CN103454423A - Up-conversion fluorescence immune chromatography test paper for quantitative detection of neomycin and preparation method thereof - Google Patents
Up-conversion fluorescence immune chromatography test paper for quantitative detection of neomycin and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an up-conversion fluorescence immune chromatography test paper for the quantitative detection of neomycin and a preparation method thereof. The up-conversion fluorescence test paper comprises a support layer, an adsorption layer and a protection layer; the absorbing layer comprises an adsorption fiber layer, a fluorescent antibody fiber layer, a cellulose membrane layer and a water adsorption material layer at a handle hand; the cellulose membrane layer is provided with detection blotting printed by a carrier protein solution coupled with NEO and contrast blotting printed by a goat anti-mouse IgG; the fluorescent antibody adopts an NEO monoclonal antibody or polyclonal antibody marked by NaYF4:Yb:Er nanoparticles. Through the up-conversion fluorescence immune chromatography test paper for the quantitative detection of the neomycin, the application of the immune chromatography marked by up-conversion fluorescence nanometer materials in the quantitative detection of NEO residual is realized, so that the detection of the NEO residual is not subjected to background interference; the up-conversion fluorescence immune chromatography test paper for the quantitative detection of the neomycin is strong in specificity, high in sensitivity, simple, intuitional and accurate in detection, low in cost, wide in range of application and easy to popularize and apply.
Description
Technical field
The present invention relates to a kind of immune chromatography test paper, particularly relate to a kind of up-conversion fluorescence immune chromatography test paper quantitatively detected for neomycin and preparation method thereof.
background technology
Neomycin (NEOenbuterol, NEO), neomycin belongs to the general class microbiotic of amino sugar, and its has a broad antifungal spectrum is usually used in suppressing the bacillary alimentary infection of livestock and poultry.Along with neomycin widespread use in aquaculture at home and abroad, its residue problem in animal-derived food also becomes increasingly conspicuous.The animal product that long-term edible Detection of neomycin residues exceeds standard, can produce chronic cumulative toxicity effect to body.No. 235 bulletin regulations that China Ministry of Agriculture announces, in the muscle of ox, pig, sheep, chicken and duck, liver and fat, neomycin maximum residue limit(MRL) (MRL) is 500 μ g/Kg, in kidney, is in 10000 μ g/Kg, milk, to be in 500 μ g/L, egg, to be 50 μ g/L.Detection of neomycin residues detection method commonly used is at present: microbial method, chromatography and immunoassay.Microbial method is simple, economical, but susceptibility is not high; Chromatography is the standard method that quantitative and qualitative analysis detects, and have higher accuracy, accuracy and susceptibility, but its flow process is loaded down with trivial details, and apparatus expensive, need the professional and technical personnel, and detection speed is slow; Immunization is simple to operate, and cost is low, and accuracy and sensitivity are preferably arranged, and also can carry out the quantitative and qualitative analysis detection, is suitable for the examination to a large amount of samples.
The immune test paper method has sxemiquantitative and certain quantitation capabilities, and the preliminary information of determinand can be provided, and this method is highly sensitive, and analytic process is simple, as examination, unique advantage is arranged, and is the detection technique that need to first develop.Up-conversion fluorescence technology (UPT) is a kind of have high sensitivity, new bio detection technique based on upper converting phosphor body (UCP), UCP has unique physical arrangement and optical characteristics, it is a kind of phosphor of complete inertia, after finishing and activation, can be used as the novel markings thing and apply in field of biological detection.Although it is wider that colloid gold test paper is applied in this regard, it can not accomplish quantitative detection, as the research of more responsive, stable, flexible, safe UPT-LF, is that the strong of colloid gold immune detection technique supplements, the research of this aspect in urgent need of strengthening and discussion.
Summary of the invention
The technical problem to be solved in the present invention: the deficiency existed for prior art, provide a kind of special, sensitive, quick, easy, can quantitatively detect up-conversion fluorescence immune chromatography test paper of Detection of neomycin residues and preparation method thereof.
Technical scheme of the present invention:
A kind of up-conversion fluorescence immune chromatography test paper quantitatively detected for neomycin, bottom is supporting layer, middle layer is adsorbed layer, protective seam is fixed on adsorbed layer, adsorbed layer is followed successively by the absorbent material layer of adsorbing fiber layer, fluorescence antibody fibrage, cellulose rete and handle end from test lead, be provided with the stealth of printing with the carrier protein solution of coupling NEO and detect trace on the cellulose rete, also be provided with the stealth contrast trace of printing with goat anti-mouse igg, the anti-mouse IgG of rabbit or goat anti-rabbit igg antibody solution; Described fluorescence antibody fibrage adopts the glass fibre cotton of absorption fluorescence antibody to make, and fluorescence antibody adopts NaYF
4: NEO monoclonal antibody or the polyclonal antibody of Yb:Er nanoparticle label.
Described NaYF
4: the Yb:Er nano particle is with NaYF
4for matrix, Yb
3+for sensitizer, the nano particle that the diameter formed by Yb and Er codope is 50-200nm.
Described NaYF
4: by following methods, prepared by NEO monoclonal antibody or the polyclonal antibody of Yb:Er nanoparticle label:
(1) surface siliconization of fluorescent nano particle: by NaYF
4: the Yb:Er fluorescent nano particle is dispersed in the ethanolic solution that mass concentration is 10%, under agitation adds the 5ml strong aqua, and reaction 10min, then add the 2ml tetraethyl orthosilicate, room temperature reaction 3h; The centrifugal 5min of 6000rpm, disperse with ethanol after washing;
(2) surface amination of fluorescent nano particle: the fluorescent nano particle of surface siliconization is added in the mixed solution of methyl alcohol that volume ratio is 3:5, acetone, through ultrasound wave, disperse, add the N-(2-aminoethyl) of 5 μ L-3-aminopropyl trimethoxysilane, 70 ℃ of water-bath 1h, the centrifugal 5min of 6000rpm, the precipitation obtained is washed to drying with ethanol;
(3) mark of NEO antibody: the NaYF that surface is modified through amination
4: the Yb:Er nano particle disperses with PBS buffer solution, the glutaraldehyde solution that adds wherein mass concentration 25%, room temperature reaction 3 hours, centrifugally wash away unnecessary glutaraldehyde, then reactant liquor is dispersed in PBS buffer solution, adds monoclonal antibody or the polyclonal antibody of NEO, 4 ℃ are reacted 3 hours, through centrifuge washing, obtain NaYF
4: NEO monoclonal antibody or the polyclonal antibody of Yb:Er nanoparticle label are placed in PBS buffer solution, 4 ℃ of preservations.
Described adsorbing fiber for layer glass fibre cotton, nylon membrane, PVDF membrane or polyester film make; Absorbent material layer is made with absorbent filter, and supporting layer is made with the toughness material do not absorbed water; Cellulose for rete nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane make;
the carrier protein of coupling NEO is bovine serum albumin(BSA), chicken ovalbumin or hemocyanin.
Described stealthy detect trace and stealthy contrast trace be arranged in parallel "
︱ ︱" the orthoscopic trace, "
10" font is arranged trace, " ┬ ┬ " font is arranged trace, " ┴ ┴ " font arrangement trace, " ├ ├ " font arrangement trace or " ┤ ┤ " font and arranged trace.
Described protective seam is the diaphragm covered on adsorbing fiber layer, fluorescence antibody fibrage and absorbent material layer; be printed with the sample mark line at the adsorbing fiber layer on the diaphragm corresponding with fluorescence antibody fibrage intersection, this mark line deflection adsorbing fiber layer one side 0.3-0.7cm.
Described NaYF4:Yb:Er nano particle adopts the coprecipitation preparation, and concrete grammar is as follows:
Y (the NO that to get respectively concentration be 0.2mol/L
3)
3yb (the NO of 20ml, 0.2mol/L
3)
3er (the NO of 2ml, 0.2mol/L
3)
30.5ml, the EDTA-Na21ml of 1mol/L, add in flask, 50 ℃ of oil bath stirring reaction 1h, obtain reactant liquor A; The NaF solution of getting 50ml, 1mol/L adds in polytetrafluoroethylcontainer container, reactant liquor A is poured in NaF solution into to reaction 1min under 50 ℃ of stirred in water bath; The centrifugal 5min of 6000rpm, abandoning supernatant must precipitate; Add water, ultrasonic washing 3 times, the centrifugal 5min of 7000rpm in precipitation; To precipitate and use absolute ethyl alcohol ultrasonic washing 2 times, the centrifugal 5min of 10000rpm, be placed in 80 ℃ of baking oven dried overnight by the solid obtained; Dried solid is put into to muffle furnace, in 500 ℃ of calcining 5h, obtain NaYF
4: the Yb:Er fluorescent nano particle.
Described NaYF
4: the Yb:Er nano particle also can adopt the hydrothermal synthesis method preparation, and concrete grammar is as follows:
Get respectively the Y (NO that concentration is 0.2mol/L
3)
34ml, Yb (NO
3)
30.5ml, Er (NO
3)
30.1ml, EDTA-Na4ml, mix and fully the reaction, obtain reactant liquor A; Take NaOH 0.7g, join in the mixed liquor of 8ml oleic acid and 12ml ethanol, mix, under agitation slowly to the NaF solution that injects 10mL, 1mol/L in mixed liquor, after filling, continue to be stirred to the solution shape that is translucent; Reactant liquor A is poured in described solution, mix, ageing 20-30min; Then potpourri is transferred in polytetrafluoroethylcontainer container, 130-150 ℃ of oil bath reaction 12-18h, the product obtained after filtration, washing, drying, obtain NaYF
4: the Yb:Er fluorescent nano particle.
The preparation method of described immune chromatography test paper comprises the following steps:
(1) preparation of NEO monoclonal antibody or polyclonal antibody;
(2) preparation of fluorescence antibody: first prepare NaYF
4: the Yb:Er fluorescent material, described fluorescent material is with NaYF
4for matrix, Yb
3+for sensitizer, formed the nano particle of diameter 50-200nm by Yb and Er codope; Then NEO monoclonal antibody or polyclonal antibody are carried out to fluorescence labeling;
(3) preparation of adsorbing fiber layer: adsorbing fiber for layer glass fibre cotton, nylon membrane, PVDF membrane or polyester film make;
(4) preparation of cellulose rete: cellulose is nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane for rete, with point sample instrument, at the diverse location difference specking of cellulose rete, detects trace and contrast trace, dries;
(5) assembling of immune chromatography test paper: adsorbing fiber layer, fluorescence antibody fibrage, cellulose rete, absorbent material layer are attached on the supporting layer that scribbles bonding agent from right to left successively, successively supporting layer, adsorbed layer and protective seam are assembled into to immune chromatography test paper.
positive beneficial effect of the present invention:
(1) high specificity, highly sensitive.The above fluorescent nano material converted NaYF of up-conversion fluorescence immune chromatography test paper of the present invention
4: the NEO antibody of Yb:Er mark is that make on basis, due to NaYF
4: the outstanding optical characteristics of Yb:Er nano particle, make the detection of NEO eliminate the interference of bias light, sensitivity improves greatly, a minimum trace residue of gram level that detects.
(2) stability is high, and dirigibility is good.In test paper, the luminescence phenomenon of UCP is that pure physical process due to its inside configuration produces, and has avoided the luminous cancellation caused from detecting sample corrosion and self decay fully; But the diversified characteristic spectrum of the independent assortment that UCP has (absorption spectrum and emission spectrum) can be applicable to multiple analysis.
(3) security is good.Up-conversion fluorescence nano material in test paper has the characteristics of inorganic inertia, infrared ray excited, VISIBLE LIGHT EMISSION, make detection based on UCP for tester, detected product and environment all without any harm.
(4) easy, quick.The up-conversion fluorescence immune chromatography test paper can carry out direct-detection to whole blood, urine, saliva, tissue homogenate etc., need not carry out the pre-service of sample.Up-conversion fluorescence immune chromatography test paper of the present invention can be read the bar instrument with fluorescence and be combined with, and directly the value of reading, realize that in situ quantitation detects.As long as test paper is inserted to 10～20 seconds of test sample, after 5 minutes, gets final product reading result.
(5) result shows image, directly perceived, accurate.Up-conversion fluorescence immune chromatography test paper of the present invention is usingd the T line and whether is occurred that absorption peak, as the positive and the negative marker that detect, contains NEO in T line place means test sample without absorption peak; The T line has absorption peak, means in test sample not containing NEO.Visual result, accurate, simple and clear, be not prone to the artificial erroneous judgement such as false positive and false negative.
(6) applied widely, easy to utilize.The present invention has realized the application of immunochromatography technique in the quantitative NEO of detection is residual based on up-conversion fluorescence nano material mark, and the residual detection of NEO is disturbed without background; This up-conversion fluorescence immune chromatography test paper is easy and simple to handle, can meet different levels personnel's needs, comprises professional chemical examination, customs quarantine control, health quarantine, quality monitoring, livestock products processing, raiser and consumer individual etc.The present invention ensuring food safety, Protection of consumer is extremely important aspect healthy, will have obvious economic benefit and social benefit.
(7), when prepared by test paper of the present invention, fluorescence antibody adopts NaYF
4: NEO monoclonal antibody or the polyclonal antibody of Yb:Er nanoparticle label, labeling process is simple, only need be by amidized NaYF
4: the Yb:Er nano particle directly is connected with antibody, avoided the loaded down with trivial details step that collaurum-antibody is connected, and can adjust by controlling volumetric molar concentration the coupling rate of fluorescent nano particle and antibody, thereby the sensitivity that effectively improves the Sidestream chromatography immune test paper based on the up-conversion fluorescence nano material.
The accompanying drawing explanation
The structural representation that Fig. 1 is immune chromatography test paper of the present invention;
The side structure schematic diagram of the immune chromatography test paper of Fig. 2 Fig. 1;
Fig. 3 is the regression curve of up-conversion fluorescence immune chromatography test paper of the present invention to NEO;
Fig. 4 is that fluorescence used in the present invention is read the bar instrument.
In figure, 1 is supporting layer, and 2 is the adsorbing fiber layer, and 3 is the fluorescence antibody fibrage, and 4 is the cellulose rete, and 5 is absorbent material layer, and 6 is the stealthy trace that detects, and 7 is stealthy contrast trace, and 8-1 is sample end diaphragm, and 8-2 is the handle end diaphragm, and 9 is the sample mark line.
Embodiment
Up-conversion fluorescence immune chromatography test paper preparation process of the present invention comprises: the steps such as the preparation of the preparation of NEO monoclonal or polyclonal antibody, the fibrolaminar preparation of fluorescence antibody, adsorbing fiber layer, the preparation of cellulose rete and test paper assembling.
1, the preparation of NEO monoclonal antibody or polyclonal antibody
The preparation of monoclonal antibody: comprise the immunogenic preparation of NEO, immune animal (mouse), Fusion of Cells, the screening of monoclonal antibody, the preparation of monoclonal antibody; The preparation of polyclonal antibody: comprise the immunogenic preparation of NEO, immune animal (rabbit), the screening of polyclonal antibody, the preparation of polyclonal antibody.
(1) preparation of NEO artificial antigen:
100 mg NEO standard items are dissolved in to the hydrochloric acid of 10mL, 1mol/L, slowly drip 30% NaNO
2solution, by starch KI detection paper, be added to when the KI test paper is brown purple and stop dripping, and stirring reaction 45 min, drip 5% sulfaminic acid ammonium salt solution, with its reaction end of starch KI detection paper.With dropper, the NEO solution of azo is slowly dripped in the PBS(pH7.4 that is dissolved with 300 mg BSA) in, and regulate pH value to 9.0 with the NaOH solution of 1 mol/L, and after 4 ℃ of lucifuges are reacted 24 h, 4 ℃ of dialysis 48 h, packing ,-20 ℃ save backup.In like manner prepare coating antigen.
(2) preparation of NEO monoclonal antibody:
With the NEO artificial antigen made with 50 μ g～100 μ g/ consumption immunity BALB/c mouse in 6～8 week age only 3～4 times, 3～5 weeks, each immune interval, determine the antibody titer rear superpower immunity that meets the requirements, 3～4 days afterwards by hole blood sampling under the immune mouse socket of the eye, separates positive serum; De-neck is lethal, and with alcohol-pickled mouse 5～10min sterilization body surface of 75%, aseptic its spleen of getting, shred spleen grind, and through 120 order nylon gauzes, filters, and the centrifugal 10min of 1000rpm, collect splenocyte.By 1 * 10
8splenocyte with NS0 myeloma cell, in the ratio of 10:1, mix, the centrifugal 10min of 1000rpm, abandon supernatant, and the cell precipitation thing slowly adds the PEG4000 effect 1min of 0.7～1.0mL 50% in 37 ℃ of water-baths, then slowly add serum-free 1640 nutrient culture media 15mL, to stop the effect of PEG; 37 ℃ of water-bath 5～10 min, 1000rpm is centrifugal, and 10min abandons supernatant, the cell precipitation thing is resuspended in to HAT and selects in nutrient culture media, and add 96 porocytes to cultivate plate hole (100 μ L～200 μ L/ holes), is placed in 37 ℃, 5%CO
2in incubator, cultivate.Cultivate 10～14 days, with indirect elisa method, carry out positive hole sizer choosing, limited dilution cloning is carried out in selection strong positive, the hole that inhibiting rate is high, Growth of Cells is vigorous 3 times, then enlarges and cultivates, and sets up hybridoma cell strain.The monoclonal antibody of prepared hybridoma secretion can be reacted with NEO specifically, and affinity constant reaches 10
10～10
12, light chain subtype is κ or λ, the heavy chain hypotype is IgG
1, IgG
2a, IgG
2b, IgG
3, for the monoclonal antibody of NEO specific antigen determinant, for the preparation of the fluorescence antibody glass fibre cotton.
(3) preparation of NEO polyclonal antibody:
By the NEO artificial antigen immunity New Zealand white rabbit made, immunizing dose is 200 μ g～500 μ g/ time, the injection of subcutaneous minute 4～6, back.Head exempts from, and with aseptic PBS, dissolves the NEO artificial antigen, and with equivalent, FCA mixes, fully emulsified; Booster immunization, dissolve NEO carrier protein couplet thing with aseptic PBS, and with equivalent, FIA mixes, fully emulsified, after exempting from, head carries out in 2～3 weeks, and continuous immunity 4～5 times, every minor tick 2～3 weeks, latter 10～15 days of last immunity, survey it with the ELISA method and surely tire and reach 10
5when above, blood sampling separated and collected hyper-immune serum.Extract IgG antibody with the saturated ammonium sulfate salting out method, get 1 portion of hyper-immune serum and add 2 parts of PBS(pH7.2) mix, adding equal-volume saturated ammonium sulfate solution mixes, put 4 ℃ of refrigerator 12h, 4 ℃, the centrifugal 15min of 2500rpm, abandon supernatant, again with appropriate PBS(pH7.2) dissolution precipitation, add saturated ammonium sulfate solution to final concentration 33%, put 4 ℃ of refrigerator 2h, 4 ℃, the centrifugal 15min of 2500rpm, abandon supernatant, with appropriate PBS(pH7.2) dissolution precipitation, putting in 4 ℃ of refrigerators and use PBS(pH7.2) 48～72h dialyses, liquid is changed for several times in centre, 4 ℃, the centrifugal 15min of 12000rpm, collect supernatant, obtain the anti-NEO polyclonal antibody of purifying,-20 ℃ frozen, for the preparation of the fluorescence antibody glass fibre cotton.
2, the fibrolaminar preparation of fluorescence antibody
The fibrolaminar preparation of fluorescence antibody, at first need to prepare NaYF
4: the Yb:Er nano particle, then with the NaYF prepared
4: Yb:Er nanoparticle label NEO antibody.
(1) NaYF
4: the preparation of Yb:Er nano particle can adopt coprecipitation or hydrothermal synthesis method.
A. coprecipitation
Y (the NO that to get respectively concentration be 0.2mol/L
3)
3yb (the NO of 20ml, 0.2mol/L
3)
3er (the NO of 2ml, 0.2mol/L
3)
30.5ml, the EDTA-Na21ml of 1mol/L, add in the 100ml flask, 50 ℃ of oil bath stirring reaction 1h, obtain reactant liquor A;
Get the NaF aqueous solution of 50ml, 1mol/L, add in polytetrafluoroethylcontainer container, under magnetic agitation, A liquid is poured in the NaF aqueous solution into to water-bath 1min in 50 ℃ of water-baths; The centrifugal 5min of 6000rpm, abandoning supernatant must precipitate, and in precipitation, adds water, ultrasonic washing 3 times, the centrifugal 5min of 7000rpm; With absolute ethyl alcohol ultrasonic washing 2 times, the centrifugal 5min of 10000rpm, be placed in 80 ℃ of baking oven dried overnight by the solid obtained; Dried solid is put into to muffle furnace, in 500 ℃ of calcining 5h, obtain NaYF
4: the Yb:Er fluorescent nano particle.
B. hydrothermal synthesis method
Get respectively the Y (NO that concentration is 0.2mol/L
3)
34ml, Yb (NO
3)
30.5ml, Er (NO
3)
30.1ml, EDTA-Na4ml, mix and fully the reaction after, obtain reactant liquor A;
Take NaOH 0.7g, join in the mixed liquor of 8ml oleic acid and 12ml ethanol and mix, under agitation, slowly to the NaF solution that injects 10mL, 1mol/L in mixed liquor, after filling, continue to stir, to the solution shape that is translucent; Reactant liquor A is poured in described solution, mix, ageing 20min; Then potpourri is transferred in polytetrafluoroethylcontainer container, 130 ℃ of oil baths reaction 12h, the product obtained after filtration, washing, drying, obtain NaYF
4: the Yb:Er fluorescent nano particle.
(2) NaYF
4: Yb:Er nanoparticle label NEO antibody
Labeling process comprises the following steps: NaYF
4: the surface siliconization of Yb:Er nano particle, NaYF
4: the surface amination of Yb:Er nano particle, NaYF
4: the coupling of Yb:Er nano particle and NEO antibody.
A. NaYF
4: the surface siliconization of Yb:Er fluorescent nano particle
By NaYF
4: the Yb:Er fluorescent nano particle is dispersed in the ethanol water of mass concentration 10%, adds 5ml strong aqua (concentration 28%) under magnetic agitation, and reaction 10min, then add the 2ml tetraethyl orthosilicate, room temperature reaction 3h; The centrifugal 5min of 6000rpm, wash 3 times, finally with ethanol, disperses;
B. NaYF
4: the surface amination of Yb:Er fluorescent nano particle
The fluorescent nano particle of surface siliconization is added in the mixed solution of methyl alcohol that volume ratio is 3:5, acetone, through ultrasound wave, disperse, add the APTSA(N-(2-aminoethyl) of 5ul-3-aminopropyl trimethoxysilane), 70 ℃ of water-bath 1h, the centrifugal 5min of 6000rpm, by the ethanol washing 3 times for precipitation obtained, drying, preserve;
C. the mark of NEO antibody (glutaraldehyde method)
The NaYF that surface is modified through amination
4: the Yb:Er nano particle disperses with the PBS buffer solution of pH=7.4, the glutaraldehyde that adds wherein mass concentration 25%, room temperature reaction 3 hours, centrifugally wash away unnecessary glutaraldehyde, then be dispersed in the PBS buffer solution of pH=7.4, then add wherein monoclonal antibody or the polyclonal antibody of NEO, 4 ℃ are reacted 3 hours, centrifuge washing for several times, obtains NaYF again
4: the antibody coupling matter of Yb:Er-NEO, with 4 ℃ of preservations of PBS buffer solution of pH=7.4.
3, the preparation of adsorbing fiber layer
The test lead adsorbing fiber is glass fibre cotton, nylon membrane, polyvinylidene fluoride pvdf membrane or polyester film preparation for layer, fibrous material is cut into to the band of wide 1.5cm specification, puts it in the sample pad confining liquid and soaks 30min, in 37 ℃ of oven dry, standby.
4, the preparation of cellulose rete
Cellulose is nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane for rete, cut into the band of wide 1.5cm specification, with point sample instrument respectively specking NEO antigen and goat anti-mouse igg antibody (or the anti-mouse IgG of rabbit, goat anti-rabbit igg antibody) of diverse location on cellulose membrane, make stealthy detection trace band and contrast trace band, in 37 ℃ of dry for standby.
Wherein the preparation method of the anti-mouse IgG of goat-anti or rabbit (or goat anti-rabbit igg) antibody is as follows:
Extract the negative mice serum IgG(of NEO or negative rabbit anteserum IgG with saturated ammonium sulfate), get 1 part of mice serum (or rabbit anteserum) and add 2 parts of PBS(pH7.2) mix, add equal-volume saturated ammonium sulfate solution and mix, put 4 ℃ of refrigerator 12h, 4 ℃, the centrifugal 15min of 2500rpm, abandon supernatant; Again with appropriate PBS(pH7.2) dissolution precipitation, add saturated ammonium sulfate solution to final concentration 33%, put 4 ℃ of refrigerator 2h, 4 ℃, the centrifugal 15min of 2500rpm, abandon supernatant; With appropriate PBS(pH7.2) dissolution precipitation, put in 4 ℃ of refrigerators, use PBS(pH7.2) dialysis 48h, and liquid is changed 3 times in centre, and 4 ℃, the centrifugal 15min of 12000rpm collect supernatant, with ultraviolet spectrophotometer, measure its protein concentration; With mice serum (or rabbit anteserum) IgG of 50 μ g～100 μ g/kg body weight through the subcutaneous and healthy goat of intramuscular injection or rabbit 3～4 times, the last injection is after 10 days, measure its serum titer with ELISA and reach 1:2000 when above, heart or arterial blood drawing, the separated and collected hyper-immune serum, extract goat-anti or the anti-mouse IgG of rabbit (or goat anti-rabbit igg) antibody (method is identical with extraction mice serum IgG, no longer repeats) with saturated ammonium sulfate, for the preparation of NEO Test paper contrast trace.
5, the assembling of immune chromatography test paper
Adsorbing fiber layer, fluorescence antibody fibrage, cellulose rete, absorbent material layer are attached on the supporting layer with bonding agent from right to left successively, and are cut into the wide test paper of 3-4cm and get final product.
6, detection reaction principle
After the test paper test lead inserts testing sample solution, the fluorescence antibody that solution to be measured drives in NEO to be measured and fluorescence antibody glass fibre cotton by syphonic effect spreads to the cellulose rete together, and finally infiltrates the absorbent material layer of handle end.In diffusion process, NEO to be measured can combine with fluorescence antibody, and then the antigen-combining site of NEO on the sealing fluorescence antibody, stop the detection trace of the NEO artificial antigen of fluorescence antibody on cellulose membrane to be combined, can not show the detection trace, the anti-mouse IgG of sheep or rabbit (or goat anti-rabbit igg) antibody can be combined with fluorescence antibody, reads bar instrument T line place by fluorescence and just do not there will be absorption peak under infrared excitation; Otherwise in sample solution during without NEO, can not stop the NEO artificial antigen of fluorescence antibody on cellulose membrane to detect trace is combined, read bar instrument T line place by fluorescence and just there will be absorption peak, same goat-anti or the anti-mouse IgG of rabbit (or goat anti-rabbit igg) antibody also are combined with golden labeling antibody, read bar instrument C line place by fluorescence and also there will be absorption peak.If show without any red trace band on cellulose membrane, by fluorescence read bar instrument T line, C line place does not occur that absorption peak shows that test paper lost efficacy.
following examples illustrate structure and the detection method of immune chromatography test paper.
Embodiment mono-: referring to Fig. 1 and Fig. 2.In figure, supporting layer 1 use plastic slice bar is made, adsorbing fiber layer 2 use glass fibre cotton are made, on fluorescence antibody fibrage 3, absorption has the fluorescence antibody glass fibre cotton of anti-NEO monoclonal antibody, cellulose rete 4 adopts nitrocellulose filter, the absorbent material layer 5 use absorbent filters of handle end are made, adsorbing fiber layer 2, fluorescence antibody fibrage 3, cellulose rete 4, absorbent material layer 5 each layers are pasted and fixed on supporting layer 1 from right to left successively to the intersection fiber infiltration that crosses one another each other.Be provided with the stealthy trace 6 that detects on cellulose rete 4, make with the bovine serum albumin solution (BSA) of coupling NEO; Stealthy contrast trace 7 use goat anti-mouse igg antibody solution trace on cellulose membrane make "
︱", two traces be arranged in parallel form combination trace band "
︱ ︱".
Cover adsorbing fiber layer 2 and the sample end diaphragm 8-1 above fluorescence antibody fibrage 3 for white; the handle end diaphragm 8-2 covered above absorbent material layer 5 is yellow; sample mark line 9 is positioned at the deflection adsorbing fiber layer about 0.5cm of 2 one side place on the white diaphragm that adsorbing fiber layer 2 is corresponding with fluorescence antibody fibrage 3 intersections, is printed on arrow and max printed words on the diaphragm of mark line right side.
The preparation of testing sample and detecting step:
Detect the meat sample: by sample shred, levigate, make the sample suspension of 1:2～10 with normal saline dilution;
Method of operating: NEO test paper sample end is inserted in testing sample, and insertion depth is no more than mark line, approximately takes out test paper 10～20 seconds, puts into fluorescence after 5min and reads directly value of reading of bar instrument.The fluorescence used is read the bar instrument and is seen Fig. 4, is existing instrument.
Result is judged: (a) positively read bar instrument T line place by fluorescence and absorption peak occurs, mean that testing result is positive, illustrate and contain NEO in testing sample; (b) negatively read bar instrument T line place by fluorescence and absorption peak do not occur, mean that testing result is negative, illustrate and do not contain NEO in testing sample; (c) lost efficacy by fluorescence read bar instrument T line, absorption peak does not appear in C line place, shows that test paper lost efficacy.
Embodiment bis-: immune chromatography test paper and embodiment mono-are basic identical; difference is: the absorption of fluorescence antibody fibrage has the polyclonal antibody of anti-NEO; the adsorbing fiber layer is made with nylon membrane; the cellulose rete adopts the pure cellulose film; the stealthy trace that detects is " ten " with stealthy contrast trace, and the handle end diaphragm covered above absorbent material layer is blue look.Detect milk sample: with physiological saline, the milk sample dilution is made to the sample suspension of 1:2～5.Result judgement, method of operating are with embodiment mono-.
Embodiment tri-: immune chromatography test paper and embodiment mono-are basic identical; difference is: the adsorbing fiber layer is made with the polyvinylidene fluoride pvdf membrane; the carrier protein solution of coupling NEO is chicken ovalbumin (OVA); stealthy contrast trace is made on cellulose membrane with the anti-mouse IgG antibody solution of rabbit; the cellulose rete adopts the carboxylation cellulose membrane; the handle end diaphragm covered above absorbent material layer is green, and the stealthy trace that detects is " ┬ " with the stealthy trace that contrasts.For detection of blood sample: extract serum and also with physiological saline, its dilution is made to the testing sample of 1:2～10.Result judgement and method of operating are all with embodiment mono-.
Embodiment tetra-: immune chromatography test paper and embodiment mono-are basic identical, and difference is: the adsorbing fiber layer is made with polyester film, and the cellulose rete adopts the carboxylation cellulose membrane, and the stealthy carrier protein solution that detects coupling NEO in trace is hemocyanin (KLH).For detection of urine sample, can directly get urine as testing sample.Detect the trace band and contrast the trace band and be " ┴ ".Method of operating and as a result decision method with example one.
Embodiment five: immune chromatography test paper and embodiment mono-are basic identical, and difference is: stealthy contrast trace is made on cellulose membrane with goat anti-rabbit igg antibody solution, and the adsorbing fiber layer is made with nylon membrane.Detect the trace band and contrast the trace band and be " ├ ".Detect sample, result judgement and method of operating with example one.
Embodiment six: and embodiment mono-is basic identical, and difference is: the absorption of fluorescence antibody fibrage has the polyclonal antibody of anti-NEO, and the detection sample is milk sample.Detect the trace band and contrast the trace band and be " ┤ ".
Embodiment seven: and embodiment mono-is basic identical, and difference is: the absorption of fluorescence antibody fibrage has the polyclonal antibody of anti-NEO, and the detection sample is blood sample.
Embodiment eight: and embodiment mono-is basic identical, and difference is: the absorption of fluorescence antibody fibrage has anti-NEO polyclonal antibody, and the detection sample is urine sample.
Embodiment nine: and embodiment mono-is basic identical, and difference is: in stealthy detection trace, coupling NEO carrier protein solution is hemocyanin (KLH), detects sample, result judgement and method of operating with embodiment mono-, does not repeat.
Embodiment ten: and embodiment mono-is basic identical, and difference is: in stealthy detection trace, coupling NEO carrier protein solution is chicken ovalbumin (OVA), detects sample, result judgement and method of operating with embodiment mono-, does not repeat.
Embodiment 11: the sensitivity of immune chromatography test paper of the present invention, specific detection
1, the detection of sensitivity: use phosphate buffered solution PBS(PH7.4) or distilled water respectively configuration concentration be 4,2,1,0.5,0.25,0.125,0.0625, the NEO standard items of 0ng/mL, loading 80-100uL on immune chromatography test paper of the present invention, after reaction 5min, read the bar instrument by fluorescence and directly read peak figure.Take peak value or peak area as ordinate, and the logarithm value of different GM concentration of take is horizontal ordinate, and drawing standard suppresses curve, carries out the correlation regression analysis, calculates the IC of this test paper to NEO
50and lowest detectable limit.After measured, this test paper to the Regression Equations of NEO is: y=-1111.8x+2836.8, related coefficient is R
2=0.9947, go out the IC of this test paper to NEO according to regression equation calculation
50for 448.84pg/mL, the lowest detection of this test paper is limited to 129.55pg/mL, shows that immune chromatography test paper has higher sensitivity to NEO.Referring to Fig. 3.
2, specific detection: using the similar drugs gentamicin, streptomysin, kanamycins, chloromycetin, terramycin, tetracycline, ampicillin of NEO as the competition thing, the concentration that configures above-mentioned mark product is 1mg/mL, detect its inhibiting rate with the up-conversion fluorescence immune chromatography test paper, the IC with this test paper to NEO
50iC with each competition thing
50number percent be its cross reacting rate.
Measurement result sees the following form 1.Can find out, the specificity of this up-conversion fluorescence immune chromatography test paper is better, with the equal no cross reaction of other drug.
The cross reactivity of table 1 neomycin up-conversion fluorescence immune chromatography test paper
| Compound | Half-inhibition concentration IC 50（ng/mL） | Cross reactivity (%) |
| Neomycin | 448.84 | 100 |
| Gentamicin | > 1.0×10 6 | < 0.045 |
| Streptomysin | > 1.0×10 6 | < 0.045 |
| Kanamycins | > 1.0×10 6 | < 0.045 |
| Chloromycetin | > 1.0×10 6 | < 0.045 |
| Terramycin | > 1.0×10 6 | < 0.045 |
| Tetracycline | > 1.0×10 6 | < 0.045 |
| Ampicillin | > 1.0×10 6 | < 0.045 |
Claims (10)
1. the up-conversion fluorescence immune chromatography test paper quantitatively detected for neomycin, bottom is supporting layer, middle layer is adsorbed layer, protective seam is fixed on adsorbed layer, adsorbed layer is followed successively by the absorbent material layer of adsorbing fiber layer, fluorescence antibody fibrage, cellulose rete and handle end from test lead, it is characterized in that: be provided with the stealth of printing with the carrier protein solution of coupling NEO and detect trace on the cellulose rete, also be provided with the stealth contrast trace of printing with goat anti-mouse igg, the anti-mouse IgG of rabbit or goat anti-rabbit igg antibody solution; Described fluorescence antibody fibrage adopts the glass fibre cotton of absorption fluorescence antibody to make, and fluorescence antibody adopts NaYF
4: NEO monoclonal antibody or the polyclonal antibody of Yb:Er nanoparticle label.
2. immune chromatography test paper according to claim 1, is characterized in that: described NaYF
4: the Yb:Er nano particle is with NaYF
4for matrix, Yb
3+for sensitizer, the nano particle that the diameter formed by Yb and Er codope is 50-200nm.
3. immune chromatography test paper according to claim 1, is characterized in that: described NaYF
4: by following methods, prepared by NEO monoclonal antibody or the polyclonal antibody of Yb:Er nanoparticle label:
(1) surface siliconization of fluorescent nano particle: by NaYF
4: the Yb:Er fluorescent nano particle is dispersed in the ethanolic solution that mass concentration is 10%, under agitation adds the 5ml strong aqua, and reaction 10min, then add the 2ml tetraethyl orthosilicate, room temperature reaction 3h; The centrifugal 5min of 6000rpm, disperse with ethanol after washing;
(2) surface amination of fluorescent nano particle: the fluorescent nano particle of surface siliconization is added in the mixed solution of methyl alcohol that volume ratio is 3:5, acetone, through ultrasound wave, disperse, add the N-(2-aminoethyl) of 5 μ L-3-aminopropyl trimethoxysilane, 70 ℃ of water-bath 1h, the centrifugal 5min of 6000rpm, the precipitation obtained is washed to drying with ethanol;
(3) mark of NEO antibody: the NaYF that surface is modified through amination
4: the Yb:Er nano particle disperses with PBS buffer solution, the glutaraldehyde solution that adds wherein mass concentration 25%, room temperature reaction 3 hours, centrifugally wash away unnecessary glutaraldehyde, then reactant liquor is dispersed in PBS buffer solution, adds monoclonal antibody or the polyclonal antibody of NEO, 4 ℃ are reacted 3 hours, through centrifuge washing, obtain NaYF
4: NEO monoclonal antibody or the polyclonal antibody of Yb:Er nanoparticle label are placed in PBS buffer solution, 4 ℃ of preservations.
4. immune chromatography test paper according to claim 1 is characterized in that: described adsorbing fiber for layer glass fibre cotton, nylon membrane, PVDF membrane or polyester film make; Absorbent material layer is made with absorbent filter, and supporting layer is made with the toughness material do not absorbed water; Cellulose for rete nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane make;
the carrier protein of coupling NEO is bovine serum albumin(BSA), chicken ovalbumin or hemocyanin.
5. immune chromatography test paper according to claim 1 is characterized in that: described stealthy detect trace and stealthy contrast trace be arranged in parallel "
︱ ︱" the orthoscopic trace, "
10" font is arranged trace, " ┬ ┬ " font is arranged trace, " ┴ ┴ " font arrangement trace, " ├ ├ " font arrangement trace or " ┤ ┤ " font and arranged trace.
6. immune chromatography test paper according to claim 1; it is characterized in that: described protective seam is the diaphragm covered on adsorbing fiber layer, fluorescence antibody fibrage and absorbent material layer; be printed with the sample mark line at the adsorbing fiber layer on the diaphragm corresponding with fluorescence antibody fibrage intersection, this mark line deflection adsorbing fiber layer one side 0.3-0.7cm.
7. according to the described immune chromatography test paper of claim 1-6 any one, it is characterized in that: described NaYF4:Yb:Er nano particle adopts the coprecipitation preparation, and concrete grammar is as follows:
Y (the NO that to get respectively concentration be 0.2mol/L
3)
3yb (the NO of 20ml, 0.2mol/L
3)
3er (the NO of 2ml, 0.2mol/L
3)
30.5ml, the EDTA-Na21ml of 1mol/L, add in flask, 50 ℃ of oil bath stirring reaction 1h, obtain reactant liquor A; The NaF solution of getting 50ml, 1mol/L adds in polytetrafluoroethylcontainer container, reactant liquor A is poured in NaF solution into to reaction 1min under 50 ℃ of stirred in water bath; The centrifugal 5min of 6000rpm, abandoning supernatant must precipitate; Add water, ultrasonic washing 3 times, the centrifugal 5min of 7000rpm in precipitation; To precipitate and use absolute ethyl alcohol ultrasonic washing 2 times, the centrifugal 5min of 10000rpm, be placed in 80 ℃ of baking oven dried overnight by the solid obtained; Dried solid is put into to muffle furnace, in 500 ℃ of calcining 5h, obtain NaYF
4: the Yb:Er fluorescent nano particle.
8. according to the described immune chromatography test paper of claim 1-6 any one, it is characterized in that: described NaYF
4: the Yb:Er nano particle adopts the hydrothermal synthesis method preparation, and concrete grammar is as follows:
Get respectively the Y (NO that concentration is 0.2mol/L
3)
34ml, Yb (NO
3)
30.5ml, Er (NO
3)
30.1ml, EDTA-Na4ml, mix and fully the reaction, obtain reactant liquor A; Take NaOH 0.7g, join in the mixed liquor of 8ml oleic acid and 12ml ethanol, mix, under agitation slowly to the NaF solution that injects 10mL, 1mol/L in mixed liquor, after filling, continue to be stirred to the solution shape that is translucent; Reactant liquor A is poured in described solution, mix, ageing 20-30min; Then potpourri is transferred in polytetrafluoroethylcontainer container, 130-150 ℃ of oil bath reaction 12-18h, the product obtained after filtration, washing, drying, obtain NaYF
4: the Yb:Er fluorescent nano particle.
9. the preparation method of the described immune chromatography test paper of claim 1, it is characterized in that: the method comprises the following steps:
(1) preparation of NEO monoclonal antibody or polyclonal antibody;
(2) preparation of fluorescence antibody: first prepare NaYF
4: the Yb:Er fluorescent material, described fluorescent material is with NaYF
4for matrix, Yb
3+for sensitizer, formed the nano particle of diameter 50-200nm by Yb and Er codope; Then NEO monoclonal antibody or polyclonal antibody are carried out to fluorescence labeling;
(3) preparation of adsorbing fiber layer: adsorbing fiber for layer glass fibre cotton, nylon membrane, PVDF membrane or polyester film make;
(4) preparation of cellulose rete: cellulose is nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane for rete, with point sample instrument, at the diverse location difference specking of cellulose rete, detects trace and contrast trace, dries;
(5) assembling of immune chromatography test paper: adsorbing fiber layer, fluorescence antibody fibrage, cellulose rete, absorbent material layer are attached on the supporting layer that scribbles bonding agent from right to left successively, successively supporting layer, adsorbed layer and protective seam are assembled into to immune chromatography test paper.
10. preparation method according to claim 9, is characterized in that: described NaYF
4: by following methods, prepared by NEO monoclonal antibody or the polyclonal antibody of Yb:Er nanoparticle label:
(1) surface siliconization of fluorescent nano particle: by NaYF
4: the Yb:Er fluorescent nano particle is dispersed in the ethanolic solution that mass concentration is 10%, under agitation adds the 5ml strong aqua, and reaction 10min, then add the 2ml tetraethyl orthosilicate, room temperature reaction 3h; The centrifugal 5min of 6000rpm, disperse with ethanol after washing;
(2) surface amination of fluorescent nano particle: the fluorescent nano particle of surface siliconization is added in the mixed solution of methyl alcohol that volume ratio is 3:5, acetone, through ultrasound wave, disperse, add the N-(2-aminoethyl) of 5 μ l-3-aminopropyl trimethoxysilane, 70 ℃ of water-bath 1h, the centrifugal 5min of 6000rpm, the precipitation obtained is washed to drying with ethanol;
(3) mark of NEO antibody: the NaYF that surface is modified through amination
4: the Yb:Er nano particle disperses with PBS buffer solution, the glutaraldehyde solution that adds wherein mass concentration 25%, room temperature reaction 3 hours, centrifugally wash away unnecessary glutaraldehyde, then reactant liquor is dispersed in PBS buffer solution, adds monoclonal antibody or the polyclonal antibody of NEO, 4 ℃ are reacted 3 hours, through centrifuge washing, obtain NaYF
4: NEO monoclonal antibody or the polyclonal antibody of Yb:Er nanoparticle label are placed in PBS buffer solution, 4 ℃ of preservations.
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