CN103439489B - Fluorescent silica labeled immunochromatographic test paper for quantitative gentamicin detection and preparation method - Google Patents
Fluorescent silica labeled immunochromatographic test paper for quantitative gentamicin detection and preparation method Download PDFInfo
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Abstract
The invention relates to a piece of fluorescent silica nano particle labeled gentamicin immunochromatographic test paper and preparation method. A bottom layer of the immunochromatographic test paper serves as a support layer; an intermediate layer of the immunochromatographic test paper serves as an adsorption layer; a protective layer is fixed on the adsorption layer; the adsorption layer comprises an adsorption fiber layer, a fluorescent antibody fiber layer, a cellulose membrane layer and a water absorption material layer at a handle end from a test end sequentially; an invisible detection blot printed by a gentamicin coupled carrier protein solution and an invisible control blot printed by a goat anti-mouse, rabbit anti-mouse or goat anti-rabbit IgG (immunoglobulin G) antibody solution, or a staphylococcus aureus protein A solution are arranged on the cellulose membrane layer; the fluorescent antibody fiber layer is made of glass fiber cotton adsorbing a fluorescent antibody; and the fluorescent antibody is made of a fluorescent silica nano particle labeled gentamicin antibody. The immunochromatographic test paper is simple, convenient and quick, realizes sensitive and fast site detection of gentamicin, and is wide in application scope, low in detection cost and easy to popularize and apply.
Description
Technical field
The present invention relates to a kind of immune chromatography test paper, particularly relating to a kind of immune chromatography test paper and preparation method for quantitatively detecting gentamicin based on fluorescent silicon dioxide nano particle.
Background technology
Gentamicin (Gentamicin, GM) be the one of aminoglycoside antibiotics, mainly act on gramnegative bacterium, because its has a broad antifungal spectrum, curative effect are good and cheap and be widely used in veterinary clinic, there is good curative effect to mastitis for milk cows, endometritis etc., be also used in mixed feed as feed medicated premix.Medication lack of standardization can cause in the milk of livestock products especially lactating mammal medicament residue, forms potential hazard, cause ototoxicity and Toxicity of Kidney to the health of the mankind.In succession define the maximum residue limit(MRL) of gentamicin in animal food both at home and abroad, regulation in " animal food herbal medicine most high residue amount " that The Ministry of Agriculture of the People's Republic of China, MOA 2002 issues, in animal food, the maximum residue limit MRL of GM is 100 ngmL
-1, the therefore method for detecting residue research of GM in urgent need of strengthening, thus provide reliable means, for the food security of the people provides safeguard for the monitoring of GM.
The domestic and international detection method remained about gentamicin etc. mainly contains microbial method, instrumental analysis and immunoassay labelling technique three kinds at present.Although microbial method principle is simple, easy to operate, the time needed for measuring is oversize, and sensitivity is not high.The physico-chemical method detecting gentamicin residue mainly contains high performance liquid chromatography (HPLC), mass spectroscopy (MS) and vapor-phase chromatography etc., although these Measures compare are sensitive, accurate, be not suitable for the screening of batch samples owing to spending the reason such as large, the time is long, technical requirement is high.Immunoassay labelling technique is just progressively applied to the residual detection of small-molecule drug as a kind of quick, special and that sensitivity is higher detection technique.
Immunoassay labelling technique refers to based on the specific reaction by between antigen-antibody, by label, certain material is carried out to the research of qualitative or quantitative detection.Labelling immunoassay is generally the label antagonists such as enzyme, fluorescein, radioactive nuclide or antigen are marked, this label maintains the activity of antibody or antigen, do not affect the activity of label, when behind it and corresponding antibodies or antigen-reactive, directly can measure the label in compound, directly quantitative test be carried out to target substance.By the signal amplification of label, the susceptibility of immunoassay can be improved.Therefore, the research and exploitation of this aspect in urgent need of strengthening.
Summary of the invention
The technical problem to be solved in the present invention: provide a kind of fluorescent silicon dioxide nano particle to be detection gentamicin immune chromatography test paper of mark and preparation method thereof, this chromatographic test paper has special, sensitive, quick, easy, quantitatively to detect micro-gentamicin feature.
Technical scheme of the present invention:
A kind of immune chromatography test paper of the fluorescent silicon dioxide mark quantitatively detected for gentamicin, bottom is supporting layer, middle layer is adsorbed layer, protective seam is fixed on adsorbed layer, adsorbed layer is followed successively by adsorbing fiber layer from test lead, fluorescence antibody fibrage, the absorbent material layer of cellulose rete and handle end, stealth cellulose rete is provided with the carrier protein solution of coupling gentamicin is printed detects trace, and with goat anti-mouse igg, the stealth contrast trace of rabbit anti-mouse IgG or goat anti-rabbit igg antibody solution or the printing of staphylococcus aureus protein A solution, described fluorescence antibody fibrage adopts the glass fibre cotton of absorption fluorescence antibody to make, and fluorescence antibody adopts the gentamicin antibody of fluorescent silicon dioxide nanoparticle label to make.
The particle diameter of described fluorescent silicon dioxide nano particle is 50-100nm.
The gentamicin antibody of described fluorescent silicon dioxide nanoparticle label is prepared by following methods:
1g nano SiO 2 particle is dispersed in toluene solution, adds 0.3-1mL 3-aminopropyl triethoxysilane, backflow 12-24h; The centrifugal 5-10min of 6500rpm after phegma cooling, by the precipitation absolute ethanol washing obtained, then is dispersed in absolute ethyl alcohol by precipitation, obtains amidized nano SiO 2 particle;
Gentamicin antibody is placed in 2-8 DEG C, concentration 0.025-0.05mol/L, pH9.5 CBS damping fluid, dialysis 8-12h; By the centrifugal 4-8min of amidized fluorescent silicon dioxide nano particle 10000-13000rpm, with described CBS buffer solution, then mix with the gentamicin antibody after dialysis, spend the night in 2-8 DEG C of reaction, obtain the antibody complex of fluorescent silicon dioxide; The sodium cyanoborohydride solution of 0.5mol/L is added by the volume ratio of 1:50-120, mixing, 2-8 DEG C of reaction 8-12h in the antibody complex of gained; Close with the BSA solution that mass concentration is 2% and spend the night, the centrifugal 4-8min of 10000-13000rpm, obtains precipitation; By the Tris-HCL buffer solution of precipitation 0.5mL concentration 0.01-0.02mol/L, pH7.8, obtain the gentamicin antibody of described fluorescent silicon dioxide nanoparticle label, in 4 DEG C of preservations, for subsequent use.
Described adsorbing fiber layer glass fibre cotton, nylon membrane, PVDF membrane or polyester film are made; Absorbent material layer absorbent filter, the supporting layer toughness material do not absorbed water is made; Cellulose rete nitrocellulose filter, pure cellulose film or carboxylated cellulose film are made; The carrier protein of coupling gentamicin is bovine serum albumin(BSA), chicken ovalbumin or hemocyanin.
Described stealth detect trace and stealthy contrast trace be arranged in parallel "
︱ ︱" orthoscopic trace, "
10" font arrangement trace, " ┬ ┬ " font arrangement trace, " ┴ ┴ " font arrangement trace, " ├ ├ " font arrangement trace or " ┤ ┤ " font arrangement trace.
Described protective seam is cover the diaphragm on adsorbing fiber layer, fluorescence antibody fibrage and absorbent material layer; The diaphragm that adsorbing fiber layer is corresponding with fluorescence antibody fibrage intersection is printed with sample mark line, this sample mark line deflection adsorbing fiber layer side 0.3-0.7cm.
Described fluorescent silicon dioxide nano particle adopts following methods preparation:
By 47mmol aminopropyl-triethoxy silicon, 7.1 mmol BHHCT and 3.55mmol EuCl
36H
2o adds in 30 μ L cyclohexanes, after sonic oscillation reacts 15 minutes, reactant liquor joins in the w/o type microemulsion containing 1.1 mL water, 4.74 g Triton X-100,3.64 g n-octyl alcohols and 14.50 g cyclohexanes, stir after 0.5 hour, add the tetraethoxysilane of 200 μ L and the strong aqua of 200 μ L, after stirring at room temperature reacts 24 hours, add 40 mL acetone and terminate reaction, by centrifugal for reactant liquor rear removing supernatant, sediment fraction ethanol and distilled water wash three times respectively, obtain described fluorescent silicon dioxide nano particle, suspend in water for subsequent use.
The preparation method of described immune chromatography test paper, comprises the following steps:
(1) preparation of gentamicin monoclonal antibody or polyclonal antibody;
(2) the fibrolaminar preparation of fluorescence antibody: comprise the preparation of fluorescent silicon dioxide nano particle, the mark of gentamicin antibody and fibrolaminar preparation;
(3) preparation of adsorbing fiber layer: adsorbing fiber layer glass fibre cotton, nylon membrane, PVDF membrane or polyester film are made;
(4) preparation of cellulose rete: cellulose rete nitrocellulose filter, pure cellulose film or carboxylated cellulose film, with diverse location respectively specking detection trace and the contrast trace of point sample instrument at cellulose rete, dries;
(5) assembling of immune chromatography test paper: adsorbing fiber layer, phosphorescent antibody fibrage, cellulose rete, absorbent material layer are attached on the supporting layer with bonding agent from left to right successively, successively supporting layer, adsorbed layer and protective seam are assembled into immune chromatography test paper.
immune chromatography test paper of the present invention has the following advantages:
(1) high specificity, highly sensitive.High-sensitive fluorescent silicon dioxide nano particle combines with immunochromatography technique by immune chromatography test paper of the present invention, fluorescent silicon dioxide nano particle just can self-luminescence without the need to wrapping up any fluorescent material, and fluorescence lifetime is long, cost of manufacture is low, light stability is strong, can obtain and detect higher sensitivity than common fluorescent.Immune chromatography test paper had both maintained the simple and rapid advantage of traditional colloidal gold strip, overcame again that the latter's sensitivity is low, shortcoming that cannot be quantitative, and detection sensitivity is higher, more stable, minimumly a trace residue for gram level detected.
(2) easy, quick.Immune chromatography test paper to direct-detections such as whole blood, urine, saliva, tissue homogenates, can need not carry out the pre-service of sample; Only test paper need be inserted test sample 10 ~ 20 seconds during detection, put into fluorescence after 5 minutes and read bar instrument and can judge testing result, can execute-in-place, time saving and energy saving, easy and simple to handle, a step completes.Read bar instrument by fluorescence and directly read value, realize quantitatively detecting.
(3) cost saving.Without the need to separately joining instrument and equipment and other reagent when this immune chromatography test paper detecting, can detect whenever and wherever possible, can qualitative detection can quantitatively detect again; Testing cost is cheap, can save a large amount of expensive instrument and equipment investment expense.
(4) applied widely, easy to utilize.This immune chromatography test paper can meet the needs of different levels personnel, comprise specialty chemical examination, customs quarantine control, health quarantine, quality monitoring, livestock products processing, raiser and consumer individual etc., both the detection of single sample had been suitable for, be suitable for again the examination of a large amount of sample, the fields such as medical diagnosis on disease, illicit drugs inspection, Bacteria Detection and environment measuring can be applied to.The present invention ensuring food safety, Protection of consumer healthy in there is meaning of crucial importance, there is obvious economic benefit and social benefit.
(5) the present invention prepares the technique simple possible of fluorescent silicon dioxide nano particle, is easy to operation.During with fluorescent silicon dioxide nanoparticle label gentamicin antibody, only need by amidized fluorescent silicon dioxide nano particle and the direct coupling of antibody, easy to implement the method controlled, by regulating the mol ratio of fluorescent silicon dioxide and antibody to control Conjugate ratio, thus effectively improve the sensitivity based on the Sidestream chromatography immune test paper of fluorescent silicon dioxide nanoparticle label.
Accompanying drawing explanation
Fig. 1 is the TEM phenogram of fluorescent silicon dioxide nano particle in the present invention;
As can be seen from Figure 1, the form of fluorescent silicon dioxide nano particle is homogeneous, in the same size, and in single dispersing shape, this is conducive to labelled antibody, lays the foundation for it is used for Sidestream chromatography immune test paper as probe.In figure, grain diameter is 50 ± 5nm.
Fig. 2 is the structural representation of immune chromatography test paper of the present invention;
Fig. 3 is the side structure schematic diagram of the immune chromatography test paper of Fig. 1;
Fig. 4 is the regression curve of immune chromatography test paper of the present invention to gentamicin;
Fig. 5 is that fluorescence used in the present invention reads bar instrument.
In figure, 1 is supporting layer, and 2 is adsorbing fiber layer, and 3 is fluorescence antibody fibrage, and 4 is cellulose rete, and 5 is absorbent material layer, and 6 is stealthy detection trace, and 7 is stealthy contrast trace, and 8-1 is sample end diaphragm, and 8-2 is handle end diaphragm, and 9 is sample mark line.
Embodiment
The preparation of the immune chromatography test paper test paper of the fluorescent silicon dioxide mark quantitatively detected for gentamicin,
Its process comprises: the steps such as the preparation of gentamicin monoclonal or polyclonal antibody, the fibrolaminar preparation of fluorescence antibody, the preparation of adsorbing fiber layer, the preparation of cellulose rete and test paper assembling.
(1) preparation of anti-gentamicin monoclonal antibody or polyclonal antibody
Prepared by monoclonal antibody: with 50 μ g ~ 100 μ g/ gentamicin carrier protein couplet thing immunity Balb/C mouse in 6 ~ 8 week age only 3 ~ 4 times, 3 ~ 5 weeks each immunization interval time, determine antibody titer meet the requirements after superpower immunity, 3 ~ 4 days afterwards, by hole blood sampling under immune mouse socket of the eye, be separated positive serum; De-neck is lethal, and the alcohol-pickled mouse 5 ~ 10min with 75% sterilizes body surface, asepticly gets its spleen, is shredded by spleen and grinds, filtering, the centrifugal 10min of 1000rpm through 120 order nylon gauzes, collection splenocyte.By 1 × 10
8splenocyte mix in the ratio of 10:1 with NS0 myeloma cell, the centrifugal 10min of 1000rpm, abandon supernatant, cell precipitation thing slowly adds the 50%PEG4000 of 0.7 ~ 1.0 mL GM in 37 DEG C of water-baths, effect 1min, then serum-free 1640 nutrient culture media 15 mL is slowly added, to stop the effect of PEG, 37 DEG C of water-bath 5 ~ 10 min, the centrifugal 10min of 1000rpm, abandons supernatant, is resuspended in by cell precipitation thing in HAT Selective agar medium, and add 96 porocytes cultivation plate hole (100 μ L/ hole, μ L ~ 200), be placed in 37 DEG C, 5%CO
2cultivate 10 ~ 14 days in incubator, carry out positive hole sizer choosing with indirect elisa method, selection strong positive, the hole that inhibiting rate is high, Growth of Cells is vigorous carry out 3 limited dilution clonings, then expand cultivation, set up hybridoma cell strain.The monoclonal antibody of prepared hybridoma secretion can be reacted with gentamicin specifically, and affinity constant reaches 10
10~ 10
12, light chain subtype is κ or λ, and heavy chain subgroup is IgG
1, IgG
2a, IgG
2b, IgG
3, for the monoclonal antibody of gentamicin specific epitope, for the mark of fluorescent silicon dioxide nano particle.
How anti-preparation: with gentamicin carrier protein couplet thing immunize New Zealand white rabbits, immunizing dose is 200 μ g ~ 500 μ g/ time, and dorsal sc divides 4 ~ 6 injections.Head exempts from, and dissolves gentamicin carrier protein couplet thing, mix with equivalent FCA with aseptic PBS, fully emulsified; Booster immunization, dissolves gentamicin carrier protein couplet thing with aseptic PBS, mixes with equivalent FIA, fully emulsified, within 2 ~ 3 weeks, carry out continuous immunity 4 ~ 5 times, every minor tick 2 ~ 3 weeks after head exempts from, after last immunity 10 ~ 15 days, survey it with ELISA method and surely tire and reach 10
5time above, blood sampling is separated and collected hyper-immune serum also.Extract IgG antibody with saturated ammonium sulfate salting out method, namely get 1 portion of hyper-immune serum and add 2 parts of PBS(pH7.2) mixing, add the mixing of equal-volume saturated ammonium sulfate solution, put 4 DEG C of refrigerator 12h, 4 DEG C, 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 DEG C of refrigerator 2h, 4 DEG C, the centrifugal 15min of 2500rpm, abandon supernatant; With appropriate PBS(pH7.2) dissolution precipitation, put 4 DEG C of interior PBS(pH7.2 of refrigerator) dialyse 48 ~ 72h, liquid is changed for several times in centre, 4 DEG C, the centrifugal 15min of 12000rpm, collect supernatant, obtain the anti-gentamicin polyclonal antibody of purifying ,-20 DEG C frozen, for the mark of fluorescent silicon dioxide nano particle.
(2) the fibrolaminar preparation of fluorescence antibody
A. the preparation of fluorescent silicon dioxide nano particle
By 47mmol aminopropyl-triethoxy silicon (APTS), 7.1 mmol BHHCT (4,4-bis (1 ", 1 ", 1 ", 2 ", 2 " and, 3 ", 3 "-heptafluoro-4 ", 6 "-hexanedion-6 "-yl)-chlorosulfo-o-terphenyl, molecular formula C
30h
15clF
14o
6s, molecular weight 804.93) and 3.55mmol EuCl
36H
2o adds in 30 μ L cyclohexanes, after sonic oscillation reacts 15 minutes, reactant liquor joins in the w/o type microemulsion containing 1.1 mL water, 4.74 g Triton X-100,3.64 g n-octyl alcohols and 14.50 g cyclohexanes, stir after 0.5 hour, add the TEOS(tetraethoxysilane of 200 μ L) and the strong aqua of 200 μ L, after stirring at room temperature reacts 24 hours, add 40 mL acetone and terminate reaction.By centrifugal for reactant liquor rear removing supernatant, sediment fraction ethanol and distilled water suspend in water for subsequent use after washing three times respectively.The nano SiO 2 particle obtained is see Fig. 1.
B. the mark of anti-gentamicin monoclonal antibody
1g nano SiO 2 particle is dispersed in toluene solution, adds 0.3-1mL 3-aminopropyl triethoxysilane, backflow 12-24h; The centrifugal 5-10min of 6500rpm after phegma cooling, by the precipitation absolute ethanol washing obtained, then is dispersed in absolute ethyl alcohol by precipitation, obtains amidized nano SiO 2 particle;
Gentamicin antibody is placed in 2-8 DEG C, concentration 0.025-0.05mol/L, pH9.5 CBS damping fluid, dialysis 8-12h; By the centrifugal 4-8min of amidized fluorescent silicon dioxide nano particle 10000-13000rpm,
With the washing of CBS buffer solution, then mix with the gentamicin antibody of dialysis, 2-8 DEG C of reaction, spend the night, obtain the antibody complex of fluorescent silicon dioxide; The sodium cyanoborohydride solution of 0.5mol/L is added by the volume ratio of antibody complex and sodium cyanoborohydride solution 1:50-120, mixing, 2-8 DEG C of reaction 8-12h in antibody complex; Close with the BSA solution of mass concentration 2% and spend the night, the centrifugal 4-8min of 10000-13000rpm must precipitate; The Tris-HCL damping fluid (0.01-0.02mol/L, pH7.8) of precipitation 0.5mL is washed, 4 DEG C of preservations, for subsequent use.
C. the fibrolaminar preparation of fluorescence antibody
Fluorescent silicon dioxide-the antibody complex prepared is diluted 100-1000 doubly with containing 2% caseic 10mM Tris pH 7.8 damping fluid, then the glass fibre as labeling pad is immersed wherein, be as the criterion to soak, then freeze-drying, for subsequent use.Maybe fluorescent silicon dioxide-the antibody complex after dilution is added in fluorescence antibody fibrage with the minim of 0.5-1.5 μ L before detection.(unit mM represents mmol/L)
(3) preparation of adsorbing fiber layer
Prepared by test lead adsorbing fiber layer glass fibre cotton, nylon membrane, polyvinylidene fluoride pvdf membrane or polyester film, fibrous material is cut into the band of wide 1.5cm, puts it in sample pad confining liquid and soak 30min, in 37 DEG C of oven dry, for subsequent use.
(4) preparation of cellulose rete
Cellulose rete nitrocellulose filter, pure cellulose film or carboxylated cellulose film, cut into the band of wide 1.5cm specification, with point sample instrument diverse location specking GM antigen and goat anti-mouse igg antibody (or rabbit anti-mouse IgG, goat anti-rabbit igg antibody) respectively on cellulose membrane, make stealthy detection trace band and contrast trace band, in 37 DEG C of dry for standby.
(5) goat-anti or rabbit anti-mouse IgG(or goat anti-rabbit igg) preparation of antibody, or the configuration of SPA solution.
Gentamicin negative mice serum IgG (or negative rabbit anteserum IgG) is extracted with saturated ammonium sulfate, namely get 1 part of mice serum (or rabbit anteserum) and add 2 parts of PBS(pH7.2) mixing, add the mixing of equal-volume saturated ammonium sulfate solution, put 4 DEG C of refrigerator 12h, 4 DEG C, 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 DEG C of refrigerator 2h, 4 DEG C, the centrifugal 15min of 2500rpm, abandon supernatant, with appropriate PBS(pH7.2) dissolution precipitation, put 4 DEG C of interior PBS(pH7.2 of refrigerator) dialyse 48h, liquid is changed 3 times in centre, 4 DEG C, the centrifugal 15min of 12000rpm, collect supernatant, its protein concentration is measured with ultraviolet spectrophotometer, with mice serum (or rabbit anteserum) IgG of 50 μ g ~ 100 μ g/kg body weight through the healthy goat of subcutaneous and intramuscular injection or rabbit 3 ~ 4 times, final injection is after 10 days, with ELISA measure its serum titer reach more than 1:2000 time, heart or arterial blood drawing, separated and collected hyper-immune serum, goat-anti or rabbit anti-mouse IgG(or goat anti-rabbit igg is extracted with saturated ammonium sulfate) (method is identical with extracting mice serum IgG for antibody, no longer repeat), for the preparation of gentamicin Test paper contrast trace.
The configuration of staphylococcus aureus protein A (SPA) solution: configuration is applicable to the SPA aqueous solution of concentration, for the preparation of gentamicin Test paper contrast trace.
(6) the detection reaction principle of immune chromatography test paper of the present invention:
After detection GM test paper test lead inserts testing sample solution, solution to be measured drives the fluorescence antibody in GM to be measured and fluorescence antibody glass fibre cotton to spread to cellulose rete together by syphonic effect, and the final absorbent material layer infiltrating handle end.In diffusion process, GM to be measured can combine with fluorescence antibody, and then the antigen-combining site of GM on closed fluorescence antibody, stop the detection trace of the artificial antigen of fluorescence antibody on cellulose membrane to be combined and can not show detection trace, sheep or rabbit anti-mouse IgG(or goat anti-rabbit igg) antibody or SPA solution then can be combined with fluorescence antibody, ultraviolet excite lower formation redness contrast trace band "
︱", namely red zone "
︱" trace is positive expression, uses fluorescence to read the absorption peak that bar instrument can read C line; Otherwise time in sample solution without GM, then the GM artificial antigen of fluorescence antibody on cellulose membrane can not be stoped to detect trace and to be combined, display red detection trace band "
︱", same goat-anti or rabbit anti-mouse IgG(or goat anti-rabbit igg) antibody is also combined with golden labeling antibody, display red contrast trace band "
︱", formed two red zones "
︱ ︱" be negative expression, use fluorescence to read the absorption peak that bar instrument can read T line, C line.If without any red zone display on cellulose membrane, fluorescence reads bar instrument can show T line, C line all without absorption peak, then show that test paper lost efficacy.
following examples illustrate structure and the detection method of test paper.
Embodiment one: see Fig. 2, Fig. 3.In figure, supporting layer 1 plastic slice bar is made, adsorbing fiber layer 2 is made with glass fibre cotton, fluorescence antibody fibrage 3 is adsorbed with the fluorescence antibody glass fibre cotton of anti-GM monoclonal antibody, cellulose rete 4 adopts nitrocellulose filter, the absorbent material layer 5 of handle end is made with absorbent filter, be pasted and fixed on supporting layer 1 from left to right by adsorbing fiber layer 2, fluorescence antibody fibrage 3, cellulose rete 4, each layer of absorbent material layer 5, the fiber of each layer intersection each other crosses one another infiltration.Cellulose rete 4 is provided with stealthy detection trace 6, makes with the bovine serum albumin solution (BSA) of coupling GM; Stealthy contrast trace 7 goat-anti antibody-solutions trace on cellulose membrane is made "
︱", two trace bands are arranged in parallel, formation combination trace band "
︱ ︱".
8-1 is the sample end diaphragm (white) covered above adsorbing fiber layer 2 and fluorescence antibody fibrage 3; 8-2 is other color handle end diaphragm (as yellow) covered above absorbent material layer 5; 9 is sample mark line; this mark line is positioned at the adsorbing fiber layer 2 white diaphragm corresponding with fluorescence antibody fibrage 3 intersection and is partial to adsorbing fiber layer 2 side and is about 0.5cm place, diaphragm is printed on arrow and max printed words on the right side of mark line.
Detect meat sample: to be shredded by sample, levigate, become the testing sample suspension of 1:5 with normal saline dilution.
Method of operating: will detect GM test paper sample end and insert in testing sample, insertion depth is no more than mark line, and about 10 seconds took out test paper, puts into fluorescence and reads bar instrument and directly read result after 5min.Fluorescence used in the present invention is read bar instrument and is seen Fig. 5, is existing instrument.
Embodiment two: test paper structure is substantially identical with embodiment one; difference is: fluorescence antibody fibrage is adsorbed with the polyclonal antibody of anti-GM; adsorbing fiber layer nylon membrane is made; cellulose rete adopts 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.
For detecting milk sample: the testing sample suspension with physiological saline, milk sample dilution being made 1:2 ~ 1:5.
Method of operating: will detect GM test paper sample end and insert in testing sample, insertion depth is no more than mark line, and about 10 seconds took out test paper, puts into fluorescence and reads bar instrument and directly read result after 5min.
Embodiment three: test paper structure is substantially identical with embodiment one; difference is: adsorbing fiber layer polyvinylidene fluoride pvdf membrane is made; the carrier protein solution of coupling GM is chicken ovalbumin (OVA); stealthy contrast trace rabbit anti-mouse IgG antibody solution is made on cellulose membrane; cellulose rete adopts carboxylated cellulose film; the handle end diaphragm covered above absorbent material layer is green, and the stealthy trace band that detects is " ┬ " with the stealthy trace band that contrasts.
For detecting blood sample: extract serum and diluted the testing sample making 1:2 ~ 10 with physiological saline.Result judgement and method of operating, all with embodiment one, do not repeat.
Embodiment four: test paper structure is substantially identical with embodiment one, and difference is: adsorbing fiber layer polyester film is made, cellulose rete adopts carboxylated cellulose film, and the stealthy carrier protein solution detecting coupling GM in trace is hemocyanin (KLH).
For detecting urine sample, can directly get urine as testing sample; Detect trace band and contrast trace band and be " ┴ ".Method of operating and result decision method are with example one.
Embodiment five: test paper structure is substantially identical with embodiment one, and difference is: stealthy contrast trace goat anti-rabbit igg antibody solution is made on cellulose membrane, and adsorbing fiber layer nylon membrane is made.Detect trace band and contrast trace band and be " ├ ".Detect sample, result judgement and method of operating with example one.
Embodiment six: substantially identical with embodiment one, difference is: fluorescence antibody fibrage is adsorbed with the polyclonal antibody of anti-GM, detection sample is milk sample; Detect trace band and contrast trace band and be " ┤ ".
Embodiment seven: substantially identical with embodiment one, difference is: fluorescence antibody fibrage is adsorbed with the polyclonal antibody of anti-GM, detection sample is blood sample.
Embodiment eight: substantially identical with embodiment one, difference is: fluorescence antibody fibrage is adsorbed with anti-GM polyclonal antibody, detection sample is urine sample.
Embodiment nine: substantially identical with embodiment one, difference is: in stealthy detection trace, coupling GM carrier protein solution is hemocyanin (KLH).
Embodiment ten: substantially identical with embodiment one, difference is: in stealthy detection trace, coupling GM carrier protein solution is chicken ovalbumin (OVA).
Embodiment 11: the sensitivity of 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 GM standard items of 0ng/mL, test paper of the present invention is put into after loading 80-100uL, 5min fluorescence read bar instrument and directly read peak figure.With peak value or peak area for ordinate, with the logarithm value of different GM concentration for horizontal ordinate, drawing standard suppresses curve, carries out correlation regression analysis, calculates the IC of this test paper to GM
50and lowest detectable limit.After measured, this test paper to the Regression Equations of GM is: y=-1153.8x+2935.5, and related coefficient is R
2=0.9927, go out the IC of this test paper to GM according to regression equation calculation
50for 475.88pg/mL, this test paper lowest detection is limited to 143.71pg/mL.Visible, this immune chromatography test paper has higher sensitivity to GM.Regression curve is see Fig. 4.
2, specific detection: using the similar drugs neomycin of GM, streptomysin, kanamycins, chloromycetin, terramycin, tetracycline, ampicillin as competitor, the concentration configuring above-mentioned mark product is 1mg/mL, its inhibiting rate is detected, with the IC of this test paper to GM with immune chromatography test paper of the present invention
50with the IC of each competitor
50number percent be its cross reacting rate.
Measurement result sees the following form 1.Can find out, the specificity of this immune chromatography test paper is better, no cross reaction equal to other drug.
The cross reactivity of table 1 gentamicin immune chromatography test paper
| Compound | Half-inhibition concentration IC 50（pg/mL） | Cross reactivity (%) |
| Gentamicin | 475.88 | 100 |
| Neomycin | > 1.0×10 6 | < 0.048 |
| Streptomysin | > 1.0×10 6 | < 0.048 |
| Kanamycins | > 1.0×10 6 | < 0.048 |
| Chloromycetin | > 1.0×10 6 | < 0.048 |
| Terramycin | > 1.0×10 6 | < 0.048 |
| Tetracycline | > 1.0×10 6 | < 0.048 |
| Ampicillin | > 1.0×10 6 | < 0.048 |
Claims (8)
1. the immune chromatography test paper of the fluorescent silicon dioxide mark quantitatively detected for gentamicin, bottom is supporting layer, middle layer is adsorbed layer, protective seam is fixed on adsorbed layer, adsorbed layer is followed successively by adsorbing fiber layer from test lead, fluorescence antibody fibrage, the absorbent material layer of cellulose rete and handle end, it is characterized in that: the stealth be provided with on cellulose rete with the carrier protein solution of coupling gentamicin is printed detects trace, and with goat anti-mouse igg, the stealth contrast trace of rabbit anti-mouse IgG or goat anti-rabbit igg antibody solution or the printing of staphylococcus aureus protein A solution, described fluorescence antibody fibrage adopts the glass fibre cotton of absorption fluorescence antibody to make, and fluorescence antibody adopts the gentamicin antibody of fluorescent silicon dioxide nanoparticle label to make,
The particle diameter of described fluorescent silicon dioxide nano particle is 50-100nm;
The gentamicin antibody of described fluorescent silicon dioxide nanoparticle label is prepared by following methods:
1g nano SiO 2 particle is dispersed in toluene solution, adds 0.3-1mL 3-aminopropyl triethoxysilane, backflow 12-24h; The centrifugal 5-10min of 6500rpm after phegma cooling, by the precipitation absolute ethanol washing obtained, then is dispersed in absolute ethyl alcohol by precipitation, obtains amidized nano SiO 2 particle;
Gentamicin antibody is placed in 2-8 DEG C, concentration 0.025-0.05mol/L, pH9.5 CBS damping fluid, dialysis 8-12h; By the centrifugal 4-8min of amidized fluorescent silicon dioxide nano particle 10000-13000rpm, with described CBS buffer solution, then mix with the gentamicin antibody after dialysis, spend the night in 2-8 DEG C of reaction, obtain the antibody complex of fluorescent silicon dioxide; The sodium cyanoborohydride solution of 0.5mol/L is added by the volume ratio of 1:50-120, mixing, 2-8 DEG C of reaction 8-12h in the antibody complex of gained; Close with the BSA solution that mass concentration is 2% and spend the night, the centrifugal 4-8min of 10000-13000rpm, obtains precipitation; By the Tris-HCL buffer solution of precipitation 0.5mL concentration 0.01-0.02mol/L, pH7.8, obtain the gentamicin antibody of described fluorescent silicon dioxide nanoparticle label, in 4 DEG C of preservations, for subsequent use.
2. immune chromatography test paper according to claim 1, is characterized in that: described adsorbing fiber layer glass fibre cotton, nylon membrane, PVDF membrane or polyester film are made; Absorbent material layer absorbent filter, the supporting layer toughness material do not absorbed water is made; Cellulose rete nitrocellulose filter, pure cellulose film or carboxylated cellulose film are made; The carrier protein of coupling gentamicin is bovine serum albumin(BSA), chicken ovalbumin or hemocyanin.
3. immune chromatography test paper according to claim 1, is characterized in that: described stealth detect trace and stealthy contrast trace be arranged in parallel "
︱ ︱" orthoscopic trace, "
10" font arrangement trace, " ┬ ┬ " font arrangement trace, " ┴ ┴ " font arrangement trace, " ├ ├ " font arrangement trace or " ┤ ┤ " font arrangement trace.
4. immune chromatography test paper according to claim 1, is characterized in that: described protective seam is cover the diaphragm on adsorbing fiber layer, fluorescence antibody fibrage and absorbent material layer; The diaphragm that adsorbing fiber layer is corresponding with fluorescence antibody fibrage intersection is printed with sample mark line, this sample mark line deflection adsorbing fiber layer side 0.3-0.7cm.
5. the immune chromatography test paper according to any one of claim 1-4, is characterized in that: described fluorescent silicon dioxide nano particle adopts following methods preparation:
By 47mmol aminopropyl-triethoxy silicon, 7.1 mmol BHHCT and 3.55mmol EuCl
36H
2o adds in 30 μ L cyclohexanes, after sonic oscillation reacts 15 minutes, reactant liquor joins in the w/o type microemulsion containing 1.1 mL water, 4.74 g Triton X-100,3.64 g n-octyl alcohols and 14.50 g cyclohexanes, stir after 0.5 hour, add the tetraethoxysilane of 200 μ L and the strong aqua of 200 μ L, after stirring at room temperature reacts 24 hours, add 40 mL acetone and terminate reaction, by centrifugal for reactant liquor rear removing supernatant, sediment fraction ethanol and distilled water wash three times respectively, obtain described fluorescent silicon dioxide nano particle, suspend in water for subsequent use.
6. the preparation method of immune chromatography test paper described in claim 1, is characterized in that: the method comprises the following steps:
(1) preparation of gentamicin monoclonal antibody or polyclonal antibody;
(2) the fibrolaminar preparation of fluorescence antibody: comprise the preparation of fluorescent silicon dioxide nano particle, the mark of gentamicin antibody and fibrolaminar preparation;
(3) preparation of adsorbing fiber layer: adsorbing fiber layer glass fibre cotton, nylon membrane, PVDF membrane or polyester film are made;
(4) preparation of cellulose rete: cellulose rete nitrocellulose filter, pure cellulose film or carboxylated cellulose film, with diverse location respectively specking detection trace and the contrast trace of point sample instrument at cellulose rete, dries;
(5) assembling of immune chromatography test paper: adsorbing fiber layer, phosphorescent antibody fibrage, cellulose rete, absorbent material layer are attached on the supporting layer with bonding agent from left to right successively, successively supporting layer, adsorbed layer and protective seam are assembled into immune chromatography test paper.
7. preparation method according to claim 6, is characterized in that: described fluorescent silicon dioxide nano particle adopts following methods to prepare:
By 47mmol aminopropyl-triethoxy silicon, 7.1 mmol BHHCT and 3.55mmol EuCl
36H
2o adds in 30 μ L cyclohexanes, after sonic oscillation reacts 15 minutes, reactant liquor joins in the w/o type microemulsion containing 1.1 mL water, 4.74 g Triton X-100,3.64 g n-octyl alcohols and 14.50 g cyclohexanes, stir after 0.5 hour, add the tetraethoxysilane of 200 μ L and the strong aqua of 200 μ L, after stirring at room temperature reacts 24 hours, add 40 mL acetone and terminate reaction, by centrifugal for reactant liquor rear removing supernatant, sediment fraction ethanol and distilled water wash three times respectively, obtain described fluorescent silicon dioxide nano particle, suspend in water for subsequent use.
8. the preparation method according to claim 6 or 7, is characterized in that: the gentamicin antibody of described fluorescent silicon dioxide nanoparticle label is by following methods:
1g nano SiO 2 particle is dispersed in toluene solution, adds 0.3-1mL 3-aminopropyl triethoxysilane, backflow 12-24h; The centrifugal 5-10min of 6500rpm after phegma cooling, by the precipitation absolute ethanol washing obtained, then is dispersed in absolute ethyl alcohol by precipitation, obtains amidized nano SiO 2 particle;
Gentamicin antibody is placed in 2-8 DEG C, concentration 0.025-0.05mol/L, pH9.5 CBS damping fluid, dialysis 8-12h; By the centrifugal 4-8min of amidized fluorescent silicon dioxide nano particle 10000-13000rpm, with described CBS buffer solution, then mix with the gentamicin antibody after dialysis, spend the night in 2-8 DEG C of reaction, obtain the antibody complex of fluorescent silicon dioxide; The sodium cyanoborohydride solution of 0.5mol/L is added by the volume ratio of 1:50-120, mixing, 2-8 DEG C of reaction 8-12h in the antibody complex of gained; Close with the BSA solution that mass concentration is 2% and spend the night, the centrifugal 4-8min of 10000-13000rpm, obtains precipitation; After the Tris-HCL buffer solution of precipitation 0.5mL concentration 0.01-0.02mol/L, pH7.8, obtain the gentamicin antibody of described fluorescent silicon dioxide nanoparticle label, in 4 DEG C of preservations, for subsequent use.
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| CN106841612B (en) * | 2016-12-27 | 2021-04-02 | 福建普立辰生物技术有限公司 | Preparation method of human lipoprotein-associated phospholipase A2 immunochromatography test strip |
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