CN104678102A - Neomycin and tylosin two-in-one gold-labeled microporous test paper strip - Google Patents
Neomycin and tylosin two-in-one gold-labeled microporous test paper strip Download PDFInfo
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- CN104678102A CN104678102A CN201310617093.8A CN201310617093A CN104678102A CN 104678102 A CN104678102 A CN 104678102A CN 201310617093 A CN201310617093 A CN 201310617093A CN 104678102 A CN104678102 A CN 104678102A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- 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
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- G01N33/531—Production of immunochemical test materials
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Abstract
The invention discloses a neomycin and tylosin two-in-one gold-labeled microporous test paper strip. A kit provided by the invention comprises a sample board and at least one test paper strip, wherein the sample board is at least provided with one sample groove; the bottom of each sample groove is attached with a colloidal gold label of a neomycin specific antibody and a colloidal gold label of a tylosin specific antibody. Each test paper strip comprises a bottom plate (1), a reaction film (2), a sample absorption pad (3) and a water absorption pad (4), wherein the corresponding sample absorption pad, the corresponding reaction film and the corresponding water absorption pad are sequentially attached to the corresponding bottom plate; each reaction film is sequentially provided with a T2 line, a T1 line and a C line from the initial end to the tail end; the corresponding T2 line and the corresponding T1 line are respectively coated with the following two kinds of conjugates, namely a tylosin-carrier protein conjugate and a neomycin-carrier protein conjugate; the corresponding C line is coated with an antiantibody. The kit provided by the invention has a broad market prospect and relatively great economic and social benefits.
Description
Technical field
The invention belongs to field of detection of food safety, be specifically related to one and detect neomycin in animal derived food (as milk etc.), the neomycin of tylosin residual quantity, tylosin two-in-one gold mark micropore test strips simultaneously.
Background technology
Neomycin (neomycin, NEO), belongs to aminoglycoside antibiotics, be by neamine and newly mould two osamines be combined into.Wherein neamine is combined into by D-Glucose diamines and deoxystreptamine, and newly mould two osamines are combined into by D-ribose and new mould osamine.Neomycin is the potpourri of two steric isomer actilines and Neomycin C, is wherein important component, the clinically sulfate of multiplex neomycin with actiline.Neomycin is important anti-infectives, and has a broad antifungal spectrum all has good inhibiting effect to Gram-negative bacteria, gram-positive bacteria and tubercle bacillus.On veterinary clinic, neomycin is one of choice drug for the treatment of fowl bacterial enteritis, in addition, and the diseases such as neomycin is also widely used in treatment mastitis, keratitis, conjunctivitis, otitis, dermatitis, trauma infection contamination, foot rot.But because it exists potential ototoxicity and kidney poison, so detect very necessary to Detection of neomycin residues in food to humans and animals.The neomycin maximum residue limit(MRL) of No. 235, the Ministry of Agriculture of China bulletin regulation is: milk, goat milk 500 μ g/kg, egg 500 μ g/kg, the muscle of ox, sheep, pig, chicken, turkey, duck, fat, liver 500 μ g/kg, the kidney 10000 μ g/kg of ox, sheep, pig, chicken, turkey, duck.In the animal food that European Union formulates, neomycin maximum residue limit(MRL) regulation muscle, liver, fat and egg are 500 μ g/kg, and kidney is 5000 μ g/kg, and milk is 1500 μ g/kg.
Tylosin is macrolide antibiotics, inhibiting effect is had to gram positive bacteria, gram-negative bacteria, class bacterium medium and some virus, especially extremely effective to Mycoplasma gallisepticum, pasteurella multocida, staphylococcus aureus, play a role mainly through the synthesis of anti-bacteria intracellular protein.As veterinary antibiotic medicine, tylosin is mainly widely used as feed addictive and treatment of animals, promotes poultry growth, improves the utilization factor of feed.But when tylosin is accumulated in vivo and reached certain concentration, can cause the infringement of vestibular and cochlea nerve, cause dizzy and dysacousis, severe patient can cause the serious damage of liver, kidney and hemopoietic system thereof.1999, European Union forbade tylosin to use as feed addictive, and from 1 day January in 2006, EU member country completely forbade and uses microbiotic as growth accelerator.No. 235th, the Ministry of Agriculture of China bulletin " animal food herbal medicine maximum residue limit(MRL) " specifies, tylosin maximum residue limit in livestock and poultry muscle tissue is 200 μ g/kg.European Union is also defined in liver, the kidney of pig, all tylosin must not be detected in the muscle of poultry, skin and fat, liver, kidney, U.S. food and drug administration (FDA) regulation, the residual safe level of tylosin in Edible tissues and egg is 0.2mg/kg, be 0.05mg/kg in Ruzhong, it is 0.05mg/kg that Japan is defined in the residual quantity allowed in muscle, pork, beef.
The detection neomycin of domestic and international report and the method for tylosin, mainly contain high performance liquid chromatography (HPLC), liquid chromatography-mass spectrography (LC-MS), Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), it is the most frequently used method of detection of antibiotics, but in practical operation, need the top-grade instruments such as LC-UV, LC-MS and LC-MS/MS, and process is complicated, loaded down with trivial details, the running time long, operative technique requires high.There is no the product that simultaneously can detect the residual quantity of neomycin and tylosin at present.
Summary of the invention
The object of this invention is to provide a kind of neomycin, tylosin two-in-one gold mark micropore test strips.
The invention provides a kind of kit for detecting neomycin and tylosin, comprising the sample panel and at least one test strips at least with a sample cell;
The Bottomattached of described sample cell has the colloid gold label thing of neomycin specific antibody and the colloid gold label thing of tylosin specific antibody;
Described test strips comprises base plate 1, reaction film 2, absorption of sample pad 3 and adsorptive pads 4; Described absorption of sample pad, described reaction film and described adsorptive pads are attached on described base plate successively, and the end of described absorption of sample pad is connected with the top of described reaction film, and the end of described reaction film is connected with the top of described adsorptive pads; Described reaction film has T by top successively to end
2line, T
1line and C line, described T
2line, T
1line and C line all with the axes normal of described reaction film; Described T
2line and T
1line wraps respectively by the following two kinds conjugate: tylosin-carrier protein couplet thing, neomycin-carrier protein couplet thing; On described C line, bag is by the antiantibody that can be combined with neomycin specific antibody and/or tylosin specific antibody.
Described neomycin specific antibody can be neomycin monoclonal antibody or neomycin polyclonal antibody.Described tylosin specific antibody can be tylosin monoclonal antibody or tylosin polyclonal antibody.Described neomycin monoclonal antibody specifically can be the monoclonal antibody that neomycin monoclonal antibody hybridoma cell strain NEO-5E9CGMCC No.8410 secretes.Described tylosin monoclonal antibody specifically can be the monoclonal antibody that tylosin monoclonal antibody hybridoma cell strain TYL-5D6CGMCC No.8409 secretes.
Arbitrary described carrier protein is bovine serum albumin(BSA), chicken ovalbumin or human serum albumins above.
In described neomycin-carrier protein couplet thing, described carrier protein specifically can be BSA, and neomycin and BSA mole combine than specifically can be 6:1.
In described tylosin-carrier protein couplet thing, described carrier protein specifically can be BSA, and tylosin and BSA mole combine than specifically can be 5:1.
The preparation method of described neomycin-carrier protein couplet thing is specific as follows:
(1) get 20mg neomycin bulk drug, be dissolved in 4ml carbonate buffer solution (pH9.5,0.1M);
(2) get BSA60mg, be dissolved in 6ml carbonate buffer solution (pH9.5,0.1M);
(3) solution that step (1) obtains to be added in the BSA solution that step (2) obtains and shaken at room temperature mixing 10min, then adds 325 μ l1%(volume ratios) glutaraldehyde water solution stirred overnight at room temperature, then drip NaBH
4solution (4mg is dissolved in 400 μ l pure water) stirring at room temperature 1h, then dialyse with distilled water, PBS damping fluid (pH7.2,0.01M) is subsequently used to dialyse, then the centrifugal 6min of 4500rpm get supernatant (neomycin-carrier protein couplet thing solution).
The preparation method of described tylosin-carrier protein couplet thing is specific as follows:
(1) get Tylosin Tartrate 1.0g, be dissolved in the aqueous sulfuric acid of 20mL pH2, add hot reflux 2h, then regulate pH to 8.8 with saturated metabisulfite solution, add methylene chloride and extract, get dichloromethane layer, after adding anhydrous sodium sulfate jolting, room temperature leaves standstill, and then filters and collects filtrate;
(2) get the filtrate that step (1) obtains, 60 DEG C of vacuum rotary steam evaporates to dryness, obtain dry;
(3) get step (2) and obtain dry 150mg, be dissolved in 4mL methyl alcohol, be called A liquid;
(4) get ethyloic azanol half hydrochloride 22mg and sodium bicarbonate 8.4mg, be dissolved in 4mL water, be called B liquid;
(5) add in A liquid by B liquid, room temperature magnetic agitation 2.5 hours, then 60 DEG C of vacuum rotary steam evaporates to dryness, obtain dry;
(6) get the dry 122mg that step (5) obtains, be dissolved in 8.3mL DMF, then add N successively, N'-dicyclohexylcarbodiimide 34.3mg and N-hydroxy-succinamide 19.1mg, room temperature magnetic agitation 12 hours;
(7) get 50mg BSA, be dissolved in 5mL sodium phosphate buffer (get 13.45g sodium hydrogen phosphate and 0.64g sodium dihydrogen phosphate, be settled to 1000mL with pure water), 400rpm stirs 10min;
(8) in ice-water bath, the solution that 3.2mL step (6) obtains is joined in the BSA solution that step (7) obtains, room temperature magnetic agitation 12 hours, then dialyses with physiological saline, then the centrifugal 6min of 4500rpm get supernatant (tylosin-carrier protein couplet thing solution).
The preparation method of the colloid gold label thing of neomycin specific antibody and the colloid gold label thing of tylosin specific antibody is all as follows:
(1) in the 0.01g/100ml aqueous solution of chloraurate of the 100mL of boiling, add 1% trisodium citrate aqueous solution of 2-4mL, obtain the colloidal gold solution (diameter of collaurum is 15-20nm) that final concentration is 0.01%;
(2) with the K of 0.1mol/L
2cO
3aqueous solution adjusts the pH to 8.5 of colloidal gold solution;
(3) the specific antibody solution (neomycin specific antibody solution or tylosin specific antibody solution) of 1 μ L10mg/mL to be added in the colloidal gold solution that 1mL step (2) obtains and to stir 30 minutes, then add the BSA aqueous solution of 10 μ L20g/100ml and stir 15min, then adding the PEG of 10 μ L10g/100ml
8000aqueous solution also stirs 15 minutes, then 4 DEG C, centrifugal 15min gets supernatant to 4000g, then 4 DEG C, 12000g gets precipitation in centrifugal 20 minutes;
(4) by the precipitation that PBS damping fluid (pH7.2,2mM) dissolving step (3) obtains, 4 DEG C, 12000g gets precipitation in centrifugal 20 minutes, then by PBS damping fluid (pH7.2,20mM) the resuspended precipitation of 200 μ L containing 1g/100ml BSA, the colloid gold label thing of specific antibody is obtained.
The colloid gold label thing of described neomycin specific antibody and the colloid gold label thing of described tylosin specific antibody all can the form of dried frozen aquatic products be attached to bottom described sample cell.The advantage done like this simplifies sample pretreatment process, improves the sensitivity of the detection of product.The preparation method of described sample panel is specific as follows: get micro reaction plate (as 8 ELISA micro reaction plates), the colloid gold label thing of neomycin specific antibody and each 25 μ l of colloid gold label thing of tylosin specific antibody are added in each sample cell, then sample panel is put into freeze drier, freeze-drying 14h after pre-freeze 4h, obtains the sample panel that sample cell Bottomattached has the colloid gold label thing of two strain specific antibodies.
Described test strips also can comprise the fixing film 5 being covered on described absorption of sample pad and being covered on described adsorptive pads.Described fixing film can promote the steadiness of each assembly in test strips, is preferably waterproof material.Described fixing film also has the effect avoiding the material in sample or reagent and external environment to react.
Described absorption of sample pad and described reaction film have the overlay region of 2mm; Described adsorptive pads and described reaction film have the overlay region of 2mm.Overlay region ensure that the continuity that sample liquid flows, and reaction is fully occurred, significantly reduces experimental error.
Described T
2line and T
1line wraps successively by the following two kinds conjugate: tylosin-carrier protein couplet thing, neomycin-carrier protein couplet thing.
In described test strips, the top of described absorption of sample pad aligns with the top of described base plate, and the end of described adsorptive pads aligns with the end of described base plate.
The top of described test strips is test side, and end is handle end.
The length (from top to end) of described absorption of sample pad can be 18mm, and the length (from top to end) of described reaction film can be 20mm, and the length (from top to end) of described adsorptive pads can be 20mm.Described T
2described in linear distance, the vertical range of absorption of sample pad can be 6mm, described T
1t described in linear distance
2the vertical range of line can be 3.5mm; Described in described C linear distance, the vertical range of adsorptive pads can be 6mm.
The bag of described neomycin-carrier protein couplet thing be can be 1mg/mL by concentration, wrap be can be 1mm by width, package amount can be 0.8 μ L/cm.The bag of described tylosin-carrier protein couplet thing be can be 1mg/mL by concentration, wrap be can be 1mm by width, package amount can be 0.8 μ L/cm.
Described antiantibody specifically can be sheep anti mouse two and resists.The bag of described antiantibody be can be 0.2mg/mL by concentration, wrap and be can be 1mm by width, and package amount can be 1 μ L/cm.
Described base plate can be PVC board.Described absorption of sample pad can be glass fibre cotton.Described reaction film can be nitrocellulose filter.Described adsorptive pads can be thieving paper.Described diaphragm can be PE diaphragm.Described sample panel can be plastic material.
Described kit also can comprise aluminium foil bag, and described sample panel and described test strips are packaged in described aluminium foil bag.
Each kit specifically can comprise the sample panel and 8 test strips with 8 sample cells.
In kit provided by the invention, formed without valence link between specific antibody and colloid gold particle, the two is combined by the Van der Waals force between the charges of different polarity, and collaurum antagonist specificity and affinity affect very little, and there is higher mark rate, therefore there is higher specificity and sensitivity.
The present invention also protects neomycin monoclonal antibody hybridoma cell strain NEO-5E9(to be called for short hybridoma NEO-5E9).Hybridoma NEO-5E9 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on October 25th, 2013, and preserving number is CGMCC No.8410.
The monoclonal antibody that the present invention also protects hybridoma NEO-5E9 to secrete.
The present invention also protects tylosin monoclonal antibody hybridoma cell strain TYL-5D6(to be called for short hybridoma TYL-5D6).Hybridoma TYL-5D6 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on October 25th, 2013, and preserving number is CGMCC No.8409.
The monoclonal antibody that the present invention also protects hybridoma TYL-5D6 to secrete.
In kit provided by the invention, the colloid gold label thing of neomycin specific antibody and the equal freeze-drying of colloid gold label thing thing of tylosin specific antibody are in micropore, and the advantage done like this simplifies sample pretreatment process, improves the sensitivity of the detection of product.
The Cleaning Principle of kit provided by the invention is based on immunoassay, whether neomycin and tylosin is contained in sample to be tested while of energy, there is pre-treatment simple, do not need any instrument, highly sensitive, high specificity, applied widely, (not having specialty to require to operating personnel) easy and simple to handle, fast (result can be obtained in 5-10min), with low cost, namely colour developing by means of only T line and C line can read result and the on-the-spot advantage such as adaptable, be applicable to very much the screening of on-the-spot sample in enormous quantities, can be used for specialty chemical examination, customs quarantine control, health and epidemic prevention, quality monitoring, dairy industry enterprise etc., there is wide market outlook and larger economy, social benefit.
Accompanying drawing explanation
Fig. 1 is the structural representation of test strips.
Fig. 2 is the using state schematic diagram of kit.
Fig. 3 is the result interpretation schematic diagram of kit.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.
Neomycin bulk drug: (Sigma-Aldrich, model: N1876).Bovine serum albumin(BSA) (BSA): Sigma-Aldrich, A2058.Tylosin Tartrate: (Sigma-Aldrich, model: T6134).
The structural formula of neomycin is as follows:
The structural formula of tylosin is as follows:
Embodiment 1, conjugate, specific antibody and specific antibody the preparation of colloid gold label thing
One, the preparation of conjugate
The preparation of 1, neomycin-carrier protein couplet thing
(1) get 20mg neomycin bulk drug, be dissolved in 4ml carbonate buffer solution (pH9.5,0.1M).
(2) get BSA60mg, be dissolved in 6ml carbonate buffer solution (pH9.5,0.1M).
(3) solution that step (1) obtains being added drop-wise in the BSA solution that step (2) obtains and shaken at room temperature mixing 10min, then adding 325 μ l1%(volume ratios) glutaraldehyde water solution stirring at room temperature (400rpm) spend the night, and then drips NaBH
4(4mg is dissolved in 400 μ l pure water to solution, brand-new) and stirring at room temperature 1h, then be placed in bag filter distilled water carry out dialysing (dialysis procedure parameter: 4 DEG C, 100rpm stirs, 2 days, every day changes liquid 2 times), subsequently use PBS damping fluid (pH7.2,0.01M) carry out dialysing (dialysis procedure parameter: 4 DEG C, 100rpm stirs, 2 days, every day changes liquid 2 times), then the centrifugal 6min of 4500rpm get supernatant (neomycin-carrier protein couplet thing solution), the packing of 1ml/ pipe ,-20 DEG C of preservations.
(4) what adopt neomycin and carrier protein in determined by ultraviolet spectrophotometry neomycin-carrier protein couplet thing solution mole combines than (method list of references: Luo Shunjing, Zhong Hanyan, Liu Chengmei, Chen Tingting, Chen Farong, Yan Jielin; The preparation of chloromycetin immunizing antigen and envelope antigen; Food Science; 2010, Vol.31, No.10,91-94), neomycin and BSA mole combine than being 6:1.
The preparation of 2, tylosin-carrier protein couplet thing
(1) Tylosin Tartrate 1.0g is got, be dissolved in the aqueous sulfuric acid of 20mL pH2, add hot reflux 2h, then regulate pH to 8.8 with saturated metabisulfite solution, proceed in separating funnel, add methylene chloride and carry out extracting (extracting twice, add 20mL methylene chloride) at every turn, combined dichloromethane layer, adds room temperature hold over night after anhydrous sodium sulfate jolting, then filters and collect filtrate.
(2) get the filtrate that step (1) obtains, 60 DEG C of vacuum rotary steam evaporates to dryness, obtain dry.
(3) get step (2) and obtain dry 150mg, be dissolved in 4mL methyl alcohol, be called A liquid.
(4) get ethyloic azanol half hydrochloride 22mg and sodium bicarbonate 8.4mg, be dissolved in 4mL water, be called B liquid.
(5) dropwise add in A liquid by B liquid, room temperature magnetic agitation 2.5 hours, then 60 DEG C of vacuum rotary steam evaporates to dryness, obtain dry.
(6) get the dry 122mg that step (5) obtains, be dissolved in 8.3mL DMF, then add N successively, N'-dicyclohexylcarbodiimide (DCC) 34.3mg and N-hydroxy-succinamide (NHS) 19.1mg, room temperature magnetic agitation 12 hours.
(7) get 50mg BSA, be dissolved in 5mL sodium phosphate buffer (get 13.45g sodium hydrogen phosphate and 0.64g sodium dihydrogen phosphate, be settled to 1000mL with pure water), 400rpm stirs 10min.
(8) in ice-water bath, the dropwise that 3.2mL step (6) obtains is joined in the BSA solution that step (7) obtains, room temperature magnetic agitation 12 hours, then be placed in bag filter physiological saline carry out dialysing (dialysis procedure parameter: room temperature, 100rpm stir, 3 days, every day changes liquid 2 times), then the centrifugal 6min of 4500rpm get supernatant (tylosin-carrier protein couplet thing solution), the packing of 0.5ml/ pipe ,-20 DEG C of preservations.
(9) what adopt tylosin and carrier protein in determined by ultraviolet spectrophotometry tylosin-carrier protein couplet thing solution mole combines than (method list of references: Luo Shunjing, Zhong Hanyan, Liu Chengmei, Chen Tingting, Chen Farong, Yan Jielin; The preparation of chloromycetin immunizing antigen and envelope antigen; Food Science; 2010, Vol.31, No.10,91-94), tylosin and BSA mole combine than being 5:1.
Two, the preparation of specific antibody
1, the preparation of neomycin mouse monoclonal antibody
(1) animal immune program
Adopt BALB/c mouse as immune animal, with neomycin-carrier protein couplet thing for immunogene, single immunization dosage is 50-100 μ g/ (in carrier protein).During first immunisation, immunogen solution and isopyknic Freund's complete adjuvant are mixed and made into emulsifying agent, neck dorsal sc multi-point injection.After first immunisation, at interval of 2 weeks by immunogen solution and isopyknic incomplete Freund's adjuvant mixing and emulsifying, carry out booster immunization, neck dorsal sc multi-point injection.Immunogenic dose reduces by half after 2 weeks with after the dilution of 0.5ml sterile saline by booster immunization for the third time, adopts the method booster immunization of lumbar injection once, extracting spleen cell after 3 days.
(2) Fusion of Cells and cloning
The splenocyte that step (1) obtains, in (4-8): the ratio of 1 and SP2/0 myeloma cell fusion, adopts indirect competitive ELISA to measure cell conditioned medium liquid, screens positive hole.Limiting dilution assay is utilized to carry out cloning to positive hole, until obtain the hybridoma cell strain of stably excreting monoclonal antibody.
(3) preservation of hybridoma cell strain
The hybridoma called after neomycin monoclonal antibody hybridoma cell strain NEO-5E9(of one strain stably excreting monoclonal antibody is called for short hybridoma NEO-5E9).Hybridoma NEO-5E9 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on October 25th, 2013, and preserving number is CGMCC No.8410.
(4) cell cryopreservation and recovery
With cryopreserving liquid, hybridoma NEO-5E9 is made cell concentration for (1-5) × 10
6the cell suspension of individual/mL, preserves for a long time in liquid nitrogen.During recovery, take out cryopreservation tube, put into 37 DEG C of water-bath middling speeds immediately and melt, move into after centrifugal segregation cryopreserving liquid and cultivate culture in glassware.
(5) preparation and purification of monoclonal antibody
The preparation method of cell culture medium (7.4): add calf serum and sodium bicarbonate in RPMI-1640 nutrient culture media, the final concentration of calf serum is 20%(mass percentage), the final concentration of sodium bicarbonate is 0.2%(mass percentage).
Hybridoma NEO-5E9 is placed in cell culture medium, cultivates 2 days, adopt sad-saturated ammonium sulfate method that the cell culture supernatant obtained is carried out purifying, obtain neomycin monoclonal antibody solution (-20 DEG C of preservations) for 37 DEG C.
Protein concentration (mg/ml)=1.45 × OD in monoclonal antibody solution
280-0.74 × OD
260.
Adopting the protein concentration in above formulae discovery neomycin monoclonal antibody solution, is 0.8mg/ml.
2, the preparation of tylosin mouse monoclonal antibody
Replace neomycin-carrier protein couplet thing with tylosin-carrier protein couplet thing, other are with step 1.
The hybridoma called after tylosin monoclonal antibody hybridoma cell strain TYL-5D6(of one strain stably excreting monoclonal antibody is called for short hybridoma TYL-5D6).Hybridoma TYL-5D6 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on October 25th, 2013, and preserving number is CGMCC No.8409.
Protein concentration (mg/ml)=0.6mg/ml in tylosin monoclonal antibody solution.
Three, the preparation of the colloid gold label thing of specific antibody
1, in the 0.01g/100ml aqueous solution of chloraurate of the 100mL of boiling, add 1% trisodium citrate aqueous solution of 2-4mL, obtain the colloidal gold solution (diameter of collaurum is 15-20nm) that final concentration is 0.01%.
2, with the K of 0.1mol/L
2cO
3aqueous solution adjusts the pH to 8.5 of colloidal gold solution.
3, by monoclonal antibody solution (neomycin monoclonal antibody solution prepared by step 2 or the tylosin monoclonal antibody solution of 1 μ L10mg/mL; Preliminary experiment has obtained optimum mark concentration) to add in the colloidal gold solution that 1mL step 2 obtains and to stir 30 minutes, then add the BSA aqueous solution of 10 μ L20g/100ml and stir 15min, then adding the PEG of 10 μ L10g/100ml
8000aqueous solution also stirs 15 minutes, then 4 DEG C, centrifugal 15min gets supernatant to 4000g, then 4 DEG C, 12000g gets precipitation in centrifugal 20 minutes.
4, by the precipitation that PBS damping fluid (pH7.2,2mM) dissolving step 3 obtains, 4 DEG C, 12000g gets precipitation in centrifugal 20 minutes, then by PBS damping fluid (pH7.2,2mM) the resuspended precipitation of 200 μ L containing 1g/100ml BSA, the colloid gold label thing of monoclonal antibody is obtained.
5,8 ELISA micro reaction plates are got, the colloid gold label thing of neomycin monoclonal antibody and each 25 μ l of colloid gold label thing of tylosin monoclonal antibody are added in each sample cell, then sample panel is put into freeze drier, freeze-drying 14h after pre-freeze 4h, obtains the sample panel that sample cell Bottomattached has the colloid gold label thing of two kinds of monoclonal antibodies.
The assembling of embodiment 2, kit
Fig. 1 is the structural representation of test strips.Fig. 2 is the using state schematic diagram of kit.Fig. 3 is the result interpretation schematic diagram of kit.
Kit for detecting neomycin and tylosin provided by the invention, sample panel prepared by the step 3 comprising embodiment 1 and 8 identical test strips.Kit also comprises aluminium foil bag, and described sample panel and described test strips are packaged in described aluminium foil bag.Each test strips comprises base plate 1, reaction film 2, absorption of sample pad 3 and adsorptive pads 4.Described absorption of sample pad, described reaction film and described adsorptive pads are attached on described base plate successively, and the end of described absorption of sample pad is connected with the top of described reaction film, and the end of described reaction film is connected with the top of described adsorptive pads.The top of described test strips is test side, and end is handle end.Described absorption of sample pad and described reaction film have the overlay region of 2mm, and described adsorptive pads and described reaction film have the overlay region of 2mm.The top of described absorption of sample pad aligns with the top of described base plate, and the end of described adsorptive pads aligns with the end of described base plate.Described test strips also comprises the fixing film 5 being covered on described absorption of sample pad and being covered on described adsorptive pads.Described reaction film has T by top successively to end
2line, T
1line and C line, described T
2line, T
1line and C line are all vertical with the axis of described reaction film (namely from top to the axis of end).Described T
2line and T
1line wraps successively by the following two kinds conjugate: (wrap by concentration is 1mg/mL to the tylosin-carrier protein couplet thing of preparation in the step one of embodiment 1, bag is 1mm by width, package amount is 0.8 μ L/cm) and embodiment 1 step one in preparation neomycin-carrier protein couplet thing (wrapping by concentration is 1mg/mL, bag is 1mm by width, and package amount is 0.8 μ L/cm).Described C line wraps by antiantibody that (sheep anti mouse two resists, Ge Langrui bio tech ltd, Hangzhou, product type: GRBT-044; Bag is 0.2mg/mL by concentration, and wrapping by width is 1mm, and package amount is 1 μ L/cm).
The length (from top to end) of described absorption of sample pad is 18mm, and the length (from top to end) of described reaction film is 20mm, and the length (from top to end) of described adsorptive pads is 20mm.Described T
2described in linear distance, the vertical range of absorption of sample pad is 6mm, described T
1t described in linear distance
2the vertical range of line is 3.5mm, and described in described C linear distance, the vertical range of adsorptive pads is 6mm.
Described base plate is PVC board.Described absorption of sample pad is glass fibre cotton.Described reaction film is nitrocellulose filter.Described adsorptive pads is thieving paper.Described diaphragm is PE diaphragm.
The application process of described kit is as follows:
Measuring samples solution 200 μ 1 is added drop-wise in a sample cell of sample panel, repeatedly blow and beat and dissolve completely to the aubergine particle at the bottom of hole, incubated at room 3min, then inserts in this sample cell by the absorption of sample pad end of test strips, reaction 5-8min, according to following standard judged result:
Negative: C line, T
1line and T
2line is all aobvious red; Not containing neomycin and tylosin in measuring samples;
Positive: aobvious red, the T of C line
2aobvious red, the T of line
1line does not develop the color, containing neomycin in measuring samples, not containing tylosin; Aobvious red, the T of C line
1aobvious red, the T of line
2line does not develop the color, containing tylosin in measuring samples, not containing neomycin; Aobvious red, the T of C line
1line and T
2line does not all develop the color, containing neomycin and tylosin in measuring samples;
Invalid: C line does not develop the color.
The described neomycin that do not contain comprises the following two kinds implication: the first, do not contain; The second, contains, but content is lower than detectability.The described tylosin class that do not contain comprises the following two kinds implication: the first, do not contain; The second, contains, but content is lower than detectability.
The Cleaning Principle of described kit is as follows:
When in measuring samples, the content of neomycin and tylosin is greater than or equal to detectability, the colloid gold label thing of the neomycin monoclonal antibody in micropore and/or the antigen of colloid gold label thing all in testing sample of tylosin monoclonal antibody are combined, the colloid gold label thing being combined with the monoclonal antibody of the antigen in testing sample cannot neomycin-carrier protein couplet thing on reaction film and/or tylosin-carrier protein couplet thing be combined, T
1line and/or T
2line is not aobvious red, and the colloid gold label thing being combined with the monoclonal antibody of the antigen in testing sample can be combined with antiantibody, and C line is aobvious red;
When in measuring samples, the content of neomycin and tylosin is lower than detectability, the infull antigen in testing sample of the colloid gold label thing of the neomycin monoclonal antibody in micropore and/or the colloid gold label thing of tylosin monoclonal antibody is combined, colloid gold label thing in conjunction with the monoclonal antibody of the antigen in testing sample can neomycin-carrier protein couplet thing on reaction film and/or tylosin-carrier protein couplet thing be combined, T
1line and/or T
2line is aobvious red, be combined with the monoclonal antibody of the antigen in testing sample colloid gold label thing or in conjunction with the antigen in testing sample the colloid gold label thing of monoclonal antibody all can be combined with antiantibody, C line is aobvious red.
Prepare gradient dilution liquid respectively with neomycin bulk drug and Tylosin Tartrate, as measuring samples solution, application mentioned reagent box detects.The solvent that each dilution adopts is PB damping fluid (pH7.2,0.02M).
Kit provided by the invention to the detectability of neomycin and tylosin in table 1.
Table 1 detectability
| Medicine name | Detectability μ g/L (kg) |
| Neomycin | 200 |
| Tylosin | 20 |
Claims (10)
1., for detecting a kit for neomycin and tylosin, comprise the sample panel and at least one test strips at least with a sample cell;
The Bottomattached of described sample cell has the colloid gold label thing of neomycin specific antibody and the colloid gold label thing of tylosin specific antibody;
Described test strips comprises base plate (1), reaction film (2), absorption of sample pad (3) and adsorptive pads (4); Described absorption of sample pad, described reaction film and described adsorptive pads are attached on described base plate successively, and the end of described absorption of sample pad is connected with the top of described reaction film, and the end of described reaction film is connected with the top of described adsorptive pads; Described reaction film has T by top successively to end
2line, T
1line and C line, described T
2line, T
1line and C line all with the axes normal of described reaction film; Described T
2line and T
1line wraps respectively by the following two kinds conjugate: tylosin-carrier protein couplet thing, neomycin-carrier protein couplet thing; On described C line, bag is by the antiantibody that can be combined with neomycin specific antibody and/or tylosin specific antibody.
2. kit as claimed in claim 1, is characterized in that: described neomycin specific antibody is neomycin monoclonal antibody or neomycin polyclonal antibody; Described tylosin specific antibody is tylosin monoclonal antibody or tylosin polyclonal antibody.
3. kit as claimed in claim 2, is characterized in that: described neomycin monoclonal antibody is the monoclonal antibody that neomycin monoclonal antibody hybridoma cell strain NEO-5E9CGMCC No.8410 secretes; Described tylosin monoclonal antibody is the monoclonal antibody that tylosin monoclonal antibody hybridoma cell strain TYL-5D6CGMCC No.8409 secretes.
4. kit as claimed in claim 1, is characterized in that: arbitrary described carrier protein is bovine serum albumin(BSA), chicken ovalbumin or human serum albumins.
5. kit as claimed in claim 1, is characterized in that: described test strips also comprises the fixing film (5) being covered on described absorption of sample pad and being covered on described adsorptive pads.
6. kit as claimed in claim 1, is characterized in that: described absorption of sample pad and described reaction film have the overlay region of 2mm; Described adsorptive pads and described reaction film have the overlay region of 2mm.
7. kit as claimed in claim 1, is characterized in that: described T
2line and T
1line wraps successively by the following two kinds conjugate: tylosin-carrier protein couplet thing, neomycin-carrier protein couplet thing.
8. neomycin monoclonal antibody hybridoma cell strain NEO-5E9, its deposit number is CGMCC No.8410.
9. tylosin monoclonal antibody hybridoma cell strain TYL-5D6, its deposit number is CGMCC No.8409.
10. described in claim 8 hybridoma cell strain secretion monoclonal antibody or claim 9 described in hybridoma cell strain secretion monoclonal antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310617093.8A CN104678102B (en) | 2013-11-27 | 2013-11-27 | Neomycin, tylosin two-in-one gold mark micropore test strips |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310617093.8A CN104678102B (en) | 2013-11-27 | 2013-11-27 | Neomycin, tylosin two-in-one gold mark micropore test strips |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104678102A true CN104678102A (en) | 2015-06-03 |
| CN104678102B CN104678102B (en) | 2016-08-31 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310617093.8A Active CN104678102B (en) | 2013-11-27 | 2013-11-27 | Neomycin, tylosin two-in-one gold mark micropore test strips |
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|---|---|---|---|---|
| CN101029893A (en) * | 2006-12-19 | 2007-09-05 | 河南省农业科学院生物技术研究所 | Quick testing strip for residual pesticide |
| CN200989901Y (en) * | 2006-12-19 | 2007-12-12 | 河南省动物免疫学重点实验室 | Tylosion residual fast detection test paper strip |
| CN202710551U (en) * | 2012-06-15 | 2013-01-30 | 北京博欧实德生物技术有限公司 | Kit capable of fast detecting tylosin residual |
| CN202720234U (en) * | 2012-08-02 | 2013-02-06 | 北京勤邦生物技术有限公司 | Test strip for detection of neomycin |
| CN202854149U (en) * | 2012-08-16 | 2013-04-03 | 北京维德维康生物技术有限公司 | Gold-marking micropore kit for rapidly detecting small molecule compound |
| CN203069593U (en) * | 2012-09-29 | 2013-07-17 | 北京勤邦生物技术有限公司 | Test strip for detecting spectinomycin, streptomycin, gentamicin and neomycin |
| CN203069592U (en) * | 2012-09-29 | 2013-07-17 | 北京勤邦生物技术有限公司 | Test strip for detecting spiramycin, streptomycin, gentamicin and neomycin |
-
2013
- 2013-11-27 CN CN201310617093.8A patent/CN104678102B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101029893A (en) * | 2006-12-19 | 2007-09-05 | 河南省农业科学院生物技术研究所 | Quick testing strip for residual pesticide |
| CN200989901Y (en) * | 2006-12-19 | 2007-12-12 | 河南省动物免疫学重点实验室 | Tylosion residual fast detection test paper strip |
| CN202710551U (en) * | 2012-06-15 | 2013-01-30 | 北京博欧实德生物技术有限公司 | Kit capable of fast detecting tylosin residual |
| CN202720234U (en) * | 2012-08-02 | 2013-02-06 | 北京勤邦生物技术有限公司 | Test strip for detection of neomycin |
| CN202854149U (en) * | 2012-08-16 | 2013-04-03 | 北京维德维康生物技术有限公司 | Gold-marking micropore kit for rapidly detecting small molecule compound |
| CN203069593U (en) * | 2012-09-29 | 2013-07-17 | 北京勤邦生物技术有限公司 | Test strip for detecting spectinomycin, streptomycin, gentamicin and neomycin |
| CN203069592U (en) * | 2012-09-29 | 2013-07-17 | 北京勤邦生物技术有限公司 | Test strip for detecting spiramycin, streptomycin, gentamicin and neomycin |
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| Publication number | Publication date |
|---|---|
| CN104678102B (en) | 2016-08-31 |
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