CN103675271B - Anaphylactia allergen colloidal gold diagnosis test strips and preparation method thereof - Google Patents

Anaphylactia allergen colloidal gold diagnosis test strips and preparation method thereof Download PDF

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CN103675271B
CN103675271B CN201310718509.5A CN201310718509A CN103675271B CN 103675271 B CN103675271 B CN 103675271B CN 201310718509 A CN201310718509 A CN 201310718509A CN 103675271 B CN103675271 B CN 103675271B
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anaphylactogen
colloidal gold
allergen
test strips
pollen
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CN103675271A (en
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白彩明
马晓晖
汤承祁
裴潇竹
姜敏
李雪妮
王兆仿
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Beijing Xinhualian Xiehe Pharmaceutical Co Ltd
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    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
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Abstract

The invention provides a kind of anaphylactia allergen colloidal gold diagnosis test strips, described test strips comprises sample well, colloidal gold pad, film bar, adsorptive pads and PVC board, 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 detection zone and a quality control band.Each allergic effect proper energy that this test strips comprises represents the allergen molecule of 80% PATIENT POPULATION, on detection film bar, is replaced allergenic extract, achieve the object of quick diagnosis anaphylactia technically with bag by significant allergen molecule.With this ELISA test strip 5 ~ 30 minutes consuming time, high specificity, highly sensitive, simple and fast, can Site Detection, and operating personnel are without the need to professional training, and by specification gets final product complete operation, realize the quick diagnosis of anaphylactia.

Description

Anaphylactia allergen colloidal gold diagnosis test strips and preparation method thereof
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 (also known as 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.Allergic disease has been classified as 21 century primary study and the disease of control by WHO.In recent years, along with SABC, Protocols in Molecular Biology and clinical techniques, as carrying out of branchofiberoscope, there is common recognition to this disease, namely it belongs to allergic inflammation, has a large amount of inflammatory cell (comprising eosinophilic granulocyte, lymphocyte, mast cell, basophil etc.) to infiltrate in inflammation district.The morbidity of this disease relates generally to allergen, antibody, cell, acceptor and medium 5 links.After anaphylactogen excites, it is main relevant with mast cell that the speed occurred at 15 ~ 20 minutes sends out phase reaction, and the delayed response occurred for 4 ~ 24 hours after excitation has then been considered to eosinophilic granulocyte and basophil participates in.The reaction of these two phases all depends on a hypotype TH in T lymphocyte, particularly helper cell (TH) 2.Allergen is the reason causing allergic inflammation, if therefore can find sensitization allergen kind, then significant to preventing and treating anaphylactia.
Anaphylactia is mostly I metallergy disease, is clinical multiple illness, has feature seasonal and that regionality is occurred frequently.Many 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%.Chinese city survey on prevalence rate of asthma in children result display in 2003: 0-15 year childhood asthma existing morbidity rate be 0.12 ~ 3.34%, national average out to 1.54%; Accumulative morbidity rate is 1.97%, with 10 years before (1988 ~ 1990 sample survey, 0.11 ~ 2.03%) compare obvious increase.70% infant pants outbreak first in 3 years old; Account for 94.62% with respiratory tract infection and allergy for inducement causes sending out an author, think that wherein part may be allergic rhinitis symptoms.This investigation shows, and asthma causes serious impact to infant, infant family and social economy.Patient and head of a family's medical demand urgent.About the risk factor of asthma, large quantity research shows, allergy causes the key factor of breathing heavily, wherein dirt mite, room dirt, pollen outbalance.
Modal a kind of chronic skin inflammation childhood that atopic dermatitis (atopicdermatitis, AD) being, whole world children 5% ~ 20% suffer from atopic dermatitis, wherein 60% adolesce after still have atopic dermatitis to show.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, Tan etc. find that dirt mite avoids improving atopic dermatitis.As can be seen here, carry out skin prick test or specific serum IgE to measure determining whether atopic dermatitis patients has gas transmissive allergen allergic reaction and take suitable allergen avoiding measures very effective.
Chronic urticaria, eczema are common recurrent, anaphylaxis dermatosis, and the cause of disease is complicated, often cannot effect a radical cure, easily recur; The former causes a disease for there is I type or type III allergic reaction and non-allergic reaction two approach after body contact allergen, and the latter is a kind of delayed allergy caused by inside and outside motivating factor.Wherein 60 ~ 80% morbidities are relevant with specific allergen, and the effect of an allergen is that morbidity is crucial, if can detect the allergen of patient in time, extremely meaningful to this kind of disease of prevention and therapy.
The diagnostic method of anaphylactia allergen is divided in vivo studies and in vitro test, and in vivo studies has intracutaneous test and Histamine positive two kinds of methods.The method of in vitro test mainly contains Western blotting, enzyme-linked immuno assay is sent out, radioactive immunoassay and fluoroimmunoassay.
The principle of Skin-test makes the harmless allergen of trace enter skin, be combined with the specific IgE antibody of subcutaneous mast cell surface, through a series of enzyme activition, make mast cell degranulation, the number of chemical media such as release histamine, thus local vessel expansion, permeability are increased, there is papule and Flush reaction.Clinically according to response situation, the cause of disease of I metallergy disease is diagnosed.The method of its test has multiple, and comparatively conventional has intracutaneous test and skin prick test (SPT).Intracutaneous test Allergen enters corium, and SPT Allergen only enters epidermis.
The diagnosis of anaphylactia allergen, be that a systematicness gets rid of Screening tests, often in order to the anaphylactogen of patient diagnosed, need to carry out the examination that tens kinds are suspected to be allergen, first examination utilizes Skin-test in body, can bring misery unnecessary very greatly to patient.In the flow process of anaphylactia allergen diagnosis, first make a definite diagnosis concrete allergen by in-vitro diagnosis test strips primary dcreening operation, then confirm irritated allergen by vivo studies.Therefore the method for anaphylactia allergen in-vivo diagnostic and in-vitro diagnosis complements each other, and in-vitro diagnosis avoids the unnecessary misery and harm brought with in vivo studies.
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, again with substrate reactions, the specific IgE in patients serum is directly proportional to the intensity of substrate reactions.
The anaphylactia allergen in-vitro diagnosis test strips of current listing is Western blotting and enzyme-linked immuno assay is the test strips of principle, running time long (150min ~ 240min), operation steps complexity (comprises and hatching, the steps such as wash-out), support equipment costliness (10 ~ 300,000 yuan), action need operates through the technician of professional training.Serious have impact on the universal of allergen external detection method, and current domestic large hospital just has the ability of allergen vitro detection, and most basic hospital and army's field hospital all can not carry 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, colloidal gold pad, film bar, adsorptive pads and PVC board, sample well in test strips, colloidal gold pad, film bar and adsorptive pads are all attached in PVC board, in PVC board, the film bar being coated with the significant molecule mixture of anaphylactogen and mouse-anti sheep IgG polyclonal antibody sticks in central authorities, film bar upper limb pastes adsorptive pads, film bar lower edge pastes colloidal gold pad, and colloidal gold pad lower edge pastes 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 detection zone 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, rod method, 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, Betv1, Betv2, Betv4, Betv6, 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, nBosd4, nBosd5, nBosd8, Arah1, Arah2, Arah3, Glym4, Cypc1, Gadc1, Pena1 etc.
The significant allergen molecule related in the present invention is separated and obtains from natural allergenic extract or by genetic engineering restructuring.
The significant allergen molecule related in the present invention purchased from companies such as Allergon, Indoor, also can prepare voluntarily.
Inhalation group anaphylactogen I, inhalation group anaphylactogen II, diet group anaphylactogen can be packaged in same packaging bag by the present invention respectively, test as one-time detection.
Sample well in colloidal gold diagnosis test strips of the present invention is glass fibre membrane.Gold mark pad in test strips, for being adsorbed with the glass fibre membrane of colloidal gold composite.Wherein, colloidal gold composite is colloid gold particle-streptomysin avidin-biotin-sheep antihuman IgE antibody.
Film bar in test strips, for being coated with the cellulose acetate film, nitrocellulose membrane, PVDF etc. 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.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.Bag is sticked by the cellulose acetate film of the significant molecule mixture of good anaphylactogen and mouse-anti sheep IgG polyclonal antibody, nitrocellulose membrane or pvdf membrane bar in PVC base plate central authorities, film bar upper limb pastes absorbent filter, film bar lower edge pastes colloidal gold pad, and colloidal gold pad lower edge pastes sample well.
In the present invention, the assembling of test bar preferably: (1) pastes adsorptive pads: absorbent filter coincide along the upper edge of the plank and must having posted film, along the 0.05cm ~ 0.1cm of necessary press mold under thieving paper; (2) colloidal gold labeled monoclonal antibody compound is pasted: the upper lower edge along film must be press against of gold mark pad, and the 0.1cm ~ 0.2cm on edge under exceeding film; (3) sample well is pasted: along press against the lower to 1/2nd places of gold mark pad in sample pad.
The present invention also provides the method preparing described anaphylactia allergen colloidal gold diagnosis test strips, comprises the following steps:
1) preparation of film bar: wrap by the significant molecule mixture of anaphylactogen, film bar forms three detection zone, i.e. inhalation group anaphylactogen I detection zone, inhalation group anaphylactogen II detection zone, diet group Allergic skin test band;
2) preparation of colloidal gold pad, namely coupling has the preparation detecting antibody colloidal gold;
3) assembling of test strips: the film bar prepared in step 1) sticks in central authorities in PVC board, pastes adsorptive pads at film bar upper limb, and film bar lower edge pastes colloidal gold pad, and colloidal gold pad lower edge pastes sample well, the test strips after assembling is sealed in aluminium foil bag.
Wherein, step 2) in the preparation method of colloidal gold pad be:
I. by streptomysin albumen coupling on colloid gold particle;
Ii. biotin is coupled in sheep people IgE antibody;
Iii. i and ii is mixed, hatch formation colloid gold particle-streptomysin avidin-biotin-sheep antihuman IgE antibody compound for 37 DEG C;
Iv. above-mentioned compound is adsorbed on glass fibre membrane, obtains final product.
Concrete preparation method comprises:
1, wrap by the significant molecule mixture of anaphylactogen, film bar formed three bands:
Wherein, inhalation group anaphylactogen I comprises: the potpourri of dermatophagoides pteronyssinus, dust mite, tropical flukeless mite, Groton bug, cat skin bits, dogskin bits, rod method, 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, Betv1, Betv2, Betv4, Betv6, 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, nBosd4, nBosd5, nBosd8, Arah1, Arah2, Arah3, Glym4, Cypc1, Gadc1, Pena1.
Significant for inhalation group anaphylactogen I allergen molecule is mixed by a certain percentage, inhalation group anaphylactogen II significant allergen molecule 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 on nitrocellulose membrane, cellulose acetate film goods pvdf membrane and forms three detection zone.
The preparation of 2, colloid gold particle-streptomysin avidin-biotin-sheep antihuman 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 1% citric acid three sodium water solution 4mL, 2.5mL, 1mL or 0.75mL as required, continue to boil about 5min, occur orange red.The colloid gold particle made so is then respectively 15nm, 18nm ~ 30nm, 40nm and 50nm.
(2) combination of streptomysin albumen and colloid gold particle
1. 0.1mol/LK 2cO 3or 0.1mol/LHCl regulates aurosol to required pH value.
2. the protein solution adding optimum mark amount in 100mL aurosol stirs 2 ~ 3 minutes.
3. 5mL1%PEG20000 solution is added.
4. within centrifugal 30 ~ 60 minutes, supernatant (must guard against and topple over) is carefully sucked in 10000 ~ 100000g.
5. precipitation is suspended in certain volume containing in the damping fluid of 0.2 ~ 0.5mg/mLPEG20000, after centrifugation, then recover with same damping fluid, concentration is with A 540nmabout=1.5 are advisable, and anticorrosionly put 4 DEG C of preservations.
6. wrap by after aurosol also can concentrate after carry out gel chromatography separation and purification in SephadexG-200 post, with containing the buffer solution wash-out of 0.1%BSA.Usually be 8.2 with the aurosol eluent pH of IgG bag quilt.
More than operation it should be noted that in all solution and does not answer impure particulate, available high speed centrifugation or miillpore filter pre-service.
(3) preparation of biotin labeled sheep antihuman IgE antibody
1. with the N-hydroxyl succinimide biotin (the biotinylation succinyl ester of different size and a brachium should be selected as required) of dimethyl sulfoxide (DMSO) preparation 10mg/mL.
2. sodium borate buffer liquid (0.1mol/L, pH8.0) is used to dilute 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. 20 μ l1mol/LNH are added by every 250 μ g biotinylation succinyl esters 4cl cessation reaction, room temperature places 10 minutes.
5. with PBS enough hemodialysis removing free biotin, labelled antibody is frozen.
(4) preparation of colloid gold particle-streptomysin avidin-biotin-sheep antihuman IgE antibody
The collaurum prepared and streptomysin albumen composition are mixed in 1:1 ratio with biotin labeled sheep antihuman IgE antibody, hatches 1h for 37 DEG C, be prepared into the remarkable IgE antibody compound of colloid gold particle-streptomysin avidin-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, vacuum drying 2h.
3, the preparation of colloidal gold film bar:
Significant for inhalation group anaphylactogen I allergen molecule mixture, 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 are diluted to finite concentration, be sprayed on nitrocellulose membrane with specking instrument, 37 DEG C of dry 4h, form detection zone and quality control band.
4, the assembling of test strips:
Absorbent filter, sample pad, PVC base plate is got out in drying room, bag is sticked by good nitrocellulose filter in PVC base plate central authorities, nitrocellulose filter upper limb pastes absorbent filter, nitrocellulose filter lower edge pastes colloidal gold pad, colloidal gold pad lower edge pastes sample pad, with guillotine, the test paper plate posted is cut into the wide test strips of 4mm after completing.Again test strips is sealed in aluminium foil bag, completes the assembling of product.
The present invention further provides the using method of described anaphylactia allergen colloidal gold diagnosis test strips, comprising:
A: test serum 100 μ l joins in the sample well of this colloidal gold strip.
Reading within B:5 ~ 30min, actual operating time is no more than 1min.
Final formation collaurum-streptomysin avidin-biotin-sheep antihuman IgE antibody-specific human IgE antibody compound, 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, and energy Site Detection, operating personnel are without the need to professional training, and by specification can complete the test strips of anaphylactia quick diagnosis.
The advantage of the relatively existing allergen in-vitro diagnosis test strips/kit of anaphylactia fast diagnose test paper bar provided by the invention is:
(1) detect fast (5 ~ 30min), easy, do not need other any instrument and equipment, simple to operate, without the need to professional, easy to carry, can carry out whenever and wherever possible, cheap, result can be taken at once, need not wait for, good stability, highly sensitive, can preserve for a long time.
(2) whether this test strips can patient diagnosed be the former anaphylactia caused of Typical allergic fast, also can the former type of fast qualitative patient of hypersensitivity, is the complementation of existing allergen vitro detection test strips.
(3) this test strips compares the test strips/kit of existing listing, instead of Allergen extract bag loaded body, achieve the object of quick diagnosis anaphylactia technically with the significant allergen of an anaphylactogen point attached bag loaded body.
(4) allergen that this test strips relates to enumerates the allergen of Typical allergic disease, comprises insect, animal skin, pollen, food allergen.
(5) this test strips film bar wraps the significant allergen molecule of quilt, for there is corresponding sIgE in corresponding anaphylactogen autopath more than 80% patient body.
Whether anaphylactia allergen colloidal gold diagnosis test strips provided by the invention, can diagnose patient to fall ill because anaphylactogen is irritated at short notice.The method of relatively existing allergen in vitro diagnosis, method provided by the invention can be carried out qualitative to the reason that patient is caused a disease in the short time, eliminates a part of non-autopath and carries out the comprehensive Screening tests of anaphylactogen, avoid medical waste.Method provided by the invention also can to some patients with severe symptoms, the confirmation cause of disease in the short time.Method provided by the invention and existing allergen in vitro diagnostic method are a kind of methods of complementation.Clinically, first use method patient diagnosed of the present invention whether to be the disease that anaphylactogen causes, for the patient being diagnosed as anaphylactogen and inducing an illness, then determine the anaphylactogen that induces an illness further by other method.
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, nature controlling line corresponds to the quality control band that mouse-anti sheep IgG antibody is formed; 1-3 corresponds respectively to three detection zone 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 for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
The preparation of embodiment 1 anaphylactia allergen colloidal gold diagnosis test strips
1, wrap by the significant molecule mixture of anaphylactogen, film bar is formed three bands
Wherein, inhalation group anaphylactogen I comprises: the potpourri of dermatophagoides pteronyssinus, dust mite, tropical flukeless mite, Groton bug, cat skin bits, dogskin bits, rod method, 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, Betv1, Betv2, Betv4, Betv6, 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, nBosd4, nBosd5, nBosd8, Arah1, Arah2, Arah3, Glym4, Cypc1, Gadc1, Pena1.
Significant for inhalation group anaphylactogen I allergen molecule is mixed by a certain percentage, inhalation group anaphylactogen II significant allergen molecule 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 on nitrocellulose membrane, cellulose acetate film goods pvdf membrane and forms three detection zone.
The preparation of 2, colloid gold particle-streptomysin avidin-biotin-sheep antihuman 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 1% citric acid three sodium water solution 4mL, 2.5mL, 1mL or 0.75mL as required, continue to boil about 5min, occur orange red.The colloid gold particle made so is then respectively 15nm, 18nm ~ 30nm, 40nm and 50nm.
(2) combination of streptomysin albumen and colloid gold particle
1. 0.1mol/LK 2cO 3or 0.1mol/LHCl regulates aurosol to required pH value.
2. the protein solution adding optimum mark amount in 100mL aurosol stirs 2 ~ 3 minutes.
3. 5mL1%PEG20000 solution is added.
4. within centrifugal 30 ~ 60 minutes, supernatant (must guard against and topple over) is carefully sucked in 10000 ~ 100000g.
5. precipitation is suspended in certain volume containing in the damping fluid of 0.2 ~ 0.5mg/mLPEG20000, after centrifugation, then recover with same damping fluid, concentration is with A 540nmabout=1.5 are advisable, and anticorrosionly put 4 DEG C of preservations.
6. wrap by after aurosol also can concentrate after carry out gel chromatography separation and purification in SephadexG-200 post, with containing the buffer solution wash-out of 0.1%BSA.Usually be 8.2 with the aurosol eluent pH of IgG bag quilt.
More than it should be noted that in operation should impure particulate, available high speed centrifugation or miillpore filter pre-service in all solution.
(3) preparation of biotin labeled sheep antihuman IgE antibody
1. with the N-hydroxyl succinimide biotin (the biotinylation succinyl ester of different size and a brachium should be selected as required) of dimethyl sulfoxide (DMSO) preparation 10mg/mL.
2. sodium borate buffer liquid (0.1mol/L, pH8.0) is used to dilute sheep antihuman IgE antibody solution 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. 20 μ l1mol/LNH are added by every 250 μ g biotinylation succinyl esters 4cl cessation reaction, room temperature places 10 minutes.
5. with PBS enough hemodialysis removing 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, vacuum drying 2h.
4, the preparation of colloidal gold film bar
Significant allergen molecule mixture and mouse-anti sheep IgG antibody are diluted to finite concentration, are sprayed on nitrocellulose membrane with specking instrument, 37 DEG C of dry 4h, form three detection zone and a quality control band.
5, the assembling of test strips
Absorbent filter, sample pad, PVC base plate is got out in drying room, bag is sticked by good nitrocellulose filter in PVC base plate central authorities, nitrocellulose filter upper limb pastes absorbent filter, nitrocellulose filter lower edge pastes colloidal gold pad, colloidal gold pad lower edge pastes sample pad, with guillotine, the test paper plate posted is cut into the wide test strips of 4mm after completing.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
The test strips quality standard of preparation in the embodiment of the present invention 1 is according to the People's Republic of China's " pharmacopeia " three, and minimum detectability, negative reference product 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 inspection
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 is placed 8 days in 37 DEG C, then uses standard serum assay, CV < 15%(table 2).
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 with skin prick, Phadia100 in-vitro diagnosis in conjunction with clinical diagnosis for contrast, include 2280 routine patients altogether in 5 clinic test center, 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 without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims (5)

1. an anaphylactia allergen colloidal gold diagnosis test strips, it is characterized in that, described test strips comprises sample well, colloidal gold pad, film bar, adsorptive pads and PVC board, sample well in test strips, colloidal gold pad, film bar and adsorptive pads are all attached in PVC board, in PVC board, the film bar being coated with the significant molecule mixture of anaphylactogen and mouse-anti sheep IgG polyclonal antibody sticks in central authorities, film bar upper limb pastes adsorptive pads, and film bar lower edge pastes colloidal gold pad, and colloidal gold pad lower edge pastes 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 detection zone 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, rod method, 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;
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;
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;
Described inhalation group anaphylactogen II is the potpourri of significant allergen molecule Artv1, Artv4, Amb1, Humj1, Humj2, Zeam12.3, Cheal, Plaa1, Betv1, Betv2, Betv4, Betv6, Popv1, Sali1, Sali3, Ulm1;
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;
Described diet group anaphylactogen is the potpourri of significant allergen molecule Gald1, Gald2, Gald3, nBosd4, nBosd5, nBosd8, Arah1, Arah2, Arah3, Glym4, Cypc1, Gadc1, Pena1.
2. test strips according to claim 1, is characterized in that, described film bar includes but not limited to cellulose acetate film, nitrocellulose membrane, PVDF.
3. test strips according to claim 1, is characterized in that, described colloidal gold pad is the glass fibre membrane being coated with colloidal gold composite; Wherein, described colloidal gold composite is colloid gold particle-streptomysin avidin-biotin-sheep antihuman IgE antibody.
4. the preparation method of test strips described in any one of claim 1-3, is characterized in that, comprise the following steps:
1) preparation of film bar: wrap by the significant molecule mixture of anaphylactogen, film bar forms three detection zone, i.e. inhalation group anaphylactogen I detection zone, inhalation group anaphylactogen II detection zone, diet group Allergic skin test band;
2) preparation of colloidal gold pad;
3) assembling of test strips: step 1 is sticked by central authorities in PVC board) the middle film bar prepared, paste adsorptive pads at film bar upper limb, film bar lower edge pastes colloidal gold pad, and colloidal gold pad lower edge pastes sample well, the test strips after assembling is sealed in aluminium foil bag.
5. preparation method according to claim 4, is characterized in that, step 2) in the preparation method of colloidal gold pad be:
I. by streptomysin albumen coupling on colloid gold particle;
Ii. biotin is coupled in sheep people IgE antibody;
Iii. i and ii is mixed, hatch formation colloid gold particle-streptomysin avidin-biotin-sheep antihuman IgE antibody compound for 37 DEG C;
Iv. above-mentioned compound is adsorbed on glass fibre membrane, obtains final product.
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CN103983789B (en) * 2014-05-28 2016-03-02 中生北控生物科技股份有限公司 For the test strips and preparation method thereof of specificity dirt mite class anaphylactogen IgE examination
CN104280555B (en) * 2014-10-24 2017-09-26 江南大学 A kind of quick detection Peanut Allergen Ara h 2 colloidal gold strip and preparation method thereof
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CN109061184B (en) * 2018-08-10 2021-07-20 杭州艾乐吉生物科技有限公司 Artemisia pollen allergen combination, application and kit
CN113552338A (en) * 2020-04-26 2021-10-26 格林生物医药科技(天津)有限公司 Allergen specificity IgE antibody detection kit and preparation method thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1731979A (en) * 2002-11-26 2006-02-08 阿尔克-阿贝洛有限公司 Allergen dosage form
CN101738474A (en) * 2009-12-16 2010-06-16 杨致亭 Combined test reagent card for cytomegalovirus and rubella virus
CN101943699A (en) * 2010-08-31 2011-01-12 广州万孚生物技术有限公司 Test strip for detecting HIV antibodies in spittle and preparation method thereof
CN103033611A (en) * 2012-12-13 2013-04-10 北京新华联协和药业有限责任公司 Chemiluminiscence diagnostic kit for sensitization allergens and preparation method thereof
CN103439502A (en) * 2013-06-28 2013-12-11 北京新华联协和药业有限责任公司 Rapid specific antibody IgE detection kit and preparation method thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1195606A1 (en) * 2000-10-03 2002-04-10 VBC-Genomics Forschungsges.m.b.H. Allergen-microarray assay
CN1930474B (en) * 2004-03-05 2012-07-04 普利玛食品株式会社 Method of detecting allergen
US20080181821A1 (en) * 2007-01-29 2008-07-31 Searete Llc, A Limited Liability Corporation Of The State Of Delaware Microfluidic chips for allergen detection

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1731979A (en) * 2002-11-26 2006-02-08 阿尔克-阿贝洛有限公司 Allergen dosage form
CN101738474A (en) * 2009-12-16 2010-06-16 杨致亭 Combined test reagent card for cytomegalovirus and rubella virus
CN101943699A (en) * 2010-08-31 2011-01-12 广州万孚生物技术有限公司 Test strip for detecting HIV antibodies in spittle and preparation method thereof
CN103033611A (en) * 2012-12-13 2013-04-10 北京新华联协和药业有限责任公司 Chemiluminiscence diagnostic kit for sensitization allergens and preparation method thereof
CN103439502A (en) * 2013-06-28 2013-12-11 北京新华联协和药业有限责任公司 Rapid specific antibody IgE detection kit and preparation method thereof

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