treatment, and sealing for 60 min; centrifuging again, sucking out supernatant, retaining precipitate, adding 1mL of microsphere preservation solution, and mixing well for later use;
the method for preparing the long-chain biotinylated florfenicol monoclonal antibody and the long-chain biotinylated trimethoprim monoclonal antibody comprises the following steps:
(1) taking out long-chain Biotin (NHS-dPEG 24-Biotin), balancing for 30min at room temperature, and dissolving with DMSO to a final concentration of 10mM for use;
(2) respectively taking 1ml of florfenicol monoclonal antibody with the concentration of 2mg/ml and 1ml of trimethoprim monoclonal antibody with the concentration of 2mg/ml into a 2ml centrifugal tube, adding 27 mu l of biotin prepared in the previous step into each centrifugal tube, uniformly mixing by vortex, and reacting for 30min on a rotary incubator;
(3) transferring the mixture to an ultrafiltration tube after the reaction is finished, centrifuging the mixture for 15min at 12000g, and discarding waste liquid;
(4) adding 2ml of PBS buffer solution into the ultrafiltration tube, 12000g, centrifuging 15ml, and discarding waste liquid;
(5) repeating the step (4) for 5 times, re-dissolving 200 mu l of PBS, collecting the labeled antibody, and measuring the concentration by using a BCA kit.
The method for preparing the streptavidin fluorescent microspheres comprises the following steps:
(1) adding 1mL of marking buffer solution 0.05M MES buffer solution into a 2mL centrifuge tube, adding 1mg of fluorescent microspheres, and uniformly mixing by vortex; adding 4 μ l NHS (25 mg/ml) and 4 μ l EDC (25 mg/ml), mixing by vortex, and reacting for 20min on a rotary incubator;
(2) centrifugally separating the activated fluorescent microspheres, discarding supernatant, taking precipitate, adding 1000 μ l of borate buffer solution, performing ultrasonic treatment for 2s, performing intermittent treatment for 5s, and repeating the steps for 5 times to break up the microspheres;
(3) adding 100 mu g of streptavidin into the activated microspheres, uniformly mixing by vortex, and placing on a rotary incubator for reaction for 60 min;
(4) adding 100 μ l of 10% casein blocking solution into each tube, mixing uniformly by vortex, placing on a rotary incubator after ultrasonic treatment, sealing for 60min, centrifuging, removing supernatant, and collecting precipitate;
(5) adding 1mL of microsphere preservation solution into the precipitate, fully and uniformly mixing, carrying out centrifugal separation, sucking supernatant, and taking the precipitate;
(6) repeating the step (5) for 2-3 times to fully remove unreacted streptavidin; then adding 1mL of microsphere storage solution, and fully and uniformly mixing for later use.
The preparation process of the florfenicol detection microsphere and the trimethoprim detection microsphere is as follows
(1) Adding 5 mu g of long-chain biotinylated antibody into 1ml of streptavidin fluorescent microspheres, uniformly mixing by vortex, and reacting for 30min on a rotary incubator;
(2) after the reaction is finished, centrifuging at 16500rpm for 20min, sucking the supernatant, and taking the precipitate;
(3) adding 1mL of microsphere preservation solution, mixing well, centrifuging at 16500rpm for 20min, and removing supernatant;
(4) repeating the step (3) for 3 times, and fully removing the unreacted biotin-labeled antibody;
(5) adding 1mL of microsphere preservation solution into the precipitate, and fully and uniformly mixing for later use;
the conjugate pad was prepared by the following steps: mixing florfenicol detection microspheres, trimethoprim detection microspheres and goat anti-chicken IgY polyclonal antibody marking microspheres to enable the final concentration of the microspheres to be 0.2mg/ml, 0.1mg/ml and 0.01mg/ml respectively, carrying out ultrasonic treatment in an ultrasonic cleaning instrument for 2min, spraying the microspheres onto a glass fiber membrane 8964 pad with the width of 1.2cm according to 4 mul/cm and 20KPa, and drying at 45 ℃.
Preparation of test paper strip
After the sample pad, the combination pad and the reaction membrane are prepared according to the method, one side of the nitrocellulose membrane is tightly pressed with the edge of the nitrocellulose membrane for about 1-2mm to be pasted with the absorbent paper pad, the other side of the nitrocellulose membrane is tightly pressed with the edge of the nitrocellulose membrane for about 1-2mm to be pasted with the combination pad, and the other side of the combination pad is tightly pressed with the edge of the combination pad for about 1-2mm to be pasted with the sample pad. Then cutting the test strip into test strips with the width of 3.95mm, and placing the test strips into the test strip grooves of the lower cover of the detection card, wherein the upper cover of the detection card is covered to form a complete detection card.
Example 2
The embodiment provides a method for synchronously detecting the content of florfenicol and trimethoprim in poultry eggs, which specifically comprises the following steps:
(1) firstly, preprocessing a sample, and specifically comprises the following steps: taking 2g of homogenized poultry eggs which do not contain florfenicol and trimethoprim and are detected by high performance liquid chromatography as negative control substances, adding 3ml of ethyl acetate (containing 1wt% of trichloroacetic acid) into each tube, manually shaking for 30s at 4000rpm, and centrifuging for 1 min. Taking 1ml of supernatant into a 15ml centrifuge tube, adding 5ml of n-hexane and 0.5ml of re-solution, standing for layering, and taking the lower-layer water phase as a solution to be detected;
(2) and adding the liquid to be detected extracted in the pretreatment into a sample hole of the detection card, horizontally standing for 10min, and then inserting the liquid into a fluorescence immunochromatographic instrument to read the result. The results were included in the standard set prepared in example 3, and the florfenicol and trimethoprim content of the sample was calculated.
Example 3
Preparation of test standards
Preparing a series of standard products: adding 16 mu l of florfenicol standard substance (10 mg/kg) and 16 mu l of trimethoprim standard substance (10 mg/kg) into 968 mu l of methanol solution, uniformly mixing to obtain a mixed standard substance 1 (the florfenicol is 160 mu g/kg, the trimethoprim is 160 mu g/kg), and then diluting for 5 times in a multiple ratio to obtain a mixed standard substance 2, a mixed standard substance 3, a mixed standard substance 4, a mixed standard substance 5 and a mixed standard substance 6; taking 7 15ml centrifuge tubes, adding 2ml blank sample without florfenicol and trimethoprim into each tube, adding 50 ul of mixed standard substance 1-6 into 6 tubes, adding 50 ul of methanol into the 7 th tube, and mixing uniformly.
The florfenicol concentration gradient is: 4. mu.g/kg, 2. mu.g/kg, 1. mu.g/kg, 0.5. mu.g/kg, 0.25. mu.g/kg, 0.125. mu.g/kg, 0. mu.g/kg;
the trimethoprim concentration gradient is as follows: 4. mu.g/kg, 2. mu.g/kg, 1. mu.g/kg, 0.5. mu.g/kg, 0.25. mu.g/kg, 0.125. mu.g/kg, 0. mu.g/kg;
adding 3ml of the extracting solution into each tube, manually shaking for 30s at 4000rpm, centrifuging for 2min, taking 1ml of supernatant, adding into a 15ml centrifuge tube, adding 5ml of n-hexane and 0.5ml of complex solution, manually shaking for 10s, and standing for layering. Adding 100 μ l of lower water phase into the detection card, standing for 10min, and then inserting into a fluorescence immunochromatography instrument for reading to obtain the ratio (T/C) of the detection line and the quality control C line. And (3) drawing a working curve by taking the concentration gradient as an abscissa and the corresponding T/C value as an ordinate:
the standard curve for florfenicol detection is:
y=3.95762/(1+(x/0.11053)^0.93504)+0.16273 R2=0.99994
linear range: 0.1-4 mug/kg
The standard curve for trimethoprim detection was: y = -1.23982-2.17326 a x R2=0.99728
Linear range: 0.2-4 mug/kg
Example 4
Minimum detection limit:
taking negative samples, repeating the steps for 10 times, calculating the T/C mean value and the Standard Deviation (SD), then subtracting 2 SD from the mean value, substituting the obtained value into a dose-response curve, and obtaining the florfenicol sensitivity of the method to be 0.05 mu g/kg; the sensitivity of trimethoprim is 0.1. mu.g/kg.
Relative standard deviation:
adding the standard substance into the negative sample until the final concentration of florfenicol is 0.2 mug/kg, 0.4 mug/kg and 1.6 mug/kg; the final concentration of trimethoprim is 0.25. mu.g/kg, 0.5. mu.g/kg, 2. mu.g/kg. The test was repeated 10 times per concentration and the Coefficient of Variation (CV) of the test concentration was calculated. The results are shown in table 1, and therefore, the coefficient of variation of the detection is less than 15%, and the detection method has high precision and meets the requirement of conventional rapid detection.
TABLE 1 relative standard deviation of florfenicol, trimethoprim at different concentrations
And (3) standard addition recovery rate:
adding the standard substance into the negative sample until the final concentration of florfenicol is 0.2 mug/kg, 0.4 mug/kg and 1.6 mug/kg; the final concentration of trimethoprim is 0.25. mu.g/kg, 0.5. mu.g/kg, 2. mu.g/kg. The test was repeated 10 times per concentration, and the recovery was calculated by dividing the test concentration by the spiked concentration. The results are shown in table 2, from which it can be seen that the present detection method has a higher accuracy.
TABLE 2 recovery of florfenicol and trimethoprim from the addition of standard
In order to prove and verify the technical scheme provided by the application, an application example of the technology provided by the application is given below.
Application example 1
Detection of different kinds of samples
Taking 5 standard adding samples of eggs of chicken, ducks, geese, pigeons and quail, wherein the standard adding final concentration of florfenicol is 0.2 mu g/kg, and the standard adding final concentration of trimethoprim is 0.25 mu g/kg. Taking 10 tubes and 2 ml/tube of each sample, adding 3ml of extracting solution into each tube, manually shaking for 30s at 4000rpm, centrifuging for 2min, taking 1ml of supernate, adding into a 15ml centrifuge tube, adding 5ml of n-hexane and 0.5ml of complex solution, manually shaking for 10s, standing and layering. Adding 100 μ l of lower water phase into the detection card, standing for 10min, inserting into a fluorescence immunochromatography instrument for reading to obtain the ratio (T/C) of two detection lines to a quality control C line, substituting the T/C value into a standard curve to obtain the feedback concentration, and further calculating the standard addition recovery rate and the repeatability. The results are shown in Table 3.
TABLE 3 results of standard recovery and repeatability experiments of florfenicol and trimethoprim in different samples
The recovery rate of various samples or different samples based on the matrix difference is different, and the overall recovery rate and repeatability can meet the requirement of on-site rapid detection.
Claims (10)
1. An immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in poultry eggs comprises a bottom plate, wherein a water absorption paper pad, a reaction membrane, a combination pad and a sample pad are sequentially connected to the bottom plate from left to right, and is characterized in that the reaction membrane is marked with three lines of a florfenicol detection line T1, a trimethoprim detection line T2 and a quality control C line, and the combination pad is coated with florfenicol detection microspheres, trimethoprim detection microspheres and goat anti-chicken IgY polyclonal antibody labeled microspheres;
the florfenicol detection microsphere is prepared by combining a long-chain biotinylation florfenicol monoclonal antibody connected with a polyethylene glycol long-chain lengthened arm-dPEG 24-and a streptavidin fluorescent microsphere through biotin and streptavidin;
the trimethoprim detection microsphere is prepared by combining a long-chain biotinylated trimethoprim monoclonal antibody connected with a polyethylene glycol long-chain lengthened arm-dPEG 24-and a streptavidin fluorescent microsphere through biotin and streptavidin.
2. The immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs according to claim 1, wherein the reaction membrane is prepared by the following steps:
(1) diluting chicken IgY to 0.1mg/mL by using 0.01M phosphate buffer solution with 3% sucrose sugar and pH7.4 to serve as quality control C-line working solution;
(2) diluting the florfenicol coupling antigen to 0.2mg/mL by using a phosphate buffer solution of 0.01M with pH7.4 and containing 3% sucrose sugar to be used as a working solution of a florfenicol detection line;
(3) diluting the trimethoprim coupling antigen to 0.2mg/mL by using a phosphate buffer solution of 0.01M and pH7.4 containing 3% sucrose sugar, and using the diluted sample as a working solution of a trimethoprim detection line;
(4) scribing three lines of a florfenicol detection line T1, a trimethoprim detection line T2 and a quality control line C on a nitrocellulose membrane by using a gold spraying membrane scribing instrument, wherein the distance between two adjacent lines is 5mm, and the scribing concentration is 1 mul/cm; after finishing the scraping, the paper is dried in a drying oven at 45 ℃ for 48 hours.
3. The immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs according to claim 1, wherein the sample pad is prepared by the following steps:
(1) preparing 0.02M phosphate buffer solution of pH7.4 containing 1% casein, 0.15M NaCl, 1% PEG 8000 and 1.5% Triton X-100 as a treatment solution;
(2) treating fluid prepared in step (1) at a concentration of 50. mu.l/cm2Uniformly coating the mixture on glass fiber RB65, and drying for 16h at 45 ℃.
4. The immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs according to claim 1, wherein the combination pad is prepared by the following steps: mixing florfenicol detection microspheres, trimethoprim detection microspheres and goat anti-chicken IgY polyclonal antibody marking microspheres, uniformly spraying onto a glass fiber membrane pad with the width of 1.2cm, and drying at 45 ℃.
5. The immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs according to claim 1, wherein the goat anti-chicken IgY polyclonal antibody labeled microsphere is prepared by the following method: :
(1) adding 1mL of 0.05M MES buffer solution into a 2mL centrifuge tube, adding 1mg of fluorescent microspheres, and uniformly mixing by vortex; adding 4 mul of NHS with the concentration of 25mg/ml and 4 mul of EDC with the concentration of 25mg/ml into the centrifuge tube, uniformly mixing by vortex, and reacting for 20min on a rotary incubator;
(2) centrifugally separating the activated fluorescent microspheres, removing supernatant, taking precipitate, adding 1000 mu l of 0.02M borate buffer solution with pH8.0, and fully and uniformly mixing;
(3) adding 100 μ g goat anti-chicken IgY, mixing uniformly by vortex, placing on a rotary incubator, and reacting for 60 min;
(4) adding 100 μ l of 10% casein blocking solution into each tube, mixing uniformly by vortex, placing on a rotary incubator after completing ultrasonic treatment, and sealing for 60 min; and centrifuging again, sucking supernatant, retaining precipitate, adding 1mL of microsphere preservation solution, and mixing completely for later use.
6. The immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs according to claim 1, wherein the preparation process of the florfenicol detection microspheres and the trimethoprim detection microspheres is divided into three steps:
the first step is as follows: the preparation method of the long-chain biotinylation florfenicol monoclonal antibody and the long-chain biotinylation trimethoprim monoclonal antibody comprises the following steps:
(1) taking out the long-chain Biotin NHS-dPEG24-Biotin, balancing for 30min at room temperature, and dissolving with DMSO to a final concentration of 10mM for later use;
(2) respectively taking 1ml of florfenicol monoclonal antibody with the concentration of 2mg/ml and 1ml of trimethoprim monoclonal antibody with the concentration of 2mg/ml into a 2ml centrifuge tube, adding 27 mu l of Biotin NHS-dPEG24-Biotin prepared in the previous step into each tube, uniformly mixing by vortex, and reacting for 30min on a rotary incubator;
(3) transferring the mixture to an ultrafiltration tube after the reaction is finished, centrifuging the mixture for 15min at 12000g, and discarding waste liquid;
(4) adding 2ml of PBS buffer solution into the ultrafiltration tube, 12000g, centrifuging 15ml, and discarding waste liquid;
(5) repeating the step (4) for 5 times, redissolving by 200 mu l of PBS, collecting the labeled antibody, and measuring the concentration by using a BCA kit;
the second step is that: the preparation method of the streptavidin fluorescent microsphere comprises the following steps:
(1) adding 1mL of marking buffer solution 0.05M MES buffer solution into a 2mL centrifuge tube, adding 1mg of fluorescent microspheres, and uniformly mixing by vortex; adding 4. mu.l NHS with concentration of 25mg/ml and 4. mu.l EDC with concentration of 25mg/ml, mixing uniformly by vortex, and reacting for 20min on a rotary incubator;
(2) centrifugally separating the activated fluorescent microspheres, removing supernatant, taking precipitate, adding 1000 mu l of borate buffer solution, and fully and uniformly mixing;
(3) adding 100 mu g of streptavidin into the activated microspheres, uniformly mixing by vortex, and placing on a rotary incubator for reaction for 60 min;
(4) adding 100 μ l of 10% casein blocking solution into each tube, mixing uniformly by vortex, placing on a rotary incubator after ultrasonic treatment, sealing for 60min, centrifuging, removing supernatant, and collecting precipitate;
(5) adding 1mL of microsphere preservation solution into the precipitate, fully and uniformly mixing, carrying out centrifugal separation, sucking supernatant, and taking the precipitate;
(6) repeating the step (5) for 2-3 times to fully remove unreacted streptavidin; adding 1mL of microsphere preservation solution, and fully and uniformly mixing for later use;
the third step: the preparation process of the florfenicol detection microsphere and the trimethoprim detection microsphere comprises the following steps:
(1) respectively adding 5 mu g of each long-chain biotinylation florfenicol monoclonal antibody and long-chain biotinylation trimethoprim monoclonal antibody into 2 tubes of fluorescent microspheres containing 1ml of streptavidin, uniformly mixing in a vortex manner, and reacting for 30min on a rotary incubator;
(2) after the reaction is finished, centrifuging at 16500rpm for 20min, sucking the supernatant, and taking the precipitate;
(3) adding 1mL of microsphere preservation solution, mixing well, centrifuging at 16500rpm for 20min, and removing supernatant;
(4) repeating the step (3) for 3 times, and fully removing the unreacted biotin-labeled antibody;
(5) adding 1mL of microsphere storage solution, and mixing well for later use.
7. A method for synchronously detecting the content of florfenicol and trimethoprim in poultry eggs is characterized by comprising the following steps:
pretreating a sample to obtain a solution to be detected;
(II) adding a solution to be detected into the detection card of claim 1, horizontally standing for 10min, and then inserting into a fluorescence immunochromatographic instrument to read the result;
thirdly, the result is brought into the standard curve, and the content of the florfenicol and the trimethoprim in the sample can be calculated;
wherein, the pretreatment method in the step (one) comprises the following steps:
(1) adding the extract into the fully homogenized egg to-be-detected liquid, manually shaking and uniformly mixing for extraction, centrifuging and taking supernatant;
(2) adding n-hexane and a redissolution into the supernatant, shaking, standing and layering to obtain a lower water phase as a solution to be detected;
the volume ratio of the poultry egg sample to be detected to the extracting solution is as follows: 2: 2-3.
8. The method for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs as claimed in claim 7, wherein the extracting solution is ethyl acetate containing 0.5-1.5wt% of trichloroacetic acid.
9. The method for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs according to claim 7, wherein the complex solution is PBS buffer solution containing 0.15M NaCl and 0.05M PB, and the pH value is 6.6.
10. The method for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs according to claim 7, wherein the volume ratio of the supernatant to the n-hexane to the complex solution is as follows: 1:5:0.5-1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110285602.6A CN112798778B (en) | 2021-03-17 | 2021-03-17 | Immunofluorescence chromatography detection card and method for synchronously detecting florfenicol and trimethoprim contents in poultry eggs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110285602.6A CN112798778B (en) | 2021-03-17 | 2021-03-17 | Immunofluorescence chromatography detection card and method for synchronously detecting florfenicol and trimethoprim contents in poultry eggs |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112798778A true CN112798778A (en) | 2021-05-14 |
| CN112798778B CN112798778B (en) | 2021-07-02 |
Family
ID=75817095
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110285602.6A Active CN112798778B (en) | 2021-03-17 | 2021-03-17 | Immunofluorescence chromatography detection card and method for synchronously detecting florfenicol and trimethoprim contents in poultry eggs |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112798778B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117074663A (en) * | 2023-10-12 | 2023-11-17 | 乐福思健康产业股份公司 | Detection method of colloidal gold immunochromatography analyzer |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103524427A (en) * | 2012-07-03 | 2014-01-22 | 北京勤邦生物技术有限公司 | Preparation method as well as application of trimethoprem hapten |
| CN103946686A (en) * | 2011-11-16 | 2014-07-23 | 皇家飞利浦有限公司 | Long rigid spacers to enhance binding kinetics in immunoassays |
| CN105652004A (en) * | 2015-12-29 | 2016-06-08 | 深圳市易瑞生物技术有限公司 | Trimethoprim hapten as well as colloidal gold detection device and preparation method of trimethoprim hapten |
| CN108982450A (en) * | 2018-07-24 | 2018-12-11 | 上海雄图生物科技有限公司 | A kind of food safety fluorescence immunoassay quantifies poct detection system |
| CN208443851U (en) * | 2018-05-04 | 2019-01-29 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting cat serum amyloid A protein chromatographs detection card |
| CN208672650U (en) * | 2018-05-04 | 2019-03-29 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting canine coronavirus antigen chromatographs detection card |
| CN110312518A (en) * | 2016-12-09 | 2019-10-08 | 昆士兰大学 | Glycoprotein Avidin Construct |
| CN110845619A (en) * | 2019-09-04 | 2020-02-28 | 山东绿都生物科技有限公司 | Monoclonal antibody for identifying florfenicol, chloramphenicol and thiamphenicol and application thereof |
| CN210690597U (en) * | 2019-05-14 | 2020-06-05 | 河南知微生物工程有限公司 | A multi-card for detection of antibiotic residues in poultry and eggs |
-
2021
- 2021-03-17 CN CN202110285602.6A patent/CN112798778B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103946686A (en) * | 2011-11-16 | 2014-07-23 | 皇家飞利浦有限公司 | Long rigid spacers to enhance binding kinetics in immunoassays |
| CN103524427A (en) * | 2012-07-03 | 2014-01-22 | 北京勤邦生物技术有限公司 | Preparation method as well as application of trimethoprem hapten |
| CN105652004A (en) * | 2015-12-29 | 2016-06-08 | 深圳市易瑞生物技术有限公司 | Trimethoprim hapten as well as colloidal gold detection device and preparation method of trimethoprim hapten |
| CN110312518A (en) * | 2016-12-09 | 2019-10-08 | 昆士兰大学 | Glycoprotein Avidin Construct |
| CN208443851U (en) * | 2018-05-04 | 2019-01-29 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting cat serum amyloid A protein chromatographs detection card |
| CN208672650U (en) * | 2018-05-04 | 2019-03-29 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting canine coronavirus antigen chromatographs detection card |
| CN108982450A (en) * | 2018-07-24 | 2018-12-11 | 上海雄图生物科技有限公司 | A kind of food safety fluorescence immunoassay quantifies poct detection system |
| CN210690597U (en) * | 2019-05-14 | 2020-06-05 | 河南知微生物工程有限公司 | A multi-card for detection of antibiotic residues in poultry and eggs |
| CN110845619A (en) * | 2019-09-04 | 2020-02-28 | 山东绿都生物科技有限公司 | Monoclonal antibody for identifying florfenicol, chloramphenicol and thiamphenicol and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| GREG T HERMANSON: "《Bioconjugate techniques》", 31 December 2013 * |
| NORA THEILACKER, ET AL.,: "Multiplexed protein analysis using encoded antibody-conjugated microbeads", 《J. R. SOC. INTERFACE》 * |
| ZHONGXING WANG等: "Development and comparison of two nanomaterial label-based lateral flow immunoassays for the detection of five antibacterial synergists", 《THE ROYAL SOCIETY OF CHEMISTRY AND THE CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117074663A (en) * | 2023-10-12 | 2023-11-17 | 乐福思健康产业股份公司 | Detection method of colloidal gold immunochromatography analyzer |
| CN117074663B (en) * | 2023-10-12 | 2024-02-06 | 乐福思健康产业股份公司 | Detection method of colloidal gold immunochromatography analyzer |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112798778B (en) | 2021-07-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112666347B (en) | Immunofluorescence chromatography kit and method for synchronously detecting contents of aflatoxin B1 and zearalenone in vegetable oil | |
| CN107765002B (en) | Colloidal gold immunochromatography test strip and preparation method and application thereof | |
| CN103792357A (en) | Spectinomycin fast time-resolved fluoroimmunoassay quantitative detection test strip | |
| CN105044346A (en) | Quantum dot fluorescence immunochromatography test strip for double quantification of mycotoxin and preparation method of quantum dot fluorescence immunochromatography test strip | |
| CN107884567A (en) | A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects Clenbuterol kit | |
| CN108398565A (en) | Immunofluorescence for detecting dog C reactive protein chromatographs detection card and preparation method | |
| US20180364227A1 (en) | Detection method with centrifuge isolation and detection device thereof | |
| CN101788560B (en) | Enzyme-linked immunoassay kit for detecting residual chloramphenicol and detection method thereof | |
| CN104076154A (en) | Enzyme linked immunosorbent assay kit detecting folic acid and application thereof | |
| CN101799469A (en) | Enzyme linked immunosorbent assay kit for detecting residual clenbuterol hydrochloride and a detection method thereof | |
| CN107271682A (en) | A kind of dog c reactive protein fluorescence detection test strip | |
| CN112798778B (en) | Immunofluorescence chromatography detection card and method for synchronously detecting florfenicol and trimethoprim contents in poultry eggs | |
| CN108614106A (en) | A kind of time-resolved fluoroimmunoassay chromatography card detecting vomitoxin and its acetyl derivatives | |
| Water et al. | Sensitive streptavidin-biotin enzyme-linked immunosorbent assay for rapid screening of chloramphenicol residues in swine muscle tissue | |
| CN104977406A (en) | Enzyme-linked immunoassay kit for detecting fluoroquinolone medicine and application of kit | |
| CN104897864A (en) | ELISA (enzyme linked immunosorbent assay) kit for detecting amantadine and application of ELISA kit | |
| CN109459570A (en) | The colloid gold immune test paper preparation method of lead ion in a kind of quick detection blood of human body | |
| CN101315379A (en) | Reagent kit for detecting Ractopamine and application thereof | |
| CN114113597A (en) | Novel coronavirus antigen detection kit and preparation method thereof | |
| CN104569398B (en) | The enzyme linked immunological kit of detection ethoxy quinoline and application thereof | |
| CN208443852U (en) | Immunofluorescence for detecting dog C reactive protein chromatographs detection card | |
| CN107727850A (en) | A kind of lateral flow chromatography detection reaction starts control method | |
| CN109975538A (en) | A kind of detection perlsucht IFN-γ and the test strips of bovine paratuberculosis antibody and preparation method thereof | |
| CN106872690B (en) | A kind of high sensitivity colloid gold test paper and the preparation method and application thereof | |
| CN208607234U (en) | Immunofluorescence for detecting Canine Parvovirus antigen chromatographs detection card |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |