CN110275013A - Cimaterol colloidal gold rapid detection card and preparation method thereof suitable for animal derived food - Google Patents
Cimaterol colloidal gold rapid detection card and preparation method thereof suitable for animal derived food Download PDFInfo
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- CN110275013A CN110275013A CN201910430806.7A CN201910430806A CN110275013A CN 110275013 A CN110275013 A CN 110275013A CN 201910430806 A CN201910430806 A CN 201910430806A CN 110275013 A CN110275013 A CN 110275013A
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- cimaterol
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- 238000001514 detection method Methods 0.000 title claims abstract description 101
- BUXRLJCGHZZYNE-UHFFFAOYSA-N 2-amino-5-[1-hydroxy-2-(propan-2-ylamino)ethyl]benzonitrile Chemical compound CC(C)NCC(O)C1=CC=C(N)C(C#N)=C1 BUXRLJCGHZZYNE-UHFFFAOYSA-N 0.000 title claims abstract description 80
- 229950010971 cimaterol Drugs 0.000 title claims abstract description 80
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 46
- 238000002360 preparation method Methods 0.000 title claims abstract description 35
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- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 description 2
- NDAUXUAQIAJITI-UHFFFAOYSA-N albuterol Chemical compound CC(C)(C)NCC(O)C1=CC=C(O)C(CO)=C1 NDAUXUAQIAJITI-UHFFFAOYSA-N 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- 229960001117 clenbuterol Drugs 0.000 description 2
- STJMRWALKKWQGH-UHFFFAOYSA-N clenbuterol Chemical compound CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 STJMRWALKKWQGH-UHFFFAOYSA-N 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
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- 238000002347 injection Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
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- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 229940074095 ractopamine Drugs 0.000 description 2
- YJQZYXCXBBCEAQ-UHFFFAOYSA-N ractopamine Chemical compound C=1C=C(O)C=CC=1C(O)CNC(C)CCC1=CC=C(O)C=C1 YJQZYXCXBBCEAQ-UHFFFAOYSA-N 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
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- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- XZMCDFZZKTWFGF-UHFFFAOYSA-N Cyanamide Chemical compound NC#N XZMCDFZZKTWFGF-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
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- 229960001399 clenbuterol hydrochloride Drugs 0.000 description 1
- OPXKTCUYRHXSBK-UHFFFAOYSA-N clenbuterol hydrochloride Chemical compound Cl.CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 OPXKTCUYRHXSBK-UHFFFAOYSA-N 0.000 description 1
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- 238000003317 immunochromatography Methods 0.000 description 1
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- 238000010348 incorporation Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
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- GOLXNESZZPUPJE-UHFFFAOYSA-N spiromesifen Chemical compound CC1=CC(C)=CC(C)=C1C(C(O1)=O)=C(OC(=O)CC(C)(C)C)C11CCCC1 GOLXNESZZPUPJE-UHFFFAOYSA-N 0.000 description 1
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled 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
- G01N33/558—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
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (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)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention is intended to provide a kind of Cimaterol colloidal gold rapid detection card and preparation method thereof for being applicable in animal derived food.The Cimaterol colloidal gold rapid detection card prepared has the characteristics that high sensitivity, high specificity, stability are high, easy to operate, easy to carry, complicated instrument and equipment and technical professional are not needed, it is analyzed result and is easy to judge, testing result can be directly observed with the naked eye in short time, it is widely used in the on-site tests analysis such as fast-food detection, there is wide applicability and Development volue in Cimaterol monitoring field.
Description
Technical field
The invention mainly relates to the technical fields of immune detection, specifically, the present invention relates to a kind of Cimaterol colloids
The technical field of golden rapid detection card and preparation method thereof.
Background technique
Cimaterol (cimaterol, CIM) also known as happiness Ma Teluo, Zeeman spy sieve, are American Cyanamid Company's products, belong to β 2
Type excitant, research are the beta-adrenalines of new generation after Ractopamine, Clenbuterol and salbutamol than wide
Receptor excitomotor.Cimaterol is used to larger dose, the fat content of trunk can also be reduced, improves lean meat percentage, is promoted
Domestic animal growth achievees the effect that improve meat, therefore is illegally used in animal derived food production as feed addictive.And
Long-time service will cause Cimaterol and accumulate residual in edibility animal tissue, and eater is caused to be poisoned.Especially to coronary disease
Sick hypertensive patient, the elderly are easy to produce harm, serious or even be in peril of one's life.The developed countries such as America and Europe before and after nineteen ninety
It completely forbids in succession and uses beta-stimulants class growth promotion drug.The Ministry of Agriculture of the People's Republic of China, MOA announces No. 193 file regulation
The same Clenbuterol of Cimaterol, salbutamol etc. are put into disabling inventory together, forbid adding in Production of Livestock and Poultry and food animal
Add.
Currently, the method for detection Ractopamine and Cimaterol mainly has both at home and abroad.In food safety detection, often
After first carrying out primary dcreening operation with ELISA, then positive sample is confirmed with HPLC or GC-MS, LC-MS.But used in the above method
Instrument and equipment costly, it is at high cost, while needing to carry out Special Training to operator, and experimental result cannot be shown immediately
Show, therefore is not suitable for commodity inspection, epidemic prevention, husbandry sector person to the quick online detection and monitoring of object of suspicion.
Cimaterol lacks as novel " clenbuterol hydrochloride " substance, detection means lag, especially quick testing product, at present
The method of established detection Cimaterol has high pressure lipuid chromatography (HPLC) (HPLC), one Mass Spectrometry (LC- of liquid chromatogram
MS), ultra performance liquid chromatography one connect mass spectrometric analysis method (μ PLC-MS/MS), gas chromatography and mass spectromentry combination method (GC-MS),
High performance capillary gas chromatography method (CZE), enzyme linked immunosorbent assay (ELISA) etc..The operation of these methods is needed to operator
Carry out Special Training, and experimental result cannot be shown immediately, therefore be not suitable for the commodity inspection producer to object of suspicion it is quick
Line detection and monitoring.Therefore it is badly in need of the rapid detection method for being suitble to batch, quickly screening, to meet the detection need of government and enterprise
It wants.
Colloidal-gold detecting-card is the indicant using colloidal gold as immunochromatography, and principle is with fibre strip chromatographic material
For solid phase, make sample solution swimming on chromatography strip through capillary action, and makes the determinand and chromatographic material in sample simultaneously
The immune response of high specificity compatibility occurs for the upper receptor (such as antibody or antigen) for determinand, is immunized in chromatography process multiple
The certain area (detection band) that object was enriched with or was trapped in chromatographic material is closed, by directly with the colloid gold label object that can be estimated
And obtain intuitive experimental result.
Summary of the invention
High sensitivity, high specificity, stability height, operation letter there is no for the detection of Cimaterol for the current country
The technical issues of single, portable detection method, the present invention is intended to provide Cimaterol colloidal gold rapid detection card and its system
Preparation Method, by preparing Cimaterol monoclonal antibody, the detection card of preparation detection Cimaterol obtains a kind of Cimaterol glue
Body gold rapid detection card, the detection card high sensitivity, high specificity, stability are high, easy to operate, easy to carry, in Xi Mate
Sieve, which monitors field, has wide applicability and Development volue.
What the invention is realized by the following technical scheme:
The present invention provides a kind of Cimaterol colloidal gold rapid detection card for being applicable in animal derived food, including contains colloid
The gold-labelled pad of the Cimaterol monoclonal antibody solution spraying of gold label, NC film, sample pad and the water suction for being painted with T line and C line
Pad, is assembled.Wherein, the Cimaterol monoclonal antibody T liquid that T line is manually prepared using 1 μ L/mL is drawn, and C line makes
It is drawn with the C liquid of 250 times of diluted PC-2 sheep anti mouses.
In the present invention, a kind of specific preparation for the Cimaterol colloidal gold rapid detection card being applicable in animal derived food
Process are as follows:
(1) it the preparation of gold-labelled pad: takes 1mL colloidal gold solution in 2mL test tube with 1.35% red gold particle label, is added
0.2M K2CO32.0 μ L/mL are mixed；1.0 μ L of antibody stoste is added, stands 5min after mixing；The closing containing 10%BSA is added
10 μ L/mL of liquid, stands 5min after mixing；4 DEG C, 12000r/min centrifugation, 5min；The redissolution liquid of 1/20 volume redissolves, and is sprayed at
It on the glass fibre element film closed, is transferred quickly to be freeze-dried in freeze drier, production becomes gold-labelled pad；
(2) preparation of C liquid and T liquid: with coating buffer by antigen diluent, 250 times of PC-2 sheep anti mouse being diluted, C liquid are made, will
50 times of dilutions of artificial antigen obtained obtain T liquid；
(3) coating of NC film: the C liquid of preparation, T liquid are crossed on NC film respectively, be sequentially followed successively by from left to right T liquid and
C liquid sets 37 DEG C of dry 20~40min, and confining liquid closing is submerged into after dry, is placed in drying for standby in 37 DEG C of insulating boxs；
(4) assembling of detection card: the sample pad prepared, gold-labelled pad, NC film and water absorption pad are cut to of same size
Rectangle, successively carry out overlapping assembling, and well is set, be assembled into detection Cimaterol detection card.
In the present invention, 0.2M K is added in the gold-labelled pad preparation process2CO3Amount be 2.0 μ L/mL.
In the present invention, the amount that antibody stoste is added in the gold-labelled pad preparation process is 1.0 μ L.
In the present invention, the T liquid carries out 50 times of diluted solution with the artificial Cimaterol antibody prepared.
In the present invention, the C liquid dilutes the solution of 250 times of acquisitions with PC-2 sheep anti mouse.
In the present invention, the detection card is detection Cimaterol content, and detection card includes using the Xi Mate prepared
Sieve monoclonal antibody solution spraying gold-labelled pad, using Cimaterol monoclonal antibody T liquid draw T line and and use
The C liquid of PC-2 sheep anti mouse draws NC film, sample pad and the water absorption pad of C line, is assembled.
In the present invention, the monoclonal antibody preparation process of the Cimaterol are as follows:
About 10 Balb/c mouse are chosen as immune animals, immunogene is Cimaterol haptens-BSA, three to four times
Subcutaneous multi-point injection is for the last time abdominal cavity booster immunization；It is thin using SP2/0 murine myeloma cell and the spleen of preferred mouse
Born of the same parents carry out cell fusion, obtain ideal cell strain and antibody through culture, observation, detection and yin and yang attribute check experiment after fusion；
Mouse ascites (antibody) preparation is carried out to special positive cell strain obtained, is detected through ascites, different cell strains generates
Titer of ascites etc. have a certain difference, highest antibody titer can reach 1:27W；Monoclonal antibody will be obtained, will be lyophilized
It is spare.
In the present invention, the detection is Cimaterol haptens-OVA with coating antigen.
In addition, the present invention provides a kind of application of Cimaterol colloidal gold rapid detection card for being applicable in animal derived food,
Specifically includes the following steps:
(1) sample treatment: will be to solid-state sample product the homogeneous 1min in homogenizer, 10000rpm；Weigh 5.0 ± 0.05g
The tissue samples being homogenized detain upper tube cap into 10mL polystyrene centrifuge tube, should not be too tight；It is put into water-bath in 80 DEG C of water-baths
It takes out and is cooled to room temperature after 10min, it is to be checked；Liquid measuring samples acquire 50mL with dry, clean centrifuge tube or appropriate containers
Left and right can be reserved for 24 hours if do not detected immediately in 2-8 DEG C of refrigeration, pay attention to that corruption is avoided to cause to fail or pollute；
(2) it detects: will test card before use and sample to be examined solution restores to room temperature, detection is taken out from original packing bag
Card, will test card and lays flat, and draw measuring samples solution with dropper, vertical 3 drops that are added dropwise start timing 3- in well after sample-adding
The appearance situation of 5 minutes observation T lines and C line；
(3) testing result: C line goes out to represent result effective in detection card；It detects T line in card and does not go out to represent, indicate
Beta-stimulants Cimaterol concentration is higher than 0.01ppb in sample；T line occurs simultaneously with C line in detection card, illustrates in measuring samples
It is lower than 0.01ppb without containing beta-stimulants Cimaterol or its concentration, C line does not occur in paper slip, and it is invalid to represent result.
By implement technical solution of the present invention, can achieve it is following the utility model has the advantages that
The present invention provides a kind of Cimaterol colloidal gold rapid detection card and preparation method thereof for being applicable in animal derived food,
Based on immunologic detection method, by preparing Cimaterol monoclonal antibody, and the detection card of point preparation detection Cimaterol,
Obtain a kind of Cimaterol colloidal gold rapid detection card for being applicable in animal derived food, the detection card can quickly, easily into
The detection of Cimaterol in row food, and preferably antibody titer reaches 1:27 ten thousand, sensitivity reaches 0.1ppb, can satisfy daily
Production and processing, the needs quickly detected.
Detailed description of the invention
Fig. 1 is shown as detection card assembling schematic diagram.
Fig. 2 is shown as manually preparing monoclonal antibody color developing effect qualification figure.
Fig. 3 is shown as manually preparing monoclonal antibody-purified effect identification figure.
Fig. 4 is shown as the effect of optimization qualification figure of colloidal gold rapid detection card PH.
Fig. 5 is shown as the effect of optimization qualification figure of antibody labelled amount.
Fig. 6 is shown as Cimaterol colloidal gold rapid detection card sample simulation detection effect figure.
Fig. 7 is shown as Cimaterol colloidal gold rapid detection card testing result process decision chart.
Specific embodiment
In the following, illustrating the present invention for embodiment, still, the present invention is not limited to following embodiments.
The main agents of use: glass fibre element film is purchased from Shanghai Jinbiao Bio-Tech Co., Ltd., NC film (cellulose nitrate
Plain film) it is purchased from Shanghai Jinbiao Bio-Tech Co., Ltd.；Female Balb/c mouse is purchased from Zhenjiang biology Co., Ltd；Sheep anti mouse
IgG is purchased from SIGMA company, K2CO3Purchased from SIGMA company.
The main instrument and equipment used: the desk-top low-temperature and high-speed centrifuge of Eppendorf, the full-automatic albumen of AKTAp μ re
Purify instrument, AL104 electronic balance, BRANSON Ultrasound Instrument, spectrophotometer.
The reagent and equipment that the present invention uses can be bought or be customized by public channel.
What all material, reagent and the instrument selected in the present invention were all well known in the art, but reality of the invention is not limited
It applies, other some reagents well known in the art and equipment are applied both to the implementation of following implementation of the present invention.
Embodiment one: it is applicable in the Cimaterol monoclonal antibody preparation of animal derived food
(1) prepared by Cimaterol monoclonal antibody:
The identical 10 Balb/c mouse of week old are chosen as immune animal, immunogene is Cimaterol haptens-BSA,
Immunizing dose: 0.2mg times/only, and three to four subcutaneous multi-point injections and the immune mouse of intraperitoneal injection, specific immunization ways such as table 1
It is shown.
Table 1: Cimaterol monoclonal antibody prepares immune programme
| Process name | Time |
| Blood sampling before immune | T=-4 days |
| It is immune for the first time | T=0 days |
| Second immune | T=14 days |
| Blood sampling detection for the first time | T=21 days |
| Third time is immune | T=28 days |
| Second of blood sampling detection | T=35 days |
| Add and exempts from | T=42 days |
| Fusion | T=exempts from+4 days eventually |
After 3rd blood sampling detection, 2 optimal mouse in preferably 10 mouse are for merging, testing result such as 2 institute of table
Show.
Table 2: Cimaterol monoclonal antibody prepares immune result detection
Cell fusion is carried out using SP2/0 murine myeloma cell and the splenocyte of preferred mouse, cultivated, seen after fusion
It examines, detect and yin and yang attribute check experiment.In terms of later period screening cell strain situation, ideal cell strain is obtained from the above 3-2 mouse
And antibody.Detection coating antigen: Cimaterol haptens-OVA is detected through the primary of limiting dilution assay, two time clonings and supernatant,
6 plants of specific positive cells strains are obtained, the results are shown in Table 3 for specific experiment.
Table 3: Cimaterol monoclonal antibody prepares fusion results detection
| Code name | Cell strain number | Extension rate | Standard concentration | OD value |
| 101 | 106-B1 | 250 | 1ng/mL | 2.107 |
| 102 | 106-D1 | 450 | 1ng/mL | 1.223 |
| 201 | 106-F1-H3 | 260 | 0.1ng/mL | 2.386 |
| 202 | 106-F1-H8 | 260 | 0.1ng/mL | 1.344 |
| 301 | 1E6-E5-H2 | 260 | 0.1ng/mL | 2.078 |
| 302 | 1B6-H5-F9 | 260 | 0.1ng/mL | 1.739 |
(2) Cimaterol antibody titer detects:
With 2 μ g/mL Cimaterol antibody coated elisa plates, 50 holes μ L/, 4 DEG C of overnight or 37 DEG C of 2~4h of coating.Incline and wraps
By liquid, after washing 3~5 times with PBST, 0.4% fishskin gelatin is added and closes, 200 holes μ L/, 37 DEG C, 1~3h.Confining liquid is discarded,
It is added after PBST washs 3~5 times, the doubling dilution antibody purification since 1000 times (mother liquid concentration 1mg/mL), blank is added
Control is PBS, 50~100 holes μ L/, 37 DEG C of 1~3h of incubation.After the artificial board-washing of PBST 3 times, the diluted alkaline phosphorus of 1:5000 is added
The goat anti-mouse IgG secondary antibody of sour enzyme label, 50 holes μ L/, 37 DEG C of 1~3h of incubation.After the artificial board-washing of PBST 5 times, colour developing is added
Substrate pNPP, 50 holes μ L/, 37 DEG C of 0.5~1h of colour developing.Measure the absorbance under wavelength 405nm.
Mouse ascites (antibody) preparation is carried out to special positive cell strain obtained, is detected through ascites, different is thin
The titer of ascites etc. that born of the same parents' strain generates has a certain difference.6 plants of cell strains obtain 15 ascites samples altogether.Highest antibody effect
Valence can reach 1:27W.Specific testing result is as shown in table 4.
Table 4: Cimaterol monoclonal antibody prepares the detection of antibody titer result
| Number | Cell strain | Standard concentration | Antibody titer | OD value |
| MC-3 | 102 | 0.1ng/mL | 1:3W | 1.983 |
| MC-4 | 101 | 0.1ng/mL | 1:27W | 1.611 |
| MC-5 | 101 | 0.1ng/mL | 1:27W | 1.066 |
| MC-8 | 102 | 0.1ng/mL | 1:3W | 1.733 |
| MC-10 | 102 | 0.1ng/mL | 1:3W | 1.928 |
| MC-13 | 201 | 0.1ng/mL | 1:27W | 1.875 |
| MC-19 | 102 | 0.1ng/mL | 1:27W | 1.512 |
| MC-20 | 201 | 0.1ng/mL | 1:27W | 2.107 |
| MC-21 | 202 | 0.1ng/mL | 1:9W | 1.879 |
| MC-22 | 202 | 0.1ng/mL | 1:9W | 1.680 |
| MC-23 | 301 | 0.1ng/mL | 1:3W | 1.112 |
| MC-24 | 301 | 0.1ng/mL | 1:27W | 1.267 |
| MC-25 | 302 | 0.1ng/mL | 1:27W | 1.375 |
| MC-26 | 302 | 0.1ng/mL | 1:27W | 1.682 |
| MC-28 | 201 | 0.1ng/mL | 1:27W | 2.101 |
Embodiment two: it is applicable in the preparation of the colloidal gold rapid detection card of animal derived food
(1) preparation of gold-labelled pad
It takes 1mL colloidal gold solution in 2mL test tube with 1.35% red gold particle label, 0.2M K is added2CO3 2.0μ
L/mL is mixed；1.0 μ L of antibody stoste is added, stands 5min after mixing；10 μ L/mL (10%BSA) of confining liquid is added, after mixing
Stand 5min；4 DEG C, 12000r/min centrifugation, 5min；The redissolution liquid of 1/20 volume redissolves, and is sprayed at the glass fibers closed
It ties up on plain film, is transferred quickly to be freeze-dried in freeze drier, production becomes gold-labelled pad.
(2) preparation of sample pad and water absorption pad
Sample pad is that glass fibre element film is submerged into confining liquid (the 0.01mol/L phosphorus containing 2~3% bovine serum albumin(BSA)s
Acid buffer) it is closed, 37 DEG C of drying for standby.Water absorption pad is the preferable absorbent filter material of water absorbing properties.
(3) coating of nitrocellulose filter (NC film)
Use coating buffer that the artificial Cimaterol antibody of preparation is diluted 50 times for as T liquid, PC-2 goat-anti murine monoclonal is anti-
Body dilutes 250 times and is used as C liquid.The C liquid of preparation and T liquid are crossed on NC film respectively, are sequentially followed successively by T liquid and C from left to right
Liquid sets 37 DEG C of dry 20~40min, and confining liquid closing is submerged into after dry, is placed in drying for standby in 37 DEG C of insulating boxs.
(4) assembling of detection card
The sample pad prepared, gold-labelled pad, NC film and water absorption pad are cut to rectangle of same size, and press attached drawing
Overlapping assembling is carried out shown in 1, after 50 times of coating buffer dilutions of artificial antigen with 1 μ L of pipettor point on blank NC film, backboard
Blotting paper is sticked in one end, the other end sticks that treated gold-labelled pad and sample pad, is connected with each other between auxiliary material, NC film is pressed at both ends
1-2mm is cut into the strip of 3.5mm wide, is assembled into the detection card of detection Cimaterol content.
Embodiment three: it is applicable in the debugging of the colloidal gold rapid detection card of animal derived food
Based on scheme provided in embodiment one to embodiment two, colloidal gold rapid detection card is debugged.Specifically
Process is as follows:
(1) manually preparation monoclonal antibody color developing effect identification
Manually antigen sieves antibody, and each cell strain takes a raw material to be detected, and testing result is as shown in Fig. 2, knot
Fruit shows that MC-20 and antigen binding color developing effect are good.
(2) monoclonal antibody-purified effect identification is manually prepared
To the antibody ascites MC-13/MC-20/MC-28 with the same cell strain of MC-20 obtained after purification MC-13-CT,
Tri- Zhi Dankang of MC-20-CT, MC-28-CT is matched with antigen again.Testing result is as shown in Fig. 3, as the result is shown same tag
Under the conditions of in three antibody the whole colour developing situation of MC-28-CT be slightly better than other two antibody.Therefore final determine matches raw material
For antigen and antibody MC-28-CT.
(3) optimization of colloidal gold rapid detection card PH
It being marked with 1.35% red gold particle, the K2CO3 of 0.2mol/L adds 1.5/2.0/2.5/3.0 μ L/mL respectively,
Antibody labelled amount is 5 μ L/mL, each condition flag 1mL, 10%BSA closing, redissolves liquid and redissolves to 100 μ L, spare.Detection knot
Fruit is as shown in Fig. 4, and potency is best when potassium carbonate adds 2 μ L/mL as the result is shown.
(4) optimization of antibody labelled amount
Under above-mentioned flag condition, antibody labelled amount is adjusted to 0.5/1.0/1.5/2.0/2.5 μ L/mL, and testing result is such as
Shown in attached drawing 5, antibody labelled amount develops the color best in 1 μ L/mL as the result is shown.
(5) Cimaterol colloidal gold rapid detection card sample simulation detects
The sample containing Cimaterol is detected by using the Cimaterol colloidal gold rapid detection card of acquisition, is examined
It is as shown in Fig. 6 to survey result, testing result display present invention Cimaterol colloidal gold rapid detection card sensitivity obtained reaches
To 0.1ppb, reach detection demand by debugging product.
Example IV: it is applicable in animal derived food colloidal gold rapid detection card and quickly detects Cimaterol
In experimental basis by embodiment one to embodiment five, the present invention further provides colloidal gold rapid detection card is fast
The method of speed detection Cimaterol, the i.e. present invention further provide a kind of colloidal gold rapid detection card of detection Cimaterol
Using, specifically includes the following steps:
(1) sample treatment: will be to solid-state sample product the homogeneous 1min in homogenizer, 10000rpm；Weigh 5.0 ± 0.05g
The tissue samples being homogenized detain upper tube cap into 10mL polystyrene centrifuge tube, should not be too tight；It is put into water-bath in 80 DEG C of water-baths
It takes out and is cooled to room temperature after 10min, it is to be checked；Liquid measuring samples acquire 50mL with dry, clean centrifuge tube or appropriate containers
Left and right can be reserved for 24 hours if do not detected immediately in 2-8 DEG C of refrigeration, pay attention to that corruption is avoided to cause to fail or pollute.
(2) it detects: will test card before use and sample to be examined solution restores to room temperature, detection is taken out from original packing bag
Card, will test card and lays flat, and draw measuring samples solution with dropper, vertical 3 drops that are added dropwise start timing 3- in well after sample-adding
The appearance situation of 5 minutes observation T lines and C line；
(3) testing result: as shown in Fig. 7, C line goes out to represent result effective in detection card；It detects T line in card and does not go out
It represents, indicates that beta-stimulants Cimaterol concentration is higher than 0.01ppb in sample；T line occurs simultaneously with C line in detection card,
Illustrate to be lower than 0.01ppb without containing β-excitant Cimaterol or its concentration in measuring samples, C line does not occur in paper slip, represents
As a result invalid.
Embodiment five: it is applicable in animal derived food colloidal gold rapid detection card and detects Cimaterol application
By Cimaterol by weight in incorporation measuring samples, the ratio for mixing Cimaterol is respectively 100 μ g/kg, 10 μ
The step of according to example IV, sample liquids are dripped by g/kg, 1 μ g/kg, 0.1 μ g/kg, and distilled water is arranged as blank control
In the sample pad part of colloidal gold rapid detection card, after waiting liquid to diffuse to water absorption pad, T line and C are observed after standing 3~5min
The appearance situation of line, test result are as shown in table 5.
Table 5: colloidal gold rapid detection card detects food different proportion Cimaterol experimental result
Note: "+" indicates occur, and "-" expression does not occur
Pass through test result shown in table 5, it can be seen that the colloidal gold rapid detection card prepared through the invention can be fast
Speed be easily applicable in animal derived food in the detection of Cimaterol, and sensitivity reaches 0.1ppb, can satisfy daily
Production and processing, the needs quickly detected.
As described above, the present invention can be realized preferably, the above embodiments are only to preferred implementation side of the invention
Formula is described, and is not intended to limit the scope of the present invention, and without departing from the spirit of the design of the present invention, this field is general
The various changes and improvement that logical technical staff makes technical solution of the present invention, should all fall into present invention determine that protection scope
It is interior.
Claims (8)
1. a kind of Cimaterol colloidal gold rapid detection card for being applicable in animal derived food, which is characterized in that including manually preparing
Cimaterol monoclonal antibody and PC-2 sheep anti-mouse antibody ingredient colloidal gold rapid detection card.
2. a kind of Cimaterol colloidal gold rapid detection card for being applicable in animal derived food as described in claim 1, feature
It is, the detection card is detection Cimaterol ingredient, and detection card includes: including the Cimaterol Dan Ke containing colloid gold label
The gold-labelled pad of grand antibody-solutions spraying, NC film, sample pad and the water absorption pad for being painted with T line and C line, are assembled.
3. a kind of preparation method for the Cimaterol colloidal gold rapid detection card for being applicable in animal derived food, which is characterized in that institute
State the preparation detailed process of colloidal gold rapid detection card are as follows:
(1) it the preparation of gold-labelled pad: takes 1mL colloidal gold solution in 2mL test tube with 1.35% red gold particle label, 0.2M is added
K2CO32.0 μ L/mL are mixed；1.0 μ L of antibody stoste is added, stands 5min after mixing；10 μ of confining liquid for containing 10% BSA is added
L/mL stands 5min after mixing；4 DEG C, 12000r/min centrifugation, 5min；The redissolution liquid of 1/20 volume redissolves, and is sprayed at and has closed
It on good glass fibre element film, is transferred quickly to be freeze-dried in freeze drier, production becomes gold-labelled pad；
(2) preparation of C liquid and T liquid: with coating buffer by antigen diluent, 250 times of PC-2 sheep anti mouse are diluted, C liquid is made, will be made
50 times of dilutions of artificial antigen obtain T liquid；
(3) coating of NC film: the C liquid of preparation, T liquid being crossed on NC film respectively, are sequentially followed successively by T liquid and C liquid from left to right,
37 DEG C of dry 20 ~ 40min are set, confining liquid closing is submerged into after dry, is placed in drying for standby in 37 DEG C of insulating boxs；
(4) sample pad prepared, gold-labelled pad, NC film and water absorption pad the assembling of detection card: are cut to length of same size
It is rectangular, successively carry out overlapping assembling, and well is set, is assembled into the detection card of detection Cimaterol.
4. a kind of preparation side for the Cimaterol colloidal gold rapid detection card for being applicable in animal derived food as claimed in claim 3
Method, which is characterized in that 0.2M K is added in the gold-labelled pad preparation process2CO3Amount be 2.0 μ L/mL.
5. a kind of preparation side for the Cimaterol colloidal gold rapid detection card for being applicable in animal derived food as claimed in claim 3
Method, which is characterized in that the amount that antibody stoste is added in the gold-labelled pad preparation process is 1.0 μ L.
6. a kind of preparation side for the Cimaterol colloidal gold rapid detection card for being applicable in animal derived food as claimed in claim 3
Method, which is characterized in that the T liquid carries out 50 times of diluted solution with the artificial Cimaterol antibody prepared.
7. a kind of preparation side for the Cimaterol colloidal gold rapid detection card for being applicable in animal derived food as claimed in claim 3
Method, which is characterized in that the C liquid, with the solution for diluting 250 times of acquisitions with PC-2 sheep anti mouse.
8. specific to wrap such as a kind of application for the Cimaterol colloidal gold rapid detection card for being applicable in animal derived food of claim 1
Include following steps:
(1) sample treatment: will be to solid-state sample product the homogeneous 1min in homogenizer, 10000rpm；Weigh 5.0 ± 0.05g homogenate
Good tissue samples detain upper tube cap into 10mL polystyrene centrifuge tube, should not be too tight；It is put into water-bath in 80 DEG C of water-baths
It takes out and is cooled to room temperature after 10min, it is to be checked；Liquid measuring samples acquire 50mL with dry, clean centrifuge tube or appropriate containers
Left and right can be reserved for 24 hours if do not detected immediately in 2-8 DEG C of refrigeration, pay attention to that corruption is avoided to cause to fail or pollute；
(2) it detects: will test card before use and sample to be examined solution restores to room temperature, detection card is taken out from original packing bag, it will
Detection card is laid flat, and draws measuring samples solution with dropper, vertical 3 drops that are added dropwise start timing 3-5 minutes after sample-adding in well
Observe the appearance situation of T line and C line；
(3) testing result: C line goes out to represent result effective in detection card；It detects T line in card and does not go out to represent, indicate sample
Middle Cimaterol concentration is higher than 0.01ppb；T line occurs simultaneously with C line in detection card, illustrates in measuring samples without containing Xi Mate
Sieve or its concentration are lower than 0.01ppb, and C line does not occur in paper slip, and it is invalid to represent result.
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| CN111154000A (en) * | 2020-01-16 | 2020-05-15 | 新乡学院 | Anti-cimaterol monoclonal antibody and application thereof |
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