CN103675271A - Anaphylactic disease allergen colloidal gold diagnostic test strip and preparation method thereof - Google Patents
Anaphylactic disease allergen colloidal gold diagnostic test strip and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an anaphylactic disease allergen colloidal gold diagnostic test strip. The test strip comprises a sample hole, a colloidal gold pad, a film strip, an absorbent pad and a PVC board, wherein the film strip is coated with inhalation group allergen I, inhalation group allergen II, a food group allergen and a mouse anti-goat IgG antibody, so as to form three detecting strips and one quality control strip. Each allergen energy included in the test strip can represent allergen molecules of 80 percent of patients, and on the detecting film strip, a symbolic allergen coated molecule replaces an allergen extract, so that the purpose of quickly diagnosing the anaphylactic disease technically is achieved. The time for detecting by using the test strip lasts for 5 to 30 minutes, the specificity is high, the sensitivity is high, the testing is simple and quick, on-site detecting is achieved, special training for operators is not required, operation can be finished according to specification, and quick diagnosis to anaphylactic diseases can be achieved.
Description
Technical field
The present invention relates to allergen detection field, specifically, relate to a kind of anaphylactia allergen colloidal gold diagnosis test strips and preparation method thereof.
Background technology
Allergic disease (claiming again anaphylactia) comprises atopic dermatitis, food hypersenstivity, allergic rhinitis and allergic asthma etc., and its incidence of disease increases day by day, and the state of an illness is by becoming complicated.WHO has classified allergic disease as 21 century primary study and the disease of control.In recent years, along with SABC, Protocols in Molecular Biology and clinical techniques, as carrying out of branchofiberoscope, this disease has been had to common recognition, be that it belongs to allergic inflammation, in inflammation district, have a large amount of inflammatory cells (comprising eosinophilic granulocyte, lymphocyte, mast cell, basophil etc.) to infiltrate.This disease morbidity relates generally to allergen, antibody, cell, acceptor and 5 links of medium.After anaphylactogen excites, it is main relevant with mast cell that the speed occurring at 15～20 minutes is sent out phase reaction, and the delayed response occurring for 4～24 hours after exciting has been considered to eosinophilic granulocyte and basophil participates in.In the time of these two, the reaction of phase all depends on a hypotype TH in T lymphocyte, particularly helper cell (TH)
2.Allergen is the reason that causes allergic inflammation, if therefore can find sensitization allergen kind, significant to preventing and treating anaphylactia.
Anaphylactia is mostly I metallergy disease, is clinical multiple illness, has seasonal and regional occurred frequently feature.How because sucking the allergens such as dirt mite, plant pollen, soft flocks or causing because of respiratory tract infection cause pathogeny imcrobe infection.
It is allergic rhinitis and bronchial astehma that respiratory tract anaphylaxis reacts modal typical disease.Bronchial astehma has the trend of increase in its morbidity rate of many countries and mortality ratio, if the asthma incidence in nearly 1 year of the states such as the U.S., Britain, Australia, New Zealand is between 10-30%.Within 2003, Chinese city survey on prevalence rate of asthma in children result shows: the existing morbidity rate of 0-15 year childhood asthma is 0.12～3.34%, national average out to 1.54%; Accumulative total morbidity rate be 1.97%, with 10 years before (1988～1990 sample survey, 0.11～2.03%) compare obvious increase.70% infant is panted outbreak first in 3 years old; Take respiratory tract infection and allergy accounts for 94.62% as inducement causes sending out an author, thinks that wherein part may be allergic rhinitis symptom.This investigates demonstration, and asthma has caused serious impact to infant, infant family and social economy.Patient and head of a family's medical demand are urgent.About the risk factor of asthma, quantity research shows greatly, and allergy is to cause the key factor of breathing heavily, wherein dirt mite, chamber dirt, pollen outbalance.
Modal a kind of chronic skin inflammation childhood that atopic dermatitis (atopic dermatitis, AD) being, whole world children 5%～20% suffer from atopic dermatitis, wherein 60% still have atopic dermatitis performance after adolescing.Nearly 80% patient has the danger that allergic airway disease occurs, and comprises allergic rhinitis and asthma.Inhalant allergen, comprises house dust mite and animal skin, is the potential inducement of atopic dermatitis.In a double blind control research, the discovery dirt mites such as Tan avoid improving atopic dermatitis.As can be seen here, carrying out skin prick test or specific serum IgE measures determining whether atopic dermatitis patients has gas transmissive allergen allergic reaction and take suitable allergen to avoid measure very effective.
Chronic urticaria, eczema are common recurrent, anaphylaxis dermatosis, and the cause of disease is complicated, often cannot effect a radical cure, easily recurrence; The former I type or III allergic reaction type and two approach of non-allergic reaction occurs after for body contact allergen causes a disease, and the latter is a kind of delayed allergy being caused by inside and outside motivating factor.Wherein 60～80% morbidities are relevant with specific allergen, and once allergenic effect is that morbidity is crucial, if can detect in time patient's allergen, to preventing and to treat this class disease extremely meaningful.
The allergenic diagnostic method of anaphylactia is divided in vivo studies and in vitro test, and in vivo studies has intracutaneous test and two kinds of methods of pricking method test.The method of in vitro test mainly contains that Western blotting, enzyme-linked immuno assay are sent out, radioactive immunoassay and fluoroimmunoassay.
The principle of Skin-test is to make the harmless allergen of trace enter skin, be combined with the specific IgE antibody on subcutaneous mast cell surface, through a series of enzyme, activate, make mast cell degranulation, discharge the number of chemical media such as histamine, thereby local vessel expansion, permeability are increased, occur papule and Flush reaction.According to response situation, the cause of disease of I metallergy disease is diagnosed clinically.The method of its test has multiple, and conventional have intracutaneous test and skin prick are tested (SPT).Intracutaneous test Allergen is to enter corium, and SPT Allergen only enters epidermis.
The allergenic diagnosis of anaphylactia, is that a systematicness is got rid of examination test, often, for patient diagnosed's anaphylactogen, need to carry out tens kinds and be suspected to be allergenic examination, and first examination utilizes Skin-test in body, can bring very large unnecessary misery to patient.In the flow process of anaphylactia allergen diagnosis, first by in-vitro diagnosis test strips primary dcreening operation, make a definite diagnosis concrete allergen, then by vivo studies, confirm irritated allergen.Therefore the method for anaphylactia allergen in-vivo diagnostic and in-vitro diagnosis is to complement each other, and in-vitro diagnosis has been avoided the unnecessary misery and the harm that with in vivo studies, bring.
The cardinal principle of anaphylactia allergen in-vitro diagnosis is that the specific IgE of allergen in patients serum is combined, and then with enzyme mark, labelled with radioisotope, fluorescein-labeled two anti-bindings, with substrate reactions, the specific IgE in patients serum is directly proportional to the intensity of substrate reactions again.
The anaphylactia allergen in-vitro diagnosis test strips of listing is the test strips that Western blotting and enzyme-linked immuno assay are principle at present, running time long (150min～240min), operation steps is complicated (to be comprised and hatching, the steps such as wash-out), support equipment expensive (10～300,000 yuan), action need operates through the technician of professional training.Serious impact allergen external detection method universal, current domestic large hospital just has the ability of allergen vitro detection, and most basic hospital and army's field hospital all can not be carried out the method for allergen in-vitro diagnosis.
Summary of the invention
The object of this invention is to provide a kind of anaphylactia allergen colloidal gold diagnosis test strips.
Another object of the present invention is to provide the preparation method of described colloidal gold diagnosis test strips.
In order to realize the object of the invention, a kind of anaphylactia allergen colloidal gold diagnosis test strips of the present invention, described test strips comprises sample well, collaurum pad, film bar, adsorptive pads and PVC plate, sample well in test strips, collaurum pad, film bar and adsorptive pads are all attached on PVC plate, in PVC plate central authorities, stick the film bar that is coated with the significant molecule mixture of anaphylactogen and mouse-anti sheep IgG polyclonal antibody, film bar upper limb is pasted adsorptive pads, film bar lower edge is pasted collaurum pad, and collaurum pad lower edge is pasted sample well.Wherein, film bar is coated with inhalation group anaphylactogen I, inhalation group anaphylactogen II, diet group anaphylactogen and mouse-anti sheep IgG antibody, forms three and detects band and a quality control band.
Described inhalation group anaphylactogen I is the potpourri of the significant allergen molecules such as dermatophagoides pteronyssinus, dust mite, tropical flukeless mite, Groton bug, cat skin bits, dogskin bits, chain lattice spore, aspergillus fumigatus and some mould.In potpourri, significant allergen molecule includes but not limited to Derp1, Derp2, Derf1, Derf2, Blot5, Blag1, Blag2, Canf1, Canf2, Feld1, Feld2, Alta1, Aspf1, Aspf2, Aspf3, Aspf4, Aspf5, Aspf6, Penl3 etc.
Described inhalation group anaphylactogen II is the potpourri of the significant allergen molecule of pollen class.Described pollen class includes but not limited to: artemisia pollen, ragweed pollen, pollen humuli scandentis, zasiokaurin, oak-leaved goosefeet pollen, London plane pollen, birch pollen, Poplar Pollen, willow pollen, elm pollen etc.Described inhalation group anaphylactogen II is the potpourri of significant allergen molecule Artv1, Artv4, Amb1, Humj1, Humj2, Zeam12.3, Cheal, Plaa1, Bet v1, Bet v2, Bet v4, Bet v6, Popv1, Sali1, Sali3, Ulm1 etc.
Described diet group anaphylactogen is the potpourri of the significant allergen molecules such as egg, milk, peanut, soybean, fresh-water fishes, ocean fish, extra large shrimp.In potpourri, significant allergen molecule includes but not limited to Gald1, Gald2, Gald3, nBos d4, nBos d5, nBos d8, Ara h1, Ara h2, Ara h3, Gly m4, Cyp c1, Gad c1, Pen a1 etc.
The significant allergen molecular separation relating in the present invention is from natural allergenic extract or by the genetic engineering acquisition of recombinating.
The significant allergen molecule relating in the present invention can, purchased from companies such as Allergon, Indoor, also can prepare voluntarily.
The present invention can be packaged in inhalation group anaphylactogen I, inhalation group anaphylactogen II, diet group anaphylactogen respectively in same packaging bag, as one-time detection, tests.
Sample well in colloidal gold diagnosis test strips of the present invention is glass fibre membrane.Gold mark pad in test strips, for adsorbing the glass fibre membrane that has colloidal gold composite.Wherein, colloidal gold composite is colloid gold particle-streptomysin albumen-biotin-sheep anti human IgE antibody.
Film bar in test strips, for being coated with cellulose acetate film, nitrocellulose membrane, PVDF of inhalation group anaphylactogen I, inhalation group anaphylactogen II, the significant allergen molecule mixture of diet group anaphylactogen and mouse-anti sheep IgG polyclonal antibody etc.Adsorptive pads in test strips is multi-layer filter paper.
Sample well in test strips, gold mark pad, film bar and adsorptive pads are all attached on PVC base plate.In PVC base plate central authorities, stick cellulose acetate film, nitrocellulose membrane or the pvdf membrane bar of the coated significant molecule mixture of good anaphylactogen and mouse-anti sheep IgG polyclonal antibody, film bar upper limb is pasted absorbent filter, film bar lower edge is pasted collaurum pad, and collaurum pad lower edge is pasted sample well.
In the present invention, the assembling of test bar is preferably: (1) subsides adsorptive pads: on absorbent filter, along the upper edge that must and post the plank of film, coincide, and the 0.05cm～0.1cm of the necessary press mold in edge under thieving paper; (2) paste colloidal gold labeled monoclonal antibody compound: gold mark pads the upper lower edge along pressing film, and surpass the 0.1cm～0.2cm on edge under film; (3) paste sample well: lower to 1/2nd places along what must press gold mark to pad in sample pad.
The present invention also provides the method for the described anaphylactia allergen colloidal gold diagnosis test strips of preparation, comprises the following steps:
1) preparation of film bar: the coated significant molecule mixture of anaphylactogen, on film bar, form three and detect band, i.e. inhalation group anaphylactogen I detection band, inhalation group anaphylactogen II detect band, diet group anaphylactogen detects and is with;
2) preparation of collaurum pad, i.e. coupling has the preparation that detects antibody colloidal gold;
3) assembling of test strips: the film bar of preparing in step 1) is sticked by PVC plate central authorities, at film bar upper limb, paste adsorptive pads, film bar lower edge is pasted collaurum pad, and collaurum pad lower edge is pasted sample well, and the test strips after assembling is sealed in aluminium foil bag.
Wherein, step 2), the preparation method of collaurum pad is:
I. by streptomysin albumen coupling to colloid gold particle;
Ii. biotin is coupled on sheep people IgE antibody;
Iii. i and ii are mixed, hatch formation colloid gold particle-streptomysin albumen-biotin-sheep anti human IgE antibody complex for 37 ℃;
Iv. above-mentioned compound is adsorbed on glass fibre membrane, obtains.
Concrete preparation method comprises:
1, the coated significant molecule mixture of anaphylactogen forms three bands on film bar:
Wherein, inhalation group anaphylactogen I comprises: the potpourri of dermatophagoides pteronyssinus, dust mite, tropical flukeless mite, Groton bug, cat skin bits, dogskin bits, chain lattice spore, aspergillus fumigatus, the significant molecule of some mould allergen; Wherein in potpourri, significant allergen molecule is: Derp1, Derp2, Derf1, Derf2, Blot5, Blag1, Blag2, Canf1, Canf2, Feld1, Feld2, Alta1, Aspf1, Aspf2, Aspf3, Aspf4, Aspf5, Aspf6, Penl3.
Inhalation group allergen II, comprising: artemisia pollen, ragweed pollen, pollen humuli scandentis, zasiokaurin, oak-leaved goosefeet pollen, London plane pollen, birch pollen, Poplar Pollen, willow pollen, elm, tree pollen; Wherein in potpourri, significant allergen molecule is: Artv1, Artv4, Amb1, Humj1, Humj2, Zeam12.3, Cheal, Plaa1, Bet v1, Bet v2, Bet v4, Bet v6, Popv1, Sali1, Sali3, Ulm1.
Diet group allergen is egg, milk, peanut, soybean, fresh-water fishes, ocean fish, the significant molecule mixture of extra large shrimp allergen; Wherein in potpourri, significant allergen molecule is Gald1, Gald2, Gald3, nBos d4, nBos d5, nBos d8, Ara h1, Ara h2, Ara h3, Gly m4, Cyp c1, Gad c1, Pen a1.
By the significant allergen molecule of inhalation group anaphylactogen I mix by a certain percentage, the significant allergen molecule of inhalation group anaphylactogen II mixes by a certain percentage, the significant molecule of the significant allergen of diet group anaphylactogen mixes by a certain percentage, wherein the protein concentration of potpourri is no more than 1mg/mL, and mixed potpourri is coated on successively and on nitrocellulose membrane, cellulose acetate film goods pvdf membrane, forms three and detect band.
2, the preparation of colloid gold particle-streptomysin albumen-biotin-sheep anti human IgE antibody:
(1) preparation of colloid gold particle
The preparation of 15nm, 18nm～30nm, 40nm or 50nm colloid gold particle: get 0.01%HAuCl
4aqueous solution 100mL, heating is boiled.Add rapidly as required 1% citric acid three sodium water solution 4mL, 2.5mL, 1mL or 0.75mL, continue to boil about 5min, occur orange red.The colloid gold particle of making so is respectively 15nm, 18nm～30nm, 40nm and 50nm.
(2) combination of streptomysin albumen and colloid gold particle
1. 0.1mol/L K
2cO
3or 0.1mol/L HCl regulates aurosol to required pH value.
2. in 100mL aurosol, add the protein solution of optimum mark amount to stir 2～3 minutes.
3. add 5mL1%PEG20000 solution.
4. in 10000～100000g, within centrifugal 30～60 minutes, carefully suck supernatant (must guard against and topple over).
5. precipitation is suspended in to certain volume containing in the damping fluid of 0.2～0.5mg/mL PEG20000, after centrifugation, then recovers with same damping fluid, concentration is with A
540nmbeing advisable in=1.5 left and right, anticorrosionly puts 4 ℃ of preservations.
6. after also can concentrating, the aurosol after being coated with carries out gel chromatography separation and purification in Sephadex G-200 post, with the buffer solution wash-out containing 0.1%BSA.Conventionally with the coated aurosol eluent pH of IgG, be 8.2.
More than it should be noted that in all solution should impure particulate, available high speed centrifugation or miillpore filter pre-service in operation.
(3) preparation of biotin labeled sheep anti human IgE antibody
1. with dimethyl sulfoxide (DMSO), prepare the N-hydroxyl succinimide biotin (should select as required the biotinylation succinyl ester of different sizes and a brachium) of 10mg/mL.
2. use sodium borate buffer liquid (0.1mol/L, pH8.0) dilution monoclonal antibody solution to 1-3mg/mL.
3. every milligram of antibody adds 25-250 μ g biotinylation succinyl ester, after mixing, and room temperature effect 4 hours.
4. by every 250 μ g biotinylation succinyl esters, add 20 μ l1mol/L NH
4cl cessation reaction, room temperature is placed 10 minutes.
5. with PBS, fully dialyse and remove free biotin, labelled antibody is frozen.
(4) preparation of colloid gold particle-streptomysin albumen-biotin-sheep anti human IgE antibody
The collaurum preparing and streptomysin albumen composition are mixed in 1:1 ratio with biotin labeled sheep anti human IgE antibody, hatch 1h for 37 ℃, be prepared into the remarkable IgE antibody complex of colloid gold particle-streptomysin albumen-biotin-goat-anti.
(5) collaurum protein conjugates glass fibre element film preparation
With specking instrument, the sheep anti human IgE of colloid gold label is sprayed on glass fibre element film to vacuum drying 2h.
3, the preparation of collaurum film bar:
By the significant allergen molecule mixture of inhalation group anaphylactogen I, the significant molecule mixture of inhalation group anaphylactogen II allergen, the significant allergen molecule mixture of diet group anaphylactogen and mouse-anti sheep IgG antibody dilution to finite concentration, with specking instrument, be sprayed on nitrocellulose membrane, 37 ℃ of dry 4h, form and detect band and quality control band.
4, the assembling of test strips:
In drying room, be ready to absorbent filter, sample pad, PVC base plate, at PVC base plate, the nitrocellulose filter being coated with sticks in central authorities, nitrocellulose filter upper limb is pasted absorbent filter, nitrocellulose filter lower edge is pasted collaurum pad, collaurum pad lower edge is pasted sample pad, after completing, with guillotine, the test paper plate posting is cut into the wide test strips of 4mm.Again test strips is sealed in aluminium foil bag, completes the assembling of product.
The using method that the present invention further provides described anaphylactia allergen colloidal gold diagnosis test strips, comprising:
A: test serum 100 μ l join in the sample well of this colloidal gold strip.
Reading within B:5～30min, actual operating time is no more than 1min.
Final collaurum-streptomysin albumen-biotin-sheep anti human IgE antibody-specific human IgE antibody complex that forms, quality control band is coated with mouse-anti sheep IgG antibody.This ELISA test strip T.T. is 5～30 minutes.This test strips is a kind of high specificity, highly sensitive, simple and fast, energy Site Detection, and operating personnel are without professional training, and by specification can complete the test strips of anaphylactia quick diagnosis.
The advantage of the relatively existing allergen in-vitro diagnosis of anaphylactia fast diagnose test paper bar provided by the invention test strips/kit is:
(1) detection quick (5～30min), easy, does not need other any instrument and equipment, simple to operate, without professional, easy to carry, can carry out whenever and wherever possible, and cheapness, can take result at once, needn't wait for, and good stability, highly sensitive, can preserve for a long time.
(2) this test strips fast patient diagnosed whether be the anaphylactia that common anaphylactogen causes, type that also can fast qualitative patient anaphylactogen, is the complementation of existing allergen vitro detection test strips.
(3) this test strips is compared the test strips/kit of existing listing, with the coated carrier of the significant allergen molecule of anaphylactogen, has replaced anaphylactogen extract to be coated with carrier, has realized technically the object of quick diagnosis anaphylactia.
(4) allergen that this test strips relates to has been included the allergen of common anaphylactia, comprises insect, animal skin, pollen, food allergen.
(5), for there is corresponding sIgE in the above patient body of corresponding anaphylactogen autopath 80% in the significant allergen molecule being coated with on this test strips film bar.
Whether anaphylactia allergen colloidal gold diagnosis test strips provided by the invention, can diagnose patient because anaphylactogen is irritated, to fall ill at short notice.The method of relatively existing allergen in vitro diagnosis, method provided by the invention can be carried out qualitative to the pathogenic reason of patient in the short time, got rid of a part of non-autopath and carried out the comprehensive examination test of anaphylactogen, avoids medical treatment waste.Method provided by the invention also can be to some patients with severe symptoms, the confirmation cause of disease in the short time.A kind of method that method provided by the invention and existing allergen in vitro diagnostic method are complementations.Clinically, first use whether method patient diagnosed of the present invention is the disease that anaphylactogen causes, for the patient who is diagnosed as anaphylactogen and induces an illness, more further determine by other method the anaphylactogen inducing an illness.
Accompanying drawing explanation
Fig. 1 is the anaphylactia allergen colloidal gold diagnosis test strips finished product of assembling in the embodiment of the present invention 1; Wherein, the quality control band that nature controlling line forms corresponding to mouse-anti sheep IgG antibody; 1-3 corresponds respectively to three detection bands of inhalation group anaphylactogen I, inhalation group anaphylactogen II, the formation of diet group anaphylactogen.
Fig. 2 is the structural representation of the anaphylactia allergen colloidal gold diagnosis test strips finished product of assembling in the embodiment of the present invention 1.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art, the raw materials used commercial goods that is.
The preparation of embodiment 1 anaphylactia allergen colloidal gold diagnosis test strips
1, the coated significant molecule mixture of anaphylactogen forms three bands on film bar
Wherein, inhalation group anaphylactogen I comprises: the potpourri of dermatophagoides pteronyssinus, dust mite, tropical flukeless mite, Groton bug, cat skin bits, dogskin bits, chain lattice spore, aspergillus fumigatus, the significant molecule of some mould allergen; Wherein in potpourri, significant allergen molecule is: Derp1, Derp2, Derf1, Derf2, Blot5, Blag1, Blag2, Canf1, Canf2, Feld1, Feld2, Alta1, Aspf1, Aspf2, Aspf3, Aspf4, Aspf5, Aspf6, Penl3.
Inhalation group allergen II, comprising: artemisia pollen, ragweed pollen, pollen humuli scandentis, zasiokaurin, oak-leaved goosefeet pollen, London plane pollen, birch pollen, Poplar Pollen, willow pollen, elm, tree pollen; Wherein in potpourri, significant allergen molecule is: Artv1, Artv4, Amb1, Humj1, Humj2, Zeam12.3, Cheal, Plaa1, Bet v1, Bet v2, Bet v4, Bet v6, Popv1, Sali1, Sali3, Ulm1.
Diet group allergen is egg, milk, peanut, soybean, fresh-water fishes, ocean fish, the significant molecule mixture of extra large shrimp allergen; Wherein in potpourri, significant allergen molecule is Gald1, Gald2, Gald3, nBos d4, nBos d5, nBos d8, Ara h1, Ara h2, Ara h3, Gly m4, Cyp c1, Gad c1, Pen a1.
By the significant allergen molecule of inhalation group anaphylactogen I mix by a certain percentage, the significant allergen molecule of inhalation group anaphylactogen II mixes by a certain percentage, the significant molecule of the significant allergen of diet group anaphylactogen mixes by a certain percentage, wherein the protein concentration of potpourri is no more than 1mg/mL, and mixed potpourri is coated on successively and on nitrocellulose membrane, cellulose acetate film goods pvdf membrane, forms three and detect band.
2, the preparation of colloid gold particle-streptomysin albumen-biotin-sheep anti human IgE antibody
(1) preparation of colloid gold particle
The preparation of 15nm, 18nm～30nm, 40nm or 50nm colloid gold particle: get 0.01%HAuCl
4aqueous solution 100mL, heating is boiled.Add rapidly as required 1% citric acid three sodium water solution 4mL, 2.5mL, 1mL or 0.75mL, continue to boil about 5min, occur orange red.The colloid gold particle of making so is respectively 15nm, 18nm～30nm, 40nm and 50nm.
(2) combination of streptomysin albumen and colloid gold particle
1. 0.1mol/L K
2cO
3or 0.1mol/L HCl regulates aurosol to required pH value.
2. in 100mL aurosol, add the protein solution of optimum mark amount to stir 2～3 minutes.
3. add 5mL1%PEG20000 solution.
4. in 10000～100000g, within centrifugal 30～60 minutes, carefully suck supernatant (must guard against and topple over).
5. precipitation is suspended in to certain volume containing in the damping fluid of 0.2～0.5mg/mL PEG20000, after centrifugation, then recovers with same damping fluid, concentration is with A
540nmbeing advisable in=1.5 left and right, anticorrosionly puts 4 ℃ of preservations.
6. after also can concentrating, the aurosol after being coated with carries out gel chromatography separation and purification in Sephadex G-200 post, with the buffer solution wash-out containing 0.1%BSA.Conventionally with the coated aurosol eluent pH of IgG, be 8.2.
More than in operation, it should be noted that should impure particulate, available high speed centrifugation or miillpore filter pre-service in all solution.
(3) preparation of biotin labeled sheep anti human IgE antibody
1. with dimethyl sulfoxide (DMSO), prepare the N-hydroxyl succinimide biotin (should select as required the biotinylation succinyl ester of different sizes and a brachium) of 10mg/mL.
2. use sodium borate buffer liquid (0.1mol/L, pH8.0) dilution sheep anti human IgE antibody-solutions to 1-3mg/mL.
3. every milligram of antibody adds the biotinylation succinyl ester of 25-250 μ g, after mixing, and room temperature effect 4 hours.
4. by every 250 μ g biotinylation succinyl esters, add 20 μ l1mol/L NH
4cl cessation reaction, room temperature is placed 10 minutes.
5. with PBS, fully dialyse and remove free biotin, labelled antibody is frozen.
3, collaurum protein conjugates glass fibre element film preparation
With specking instrument, the sheep anti human IgE of colloid gold label is sprayed on glass fibre element film to vacuum drying 2h.
4, the preparation of collaurum film bar
Significant allergen molecule mixture and mouse-anti sheep IgG antibody dilution, to finite concentration, are sprayed on nitrocellulose membrane with specking instrument, and 37 ℃ of dry 4h, form three and detect band and a quality control band.
5, the assembling of test strips
In drying room, be ready to absorbent filter, sample pad, PVC base plate, at PVC base plate, the nitrocellulose filter being coated with sticks in central authorities, nitrocellulose filter upper limb is pasted absorbent filter, nitrocellulose filter lower edge is pasted collaurum pad, collaurum pad lower edge is pasted sample pad, after completing, with guillotine, the test paper plate posting is cut into the wide test strips of 4mm.Again test strips is sealed in aluminium foil bag, completes the assembling (Fig. 1 and Fig. 2) of product.
The quality inspection of embodiment 2 anaphylactia allergen colloidal gold diagnosis test strips
In the embodiment of the present invention 1, the test strips quality standard of preparation is according to tri-of the < < of People's Republic of China (PRC) pharmacopeia > >, and minimum detectability, negative reference material coincidence rate, positive reference material coincidence rate, accuracy all meet national standard (table 1).
Table 1 anaphylactia fast diagnose test paper bar quality standard and check
The study on the stability of embodiment 3 anaphylactia allergen colloidal gold diagnosis test strips
The test strips of preparation in the embodiment of the present invention 1, in 37 ℃ of placements 8 days, is then tested to CV < 15%(table 2 with standard serum).
The stability test result of table 2 test strips
The clinical performance assessment of embodiment 4 anaphylactia allergen colloidal gold diagnosis test strips
The clinical performance assessment of anaphylactia allergen colloidal gold diagnosis test strips, mainly take skin prick, Phadia100 in-vitro diagnosis is contrast in conjunction with clinical diagnosis, 5 clinic test center, include altogether 2280 routine patients in, evaluate sensitivity and the specificity of this test strips.Result shows, the sensitivity of described test strips is 90%, and specificity is 85%.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. an anaphylactia allergen colloidal gold diagnosis test strips, it is characterized in that, described test strips comprises sample well, collaurum pad, film bar, adsorptive pads and PVC plate, sample well in test strips, collaurum pad, film bar and adsorptive pads are all attached on PVC plate, in PVC plate central authorities, stick the film bar that is coated with the significant molecule mixture of anaphylactogen and mouse-anti sheep IgG polyclonal antibody, film bar upper limb is pasted adsorptive pads, and film bar lower edge is pasted collaurum pad, and collaurum pad lower edge is pasted sample well;
Wherein, film bar is coated with inhalation group anaphylactogen I, inhalation group anaphylactogen II, diet group anaphylactogen and mouse-anti sheep IgG antibody, forms three and detects band and a quality control band;
Described inhalation group anaphylactogen I is the potpourri of dermatophagoides pteronyssinus, dust mite, tropical flukeless mite, Groton bug, cat skin bits, dogskin bits, chain lattice spore, aspergillus fumigatus and the significant allergen molecule of some mould;
Described inhalation group anaphylactogen II is the potpourri of the significant allergen molecule of pollen class.
2. test strips according to claim 1, it is characterized in that, described inhalation group anaphylactogen I is the potpourri of significant allergen molecule Derp1, Derp2, Derf1, Derf2, Blot5, Blag1, Blag2, Canf1, Canf2, Feld1, Feld2, Alta1, Aspf1, Aspf2, Aspf3, Aspf4, Aspf5, Aspf6, Penl3.
3. test strips according to claim 1, it is characterized in that, described pollen class includes but not limited to: artemisia pollen, ragweed pollen, pollen humuli scandentis, zasiokaurin, oak-leaved goosefeet pollen, London plane pollen, birch pollen, Poplar Pollen, willow pollen, elm pollen.
4. test strips according to claim 3, it is characterized in that, described inhalation group anaphylactogen II is the potpourri of significant allergen molecule Artv1, Artv4, Amb1, Humj1, Humj2, Zeam12.3, Cheal, Plaa1, Bet v1, Bet v2, Bet v4, Bet v6, Popv1, Sali1, Sali3, Ulm1.
5. test strips according to claim 1, is characterized in that, described diet group anaphylactogen is the potpourri of egg, milk, peanut, soybean, fresh-water fishes, ocean fish, the significant allergen molecule of extra large shrimp.
6. test strips according to claim 5, it is characterized in that, described diet group anaphylactogen is the potpourri of significant allergen molecule Gald1, Gald2, Gald3, nBos d4, nBos d5, nBos d8, Ara h1, Ara h2, Ara h3, Gly m4, Cyp c1, Gad c1, Pen a1.
7. test strips according to claim 1, is characterized in that, described film bar includes but not limited to cellulose acetate film, nitrocellulose membrane, PVDF.
8. test strips according to claim 1, is characterized in that, described collaurum pad is the glass fibre membrane that is coated with colloidal gold composite; Wherein, described colloidal gold composite is colloid gold particle-streptomysin albumen-biotin-sheep anti human IgE antibody.
9. the preparation method of test strips described in claim 1-8 any one, is characterized in that, comprises the following steps:
1) preparation of film bar: the coated significant molecule mixture of anaphylactogen, on film bar, form three and detect band, i.e. inhalation group anaphylactogen I detection band, inhalation group anaphylactogen II detect band, diet group anaphylactogen detects and is with;
2) preparation of collaurum pad;
3) assembling of test strips: the film bar of preparing in step 1) is sticked by PVC plate central authorities, at film bar upper limb, paste adsorptive pads, film bar lower edge is pasted collaurum pad, and collaurum pad lower edge is pasted sample well, and the test strips after assembling is sealed in aluminium foil bag.
10. preparation method according to claim 9, is characterized in that step 2) in the preparation method of collaurum pad be:
I. by streptomysin albumen coupling to colloid gold particle;
Ii. biotin is coupled on sheep people IgE antibody;
Iii. i and ii are mixed, hatch formation colloid gold particle-streptomysin albumen-biotin-sheep anti human IgE antibody complex for 37 ℃;
Iv. above-mentioned compound is adsorbed on glass fibre membrane, obtains.
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