CN102156191A - Magnetic immune chromatography method capable of synchronously detecting Tm and Pa allergens - Google Patents
Magnetic immune chromatography method capable of synchronously detecting Tm and Pa allergens Download PDFInfo
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- CN102156191A CN102156191A CN2011101290845A CN201110129084A CN102156191A CN 102156191 A CN102156191 A CN 102156191A CN 2011101290845 A CN2011101290845 A CN 2011101290845A CN 201110129084 A CN201110129084 A CN 201110129084A CN 102156191 A CN102156191 A CN 102156191A
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Abstract
The invention discloses a magnetic immune chromatography method capable of synchronously detecting Tm and Pa allergens. The method is characterized by comprising the following steps of: sequentially sticking a sample pad, a combination pad combining anti-Tm and anti-Pa immune magnetic beads, a chromatography film and a water absorption pad to a bottom plate crossly at intervals of about 2 millimeters, and then covering a transparent plastic sealing film on the upper layer to construct a magnetic immune chromatography test strip capable of synchronously detecting the Tm and Pa allergens. A Pa antigen detection line T1, a Tm antigen detection line T2 and a goat anti-mouse IgG quality control line C are pre-coated on the chromatography film, the immune magnetic beads can be captured by chromatography, and quick qualitative detection of the Tm and Pa allergens is realized according to 1 to 3 macroscopic color development strips formed after the chromatography of a sample; or quantitative detection of single allergen or synchronous quantitative detection of two allergens is realized by detecting the constructed magnetic immune test strips through a magnetic analyzer according to the magnetic signal detection values of the detection lines and the control line formed after the chromatography of the sample.
Description
Technical field
The present invention relates to a kind of magnetic immuno detection technique of anaphylactogen, but the magnetic immuno-chromatographic method of especially a kind of synchronous detection Tm and Pa anaphylactogen, the check field that belongs to food relates to the detection technique field of food allergen, also relates to the clinical detection field of anaphylactogen.
Background technology
Food hypersenstivity is a worldwide public health problem.Investigation shows that about in the world 4% population is to food hypersenstivity.In order to protect susceptible person's health, strict regulations have been carried out to the label for labelling of food allergen in the part countries and regions, and list the legislation scope in, so the detection of food allergen is more and more important.Food hypersenstivity be the body that causes by food to immune abnormal response, food allergen is to cause or evoke anaphylactoid material in the food, the content that normally contains in specific food is abundant, naturally occurring protein.Speed, mechanism and clinical characters according to allergenic response are divided into I type, II type, III type, IV type with sensitization mechanism, wherein most of food hypersenstivity is the I type hypersensitivity by the IgE mediation, general symptom comprises vomiting, stomachache, diarrhoea etc., also comprise dermoreaction and respiratory symptom, under the serious situation even anaphylactic shock or death can occur, still there is not effective methods of treatment at present.In order to protect susceptible person's health, the part countries and regions have been carried out strict regulations and have been listed the legislation scope in the label for labelling of food allergen.
At present, the allergic food that has been determined is nearly more than 180 kinds, (the Food and Agriculture Organization of FAO (Food and Agriculture Organization of the United Nation), FAO) reported that 90% above food allergen is present in the eight big group foods, be milk, egg, fish, shell-fish aquatic products, peanut, soybean, nut fruits and wheat, wherein just comprise fish and shell-fish aquatic products and goods thereof.According to investigations, in 15～24 years old age bracket crowd of China, about 6% people had the experience of food hypersenstivity, in the irritated crowd of China teenager to the 3-6% that accounts for of marine product allergy such as fish, the first place that occupies other sensitization foods.The main anaphylactogen of aquatic products can be divided into two big classes, and one is the tropomyosin (Tropomyosin, be called for short Tm) in the shellfish such as shellfish and shrimp crab, and another then is the parvalbumin (Parvalbumin is called for short Pa) in the fish such as cod.Because the complicacy of composition of food, use various batchings and adjuvant in the food processing process in addition, traditional chemical analysis is difficult to satisfy the detection requirement of food allergen, and therefore, the Fast Detection Technique research of food allergen is domestic and international scientist's research focus always.
At present, the detection technique of food allergen with the mass-spectrometric technique of analyzing peptide section amino acid sequence behind holoprotein or the enzymolysis, (Polymerase Chain Reaction, PCR) the immunology detection technology of technology and diagnosis allergy originality is representative in the PCR of detecting coding anaphylactogen dna fragmentation.Mass-spectrometric technique can accurately be measured the amino acid sequence of peptide section behind holoprotein or the enzymolysis, determines allergic protein according to associated databases again, but mass-spectrometric technique exists time-consuming, used instrument costliness, the more high relatively shortcoming of testing cost.PCR method then easily produces false positive results because of misoperation is subjected to polluting in testing process, existing operation to require technical real anaphylaxis strong, that can not reflect food is that meeting produced defectives such as allergic reaction after people took in this food.The immunology detection technology of food allergen has enzyme linked immunosorbent assay (ELISA), biology sensor method and immunochromatographic method.Wherein the ELISA method is the comparatively ripe method of present technology, the ELISA detection kit of food allergens such as existing almond, soybean, peanut, shell-fish, sesame, mustard, lupin, milk on the market, these kits are realized qualitative or half-quantitative detection in can be in 30～60min, but have false positive and false negative reaction.
The biology sensor method is according to test substance and the combination of molecular recognition elements specificity, biochemical reaction takes place, the biological information that produces by signal converter be converted into can quantitative Treatment information such as electricity, light, amplify and output through instrument again, thereby reach the purpose of analyzing and detecting.At present, miniature SPR biosensors has been applied to the detection of peanut anaphylactogen in the food production, and this detecting device can be realized online detection by quantitative.But the biology sensor method needs specific equipment and accessory, detects the cost height, requires high to hardware facility.Because food industry circle needs is quick, easy, efficient, high specificity and detection method with low cost, and therefore, above-mentioned these methods all can't satisfy the demand of food industry circle.
Immunochromatography technique is the expanded application of elisa technique principle, generally uses latex particle, electroselenium, collaurum, liposome and last commentaries on classics phosphorus etc. as colored marker.When chromatography, the compound that forms between label and the determinand can be caught and be gathered in the detection line on the nitrocellulose membrane and present the color that label has by corresponding part, therefore thereby can realize testing goal by the having or not of colour developing bar on the tunica fibrosa, shade and reflection ray etc., have easy and simple to handle, advantage fast.In present disclosed report, mostly with collaurum as indicator, therefore the collaurum detection method is otherwise known as.
Publication number is that the Chinese patent literature of 101881769A discloses a kind of immunity colloidal gold test paper strip that detects shrimp allergen and preparation method thereof, can realize the qualitative detection of anaphylactogen, but be difficult to carry out detection by quantitative, and visual detection makes testing result be difficult for record and preservation.
In addition, aspect the high flux fast detecting of test substance, also many is label with the collaurum, discloses the test strips that detects one or more porcine virus diarrhea disease antibodies for the CN101358972 Chinese patent literature as publication number.Publication number is a kind of quick detection test paper bar that detects measles, rubella virus specific antibody IgG antibody simultaneously for the CN101363856 Chinese patent literature discloses.Publication number is the CN1866021 Chinese patent literature, has proposed the test strips of a kind of while fast detecting vibrio cholerae 01 group, 0139 crowd and cholera toxin.Publication number provides a kind of quick detection test paper bar that can detect influenza A, B viral antigen simultaneously for the Chinese patent literature of CN1904615.
In immuno-chromatographic assay technology,, also have and adopt the report of other label as indicator except adopting the collaurum beyond the region of objective existence that serves as a mark.The patent No. be US5753517 U.S. Patent Publication a kind of latex particle the serve as a mark quantitative immune chromatography method and instrument of thing of using.Publication number is that the Chinese patent literature of CN201429607 discloses more than one to change phosphorus be the immune chromatography method of markers tests virus.Publication number is a kind of with fluorescent rare earth nanometer particle the serve as a mark immune chromatography method and the test strip thereof of thing for the CN1645146A Chinese patent literature discloses, and can be used for quantitative novel immunochromatography and detects.
In addition, the immunochromatography with magnetic nanoparticle (abbreviation magnetic bead) thing that serves as a mark detects report also appearance successively in recent years.As publication number is the CN101750494A Chinese patent literature, and disclosed is the magnetic immuno-chromatographic test paper strip of hepatitis B surface antibody.Publication number CN101762690A Chinese patent literature, disclosed is the magnetic immuno-chromatographic test paper strip and the preparation method of c reactive protein in a kind of detection by quantitative blood.Publication number CN101551391 Chinese patent literature, disclosed is the immuomagnetic bead chromatographic test strip technology that is used for the chlorine detection mycin.Publication number US2008/013884A1 american documentation literature discloses coupling biotin on label, utilizes Avidin combining the test strips detection method of double antibodies sandwich Design Pattern with affinity interaction between the biotin, comprises magnetic-particle label chromatography.In addition, also obtaining certain progress aspect the high throughput testing that realizes test substance in recent years, as publication number CN101566631A Chinese patent literature, disclosed is the magnetic particle mark chromatograph test strip that is used for joint-detection HIV-1+2 antibody and P24 antigen.
Above-mentioned these disclosed patented technologies have all adopted the magnetic immuno-chromatographic technology, it replaces traditional label to carry out immunochromatography with supperparamagnetic particles, be combined in object on the superparamagnetic nano particle by detection detection by quantitative data to biological specimen are provided, utilize the immune complex of being caught and the linear relationship between the magnetic signal can realize fast quantification testing goal again, advantage such as that the magnetic immuno-chromatographic technology has is highly sensitive, stability is strong, the range of linearity is wide, easy and simple to handle, quick to biological specimen by magnetic particle labelled antibody.
At present, have 14 kinds of commercial immunochromatographytest test kits on the market at least, detection material comprises the product of anaphylactogens such as milk, peanut, fibert, shell-fish aquatic products, but the general employing of these products all is colloidal gold immunity chromatography, can only carry out the qualitative or sxemiquantitative monitoring of one matter, still not have the quick on-the-spot detection technique that can realize detecting simultaneously the main anaphylactogen of shell-fish and fish in the aquatic products at present both at home and abroad.
Summary of the invention
Purpose of the present invention: the deficiency and the defective that can not detect anaphylactogen Tm and Pa at prior art simultaneously, providing a kind of is the immune chromatography method of label with the immunomagnetic beads, both can catch Tm or Pa anaphylactogen separately, also Tm and Pa anaphylactogen be can catch simultaneously, thereby main anaphylactogen Tm of synchronous detection aquatic products and Pa purpose realized.
But this synchronous detection Tm that the present invention proposes and the magnetic immuno method of Pa anaphylactogen, its technical solution is:
With sample pad, combine anti--Tm and the anti--pad of Pa immunomagnetic beads, chromatographic film, the interlaced successively about 2mm of adsorptive pads stick on the base plate, cover the transparent plastic diaphragm seal then on the upper strata, but make up the magnetic immuno-chromatographic test paper strip of a kind of synchronous detection Tm and Pa anaphylactogen; Be coated with Pa Detection of antigen line T1, Tm Detection of antigen line T2 and goat anti-mouse IgG nature controlling line C on the wherein said chromatographic film in advance, can catch immunomagnetic beads by the chromatography effect, according to forming macroscopic 1-3 bar colour developing band behind the sample chromatography, the fast qualitative of realizing Tm and Pa anaphylactogen detects, perhaps constructed magnetic immuno-chromatographic test paper strip is detected by the magnetometric analysis instrument, realize the quantitative test of single anaphylactogen or the synchronous detection by quantitative of two kinds of anaphylactogens according to the magnetic signal detected value of detection line that forms behind the sample chromatography and control line.
Wherein: described immunomagnetic beads is that the specific murine of Tm anaphylactogen is that the specific murine that monoclonal antibody (abbreviation monoclonal antibody) is modified magnetic bead and Pa anaphylactogen is the potpourri of monoclonal antibody modification magnetic bead; Described Tm specific monoclonal antibody is to adopt hybridoma technology preparation screening gained, and crustacean Tm anaphylactogen and the molluscan Tm anaphylactogen of part are all had specific reaction; Described Pa specific monoclonal antibody is to adopt hybridoma technology preparation screening gained, and the Pa anaphylactogens of fish is had specific reaction.
Described magnetic bead is the superparamagnetic nano particle that finishing has carboxyl, and the hydraulics average-size is 50 ~ 200nm.
Described shell-fish aquatic products are shrimps and crab class, and described part mollusc aquatic products are oyster, the clam of biped guiding principle, the squid of Cephalopoda, octopus etc.
The hard plastic bar of described base plate for not absorbing water, single face has gum; Described sample pad is a glass fibre membrane; Described pad is a glass fibre membrane; Described chromatographic film can be selected nylon membrane, PVDF membrane, polyester film, nitrocellulose filter, cellulose acetate membrane for use; Described adsorptive pads is an absorbent filter.
But the preparation method of the magnetic immuno-chromatographic test paper strip of synchronous detection Tm and Pa anaphylactogen may further comprise the steps:
The preparation of A, antigen and purifying: the Tm anaphylactogen is raw material with the shrimp, and the Pa anaphylactogen with crucian is
Raw material, the method that adopts the laboratory to design voluntarily is prepared respectively and purifying.
The preparation of B, antibody and purifying: the specific antibody cell line with Tm and Pa anaphylactogen increases earlier
Grow cultivation, be then injected into and prepare ascites in the BALB/C mice; Preparation gained ascites is through 50%(w/v) the sulphur ammonium adopts business-like protein-G affinity column after concentrating again, carries out purifying according to appended product operation instructions.
The preparation of C, immunomagnetic beads: selecting diameter for use is the magnetic bead of 50-200nm, use the mode of carbon dimethylamine (EDC) and succinimide (NHS) covalent cross-linking will resist respectively-and Tm monoclonal antibody and anti--Pa monoclonal antibody be marked at and form immunomagnetic beads on the magnetic bead, will resist-stand-by after Tm immunomagnetic beads and anti--Pa immunomagnetic beads 1:1 evenly mix.
The processing of D, pad: with the immunomagnetic beads mixed liquor specking of the Tm for preparing and Pa on pad to form immunomagnetic beads mark pad, can adopt during specking manually hydrojet or quantitatively liquid-jet device carry out.
E, carry out the bag quilt of chromatographic film: use 20mM PBS, the pH7.0 bag is cushioned the Tm of liquid with purifying
Antigen, Pa antigen, goat anti-mouse IgG are diluted to 0.05mg/mL, 0.2mg/mL, 2mg/mL concentration respectively; On chromatographic film, carry out the bag quilt of chromatographic film with the manual evenly specking of the amount of 3-4 μ L/ test strips respectively then, form Pa Detection of antigen line T1, Tm Detection of antigen line T2 and goat anti-mouse IgG nature controlling line C; Bag by the time spacing distance between line and line be 0.5-1.0cm; Chromatographic film (abbreviation coated film) behind the bag quilt was dried 4-6 hour in 37 ℃ drying box, and it is standby to place drying bottle to preserve.
The assembling of F, test strips and preparation: with sample pad, combine anti--Tm and the anti--pad of Pa immunomagnetic beads, coated film, the interlaced successively about 2mm of adsorptive pads stick on the base plate, then at upper strata covering transparent plastic diaphragm seal; The width cutting can obtain magnetic immuno-chromatographic test paper strip as requested.
The purification process of Tm and Pa anaphylactogen in the described steps A:
1) purifying of the main anaphylactogen Tm of shell-fish: the musculature of getting shrimp is prepared into acetone powder,
In acetone powder, add 20mmolL Tris-HCl (the pH 7. 5) solution that the contains 1 mol/L KCl extracting of spending the night in the ratio of 1:10～1:15, get supernatant after centrifugal to place 100 ℃ of water-baths to boil 10min centrifugal then recovery supernatant; The gained supernatant is handled through PI 4.5 isoelectric precipitations, precipitation is dissolved in 20mmol/L Tris-HCl (pH 7.5), behind 1 mol/L N aOH adjusting pH to 7.5, carry out 40% ammonium sulfate precipitation, centrifugal back is reclaimed supernatant and is repeated the isoelectric point operation and carry out 60% ammonium sulfate precipitation, centrifugal back gained precipitation is dissolved among the PBS, and the freezing preservation of-20 degree is standby.
2) purifying of the main anaphylactogen Pa of fish: get fresh crucian white muscle, blend the back and add little clear egg
White damping fluid (the 10mM CaCl that extracts
2, 100mM PMSF, 10mM Tris-HCl, pH7.5), supernatant is reclaimed in homogenate, centrifugal back; In supernatant, slowly add 100%(w/v) trichloroacetic acid (being called for short TCA), make the TCA final concentration reach 3%(v/v), ice bath stirs 10min down.Regulate pH to 5.2 with 6M NaOH solution then, ice bath stirs 1h down, and centrifugal again recovery supernatant adds 3% TCA once more in supernatant, stirred 10 minutes, and getting precipitation after centrifugal dissolves with PBS, and-20 degree preservations are standby.
The preparation method of immunomagnetic beads is among the described C:
1), draws 2mg carboxyl magnetic bead in centrifuge tube, contain 0.5%(v/v with 500 μ L 0.01M) Tween-20, pH is 5.0 MES solution (being called for short MEST) conduct activation buffer solution cleaning magnetic bead, centrifuge tube placed make on the magnetic separator frame that magnetic bead separates with activated solution, repeated washing several times, last resuspended magnetic bead.
2), subsequently freshly prepared 2.6M EDC and NHS are added activated carboxyl 30min in the magnetic bead suspension, reaction finishes the back earlier with MEST damping fluid washing magnetic bead, uses 0.005M borate tween solution (being called for short BST) as coupling buffer washing magnetic bead 2 times again.
3), add anti-Tm monoclonal antibody of 100 μ g or the anti-Pa monoclonal antibody of 100 μ g, reaction is 3 hours on impeller.
4) use 1% (w/v) BSA solution not have the activated group of complete reaction to seal, then, with contingent non-specific adsorption in the test after being reduced in, capping 30min under the room temperature to the immunomagnetic beads surface.
5), with the immunomagnetic beads after the sealing of BST solution washing 4 times, discard cleansing solution, magnetic bead is resuspended, and 4 ℃ of preservations are standby.
The present invention compared with prior art has the following advantages:
(1) by the Tm of pad and the double-tagging of Pa immunomagnetic beads are handled and default two these innovative approachs of detection line on chromatographic film, realized the synchronous detection of Tm and Pa.
(2) superparamagnetic nanomaterial, immunochromatography technique and magnetism detector being combined, is the immuno-chromatographic test paper strip of label by making up with the immunomagnetic beads, both can satisfy the qualitative detection demand, can realize that again fast quantification detects.Stability is by force, accurately and reliably as a result for detection by quantitative.
Description of drawings
Fig. 1 is the structural representation of the immuno-chromatographic test paper strip of the thing that serves as a mark with immunomagnetic beads.
Magnetic immuno-chromatographic test paper strip shown in Figure 1 is by sample pad 1, pad 2, the chromatographic film 3(nitrocellulose filter arranged in regular turn), adsorptive pads 4 and PVC base plate 5 form.Be coated with three lines on the chromatographic film 3, wherein that detection line T1 place specking is the main anaphylactogen Pa of fish of purifying, detection line T2 place specking be the main anaphylactogen Tm of shell-fish of purifying, control line C place specking be goat anti-mouse IgG.
Fig. 2-5 is for using the testing result setting synoptic diagram that constructed magnetic immuno-chromatographic test paper strip carries out anaphylactogen Tm and Pa.
Wherein:
Fig. 2 is for adopting the testing result of described magnetic immuno-chromatographic test paper strip to negative sample: promptly detection line T1, detection line T2 and control line C all have the colour developing band.
Fig. 3 is for adopting the strong positive testing result of described magnetic immuno-chromatographic test paper strip to anaphylactogen Pa: promptly detection line T2 and control line C have the colour developing band, and detection line T1 place is not for there being the colour developing band.
Fig. 4 is for adopting the strong positive testing result of described magnetic immuno-chromatographic test paper strip to anaphylactogen Tm: promptly detection line T1 and control line C have the colour developing band, and detection line T2 place is not for there being the colour developing band.
Fig. 5 is for adopting the strong positive synchronous detection result of described magnetic immuno-chromatographic test paper strip to anaphylactogen Pa and Tm: promptly detection line T1 and detection line T2 place be not for there being the colour developing band, and control line C has the colour developing band.
Fig. 6 is for adopting the detection by quantitative curve of described magnetic immuno-chromatographic test paper strip to shell-fish and the main anaphylactogen Tm of mollusc aquatic products.
Fig. 7 detects the detection by quantitative curve of the main anaphylactogen Pa of fish aquatic products for adopting described magnetic immuno-chromatographic test paper strip.
Fig. 8 detects the qualitative detection result of shell-fish, mollusc and fish aquatic products for adopting described magnetic immuno-chromatographic test paper strip.
Wherein:
1. the negative contrast of number test sample;
2. number be the muscle protein extract sample of crucian;
3. number be the muscle protein extract sample of shrimp;
4. number be the muscle protein extract sample of clam;
5. number be the biased sample of shrimp and crucian muscle protein extract.
But be magnetic immuno-chromatographic test paper strip of further specifying main anaphylactogen Tm of the present invention's synchronous detection shell-fish and part mollusc and the main anaphylactogen Pa of fish and preparation method thereof, describe especially exemplified by following embodiment, this embodiment is in order to explain rather than limit by any way the present invention.
Embodiment
But the magnetic immuno-chromatographic method of this synchronous detection Tm of the present invention and Pa anaphylactogen as
Down:
With sample pad, combine anti--Tm and the anti--pad of Pa immunomagnetic beads, chromatographic film, the interlaced successively about 2mm of adsorptive pads stick on the base plate, cover the transparent plastic diaphragm seal then on the upper strata, but be built into the magnetic immuno-chromatographic test paper strip of a kind of synchronous detection Tm and Pa anaphylactogen; Be coated with Pa Detection of antigen line T1, Tm Detection of antigen line T2 and goat anti-mouse IgG nature controlling line C on the wherein said chromatographic film in advance, can catch immunomagnetic beads by the chromatography effect, according to forming macroscopic 1-3 bar colour developing band behind the sample chromatography, realize that the fast qualitative of Tm and Pa anaphylactogen detects; Perhaps constructed magnetic immuno test strips is detected by the magnetometric analysis instrument, realize the quantitative test of single anaphylactogen or the synchronous detection by quantitative of two kinds of anaphylactogens according to the magnetic signal detected value of detection line that forms behind the sample chromatography and control line.
Described immunomagnetic beads is that the specific murine of Tm anaphylactogen is that the specific murine that monoclonal antibody (abbreviation monoclonal antibody) is modified magnetic bead and Pa anaphylactogen is the potpourri of monoclonal antibody modification magnetic bead; Described Tm specific monoclonal antibody is to adopt hybridoma technology preparation screening gained, and crustacean Tm anaphylactogen and the molluscan Tm anaphylactogen of part are all had specific reaction; Described Pa specific monoclonal antibody is to adopt hybridoma technology preparation screening gained, and the Pa anaphylactogens of fish is had specific reaction.
Described magnetic bead is the superparamagnetic nano particle that finishing has carboxyl, and the hydraulics average-size is 50 ~ 200nm.
Described shell-fish aquatic products are shrimps and crab class, and described part mollusc aquatic products are oyster, clam, the scallop of biped guiding principle, the squid of Cephalopoda, octopus etc.
The hard plastic bar of described base plate for not absorbing water, single face has gum; Described sample pad is a glass fibre membrane; Described pad is a glass fibre membrane; Described chromatographic film can be selected nylon membrane, PVDF membrane, polyester film, nitrocellulose filter, cellulose acetate membrane for use; Described adsorptive pads is an absorbent filter.
But the preparation method of the magnetic immuno-chromatographic test paper strip of synchronous detection Tm and Pa anaphylactogen may further comprise the steps:
The preparation of C, antigen and purifying: the Tm anaphylactogen is raw material with the shrimp, and the Pa anaphylactogen with crucian is
Raw material, the method that adopts the laboratory to design voluntarily is prepared respectively and purifying.
The preparation of D, antibody and purifying: the specific antibody cell line with Tm and Pa anaphylactogen increases earlier
Grow cultivation, be then injected into and prepare ascites in the BALB/C mice; Preparation gained ascites is through 50%(w/v) the sulphur ammonium adopts business-like protein-G affinity column after concentrating again, carries out purifying according to appended product operation instructions.
The preparation of C, immunomagnetic beads: selecting diameter for use is the magnetic bead of 50-200nm, use the mode of carbon dimethylamine (EDC) and succinimide (NHS) covalent cross-linking will resist respectively-and Tm monoclonal antibody and anti--Pa monoclonal antibody be marked at and form immunomagnetic beads on the magnetic bead, will resist-stand-by after Tm immunomagnetic beads and anti--Pa immunomagnetic beads 1:1 evenly mix.
The processing of D, pad: with the immunomagnetic beads mixed liquor specking of the Tm for preparing and Pa on pad to form immunomagnetic beads mark pad, can adopt during specking manually hydrojet or quantitatively liquid-jet device carry out.
E, carry out the bag quilt of chromatographic film: use 20mM PBS, the pH7.0 bag is cushioned the Tm of liquid with purifying
Antigen, Pa antigen, goat anti-mouse IgG are diluted to 0.05mg/mL, 0.2mg/mL, 2mg/mL concentration respectively; On chromatographic film, carry out the bag quilt of chromatographic film with the manual evenly specking of the amount of 3-4 μ L/ test strips respectively then, form Pa Detection of antigen line T1, Tm Detection of antigen line T2 and goat anti-mouse IgG nature controlling line C; Bag by the time spacing distance between line and line be 0.5-1.0cm; Chromatographic film (abbreviation coated film) behind the bag quilt was dried 4-6 hour in 37 ℃ drying box, and it is standby to place drying bottle to preserve.
The assembling of F, test strips and preparation: with sample pad, combine anti--Tm and the anti--pad of Pa immunomagnetic beads, coated film, the interlaced successively about 2mm of adsorptive pads stick on the base plate, then at upper strata covering transparent plastic diaphragm seal; The width cutting can obtain magnetic immuno-chromatographic test paper strip as requested.
The purification process of Tm and Pa anaphylactogen in the described steps A:
3) purifying of the main anaphylactogen Tm of shell-fish: the musculature of getting shrimp is prepared into acetone powder,
In acetone powder, add 20mmolL Tris-HCl (the pH 7. 5) solution that the contains 1 mol/L KCl extracting of spending the night in the ratio of 1:10～1:15, get supernatant after centrifugal to place 100 ℃ of water-baths to boil 10min centrifugal then recovery supernatant; The gained supernatant is handled through PI 4.5 isoelectric precipitations, precipitation is dissolved in 20mmol/L Tris-HCl (pH 7.5), behind 1 mol/L N aOH adjusting pH to 7.5, carry out 40% ammonium sulfate precipitation, centrifugal back is reclaimed supernatant and is repeated the isoelectric point operation and carry out 60% ammonium sulfate precipitation, centrifugal back gained precipitation is dissolved among the PBS, and the freezing preservation of-20 degree is standby.
4) purifying of the main anaphylactogen Pa of fish: get fresh crucian white muscle, blend the back and add little clear egg
White damping fluid (the 10mM CaCl that extracts
2, 100mM PMSF, 10mM Tris-HCl, pH7.5), supernatant is reclaimed in homogenate, centrifugal back; In supernatant, slowly add 100%(w/v) trichloroacetic acid (being called for short TCA), make the TCA final concentration reach 3%(v/v), ice bath stirs 10min down.Regulate pH to 5.2 with 6M NaOH solution then, ice bath stirs 1h down, and centrifugal again recovery supernatant adds 3% TCA once more in supernatant, stirred 10 minutes, and getting precipitation after centrifugal dissolves with PBS, and-20 degree preservations are standby.
The preparation method of immunomagnetic beads is among the described C:
1), draws 2mg carboxyl magnetic bead in centrifuge tube, contain 0.5%(v/v with 500 μ L 0.01M) Tween-20, pH is 5.0 MES solution (being called for short MEST) conduct activation buffer solution cleaning magnetic bead, centrifuge tube placed make on the magnetic separator frame that magnetic bead separates with activated solution, repeated washing several times, last resuspended magnetic bead.
2), subsequently freshly prepared 2.6M EDC and NHS are added activated carboxyl 30min in the magnetic bead suspension, reaction finishes the back earlier with MEST damping fluid washing magnetic bead, uses 0.005M borate tween solution (being called for short BST) as coupling buffer washing magnetic bead 2 times again.
3), add anti-Tm monoclonal antibody of 100 μ g or the anti-Pa monoclonal antibody of 100 μ g, reaction is 3 hours on impeller.
4) use 1% (w/v) BSA solution not have the activated group of complete reaction to seal, then, with contingent non-specific adsorption in the test after being reduced in, capping 30min under the room temperature to the immunomagnetic beads surface.
5), with the immunomagnetic beads after the sealing of BST solution washing 4 times, discard cleansing solution, magnetic bead is resuspended, and 4 ℃ of preservations are standby.
Tm that is adopted in specific embodiment and Pa antigen and specific monoclonal antibody thereof all are non-commercially produced products, by monoclonal antibody cell line oneself preparation purifying gained.What described magnetic immuno-chromatographic test paper strip adopted is that competition law detects principle, when containing Tm or Pa anaphylactogen in the sample to be tested, immunomagnetic beads on irritated original combined pad is caught, carrying out along with the chromatography effect, when through detection line T1 and T2, thereby can not caught by the anaphylactogen on T1 or the T2 line respectively and rest on the detection line place in conjunction with the immunomagnetic beads of anaphylactogen, the immunomagnetic beads that not tested survey line is caught can continue to move ahead when arriving nature controlling line C, is rested on C line place by anti-the catching of two on the C line.Whole chromatography is reflected in 30 minutes and finishes, and generally reacts after 20 minutes on detection line and nature controlling line naked eyes and can clearly see the colour developing band.Test strips is put into the card reading slot of magnetometric analysis instrument, can obtain corresponding magnetic signal detected value at T1, T2 and C line place.The T1 of positive and T2 place magnetic signal value generally are weaker than negative sample, and difference is big more just represents that positive reaction is strong more.
Embodiment 1: the magnetic immuno-chromatographic method detects the main anaphylactogen Tm of shell-fish
(1) preparation and the purifying of main anaphylactogen Tm of shell-fish and the main anaphylactogen Pa of fish: get shrimp acetone
Powder 2g adds the extracting of spending the night of 20mmol/L Tris-HCl (pH 7. 5) solution that 30mL contains 1 mol/L KCl, gets supernatant after centrifugal to place 100 ℃ of water-baths to boil 10min centrifugal then recovery supernatant.The gained supernatant is handled through PI 4.5 isoelectric precipitations, precipitation is dissolved in 20mmol/L Tris-HCl (pH 7.5), behind 1 mol/L NaOH adjusting pH to 7.5, carry out 40% ammonium sulfate precipitation, centrifugal back is reclaimed supernatant and is repeated the isoelectric point operation and carry out 60% ammonium sulfate precipitation, centrifugal back gained precipitation is dissolved among the PBS, and the freezing preservation of-20 degree is standby.
The purification process of the main anaphylactogen Pa of fish is: get fresh crucian white muscle, blend the back and add parvalbumin extraction damping fluid ((10mM CaCl
2, 100mM PMSF, 10mM Tris-HCl, pH7.5)), supernatant is reclaimed in homogenate, centrifugal back.Slowly add 100% trichloroacetic acid (being called for short TCA) in supernatant, make the TCA final concentration reach 3%, ice bath stirs 10min down.Regulate pH to 5.2 with 6M NaOH solution then, ice bath stirs 1h down, and centrifugal again recovery supernatant adds 3% TCA once more in supernatant, stirred 10 minutes, and getting precipitation after centrifugal dissolves with PBS, and-20 degree preservations are standby.
(2) preparation of antibody and purifying:, be then injected into and prepare ascites in the BALB/C mice earlier with the specific antibody cell line enrichment culture of Tm and Pa anaphylactogen.Preparation gained ascites adopts business-like protein-G affinity column again after 50% sulphur ammonium concentrates, carry out purifying according to appended product operation instructions.
(3) preparation of immunomagnetic beads: selecting diameter for use is the magnetic bead of 200nm, draw 2mg carboxyl magnetic bead in centrifuge tube, contain 0.5%(v/v with 500 μ L 0.01M) Tween-20, pH is 5.0 MES solution (being called for short MEST) conduct activation buffer solution cleaning magnetic bead, centrifuge tube placed make on the magnetic separator frame that magnetic bead separates with activated solution, repeated washing is the resuspended magnetic bead in back several times.Subsequently freshly prepared 2.6M EDC and NHS are added activated carboxyl 30min in the magnetic bead suspension, earlier with MEST damping fluid washing magnetic bead, use 0.005M borate tween solution (being called for short BST) to wash magnetic bead 2 times again after reaction finishes as coupling buffer.Then add anti-Tm monoclonal antibody of 100 μ g or the anti-Pa monoclonal antibody of 100 μ g, reaction is 3 hours on impeller.Use 1% (w/v) BSA solution not have the activated group of complete reaction to seal then, with contingent non-specific adsorption in the test after being reduced in, capping 30min under the room temperature to the immunomagnetic beads surface.Immunomagnetic beads after reaction end back is sealed with the BST solution washing 4 times discards cleansing solution, and magnetic bead is resuspended, and 4 ℃ of preservations are standby.
(4) immunomagnetic beads for preparing manually is sprayed on is prepared into the magnetic pad on the pad.
(5) carry out the bag quilt of chromatographic film: use 20mM PBS, the pH7.0 bag is cushioned liquid Tm antigen, Pa antigen, the goat anti-mouse IgG of purifying is diluted to 0.05mg/mL, 0.2mg/mL, 2mg/mL concentration respectively.Carry out the bag quilt of chromatographic film with the manual evenly specking of the amount of 3-4 μ L/ test strips on chromatographic film respectively then, to form Pa Detection of antigen line T1, Tm Detection of antigen line T2 and goat anti-mouse IgG nature controlling line C, the spacing distance between line and line is 0.5-1.0cm.Chromatographic film (abbreviation coated film) behind the bag quilt was dried 4-6 hour in 37 ℃ drying box, and it is standby to place drying bottle to preserve.
(6) assembling of test strips and preparation: with sample pad, combine anti--Tm and the anti--pad of Pa immunomagnetic beads, coated film, the interlaced successively about 2mm of adsorptive pads stick on the base plate, cover the transparent plastic diaphragm seal then on the upper strata, cut into the wide test strips of 1cm.
(7) detection of sample: constitute add on the sample pad of immunochromatographydetecting detecting test strip 100 μ L with PBS-T(1%Tween-20) gradient dilution Tm purification of samples.Behind the chromatography 20min, observations.Negative findings is three tangible bands; The Tm positive findings is at the T1 line, and band or T2 line appear in control line C place, the T1 line, and band all appears in control line C, but T2 line place band is more shallow than negative sample place color; Also can read magnetic signal in the magnetic signal reader MAR instrument and carry out quantitative test, the drawing standard curve dripping the magnetic immuno-chromatographic test paper strip that analyte sample fluid the is arranged special-purpose test strips draw-in groove of packing into, inserting.
Embodiment 2:The magnetic immuno-chromatographic method detects the main anaphylactogen Pa of fish
In the sample detection step, test sample is that other step is with example 1 outside the main anaphylactogen Pa of the fish of purifying.
Embodiment 3:The magnetic immuno-chromatographic method detects anaphylactogen Tm and the Pa in the aquatic products
(1) preparation of foodstuff samples extract
With the shrimp in the shell-fish in the aquatic products, the crucian in the fish, the clam of mollusc is example.Get the musculature of 2g aquatic products respectively, after stirring evenly with 40mL PBS-T, boiling water boils 15min, and 10, get supernatant behind the centrifugal 20min of 000g, freezing preservation-20 ℃, stand-by.
(2) sample detection
The foodstuff samples to be measured that on the sample pad of test strips, adds 100 μ L.Behind the chromatography 20min, observations.Negative findings is three tangible bands; Band or T2 line, T1 line appear in the Tm positive findings at T1 line, control line C place, band all appears in control line C, but T2 line place band is more shallow than negative sample place color; Also can read magnetic signal among the magnetic signal reader MAR and carry out detection by quantitative dripping the magnetic immuno-chromatographic test paper strip that analyte sample fluid the is arranged special-purpose test strips draw-in groove of packing into, inserting, the combined standard curve calculates the concentration of Tm in the testing sample or Pa.
Constructed magnetic immuno-chromatographic test paper strip is by using magnetometric analysis instrument (Magnetic Assay Reader, be called for short the MAR instrument) detect, according to the magnetic signal detected value of detection line that forms behind the sample chromatography and control line, can realize the quantitative test of single anaphylactogen or the synchronous detection by quantitative of two kinds of anaphylactogens.
Claims (6)
1. the magnetic immuno-chromatographic method of synchronous detection Tm and Pa anaphylactogen is characterized in that:
With sample pad, combine anti--Tm and the anti--pad of Pa immunomagnetic beads, chromatographic film, the interlaced successively about 2mm of adsorptive pads stick on the base plate, cover the transparent plastic diaphragm seal then on the upper strata, but be built into the magnetic immuno-chromatographic test paper strip of a kind of synchronous detection Tm and Pa anaphylactogen; Be coated with Pa Detection of antigen line T1, Tm Detection of antigen line T2 and goat anti-mouse IgG nature controlling line C on the wherein said chromatographic film in advance, can catch immunomagnetic beads by the chromatography effect, according to forming macroscopic 1-3 bar colour developing band behind the sample chromatography, realize that the fast qualitative of Tm and Pa anaphylactogen detects; Perhaps constructed magnetic immuno test strips is detected by the magnetometric analysis instrument, realize the quantitative test of single anaphylactogen or the synchronous detection by quantitative of two kinds of anaphylactogens according to the magnetic signal detected value of detection line that forms behind the sample chromatography and control line.
2. the magnetic immuno layer of a kind of synchronous detection Tm as claimed in claim 1 and Pa anaphylactogen
Analysis method is characterized in that: described immunomagnetic beads is that the specific murine of Tm anaphylactogen is that the specific murine that monoclonal antibody is modified magnetic bead and Pa anaphylactogen is the potpourri of monoclonal antibody modification magnetic bead; Described Tm specific monoclonal antibody is to adopt hybridoma technology preparation screening gained, and crustacean Tm anaphylactogen and the molluscan Tm anaphylactogen of part are all had specific reaction; Described Pa specific monoclonal antibody is to adopt hybridoma technology preparation screening gained, and the Pa anaphylactogens of fish is had specific reaction.
3. the magnetic immuno layer of a kind of synchronous detection Tm as claimed in claim 1 and Pa anaphylactogen
Analysis method is characterized in that: described magnetic bead is the superparamagnetic nano particle that finishing has carboxyl, and the hydraulics average-size is 50 ~ 200nm.
4. the magnetic immuno layer of a kind of synchronous detection Tm as claimed in claim 1 and Pa anaphylactogen
Analysis method is characterized in that: described shell-fish aquatic products are shrimps and crab class, and described part mollusc aquatic products are oyster, clam, the scallop of biped guiding principle, the squid of Cephalopoda, octopus etc.
5. the magnetic immuno layer of a kind of synchronous detection Tm as claimed in claim 1 and Pa anaphylactogen
Analysis method is characterized in that, the preparation method of chromatograph test strip may further comprise the steps:
The preparation of A, antigen and purifying: the Tm anaphylactogen is raw material with the shrimp, and the Pa anaphylactogen is raw material with the crucian, and the method that adopts the laboratory to design voluntarily is prepared respectively and purifying;
The preparation of B, antibody and purifying:, be then injected into and prepare ascites in the BALB/C mice earlier with the specific antibody cell line enrichment culture of Tm and Pa anaphylactogen;
Preparation gained ascites adopts business-like protein-G affinity column again after 50% sulphur ammonium concentrates, carry out purifying according to appended product operation instructions;
The preparation of C, immunomagnetic beads: selecting diameter for use is the magnetic bead of 50-200nm, use the mode of carbon dimethylamine (EDC) and succinimide (NHS) covalent cross-linking will resist respectively-and Tm monoclonal antibody and anti--Pa monoclonal antibody be marked at and form immunomagnetic beads on the magnetic bead, will resist-stand-by after Tm immunomagnetic beads and anti--Pa immunomagnetic beads 1:1 evenly mix;
The processing of D, pad: with the immunomagnetic beads mixed liquor specking of the Tm for preparing and Pa on pad to form immunomagnetic beads mark pad, can adopt during specking manually hydrojet or quantitatively liquid-jet device carry out;
E, the bag quilt of chromatographic film: use 20mM PBS, the pH7.0 bag is cushioned the Tm antigen of liquid with purifying, Pa antigen, goat anti-mouse IgG is diluted to 0.05mg/mL respectively, 0.2mg/mL, 2mg/mL concentration, then respectively with the manual evenly specking of the amount of 3-4 μ L/ test strips on chromatographic film, carry out the bag quilt of chromatographic film, to form Pa Detection of antigen line T1, Tm Detection of antigen line T2 and goat anti-mouse IgG nature controlling line C, spacing distance between line and line is 0.5-1.0cm, chromatographic film behind the bag quilt was dried 4-6 hour in 37 ℃ drying box, and it is standby to place drying bottle to preserve;
The assembling of F, test strips and preparation: with sample pad, combine anti--Tm and the anti--pad of Pa immunomagnetic beads, coated film, the interlaced successively about 2mm of adsorptive pads stick on the base plate, then at upper strata covering transparent plastic diaphragm seal; The width cutting magnetic immuno-chromatographic test paper strip that can obtain being suitable for as requested.
6. as the magnetic immuno-chromatographic method of claim 3 synchronous detection Tm and Pa anaphylactogen, it is characterized in that: the preparation method of the immuno-chromatographic test paper strip simultaneously and rapidly of described Tm and Pa anaphylactogen may further comprise the steps:
1), draws 2mg carboxyl magnetic bead in centrifuge tube, contain 0.5%(v/v with 500 μ L 0.01M) Tween-20, pH is 5.0 MES solution (being called for short MEST) conduct activation buffer solution cleaning magnetic bead, centrifuge tube placed make on the magnetic separator frame that magnetic bead separates with activated solution, repeated washing several times, last resuspended magnetic bead;
2), subsequently freshly prepared 2.6M EDC and NHS are added activated carboxyl 30min in the magnetic bead suspension, reaction finishes the back earlier with MEST damping fluid washing magnetic bead, uses 0.005M borate tween solution (being called for short BST) as coupling buffer washing magnetic bead 2 times again;
3), add anti-Tm monoclonal antibody of 100 μ g or the anti-Pa monoclonal antibody of 100 μ g, reaction is 3 hours on impeller;
4) use 1% (w/v) BSA solution not have the activated group of complete reaction to seal, then, with contingent non-specific adsorption in the test after being reduced in, capping 30min under the room temperature to the immunomagnetic beads surface;
5), with the immunomagnetic beads after the sealing of BST solution washing 4 times, discard cleansing solution, magnetic bead is resuspended, and 4 ℃ of preservations are standby.
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Application publication date: 20110817 |