CN203037658U - Phosphorescent silicon dioxide nanoparticle labeled immunochromatographic test strip for quantitatively detecting cimaterol - Google Patents
Phosphorescent silicon dioxide nanoparticle labeled immunochromatographic test strip for quantitatively detecting cimaterol Download PDFInfo
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- CN203037658U CN203037658U CN 201220392862 CN201220392862U CN203037658U CN 203037658 U CN203037658 U CN 203037658U CN 201220392862 CN201220392862 CN 201220392862 CN 201220392862 U CN201220392862 U CN 201220392862U CN 203037658 U CN203037658 U CN 203037658U
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Abstract
The utility model relates to a phosphorescent silicon dioxide nanoparticle labeled immunochromatographic test strip for cimaterol. The test strip comprises a support layer, an adsorption layer and a protective layer, wherein the adsorption layer comprises an adsorption fiber layer, a phosphorescent antibody fiber layer and a cellulose membrane layer from a test end in sequence, and a water absorption material layer at a handle end; an invisible detection blot printed by a carrier protein solution coupled with cimaterol, and an invisible control blot printed by a goat or rabbit anti-mouse IgG (immunoglobulin G) antibody solution are arranged on the cellulose membrane layer; the phosphorescent antibody fiber layer is made of glass fiber cotton absorbing a phosphorescent antibody; and the phosphorescent antibody is a phosphorescent silicon dioxide nanoparticle labeled cimaterol antibody. The test strip is high in specificity and sensitivity, simple, convenient, visual and accurate, can be used for quantitative and qualitative detection, can detect pg-level trace residuals to the greatest extent, and is wide in application scope, low in cost and easy to popularize and apply.
Description
Technical field
The utility model relates to a kind of immune chromatography test paper, and particularly relating to a kind of is the immuno-chromatographic test paper strip that is used for quantitatively detecting Cimaterol of mark with the phosphorescence nano SiO 2 particle.
Background technology
(cimaterol CIM) has another name called happiness Ma Teluo to Cimaterol, and special sieve of Zeeman belongs to a kind of of phenyl ethylamine class medicine, is a kind of potent broxaterol.CIM is mainly used in expanding tracheae and increases pulmonary ventilation volume in medical science and veterinary clinic, can be used for diseases such as asthma, obstructive pneumonia, smooth muscle spasm and shock.Studies show that the CIM that feeds can increase the price of deed of fattening pig and increase lean meat percentage, and animals such as ox, chicken, duck and sheep are had certain effect, illegally be used for animal derived food production so CIM often is used as feed addictive.Long-term use then may cause CIM property accumulated in edible animal tissue residual, usually cause that clinical symptoms such as palpitaition, muscular tremor, pain, nervous symptoms, dizziness headache, n and V, heating shiver with cold take place the eater, bigger to patients such as heart disease, diabetes and hypertension harm especially.In view of the obvious harm of CIM, China forbids that CIM uses in Production of Livestock and Poultry, and is defined in the edible tissue of all animals and must not detects.
At present, the method that is used for the Cimaterol residue detection is more, as (1) physico-chemical analysis method, as high pressure lipuid chromatography (HPLC), vapor-phase chromatography, thin layer chromatography, electrophoresis etc.; (2) immunoassay is as radioimmunology, enzyme linked immunosorbent assay etc.; (3) bioassay method is as microbiological assay, radioreceptor assay etc.These methods need expensive instrument and equipment, need skilled professional's operation, the process complexity, and the time is longer, has limited its range of application, is difficult to apply aborning.
The immunoassay labelling technique refers to based on the specific reaction between antigen-antibody, certain material is carried out the research of qualitative or quantitative detection by label.The labelled immune analysis generally is that label antagonists such as enzyme, fluorescein, radioactive nuclide or antigen are carried out mark, this label has kept the activity of antibody or antigen, do not influence the activity of label, behind it and corresponding antibodies or antigen-reactive, can directly measure the label in the compound, directly target substance be carried out quantitative test.By the signal amplification of label, can improve the susceptibility of immunoassay.
Fluoroimmunoassay is to be label with fluorescein, fluorescent dye, quantum dot, lanthanide series etc., and antigen or antibody labeling are combined a kind of detection method of fluorescence intensity under fluorescent microscope with fluorescent material with corresponding antigen or antibody.Characteristics highly sensitive, high specificity that this method has, but some fluorescein can produce biology toxicity, causes the sensitivity of antigen or antibody and selectivity to descend.
The utility model content
The technical problems to be solved in the utility model: providing a kind of is the detection Cimaterol immuno-chromatographic test paper strip of mark with the phosphorescence nano SiO 2 particle, and this test strips has the characteristics of the micro-Cimaterol of special, sensitive, quick, easy quantitative detection.
The technical solution of the utility model:
A kind of immuno-chromatographic test paper strip of detection Cimaterol of phosphorescence nano SiO 2 particle mark, bottom is supporting layer, the middle layer is adsorbed layer, protective seam is fixed on the adsorbed layer, adsorbed layer is followed successively by the absorbent material layer of adsorbing fiber layer, phosphorescence antibody fibrage, cellulose rete and handle end from test lead, be provided with the stealth of printing with the carrier protein solution of coupling Cimaterol at the cellulose rete and detect trace, 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 phosphorescence antibody fibrage adopts the glass fibre cotton of absorption phosphorescence antibody to make, and phosphorescence antibody adopts the Cimaterol antibody of phosphorescence nano SiO 2 particle mark.
The particle diameter of described phosphorescence nano SiO 2 particle is 100-220nm.
Described adsorbing fiber layer glass fibre cotton, nylon membrane, PVDF membrane or polyester film; The absorbent material layer absorbent filter, the supporting layer toughness material that does not absorb water; Cellulose rete nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane; The carrier protein of coupling Cimaterol is bovine serum albumin(BSA), the pure albumen of ovum gallinaceum or hemocyanin.
The described stealthy trace that detects is " ︱ ︱ " the orthoscopic trace that is arranged in parallel with stealthy contrast trace, or " 10 " font is arranged trace, or " ┬ ┬ " font is arranged trace, or " ┴ ┴ " font is arranged trace, or " ├ ├ " font arrangement trace, or " ┤ ┤ " font is arranged trace.
Described protective seam is the diaphragm that covers on adsorbing fiber layer, phosphorescence antibody fibrage and the absorbent material layer; The diaphragm corresponding with phosphorescence antibody fibrage intersection at the adsorbing fiber layer is printed with the sample mark line, this sample mark line deflection adsorbing fiber layer one side 0.3-0.7cm.
Test strips of the present utility model has the following advantages:
(1) high specificity, highly sensitive.The utility model test strips combines high-sensitive phosphorescence nano SiO 2 particle with immunochromatography technique, the phosphorescence nano SiO 2 particle need not to wrap up any fluorescent material just can be luminous voluntarily, and phosphorescent lifetime is long, cost of manufacture is low, light stability is strong, can obtain to detect higher sensitivity than common phosphorescence.This test strips had both kept the simple and rapid advantage of traditional colloidal gold strip, had overcome low, the shortcoming that can't be quantitative of latter's sensitivity again, and detection sensitivity is higher, more stable, a minimum trace residue of gram level that detects.
(2) easy, quick.This test strips can directly detect whole blood, urine, saliva, tissue homogenate etc., need not carry out The pretreatment, and direct observed result under ultraviolet light also can be realized quantitatively detection by the direct value of reading of fluorescence reader.Only need during detection test strips was inserted test sample 10～20 seconds, can judge testing result in 5 minutes, but execute-in-place is time saving and energy saving, easy and simple to handle, a step finishes.
(3) result shows image, directly perceived, accurate.Test strips with show blue and white "
︱" and "
︱ ︱" (or " ten ", " ┬ ", " ┴ ", " ├ ", " ┤ ") trace is as the positive and the negative marker that detect, namely cellulose membrane show a blue and white "
︱" trace, contain CIM in the expression test sample; Show two blue and whites "
︱ ︱" trace, do not contain CIM in the expression test sample.Testing result can with the naked eye directly be observed, and image, directly perceived, accurate, simple and clear is not prone to artificial erroneous judgement such as false positive and false negative as a result.
(4) cost saving.This test strips need not to join in addition instrument and equipment and other reagent when detecting, can detect whenever and wherever possible, can qualitative detection can quantitatively detect again; Testing cost is cheap, can save the input expense of a large amount of expensive instruments and equipment.
(5) applied widely, easy to utilize.This test strips can satisfy different levels personnel's needs, comprise professional chemical examination, customs quarantine control, health quarantine, quality monitoring, livestock products processing, raiser and consumer individual etc., both be suitable for the detection of single sample, be suitable for the examination of a large amount of samples again, can be applied to fields such as medical diagnosis on disease, drugs detection, Bacteria Detection and environment measuring.The utility model has meaning of crucial importance aspect the consumer health ensuring food safety, protect, and has tangible economic benefit and social benefit.
Description of drawings
Fig. 1 is the structural representation of immuno-chromatographic test paper strip of the present utility model;
Fig. 2 is the vertical view structural representation of immuno-chromatographic test paper strip of the present utility model;
Fig. 3 is that immuno-chromatographic test paper strip of the present utility model is to the regression curve of CIM.
Embodiment
The preparation process of the utility model test strips comprises: the preparation of Cimaterol monoclonal or Polyclonal Antibody Preparation, the fibrolaminar preparation of phosphorescence antibody, adsorbing fiber layer, the preparation of cellulose rete and the steps such as assembling of test strips.
(1) anti-Cimaterol monoclonal antibody or Polyclonal Antibody Preparation
Monoclonal antibody preparation: with 50 μ g～100 μ g/ Cimaterol carrier protein couplet thing immunity Balb/C mouse in 6～8 age in week only 3～4 times, each immunity 3～5 weeks of interval time, determine antibody titer meet the requirements the back superpower immunity, 3～4 days afterwards, with hole blood sampling under the immune mouse socket of the eye, separate positive serum; Take off neck and cause death, with alcohol-pickled mouse 5～10min sterilization body surface of 75%, aseptic its spleen of getting shreds spleen and grind, and filters through 120 order nylon gauzes, and the centrifugal 10min of 1000rpm collects splenocyte.With 1 * 10
8Splenocyte mix in the ratio of 10:1 with NS0 myeloma cell, the centrifugal 10min of 1000rpm, abandon supernatant, the cell precipitation thing slowly adds the 50%PEG4000 of 0.7～1.0 mL CIM in 37 ℃ of water-baths, effect 1min, slowly add serum-free 1640 nutrient culture media 15 mL then, to stop the effect of PEG, 37 ℃ of water-bath 5～10 min, the centrifugal 10min of 1000rpm abandons supernatant, the cell precipitation thing is resuspended in HAT selects in the nutrient culture media, and add 96 porocytes cultivation plate hole (100 μ L～200 μ L/ holes), place 37 ℃, 5%CO
2Cultivated in the incubator 10～14 days, and carried out positive hole sizer choosing with indirect elisa method, select strong positive, inhibiting rate height, the eugonic hole of cell to carry out limited dilution cloningization 3 times, then enlarge and cultivate, set up hybridoma cell strain.The monoclonal antibody of prepared hybridoma secretion can be reacted with Cimaterol 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, at the monoclonal antibody of Cimaterol specific antigen determinant, be used for the mark of phosphorescence nano SiO 2 particle.
Many anti-preparations: with Cimaterol carrier protein couplet thing immunity New Zealand white rabbit, immunizing dose is 200 μ g～500 μ g/ time, the subcutaneous branch 4 in back～6 injections.Head exempts from, and with aseptic PBS dissolving Cimaterol carrier protein couplet thing, FCA mixes with equivalent, and is fully emulsified; Booster immunization, with aseptic PBS dissolving Cimaterol carrier protein couplet thing, FIA mixes with equivalent, fully emulsified, head exempts from the back and carries out continuous immunity 2～3 weeks 4～5 times, each 2～3 weeks at interval, last immunity back 10～15 days is surveyed it with the ELISA method and is tired surely and reach 10
5When above, blood sampling and separated and collected hyper-immune serum.Extract IgG antibody with the saturated ammonium sulfate salting out method, namely get 1 portion of hyper-immune serum and add 2 parts of PBS(pH7.2) mixing, add equal-volume saturated ammonium sulfate solution mixing, put 4 ℃ of refrigerator 12h, 4 ℃, the centrifugal 15min of 2500rpm abandon supernatant; Again with an amount of 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 an amount of PBS(pH7.2) dissolution precipitation, put and use PBS(pH7.2 in 4 ℃ of refrigerators) dialysis 48～72h, liquid is changed for several times in the centre, 4 ℃, the centrifugal 15min of 12000rpm, collect supernatant, get the anti-Cimaterol polyclonal antibody of purifying ,-20 ℃ frozen, is used for the mark of phosphorescence nano SiO 2 particle.
(2) the fibrolaminar preparation of phosphorescence antibody
A. the preparation of phosphorescence nano SiO 2 particle
Adopt the reverse microemulsion legal system to be equipped with blank nano SiO 2 particle, the proportioning by optimizing each component in the microemulsion and the amount of ammoniacal liquor are controlled the size of nano particle.Dried nano SiO 2 particle is put in high-temperature calcination in the muffle furnace, according to different calcining heats, obtains the phosphorescence nano SiO 2 particle of different phosphate light intensity and color.Detailed process is as follows:
Strong aqua (mass content 28%), ethanol, water are pressed: the volume ratio of 1:2:3 places beaker, evenly stirs, and obtains reactant liquor A; Positive tetraethyl orthosilicate, ethanol are mixed by the volume ratio of 1:11, obtain B solution; B solution is joined among the reactant liquor A room temperature reaction 2h rapidly; The centrifugal 5min of 6500rpm gets precipitation, will precipitate to use ethanol ultrasonic washing 3 times, and 80 ℃ of oven dry, calcining 2h namely obtains the phosphorescence nano SiO 2 particle under 600 ℃.
B. the mark of anti-Cimaterol monoclonal antibody
The first step, the amination of phosphorescence nano SiO 2 particle: 1g phosphorescence nano SiO 2 particle is dispersed in the toluene solution, adds 0.3-1mL APTES(3-aminopropyl triethoxysilane), backflow 12-24h; The centrifugal 5-10min of phegma cooling back 6500rpm will precipitate and use absolute ethanol washing, be dispersed at last in the absolute ethyl alcohol;
Second step, the mark of Cimaterol antibody: Cimaterol antibody is placed 2-8 ℃, the CBS damping fluid of concentration 0.025-0.05mol/L, the pH9.5 8-12h that dialyses; With the centrifugal 4-8min of silica dioxide granule 10000-13000rpm, with the washing of CBS buffer solution, the Cimaterol antibody with dialysis mixes then, 2-8 ℃ of reaction, spends the night, and obtains the antibody complex of phosphorescence silicon dioxide; The volume ratio of pressing antibody complex and sodium cyanoborohydride solution 1:50-120 in the antibody complex adds the sodium cyanoborohydride solution of 0.5mol/L, mixing, 2-8 ℃ of reaction 8-12h; BSA solution sealing with mass concentration 2% is spent the night, and the centrifugal 4-8min of 10000-13000rpm must precipitate; (0.01-0.02mol/L pH7.8) washs, and 4 ℃ of preservations are standby with precipitating the Tris-HCL damping fluid of using 0.5mL.
C. the fibrolaminar preparation of phosphorescence antibody
Phosphorescence silicon dioxide-the antibody complex for preparing is diluted 100-1000 doubly with containing 2% caseic 10 mM Tris pH, 7.8 damping fluids, and the glass fibre of the thing pad that will serve as a mark then immerses wherein, be as the criterion to soak, and freeze-drying then, standby.Phosphorescence silicon dioxide-antibody complex after maybe will diluting minim with 0.5-1.5 μ L before detection is added in the phosphorescence antibody fibrage.
(3) preparation of adsorbing fiber layer
Test lead adsorbing fiber layer is with glass fibre cotton, nylon membrane, polyvinylidene fluoride pvdf membrane or polyester film preparation, and fibrous material is cut into the band of wide 1.5cm, puts it in the sample pad confining liquid and soaks 30min, and is in 37 ℃ of oven dry, standby.
(4) preparation of cellulose rete
Cellulose rete nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane, cut into the band of wide 1.5cm specification, with point sample instrument respectively specking CIM 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.
(5) the anti-mouse IgG(of goat-anti or rabbit or goat anti-rabbit igg) preparation of antibody
Extract the negative mice serum IgG(of Cimaterol or negative rabbit anteserum IgG with saturated ammonium sulfate), namely get 1 part of mice serum (or rabbit anteserum) and add 2 parts of PBS(pH7.2) mixing, add equal-volume saturated ammonium sulfate solution mixing, put 4 ℃ of refrigerator 12h, 4 ℃, the centrifugal 15min of 2500rpm, abandon supernatant, again with an amount of 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 an amount of PBS(pH7.2) dissolution precipitation, put and use PBS(pH7.2 in 4 ℃ of refrigerators) dialysis 48h, liquid is changed 3 times in the centre, 4 ℃, the centrifugal 15min of 12000rpm, collect supernatant, measure its protein concentration with ultraviolet spectrophotometer, 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, extracting goat-anti or the anti-mouse IgG(of rabbit or goat anti-rabbit igg with saturated ammonium sulfate) (method is identical with extraction mice serum IgG for antibody, no longer repeat), be used for the preparation of Cimaterol test strip contrast trace.
(6) the detection reaction principle of the utility model test strips:
After detecting CIM test paper test lead insertion testing sample solution, solution to be measured spreads to the cellulose rete together by the phosphorescence antibody that syphonic effect drives in CIM to be measured and the phosphorescence antibody glass fibre cotton, and finally infiltrates the absorbent material layer of handle end.In diffusion process, CIM to be measured can combine with phosphorescence antibody, and then the antigen-combining site of CIM on the sealing phosphorescence antibody, the detection trace of the artificial antigen of prevention phosphorescence antibody on cellulose membrane is combined and can not be shown the detection trace, the anti-mouse IgG(of sheep or rabbit or goat anti-rabbit igg) antibody then can be combined with phosphorescence antibody, ultraviolet ray excited (or using directly value of reading of fluorescence reader) down form blue and white contrast the trace band "
︱", namely blue and white band "
︱" the positive expression of trace; Otherwise during no CIM, then can not stop the CIM artificial antigen of phosphorescence antibody on cellulose membrane to detect trace and be combined in the sample solution, show blue and white detect the trace band "
︱", same goat-anti or the anti-mouse IgG(of rabbit or goat anti-rabbit igg) antibody also is combined with golden labeling antibody, show blue and white contrast trace band "
︱", form two blue and white bands "
︱ ︱" negative expression.If show without any the blue and white band on the cellulose membrane, show that then test strips lost efficacy.
Following examples specify structure and the detection method of test strips.
Embodiment one: referring to Fig. 1, Fig. 2.1 is supporting layer among the figure, make with the plastic slice bar, 2 is the adsorbing fiber layer, make with glass fibre cotton, be adsorbed with the phosphorescence antibody glass fibre cotton of anti-CIM monoclonal antibody on the phosphorescence antibody fibrage 3, cellulose rete 4 adopts nitrocellulose filter, the absorbent material layer 5 usefulness absorbent filters of handle end are made, adsorbing fiber layer 2, phosphorescence antibody fibrage 3, cellulose rete 4, absorbent material layer 5 each layers are pasted and fixed on the supporting layer 1 from right to left each layer fiber of intersection infiltration that crosses one another each other.Be provided with the stealthy trace 6 that detects at cellulose rete 4, make with the bovine serum albumin solution (BSA) of coupling CIM; Stealthy contrast trace 7 usefulness goat-anti antibody-solutions trace on cellulose membrane make "
︱", two trace bands are arranged in parallel, formation combination trace band "
︱ ︱".
8-1 covers adsorbing fiber layer 2 and the white of the sample end above the phosphorescence antibody fibrage 3 diaphragm; 8-2 is other color diaphragm (as yellow) that covers above the absorbent material layer 5; 9 is the sample mark line; this mark line is positioned at the adsorbing fiber layer 2 white diaphragm deflection adsorbing fiber layer 2 one side about 0.5cm place corresponding with phosphorescence antibody fibrage 3 intersections, is printed on arrow and max printed words at mark line right side diaphragm.
The preparation of testing sample and detection operation steps:
Detect the meat sample: with sample shred, levigate, be diluted to the testing sample suspension of 1:5 with physiological saline.
Method of operating: will detect CIM test strips sample end and insert in the testing sample, insertion depth is no more than mark line, takes out test strips, observed result under ultraviolet excitation about 10 seconds.
The result judges: if (a) positive cellulose membrane show a blue and white trace band "
︱", the expression testing result is positive, illustrates and contain CIM in testing sample; (b) if negative cellulose membrane show two blue and white trace bands "
︱ ︱", the expression testing result is negative, illustrates not contain CIM in testing sample; (c) do not have the blue and white band to show if lose efficacy at cellulose membrane, showed then that test strips lost efficacy.
Embodiment two: test strips structure and embodiment one are basic identical; difference is: phosphorescence antibody fibrage 3 is adsorbed with the polyclonal antibody of anti-CIM; adsorbing fiber layer 2 usefulness nylon membrane are made; cellulose rete 4 adopts the pure cellulose film; the stealthy trace that detects is " ten " with stealthy contrast trace, and 8-2 is the blue look diaphragm of handle end that covers above the absorbent material layer 5.
For detection of the milk sample: dilute the testing sample suspension make 1:2～1:5 with the physiological saline sample of will suckle.
Method of operating: will detect CIM test strips sample end and insert in the testing sample, insertion depth is no more than mark line, takes out test strips about 10 seconds, by the direct value of reading of fluorescence reader, numerical value is the fluorescence intensity of T line and C line, according to peak value or peak area drawing standard curve, calculates actual content.
Embodiment three: test strips structure and embodiment one are basic identical; difference is: adsorbing fiber layer 2 usefulness polyvinylidene fluoride pvdf membrane are made; the carrier protein solution of coupling CIM is the pure albumen of ovum gallinaceum (OVA); the anti-mouse IgG antibody solution of stealthy contrast trace 7 usefulness rabbits is made at cellulose membrane; cellulose rete 4 adopts the carboxylation cellulose membrane; 8-2 is the handle end greenism film that covers above the absorbent material layer 5, and the stealthy trace band that detects is " ┬ " with stealthy contrast trace band.
For detection of blood sample: extract serum and also its dilution is made the testing sample of 1:2～10 with physiological saline.Result's judgement and method of operating do not repeat all with embodiment one.
Embodiment four: test strips structure and embodiment one are basic identical, difference is: adsorbing fiber layer 2 usefulness polyester film are made, cellulose rete 4 adopts the carboxylation cellulose membrane, and the stealthy carrier protein solution that detects coupling CIM in the trace 6 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: test strips structure and embodiment one are basic identical, and difference is: stealthy contrast trace 7 usefulness goat anti-rabbit igg antibody solution are made at cellulose membrane, and adsorbing fiber layer 2 usefulness nylon membrane are made.Detect the trace band and contrast the trace band and be " ├ ".Test sample, result's judgement and method of operating are with example one.
Embodiment six: and embodiment one is basic identical, difference is: phosphorescence antibody fibrage 3 is adsorbed with the polyclonal antibody of anti-CIM, and test sample is the milk sample; Detect the trace band and contrast the trace band and be " ┤ ".
Embodiment seven: and embodiment one is basic identical, difference is: phosphorescence antibody fibrage 3 is adsorbed with the polyclonal antibody of anti-CIM, and test sample is blood sample.
Embodiment eight: and embodiment one is basic identical, difference is: phosphorescence antibody fibrage 3 is adsorbed with anti-CIM polyclonal antibody, and test sample is urine sample.
Embodiment nine: and embodiment one is basic identical, and difference is: coupling CIM carrier protein solution is hemocyanin (KLH) in the stealthy detection trace 6.
Embodiment ten: and embodiment one is basic identical, and difference is: coupling CIM carrier protein solution is the pure albumen of ovum gallinaceum (OVA) in the stealthy detection trace 6.
Embodiment 11: the sensitivity of the utility model test strips, 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 CIM standard items of 0ng/mL, test strips with embodiment one is example, sample 80-100uL on the test strips, react after 5-10 minute observations under ultraviolet light.Reacted test paper under the ultraviolet light is taken pictures, picture is opened with drawing tools, select T line zone with the rectangle tool, measure its shading value (L
t), deduct the background shading value (L between T line (detection line) and the C line (control line)
b), with inhibiting rate L
t-L
bBeing ordinate, is horizontal ordinate with the logarithm value of different CIM concentration, and drawing standard suppresses curve, carries out the correlation regression analysis, calculates the IC of this test strips to CIM
50And lowest detectable limit.After measured, this test strips to the Regression Equations of CIM is: y=-50.637x+133.1, related coefficient is R
2=0.9923, go out this test paper to the IC of CIM according to regression equation calculation
50Be 437.62pg/mL, the lowest detection of this test strips is limited to 111.85pg/mL.As seen, this test strips has higher sensitivity to CIM.The testing result of test strips is similar with this in other examples.Referring to Fig. 3.
2, specific detection: with similar medicine Ractopamine, clenobuterol hydrochloride and the daimeton of CIM, sulphadiazine, oxolinic acid, penicillin, tetracycline chloromycetin, neomycin, olaquindox as the competition thing, the concentration that disposes above-mentioned mark product is 1mg/mL, test strips with embodiment one detects its inhibiting rate, with the IC of test strips to CIM
50IC with each competition thing
50Number percent be its cross reacting rate.
Measurement result sees the following form 1.Can find out that the specificity of test strips is better, with the equal no cross reaction of other drug.The testing result of test strips is similar with this in other examples.
| Compound | Half-inhibition concentration IC 50（ng/mL） | Cross reactivity (%) |
| Cimaterol | 0.44 | 100 |
| Ractopamine | > 1.0×10 3 | < 0.04 |
| Clenobuterol hydrochloride | > 1.0×10 3 | < 0.04 |
| Daimeton | > 1.0×10 3 | < 0.04 |
| Sulphadiazine | > 1.0×10 3 | < 0.04 |
| Oxolinic acid | > 1.0×10 3 | < 0.04 |
| Penicillin | > 1.0×10 3 | < 0.04 |
| Tetracycline | > 1.0×10 3 | < 0.04 |
| Chloromycetin | > 1.0×10 3 | < 0.04 |
| Neomycin | > 1.0×10 3 | < 0.04 |
| Olaquindox | > 1.0×10 3 | < 0.04 |
Claims (3)
1. the immuno-chromatographic test paper strip of the detection Cimaterol of a phosphorescence nano SiO 2 particle mark; bottom is supporting layer; the middle layer is adsorbed layer; protective seam is fixed on the adsorbed layer; adsorbed layer is followed successively by the absorbent material layer of adsorbing fiber layer, phosphorescence 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 Cimaterol at the cellulose rete and detect trace, 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.
2. immuno-chromatographic test paper strip according to claim 1 is characterized in that: described adsorbing fiber layer glass fibre cotton, nylon membrane, PVDF membrane or polyester film; The absorbent material layer absorbent filter, the supporting layer toughness material that does not absorb water; Cellulose rete nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane;
The carrier protein of coupling Cimaterol is bovine serum albumin(BSA), the pure albumen of ovum gallinaceum or hemocyanin.
3. immuno-chromatographic test paper strip according to claim 1, it is characterized in that: described protective seam is the diaphragm that covers on adsorbing fiber layer, phosphorescence antibody fibrage and the absorbent material layer; The diaphragm corresponding with phosphorescence antibody fibrage intersection at the adsorbing fiber layer is printed with the sample mark line, this sample mark line deflection adsorbing fiber layer one side 0.3-0.7cm.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110275013A (en) * | 2019-05-22 | 2019-09-24 | 克拉玛依市市场监督管理局 | Cimaterol colloidal gold rapid detection card and preparation method thereof suitable for animal derived food |
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2012
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110275013A (en) * | 2019-05-22 | 2019-09-24 | 克拉玛依市市场监督管理局 | Cimaterol colloidal gold rapid detection card and preparation method thereof suitable for animal derived food |
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