CN102043055B - Rapid immunomagnetic bead chromatographic method for main allergens in aquatic products - Google Patents
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Abstract
The invention relates to a rapid immunomagnetic bead chromatographic method for main allergens in aquatic products. A rat monoclonal antibody which has specific reaction with a tropomyosin (Tm) allergen of edible crustacean aquatic organisms and edible soft-bodied aquatic organisms, has specific reaction with a parvalbumin (Pa) allergen of fishes, frogs, rats and sea turtle vertebrates, and is prepared and screened by hybridoma technology is taken as a specific antibody and coupled to the surface of a magnetic nanometer material in a chemical bonding mode to serve as a marker so as to prepare a specific nanometer detection probe suitable for magnetic bead chromatography; the allergen specific nanometer detection probe and sample solution are added into a sample pad of an allergen magnetic immunochromatographic test strip, the sample pad is stood at room temperature for 20 minutes, the allergens are captured by a detection strip and a control strip through chromatography to form a macroscopic colored strip, so that immunochromatography is realized; and a test card is put on a magnetic assay reader, the content of the allergens in a sample to be detected is calculated according to a marked line, comparison is performed and the immunochromatography is finished.
Description
Technical field
The present invention relates to the detection method of food allergen, particularly relate to a kind of immuomagnetic bead chromatographic method that detects main allergen in aquatic products, the method is mainly the main allergen tropomyosin (Tropomyosin that uses shell-fish and shellfish, be called for short Tm), the immuomagnetic bead chromatographic detection method of fish main allergen parvalbumin (Parvalbumin, be called for short Pa).
Background technology
Food hypersenstivity is that the body that caused by food is to immune abnormal response, in recent decades, in world wide, food hypersenstivity reaction disease patient presents ascendant trend year by year, according to WHO, by 2010, the whole world may have 40% to 50% people to suffer from allergy, and food hypersenstivity has now become an emerging food-safety problem.Food allergen is in food, to cause or evoke anaphylactoid material, the rich content normally containing in specific food, naturally occurring protein.According to the speed of allergenic response, mechanism and clinical characters, sensitization mechanism is divided into I type, II type, III type, IV type, wherein most foods allergy is the I type hypersensitivity being mediated by IgE, general symptom comprises vomiting, stomachache, diarrhoea etc., also comprise dermoreaction and respiratory symptom, in serious situation, even there will be anaphylactic shock or death, there is no at present effective methods for the treatment of.In order to protect susceptible person's health, Countries and area have been carried out strict regulations and have been listed legislation scope in the label for labelling of food allergen.
Fish and Species of Crustacea and goods thereof are the main sensitization food that two generally acknowledged classes of FAO (Food and Agriculture Organization of the United Nation) easily cause food hypersenstivity.According to investigations, in 15～24 years old age bracket crowd of China, approximately 6% people had the experience of food hypersenstivity, the 3-6% that accounts for to marine product allergy such as fish in the irritated crowd of China teenager, the first place that occupies other sensitization food.The main allergen of aquatic products can be divided into two large classes, and one is the tropomyosin (Tropomyosin) in the shellfish such as shellfish and shrimp crab, and another is the parvalbumin (Parvalbumin) in the fish such as cod.Due to the complicacy of composition of food, in addition in food processing process, use various batchings and adjuvant, traditional chemical analysis is difficult to meet the testing requirement of food allergen, and therefore, the Fast Detection Technique research of food allergen is domestic and international scientist's study hotspot always.
At present, the detection technique of food allergen taking analyze peptide section amino acid sequence after holoprotein or enzymolysis mass-spectrometric technique, detect PCR (Polymerase Chain Reaction, the PCR) technology of coding anaphylactogen DNA fragmentation and the immunology detection technology of diagnosis allergy originality as representative.
The amino acid sequence of peptide section after mass-spectrometric technique energy Accurate Measurement holoprotein or enzymolysis, then determine allergic protein according to associated databases, but the shortcoming such as that mass-spectrometric technique exists is time-consuming, instrument is expensive, testing cost is relatively high.
PCR method easily produces false positive results because misoperation is subject to polluting in testing process, and having the technical real anaphylaxis strong, that can not reflect food of operation requirements is can produce the defects such as allergic reaction after people take in this food.
The immunology detection technology of food allergen has enzyme linked immunosorbent assay (ELISA), biology sensor method and immunochromatographic method.Wherein ELISA method is the method that current technology is comparatively ripe, on market, have the ELISA detection kit of the food allergens such as almond, soybean, peanut, shell-fish, sesame, mustard, lupin, milk, these kits are realized qualitative or half-quantitative detection in can be in 30～60min, but have false positive and false negative reaction.In addition, publication number CN101285840A Patent Application Publication the solid-phase immunity detection method of anaphylactogen in a kind of food, adopt removable ELISA Plate as detecting carrier, utilize double antibodies sandwich pattern, directly marker enzyme on allergen specificity monoclonal antibody, then realize detection by substrate chromogenic reaction.The method detect principle with ELISA method, with unique difference of double antibodies sandwich ELISA be not need to use ELIAS secondary antibody.Detect advantage although therefore have multisample simultaneously, also exist false negative or false positive reaction, testing result is easy to be subject to the shortcomings such as the impact (as food substrate, the processing stage of food etc.) of extraneous factor.
Biology sensor method is according to test substance and molecular recognition elements specific binding, there is biochemical reaction, the biological information producing by signal converter be converted into can quantitative Treatment the information such as electricity, light, then amplify and output through instrument, thereby reach the object of analyzing and detecting.At present, miniature SPR biosensors has been applied to the detection of Peanut Allergen in food production, and this detecting device can be realized online quantitatively detection.But biology sensor method needs specific equipment and accessory, and testing cost is high, require high to hardware facility.What food industry circle needed is quick, easy, efficient, high specificity and detection method with low cost, and above-mentioned these methods all cannot meet the demand of food industry circle.
Immunochromatography technique is the expanded application of elisa technique principle, generally uses latex particle, electroselenium, collaurum and liposome etc. as colored marker.In the time of chromatography, the compound forming between label and determinand can be caught by corresponding part and be gathered in the detection line on nitrocellulose membrane and present label with color, thereby therefore can realize testing goal by the having or not of the bar that develops the color on tunica fibrosa, shade and reflection ray etc., there is easy and simple to handle, advantage fast.In current disclosed report, mostly using collaurum as indicator, collaurum detection method is therefore otherwise known as.In open source literature, related to a kind of immune colloidal gold chromatography method that detects food allergy to aquatic products, can realize the qualitative detection of anaphylactogen, but be difficult to quantitatively detect, and visual detection makes testing result be difficult for record and preserve.
In addition,, in immuno-chromatographic assay technology, except adopting the collaurum beyond the region of objective existence that serves as a mark, also have and adopt the report of other label as indicator.The patent No. is that the patent of US5753517 discloses a kind of latex particle serve as a mark quantitative immune chromatography method and instrument of thing of using.The Patent Application Publication of publication number CN1480391A a kind ofly prepare at normal temperatures colloidal nano granules of selenium and with the serve as a mark immunochromatography detection method of thing of this material, obtained the detection method higher than the sensitivity of collaurum method.Publication number CN1645146A discloses a kind of by fluorescent rare earth nanometer particle serve as a mark immune chromatography method and the test strip thereof of thing, can be used for quantitative Novel immune chromatography and detects.
In recent years, with magnetic nanoparticle serve as a mark thing immunochromatography detect report also successively occur.As disclosed in publication number CN101566636A is magnetic-particle mark chromatograph test strip for alpha-fetoprotein in blood, publication number CN101551390A is disclosed is a kind of immuomagnetic bead chromatographic test strip of fast detecting algae toxin, publication number CN101566631A is disclosed is the magnetic particle marker chromatograph test strip for joint-detection HIV-1+2 antibody and P24 antigen, and publication number CN101561437A is disclosed is the immuomagnetic bead chromatographic test strip technology for detection of phenol adrenaline hormone Ractopamine.Publication number US2008/013884A1 discloses coupling biotin on label, utilizes affinity interaction between Avidin and biotin in the ELISA test strip method in conjunction with double antibodies sandwich Model Design, comprises magnetic-particle label chromatography.
Above-mentioned these disclosed technology have all adopted magnetic immuno-chromatographic technology, it replaces traditional label to carry out immunochromatography with supperparamagnetic particles, the object being combined on superparamagnetic nano particle by detection improves the quantitative detection data to biological specimen, linear relationship between recycling immune complex and the magnetic signal of being caught by magnetic particle marker antibody can realize the Quantitative detection object to biological specimen, has highly sensitive, high specificity, the advantage such as easy and simple to handle, quick.
At present, on market, have at least 14 kinds of detections to comprise the business immunochromatographytest test kit of the anaphylactogens such as milk, peanut, fibert, Species of Crustacea, what but these products adopted is all colloidal gold immunity chromatography, can only carry out qualitative or sxemiquantitative monitoring, there is no both at home and abroad at present and can realize the quick onthe technology of site test quantitatively detecting to aquatic products main allergen.
Summary of the invention
Object of the present invention: be intended to solve and lack in the market this defect of fast quantification onthe technology of site test of aquatic products main allergen, provide a kind of and have more sensitivity and can realize the Novel immune chromatography detection method of quantitative detection than colloidal gold strip; The immunochromatographydetecting detecting test strip that simultaneously provides a kind of magnetic Nano detector probe label with allergen specificity to make, to reach quick, sensitive and can in situ quantitation to detect the object of aquatic products main allergen Tm and Pa.
Technical solution of the present invention is:
The tachysynthesis bead chromatograph method of main allergen in this aquatic products, adopt hybridoma technology preparation to screen, can be monoclonal antibody with the mouse of edible shell-fish hydrobiont and the irritated original specific reaction of edible software hydrobiont Tm, respectively can be to the parvalbumin of fish and frog, rat, the mouse that the vertebrate Pa of green turtle has specific reaction is that monoclonal antibody is as specific antibody, and be surface that monoclonal anti the is coupled to magnetic Nano material thing that serves as a mark by chemical bond mode by mouse, be made into the specific nano detector probe that is applicable to bead chromatograph method, then in the sample pad of the magnetic immuno-chromatographic test paper strip of anaphylactogen, add allergen specificity nanometer detection probe and sample liquid, under room temperature, place after 20min, because the immune complex that specific reaction between Ag-Ab forms is caught through the tested measuring tape of chromatography effect and control band institute, form macroscopic coloured band, realize immunochromatography, finally test card is put on magnetic signal detector (MAR instrument: Magnetic Assay Reader), the detection band forming after immunochromatography and control band are detected to obtain biologically signal, then draw the typical curve of sample to be checked, obtain the allergen content of testing sample according to graticule, after comparing, complete immunochromatography.
The preparation method of described specific nano detector probe is:
A, draw appropriate carboxyl magnetic bead in centrifuge tube, with 500 μ L 0.01M containing 0.5%v/v Tween-20, pH is that 5.0 MEST solution cleans magnetic bead as activation buffer solution, centrifuge tube is placed on magnetic separator frame and makes magnetic bead and separate with activated solution, repeated washing several times, last resuspended magnetic bead;
B, then add freshly prepared 2.6M carbodiimides (EDC) and 2.2M N-hydroxyl succinyl
Imines (NHS) adds activated carboxyl 30min in magnetic bead suspension, then with MEST damping fluid washing magnetic bead; Use again subsequently 0.005M borate tween (being called for short BST) solution to wash magnetic bead 2 times as coupling buffer;
C, add parvalbumin monoclonal antibody subsequently, on impeller, react 3 hours;
D, then use 1%w/v BSA solution (BSA is dissolved in BST damping fluid in advance) not have the activated group of complete reaction to seal to immunomagnetic beads surface, and by the physisorption of BSA, seal other site, space, with contingent non-specific adsorption in test after being reduced in, capping 30min under room temperature;
E, finally with the immunomagnetic beads after BST washing sealing 4 times, discard cleansing solution, magnetic bead is resuspended in
Preserve in liquid for subsequent use.
Described edible shell-fish hydrobiont refers to: shrimps, crab class, oyster etc., described edible software
Hydrobiont refers to conch, abalone, squid, octopus, clam etc.
Described magnetic Nano material is the superparamagnetism magnetic nanoparticle that finishing has carboxyl, and particle diameter is 50 ~ 300nm.
The present invention due to adopt hybridoma technology preparation screening, respectively can with edible shell-fish hydrobiont,
Also can with drinkable water mollush class in the mouse of the irritated original specific reaction of Tm be monoclonal antibody, the mouse that simultaneously also can there is specific reaction to vertebrate Pa such as the parvalbumin of fish and frog, rat, green turtles be monoclonal anti as detection specific antibody, compared with prior art have the following advantages:
(1) superparamagnetic nanomaterial, immunochromatography technique and magnetometric analysis system are being combined, logical
Cross preparation test strip, can meet can qualitative Site Detection demand that again can be quantitative.
(2) use the test strips prepared of the technology of the present invention, easy and simple to handle, detect fast, only need 10 ~ 20min
Can provide result.
(3) high, the high specificity of detection sensitivity.Detection to aquatic products main allergen Tm is limited to
0.15 μ g/mL, Pa detects and is limited to 0.16 μ g/mL.
Brief description of the drawings
Fig. 1 is the serve as a mark structural representation of magnetic immuno-chromatographic test paper strip of thing of allergen specificity nanometer detection probe;
Formed by sample pad, pad, nitrocellulose filter (NC film), thieving paper and base plate respectively.
Fig. 2 adopts competition law to detect the qualitative detection result of aquatic products main allergen Tm;
Negative findings presents two bands, and positive findings presents a band.As seen from the figure, anaphylactogen Tm is in the time of 10mg/mL, and T line color is still obviously shallow than negative band.
Fig. 3 adopts competition law to detect the qualitative detection result of aquatic products main allergen Pa;
Negative findings presents two bands, and positive findings presents a band.As seen from the figure, anaphylactogen Pa is in the time of 10mg/mL, and T line color is still obviously shallow than negative band.
Fig. 4 is the thing that serves as a mark with allergen specificity nanometer detection probe, adopts competition law to detect the quantitative criterion curve of aquatic products main allergen Tm.
Fig. 5 is the thing that serves as a mark with allergen specificity nanometer detection probe, adopts competition law to detect the quantitative criterion curve of aquatic products main allergen Pa.
In figure: 1-sample pad 2-pad 3-NC film 4-thieving paper 5-base plate 6-C line 7-T line.
Embodiment
The tachysynthesis bead chromatograph method of main allergen in this aquatic products, what adopt hybridoma technology preparation screening can be monoclonal antibody with the mouse of edible shell-fish hydrobiont and the irritated original specific reaction of edible software hydrobiont Tm, with can be to the parvalbumin of fish and frog, rat, the mouse that the vertebrate Pa of green turtle has specific reaction is that monoclonal antibody is as specific antibody, and be surface that monoclonal anti the is coupled to magnetic Nano material thing that serves as a mark by chemical bond mode by mouse, be made into the specific nano detector probe that is applicable to bead chromatograph method, then in the sample pad of the magnetic immuno-chromatographic test paper strip of anaphylactogen, add allergen specificity nanometer detection probe and sample liquid, under room temperature, place after 20min, because the immune complex that specific reaction between Ag-Ab forms is caught through the tested measuring tape of chromatography effect and control band institute, form macroscopic coloured band, realize immunochromatography, finally test card is put on magnetic signal detector (MAR instrument: Magnetic Assay Reader), the detection band forming after immunochromatography and control band are detected to obtain biologically signal, then draw the typical curve of sample to be checked, obtain the allergen content of testing sample according to graticule, after comparing, obtain final immunochromatography result.
Described edible shell-fish hydrobiont refers to: shrimps, crab class and oyster etc., and described edible software hydrobiont refers to conch, belongs to the abalone of Gastropoda, the squid that belongs to Cephalopoda, octopus, and bivalve shellfish clam etc.
Described magnetic Nano material is the superparamagnetism magnetic nanoparticle that finishing has carboxyl, and particle diameter is 50 ~ 300nm.
Described chromatographic film is the one in hybrid films of nylon membrane, PVDF membrane, polyester film, nitrocellulose filter, cellulose acetate membrane or cellulose nitrate and cellulose acetate etc.
Described antigen or antibody is coated to be referred to by a film device and is wire and draws the T line as immune chromatography test paper in chromatographic film catching antibody or antigen, generally automatically completes by machine; In the downstream of detecting band,,, generally automatically complete by machine as the C line of immune chromatography test paper being wire or banded drawing on chromatographic film with two anti-(as sheep anti mouse or rabbit anti-mouse iggs) of antibody specific reaction by a film instrument.
The preparation method of described specific nano detector probe is:
A, draw appropriate carboxyl magnetic bead in centrifuge tube, with 500 μ L 0.01M containing 0.5% (v/v) Tween-20, the MEST solution of pH5.0 cleans magnetic bead as activation buffer solution, centrifuge tube is placed on magnetic separator frame and makes magnetic bead and separate with activated solution, repeated washing several times, last resuspended magnetic bead;
B, then add freshly prepared 2.6M carbodiimides (EDC) and 2.2M N-hydroxyl succinyl
Imines (NHS) adds activated carboxyl 30min in magnetic bead suspension, then with MEST damping fluid washing magnetic bead; Use again subsequently 0.005M borate tween (being called for short BST) solution to wash magnetic bead 2 times as coupling buffer;
C, add parvalbumin monoclonal antibody subsequently, on impeller, react 3h;
D, then use 1% w/v BSA solution (BSA is dissolved in BST damping fluid in advance) not have the activated group of complete reaction to seal to immunomagnetic beads surface, and by the physisorption of BSA, seal other site, space, with contingent non-specific adsorption in test after being reduced in, capping 30min under room temperature;
E, finally with the immunomagnetic beads after BST washing sealing 4 times, discard cleansing solution, magnetic bead is resuspended in
Preserve in liquid for subsequent use.
Immunochromatographydetecting detecting test strip of the present invention is made up of liner plate, sample pad, pad, chromatographic film and adsorptive pads.Sample pad, pad, chromatographic film and adsorptive pads are engaged on liner plate successively, chromatographic film is placed in the middle part of liner plate, sample pad and adsorptive pads are placed in respectively the both sides of liner plate, labeling pad is placed between sample pad and chromatographic film, is assembled into the magnetic immuno-chromatographic test strip of aquatic products main allergen.Described liner plate is the hard plastic bar not absorbing water, and one side has gum.Sample pad and pad are; Glass fibre membrane; Adsorptive pads is absorbent filter;
The preparation process of immunochromatographydetecting detecting test strip of the present invention is as follows:
(1) prepare allergen specificity nanometer detection probe;
(2) in chromatographic film, fix capture antigen or antibody and control line antibody;
(3) assembling test strips;
(4) allergen specificity nanometer detection probe on labeling pad point.
Embodiment 1: the preparation of aquatic products main allergen Tm Rapid detection test strip
This embodiment is taking Tm as detected object, select finishing to have the magnetic nanoparticle (particle diameter is 200nm) of carboxyl, employing can detect shell-fish edible aquatic organism as the Tm of shrimp, crab and clam, the mouse that also can detect the irritated original specific reaction of Tm in the software class edible aquatic organisms such as conch, abalone, squid and octopus is the specific probe of monoclonal antibody preparation marine product main allergen Tm, utilizes competitive reaction principle to design detection line.
(1) the specific nano detector probe of anaphylactogen Tm preparation
EDC/NHS coupling
The nanometer magnetic bead of the monoclonal antibody specific of Tm and carboxyl modified (particle diameter is 200nm) is carried out to coupling, and preparation is containing the immunomagnetic beads of Tm monoclonal antibody, i.e. Tm magnetic labeling antibody.Using the MEST solution (0.05%Tween-20) of pH5.0 as activation buffer solution, get 2mg carboxyl magnetic bead in 2mL centrifuge tube, add 500 μ L activation damping fluids, on vortex oscillation device, mix, again centrifuge tube is positioned on magnetic separator frame, treat that magnetic bead is adsorbed completely, with mini desktop vacuum pump extracting supernatant; Relaunder after twice of magnetic bead with 500 μ L activation damping fluids, EDC and NHS solution, adjust volume to 500 μ L with activation damping fluid, on vortex oscillation device, mixes, and room temperature activates 30min.After activation, with activation damping fluid washing magnetic bead twice, to remove unreacted activator.Borate tween damping fluid (0.05%Tween-20) solution with pH9.0 is made coupling buffer, washs magnetic bead twice with coupling buffer; Then add the 150 μ g anti-Pa monoclonal antibody of purifying, make the carboxyl of magnetic bead surfaces activation and the amino room temperature reaction 5h of antibody, antibody coupling, in magnetic bead surfaces, is obtained to immunomagnetic beads.After coupling completes, add the 1%BSA(w/v of 500 μ L), do not have the activated group of complete reaction to seal to immunomagnetic beads surface, capping 30min under room temperature.Finally, with after the magnetic bead after coupling buffer washing sealing four times, be stored in 500 μ L and preserve in liquid.
(2) immobilization of Tm nano-probe
Suspended state preservation method
The magnetic labeling antibody preparing is resuspended with the coupling buffer containing 1% (w/v) sucrose, in centrifuge tube, be stored in 4 DEG C, before detection, get 1.5 ~ 2 μ L allergen specificity immunomagnetic beadses and be added on pad place.
(3) capture agent is coated
With the speed of 1 μ L/cm, capture agent is coated on nitrocellulose filter with a film instrument.On T line, to be coated with concentration be 0.05mg/mL to Tm; Apart from T line 7mm, be C line near thieving paper place, being coated with concentration is 2mg/mL goat anti-mouse IgG.After being coated with, in 37 DEG C of baking ovens, dry 2 hours.
(4) assembling of test strips
Sample pad, pad, the nitrocellulose filter, the adsorptive pads that are embedded with T line and C line are engaged on liner plate successively, and are cut into the wide Tm immuomagnetic bead chromatographic test strip of 0.5cm.Between each parts, all there is the lap of 1mm.
(5) sample detection
First on the pad of test strips, drip 1.5 μ L immunomagnetic beadses, then in the sample pad of test strips, add the testing sample of 100 μ L.Wait for after 20min observations.Negative findings is two obvious bands, and a band only appears in positive findings, or T lines band is shallow compared with negative sample.Also test strips can be packed into special test strips draw-in groove, in insertion magnetic signal reader MAR, read magnetic signal and carry out quantitative test, compare with typical curve, draw the concentration of antigenic substance in sample.
Embodiment 2: the preparation of aquatic products main allergen Pa Rapid detection test strip
This embodiment is taking Pa as detected object, select finishing to have the magnetic nanoparticle (particle diameter is 100nm) of carboxyl, the mouse in conjunction with vertebrate Pa such as the parvalbumin to fish and frog, rat, green turtles with specific reaction is the specific nano probe that monoclonal antibody is prepared fish main allergen Pa, and recycling competitive reaction principle designs detection line.
(1) the specific nano probe of anaphylactogen Pa preparation
EDC/NHS coupling
The nanometer magnetic bead of parvalbumin monoclonal antibody and carboxyl modified is carried out to coupling, and preparation is containing the immunomagnetic beads of parvalbumin monoclonal antibody, i.e. parvalbumin magnetic labeling antibody.Using the MEST solution (0.05%Tween-20) of pH5.0 as activation buffer solution, get 2mg carboxyl magnetic bead in 2mL centrifuge tube, add 500 μ L activation damping fluids, on vortex oscillation device, mix, again centrifuge tube is positioned on magnetic separator frame, treat that magnetic bead is adsorbed completely, with mini desktop vacuum pump extracting supernatant; Relaunder after twice of magnetic bead with 500 μ L activation damping fluids, EDC and NHS solution, adjust volume to 500 μ L with activation damping fluid, on vortex oscillation device, mixes, and room temperature activates 30min.After activation, with activation damping fluid washing magnetic bead twice, to remove unreacted activator.Borate tween damping fluid (0.05%Tween-20) solution with pH9.0 is made coupling buffer, washs magnetic bead twice with coupling buffer; Then add the 150 μ g anti-Pa monoclonal antibody of purifying, make the carboxyl of magnetic bead surfaces activation and the amino room temperature reaction 3h of antibody, antibody coupling, in magnetic bead surfaces, is obtained to immunomagnetic beads.After coupling completes, add the 1%BSA (w/v) of 500 μ L, do not have the activated group of complete reaction to seal to immunomagnetic beads surface, capping 30min under room temperature.Finally, with after the magnetic bead after coupling buffer washing sealing four times, be stored in 500 μ L and preserve in liquid.
(2) immobilization of Pa nano-probe
Suspended state preservation method
By the magnetic labeling antibody preparing with containing 1%(w/v) coupling buffer of sucrose is resuspended, is stored in 4 DEG C in centrifuge tube.Before detection, get 1 ~ 2 μ L immunomagnetic beads and be added on pad place.
(3) capture agent is coated
With the speed of 1 μ L/cm, capture agent is coated on nitrocellulose filter with a film instrument.On T line, be coated with the Pa that concentration is 0.2mg/mL; Apart from T line 7mm, be C line near thieving paper place, being coated with concentration is 2mg/mL goat anti-mouse IgG.After being coated with, in 37 DEG C of baking ovens, dry 2h.
(4) assembling of test strips
Sample pad, pad, the nitrocellulose filter, the adsorptive pads that are embedded with T line and C line are engaged on liner plate successively, and are cut into the wide parvalbumin immuomagnetic bead chromatographic test strip of 0.5cm.Between each parts, all there is the lap of 1mm.
(5) sample detection
First on the pad of test strips, drip 2 μ L immunomagnetic beadses, then in the sample pad of test strips, add the testing sample of 100 μ L.Wait for after 20min observations.Negative findings is two obvious bands, and a band only appears in positive findings, or T lines band is shallow compared with negative sample.Also test strips can be packed into special test strips draw-in groove, in insertion magnetic signal reader MAR, read magnetic signal and carry out quantitative test, compare with typical curve, draw the concentration of antigenic substance in sample.
Following table is with the allergen specificity nanometer detection probe test strip of thing and commercially available commercialization colloidal gold colloidal gold detection test paper strip (Japanese Shui drugmaker) the testing result comparative analysis table to aquatic products main allergen Tm that serves as a mark.As seen from table, the consistance of two kinds of methods is 90%.
Remarks: IMNP-LFA: immunomagnetic beads probe mark chromatograph test strip; LFA: chromatograph test strip.
Claims (2)
1. the tachysynthesis bead chromatograph method of main allergen in aquatic products, it is characterized in that: adopting the mouse that can have a specific reaction with the parvalbumin of fish and vertebrate parvalbumin of hybridoma technology preparation screening is monoclonal antibody, or can be that monoclonal antibody is as specific antibody with the mouse of edible shell-fish hydrobiont and the irritated original specific reaction of edible software hydrobiont tropomyosin, and be surface that monoclonal antibody the is coupled to magnetic Nano material thing that serves as a mark by chemical bond mode by mouse, be made into the specificity magnetic Nano probe that is applicable to bead chromatograph method, then in the sample pad of the magnetic immuno-chromatographic test paper strip of anaphylactogen, add allergen specificity magnetic Nano probe and sample liquid, under room temperature, place after 20min, because the immune complex that specific reaction between Ag-Ab forms is caught through the tested measuring tape of chromatography effect and control band institute, form macroscopic coloured band, realize immunochromatography, finally test card is put on magnetic signal detector, the detection band forming after immunochromatography and control band are detected to obtain biologically signal, then draw the typical curve of sample to be checked, establishing criteria curve is obtained the allergen content of testing sample, obtains final immunochromatography result after comparing,
Wherein, the preparation method of the magnetic Nano probe of anti-parvalbumin is:
Draw 2mg carboxyl magnetic bead in 2mL centrifuge tube, clean magnetic bead with the MEST solution of 500 μ L0.01M as activation buffer solution, wherein the pH of MEST solution is 5.0, contains 0.5%Tween-20; Centrifuge tube is placed in and on magnetic separator frame, makes magnetic bead separate with activation damping fluid, repeated washing several times, last resuspended magnetic bead; Then add freshly prepared 2.6M EDC and 2.2M NHS to activated carboxyl 30min in magnetic bead suspension, then with MEST damping fluid washing magnetic bead; Wash magnetic bead 2 times with 0.005M borate tween solution as coupling buffer more subsequently; Add subsequently the 150 μ g anti-parvalbumin monoclonal antibody of purifying, on impeller, react 3 hours; Then use 1% (w/v) BSA solution not have the activated group of complete reaction to seal to immunomagnetic beads surface, and by the physisorption of BSA, seal other site, space, with contingent non-specific adsorption in test after being reduced in, capping 30min under room temperature; Described BSA solution is dissolved in borate tween solution in advance; Immunomagnetic beads after finally sealing with the washing of borate tween solution 4 times, discards cleansing solution, and magnetic bead is resuspended in and is preserved in liquid;
Wherein, the preparation method of the magnetic Nano probe of antigen myosin is:
Using pH5.0, containing the MEST solution of 0.05%Tween-20 as activation buffer solution, get 2mg carboxyl magnetic bead in 2mL centrifuge tube, add 500 μ L activation damping fluids, on vortex oscillation device, mix, again centrifuge tube is positioned on magnetic separator frame, treat that magnetic bead is adsorbed completely, with mini desktop vacuum pump extracting supernatant; Relaunder after twice of magnetic bead with 500 μ L activation damping fluids, add EDC and NHS solution, adjust volume to 500 μ L with activation damping fluid, on vortex oscillation device, mix, room temperature activates 30min; After activation, with activation damping fluid washing magnetic bead twice, to remove unreacted activator; With pH9.0, make coupling buffer containing the borate tween damping fluid of 0.05%Tween-20, with twice of coupling buffer washing magnetic bead; Then add the 150 μ g anti-tropomyosin monoclonal antibody of purifying, make the carboxyl of magnetic bead surfaces activation and the amino room temperature reaction 5h of antibody, antibody coupling, in magnetic bead surfaces, is obtained to immunomagnetic beads; After coupling completes, add the 1%BSA (w/v) of 500 μ L, do not have the activated group of complete reaction to seal to immunomagnetic beads surface, capping 30min under room temperature;
Wherein, magnetic Nano material is the superparamagnetic nano particle that finishing has carboxyl, and particle diameter is 50-300nm.
2. the tachysynthesis bead chromatograph method of main allergen in aquatic products as claimed in claim 1, it is characterized in that: described edible shell-fish hydrobiont refers to: shrimps, crab class and oyster, described edible software hydrobiont refers to conch, belongs to the abalone of Gastropoda, the squid that belongs to Cephalopoda, octopus, and bivalve shellfish clam.
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| CN108445218A (en) * | 2018-03-21 | 2018-08-24 | 浙江艾明德生物科技有限公司 | The kit and preparation method thereof of joint-detection CRP, PCT and SAA |
| CN110684094A (en) * | 2019-08-30 | 2020-01-14 | 集美大学 | Ostrea dentata allergic protein and application thereof |
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