CN105044326A - Kit and method for achieving highly-sensitive multiterm joint inspection of allergen-specific IgE antibodies - Google Patents
Kit and method for achieving highly-sensitive multiterm joint inspection of allergen-specific IgE antibodies Download PDFInfo
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Abstract
The invention discloses a kit and method for achieving highly-sensitive multiterm joint inspection of allergen-specific IgE antibodies. The kit comprises a membrane strip, the polymerase coupling anti-human IgE antibodies and a substrate color development agent matched with the polymerase coupling anti-human IgE antibodies; str-bio-allergens are fixed on the membrane strip, and the allergen-specific IgE antibodies are detected by adopting the kit. In this way, According to the kit and method for achieving highly-sensitive multiterm joint inspection of the allergen-specific IgE antibodies, the concentration of the allergen-specific antibodies IgE in the human serum or plasma can be detected highly sensitively, qualitatively or semi-quantitatively and quickly, the allergen usage amount can be reduced, dozens of allergens can be screened for one time, the advantages of being quick, accurate and little in sample usage amount are achieved, and high throughput detection is suitably performed.
Description
Technical field
The present invention relates to a kind of high-sensitive qualitative or semi-quantitatively sieve Check or detect the detection kit of the multiple allergenic specific IgE antibody levels in human serum or blood plasma, and preparation method thereof and detection method.
Background technology
Point out in 2011 ~ 2012 years world's allergic reaction tissue (WAO) white paper about allergic reaction (also claiming allergic reaction, Allergy): " the popular of anaphylactia rises just dramatically in developed country and developing country's worldwide.Anaphylactia comprises allergic asthma, allergic rhinitis, severe allergic reaction, and medicine, food and insect allergy react, eczema, nettle rash (hay fever) and angioedema etc.The increase that especially children suffer from anaphylactia number of past Two decades years has the trend broken out." prevalence rate of anaphylactia that only food causes just reaches 3% ~ 6%.Anaphylactia is that patient sucks or ingests and (is called anaphylactogen or allergen into the material containing allergen, Allergen) B cell triggering body afterwards produces excessive immunoglobulin E (ImmunoglobulinE, IgE), just be attached on the high affinity receptor Fc ε R1 on mast cell and basophilic granulocyte surface with Allergens cross-link when contact allergy is former again in vivo for IgE antibody, cause the gathering of Fc ε R1 acceptor, make mast cell and Basohil activation.Mast cell retting conditions in reactivation process also discharges the inflammatory mediator be stored in cytoplasmic granules: histamine, with cell factor and chemotactic factor (CF)s such as the leukotriene synthesized by arachidonic acid pathway, immunoreactivity prostaglandin and IL4, IL5, cause anaphylactoid disease symptoms.The generation of anaphylactia, IgE antibody plays a crucial role, and is called the allergic reaction (i.e. Gell-CoombsI type hypersensitivity, or the speed property the sent out hypersensitivity of title IgE mediation) that IgE mediates.The feature of anaphylactia of IgE mediation is high under allergenic specific IgE (sIgE) the antibody concentration compared with normal situation in patient body Inner eycle blood, and illness is more serious, and sIgE antibody concentration is higher.The clinical diagnosis of anaphylactia is pointed out in U.S. clinical doctor practical guide, and according to patient's medical history binding site thorn or blood testing allergenic specific IgE (sIgE) antibody concentration, examination is caused a disease anaphylactogen.At present, detect allergenic specific IgE (sIgE) antibody concentration in blood, the cause a disease method of anaphylactogen of examination has enzyme immunoassay (EIA), Western blot (ImmunoblottingAssay), collaurum Sidestream chromatography method (LFA), protein chip (Proteinsmicroarray) etc., development trend also meets market demands: robotization, fast, accurately, amount of samples is few, an examination tens kinds of anaphylactogens.On market, product is many, wherein, Pharmacia Inc., Sweden's product I mmunoCAP250 system is the representative of enzyme immunoassay, and ImmunoCAPRapid is the representative of collaurum Sidestream chromatography method, ImmunoCAPISAC is the representative of protein chip, has started allergen molecule diagnosis; The AllergyScreen of German Mediwiss-analytic company is then the representative (ImmunoblotforanalysingspecificIgEinhumanserum) of Western blot.Because of IgE antibody mean concentration ~ 0.005ug/ml in human blood, 0.002% of total immunoglobulin (Ig) mean concentration, sIgE antibody concentration is lower, for meet robotization, fast, accurately, few a, the examination tens kinds of anaphylactogens of amount of samples, sxemiquantitative or quantitatively detect the requirement of allergenic specific IgE (sIgE) antibody concentration in sample, the detector that photosignal amplifies must be configured, or the signal amplifying system of biochemical method.As, Pharmacia Inc., Sweden product I mmunoCAPRapid once can only examination about ten kinds of anaphylactogens, be unworthy of reading apparatus, the sensitivity of naked eyes interpretation sIgE antibody concentration only has 1.0IU/ml(1IUIgE=2.44ngIgE), distinguish negative and positive findings with 1.49IU/ml.
Summary of the invention
The technical matters that the present invention mainly solves there is provided a kind of kit and method, be use biological respinse amplification system and biotin-avidin system to improve Allergic skin test sensitivity and utilize Biodot chip point sample instrument binary channels simultaneously method for coating to improve the bag of anaphylactogen by efficiency, the bag reducing anaphylactogen is by concentration and anaphylactogen and antibody combining site are more fully exposed, thus detect allergen specificity antibody IgE with sensitivity, in order to allergen specificity antibody IgE concentration that is qualitative or that semi-quantitatively detect fast in human serum or blood plasma, and can an examination tens of kinds of anaphylactogens.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is: the kit providing a kind of highly sensitive multinomial joint inspection allergenic specific IgE antibody, comprise film bar, the antihuman IgE antibody of polymerase coupling and the substrate developer that matches with the antihuman IgE antibody of described polymerase coupling, described film bar is fixed with str-bio-anaphylactogen.
In a preferred embodiment of the present invention, described polymerase is poly alkaline phosphatase or poly horseradish peroxidase, and described antihuman IgE antibody is sheep antihuman IgE antibody, rabbit antihuman IgE antibody or mouse antihuman IgE antibody.
In a preferred embodiment of the present invention, the manufacturing process of described film bar is: anaphylactogen is dissolved in phosphate buffer and obtains the first solution by (1), the second solution is obtained by soluble in water for Sulfo-NHS-Biotin, by the first solution and the second solution mixing reaction, and column chromatography obtains biotinylated allergen solution; (2) Streptavidin Str is dissolved with antigen coated liquid obtain the 3rd solution, the allergen solution of bioid is dissolved with antigen coated liquid and obtains the 4th solution, by the 3rd solution and the 4th solution spraying on film article; (3) described film bar is dry, more dried film bar and digital label assembling are affixed on film, cut and obtain film bar.
In a preferred embodiment of the present invention, described in step (1), the ratio of anaphylactogen and described phosphate buffer is: 2 ~ 10mg:1ml; Calculate the mM number of described first solution, computing method are: the mM number of albumen quality (mg)/molecular weight of albumen (MW)=protein; When described anaphylactogen to be dissolved in before phosphate buffer containing inappropriate damping fluid, by dialysis in phosphate buffer or the removing of concentrated method; The molar ratio of described biotin and described anaphylactogen is 2 ~ 4:1; Add Sodium azide in described biotinylation allergen solution to deposit.
In a preferred embodiment of the present invention, the spraying of the 3rd solution and the 4th solution described in step (2) adopts BIODOTAD1510 chip point sample instrument to carry out, the first passage of described chip point sample instrument draws the 3rd solution, the 4th solution drawn by second channel, first passage is first rule, and second channel is rule again, and the discharge rate of first passage is 0.5ul/cm, the discharge rate of second channel is 1ul/cm, is sprayed on nitrocellulose filter.
In a preferred embodiment of the present invention, in step (3) by described film bar in humidity be after placing 15min in the constant humidity cabinet of 40%-60% at 37 DEG C dry 12-16 hour, dried film bar and digital label are attached on transparent film by rigging, used by the film bar posted numeral induction cutting machine to cut by 1.88mm, the film bar cut is put into the aluminium foil valve bag that drying agent is housed and seals.
There is provided a kind of detection method of highly sensitive multinomial joint inspection allergenic specific IgE antibody, comprising step is: (1) sample buffer adds in the incubation slot being equipped with film bar, then is sopped up by described sample buffer; (2) blood serum sample after dilution add hatch in incubation slot after sop up, then add cleaning buffer solution and wash; (3) suck liquid in described incubation slot, add enzyme conjugates and hatch; (4) blood serum sample after adding dilution again sops up after hatching; (5) add substrate solution hatch and add water washing again after sopping up; (6) detect after taking out the drying of film bar.
In a preferred embodiment of the present invention, film bar described in step (1) to face up placement with one of word marking, adds after described sample buffer rocks 5 minutes and sop up; Add in step (2) blood serum sample rock hatch 30 minutes after sop up; Add in step (2) after described cleaning buffer solution rocks 5 minutes and sop up, repeated washing 2 times; Enzyme conjugates after adding dilution in step (3) rocks hatches 30 minutes; Add in step (4) blood serum sample rock hatch 30 minutes after sop up; Add substrate solution in step (5) to rock and hatch 10 minutes; Described in step (5), water is distilled water or purified water, adds water and rocks 1 minute, repeated washing 2 times; In step (6), value is read and logging test results to described film bar Reader.
In a preferred embodiment of the present invention, the process being placed in incubation slot at described film bar will prevent film bar dry; With film bar described in fixture gripping; Cross pollution is prevented in testing process.
The invention has the beneficial effects as follows: the kit of highly sensitive multinomial joint inspection allergenic specific IgE antibody of the present invention and method, can the qualitative or allergen specificity antibody IgE concentration that semi-quantitatively detects fast in human serum or blood plasma with sensitivity, and anaphylactogen consumption can be reduced, and can an examination tens of kinds of anaphylactogens, there is quick, that accurate, amount of samples is few advantage, be suitable for carrying out high flux detection.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing used required in describing embodiment is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings, wherein:
Fig. 1 is the structural representation of film bar one preferred embodiment in the kit of highly sensitive multinomial joint inspection allergenic specific IgE antibody of the present invention.
Embodiment
Be clearly and completely described to the technical scheme in the embodiment of the present invention below, obviously, described embodiment is only a part of embodiment of the present invention, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making other embodiments all obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment one:
A kind of kit of highly sensitive multinomial joint inspection allergenic specific IgE antibody is provided, comprises film bar, the antihuman IgE antibody of polymerase coupling and the substrate developer that matches with the antihuman IgE antibody of described polymerase coupling.Described film bar is the fibrous material film being solidified with str-bio-anaphylactogen.The commercially available acquisition of antihuman IgE antibody of described polymerase coupling, during use pH value be 7.4 be the working fluid that the PBS of the TritonX-100 of 0.05% is mixed with concentration 1:100 ~ 10000 containing volume fraction.The substrate developer commercially available acquisition corresponding with the antihuman IgE antibody of described polymerase coupling, also can prepare voluntarily.Described antihuman IgE antibody is sheep antihuman IgE antibody, rabbit antihuman IgE antibody or mouse antihuman IgE antibody, and described polymerase is poly alkaline phosphatase or poly horseradish peroxidase.Above-mentioned is the principal ingredient of reagent, does not comprise and detects conventional reagent used as dilution, cleansing solution etc.
The manufacturing process of described film bar is:
One, biotinylation anaphylactogen preparation
(1) be dissolved in by 2 ~ 10mg anaphylactogen in the phosphate buffer of 1ml and obtain the first solution, containing 100mM phosphate, 150mMNaCl in described phosphate buffer, pH value is 7.2.Calculate the mM number of the first solubilize, computing method are: the mM number of albumen quality (mg)/molecular weight of albumen (MW)=protein.If containing inappropriate damping fluid in anaphylactogen, as Tris or glycocoll, can by dialysis in PBS or the removing of concentrated method.
Anaphylactogen can elect original soybean sensitive as, is to be dissolved in the phosphate buffer of 1ml by 10mg original soybean sensitive, and computing method are: the mM number (0.001mmol) of molecular weight (the MW:10KD)=soybean of quality (the 10mg)/soybean of soybean.
(2) biotin before opening, is balanced to room temperature.Add 2mgSulfo-NHS-Biotin and obtain the second solution in 100ul ultrapure water.
(3) the second solution is slowly joined in the first solution, mixing, room temperature magnetic agitation is reacted and within 30 minutes, is obtained the 3rd solution, and the mol ratio of wherein said described biotin and described anaphylactogen is 2:1.
(4) G25 post on the 3rd solution reacted completely, use phosphate buffer wash-out, collect and obtain biotinylated allergen solution, containing 100mM phosphate, 150mMNaCl in wherein said phosphate buffer, pH value is 7.2.
(5) measure protein content with the absorption value of 280nm, the protein content of original soybean sensitive is 5mg/ml.
(6) biotinylated albumen and biotinylated soybean allergy original solution are deposited to use at 4 DEG C, can add the Sodium azide that mass content is 0.05%.
Two, Str-Bio-anaphylactogen antigenic membrane bar preparation
(1) Streptavidin Str is dissolved to 0.5mg/ml with antigen coated liquid and obtains the 4th solution, be the sucrose of 10% containing 100mM phosphate, 150mMNaCl, massfraction in described antigen coated liquid, pH value is 7.2.
(2) biotinylated soybean allergy original solution is dissolved to 1mg/ml with antigen coated liquid and obtains the 5th solution.
(3) draw the 4th solution and the 5th solution respectively with BIODOTAD1510 chip point sample instrument, the first passage of described chip point sample instrument draws the 4th solution, and the 5th solution drawn by second channel.During line, first passage is first drawn, and second channel is drawn again, and the discharge rate of first passage is 0.5ul/cm, and the discharge rate of second channel is 1ul/cm, is sprayed on nitrocellulose filter.
(4) nitrocellulose filter sprayed after humidity is place 15min in 40%-60% constant humidity cabinet at 37 DEG C dry 12-16 hour.Drying 12 hours during employing original soybean sensitive.
(5) according to the form assembling film forming bar of Fig. 1.
Dried film bar and digital label are attached to (as Fig. 1 measuring ability line (PC) the carbohydrate cross reaction factor (CCD) dermatophagoides pteronyssinus (D1) dust mite (D2) cat skin bits (E1) dogskin bits (E5) egg white (F1) milk (F2) peanut (F13) soybean (F14) wheat (F4) fibert (F17) carrot (F31) Kiwi berry (F84) beef (F27) rod method (M6) argy wormwood (W6) Asiatic plantain (W9) common silver birch (T3) Chinese olive tree pollen (T9) ash end (T25) Gramineae combination (Gx)) on transparent film by rigging, by the film bar posted, numeral induction cutting machine is used to cut by 1.88mm.The film bar cut is put into the aluminium foil valve bag that drying agent is housed and seals.
Embodiment two:
There is provided a kind of detection method of highly sensitive multinomial joint inspection allergenic specific IgE antibody, comprising step is:
(1) antigenic membrane bar is placed in incubation slot, please faces up with one of word marking; Every groove adds 1ml sample buffer (50mMPH7.4Tris damping fluid) and waves shaking table weak vibrations 5 minutes by under film bar room temperature, is sopped up thereafter.
(2) every groove adds 1ml dilute serum sample (with Sample dilution double dilute serum sample), hatches 30 minutes waving weak vibrations on shaking table under room temperature.
(3) liquid in incubation slot is sucked.Then, every groove adds 1ml cleaning buffer solution (0.05% polysorbas20,50mMPH7.4Tris damping fluid, 0.05% Sodium azide), waving on shaking table weak vibrations 5 minutes under room temperature, sucks liquid in incubation slot.Repeated washing 2 times again.
(4) liquid in incubation slot is sucked.Every groove adds 1ml enzyme conjugates (the mouse-anti people IgE(FC fragment of 0.1ug/ml) antibody), hatch 30 minutes waving weak vibrations on shaking table under room temperature.
(5) step 3 is repeated.
(6) suck liquid in incubation slot, every groove adds 1ml substrate solution NBT/BCIP (the bromo-4-of NBT/5-chloro-3-indolyl phosphate toluene amine salt), hatches 10 minutes waving weak vibrations on shaking table under room temperature.
(7) liquid in incubation slot is sucked.Then, every groove adds 1ml distilled water or purified water, waving on shaking table weak vibrations 1 minute under room temperature, sucks liquid in incubation slot.Repeated washing 2 times again..
(8), after the drying of film bar, value is read and logging test results with Reader.
To note in testing process: hatch in process at antigenic membrane bar, film bar please don't be made dry.Please don't directly contact antigenic membrane bar with hand, please tweezers gripping film bar be use.After completing sera incubation, should note avoiding cross pollution when toppling over reactant liquor.In the process of hatching, weak vibrations antigenic membrane bar on shaking table please waved under room temperature.The antigenic membrane bar that PLSCONFM is all and sample meet testing requirement.
Test result sees the following form:
| Concentration (IU/mL) | Rank | Result is explained |
| ＜0.35 | 0 | Specific antibodies do not detected |
| 0.35-0.7 | + | Slight allergy, usually possesses certain susceptibility without clinical symptoms |
| 0.7-3.5 | ++ | Moderate is irritated, usually there will be clinical symptoms after a large amount of contact |
| 3.5-17.5 | +++ | Hyperirritability, usual clinical symptoms also there will be |
| 17.5-50 | ++++ | Very the allergy of height, has clinical symptoms usually |
| 50-100 | +++++ | The very allergy of high level |
According to the inventive method, according to operation steps operation, just can easily with the naked eye qualitative or semi-quantitatively interpretation detection human serum or blood plasma in multiple allergenic specific IgE antibody levels; Use the interpretoscope of band data processing function, the multiple allergenic specific IgE antibody concentration in human serum or blood plasma can be detected quantitatively.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize description of the present invention to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other relevant technical field, be all in like manner included in scope of patent protection of the present invention.
Claims (9)
1. the kit of a highly sensitive multinomial joint inspection allergenic specific IgE antibody, it is characterized in that, comprise film bar, the antihuman IgE antibody of polymerase coupling and the substrate developer that matches with the antihuman IgE antibody of described polymerase coupling, described film bar is fixed with str-bio-anaphylactogen.
2. kit according to claim 1, is characterized in that, described polymerase is poly alkaline phosphatase or poly horseradish peroxidase, and described antihuman IgE antibody is sheep antihuman IgE antibody, rabbit antihuman IgE antibody or mouse antihuman IgE antibody.
3. kit according to claim 1, it is characterized in that, the manufacturing process of described film bar is: anaphylactogen is dissolved in phosphate buffer and obtains the first solution by (1), the second solution is obtained by soluble in water for Sulfo-NHS-Biotin, by the first solution and the second solution mixing reaction, and column chromatography obtains biotinylated allergen solution; (2) Streptavidin Str is dissolved with antigen coated liquid obtain the 3rd solution, the allergen solution of bioid is dissolved with antigen coated liquid and obtains the 4th solution, by the 3rd solution and the 4th solution spraying on film article; (3) described film bar is dry, more dried film bar and digital label assembling are affixed on film, cut and obtain film bar.
4. kit according to claim 3, is characterized in that, described in step (1), the ratio of anaphylactogen and described phosphate buffer is: 2 ~ 10mg:1ml; Calculate the mM number of described first solution, computing method are: the mM number of albumen quality (mg)/molecular weight of albumen (MW)=protein; When described anaphylactogen to be dissolved in before phosphate buffer containing inappropriate damping fluid, by dialysis in phosphate buffer or the removing of concentrated method; The molar ratio of described biotin and described anaphylactogen is 2 ~ 4:1; Add Sodium azide in described biotinylation allergen solution to deposit.
5. kit according to claim 3, it is characterized in that, the spraying of the 3rd solution and the 4th solution described in step (2) adopts BIODOTAD1510 chip point sample instrument to carry out, the first passage of described chip point sample instrument draws the 3rd solution, and the 4th solution drawn by second channel, and first passage is first rule, second channel is rule again, the discharge rate of first passage is 0.5ul/cm, and the discharge rate of second channel is 1ul/cm, is sprayed on nitrocellulose filter.
6. kit according to claim 3, it is characterized in that, in step (3) by described film bar in humidity be after placing 15min in the constant humidity cabinet of 40%-60% at 37 DEG C dry 12-16 hour, dried film bar and digital label are attached on transparent film by rigging, used by the film bar posted numeral induction cutting machine to cut by 1.88mm, the film bar cut is put into the aluminium foil valve bag that drying agent is housed and seals.
7. a detection method for highly sensitive multinomial joint inspection allergenic specific IgE antibody, is characterized in that, comprises step and is: (1) sample buffer adds in the incubation slot being equipped with film bar, then is sopped up by described sample buffer; (2) blood serum sample after dilution add hatch in incubation slot after sop up, then add cleaning buffer solution and wash; (3) suck liquid in described incubation slot, add enzyme conjugates and hatch; (4) blood serum sample after adding dilution again sops up after hatching; (5) add substrate solution hatch and add water washing again after sopping up; (6) detect after taking out the drying of film bar.
8. detection method according to claim 7, is characterized in that, film bar described in step (1) to face up placement with one of word marking, adds after described sample buffer rocks 5 minutes and sop up; Add in step (2) blood serum sample rock hatch 30 minutes after sop up; Add in step (2) after described cleaning buffer solution rocks 5 minutes and sop up, repeated washing 2 times; Enzyme conjugates after adding dilution in step (3) rocks hatches 30 minutes; Add in step (4) blood serum sample rock hatch 30 minutes after sop up; Add substrate solution in step (5) to rock and hatch 10 minutes; Described in step (5), water is distilled water or purified water, adds water and rocks 1 minute, repeated washing 2 times; In step (6), value is read and logging test results to described film bar Reader.
9. detection method according to claim 7, is characterized in that, the process being placed in incubation slot at described film bar will prevent film bar dry; With film bar described in fixture gripping; Cross pollution is prevented in testing process.
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| CN106596929A (en) * | 2016-12-27 | 2017-04-26 | 斯德润(北京)医疗诊断用品有限公司 | Kit for detecting IgG antibodies of allergens of 30 types of foods and preparation method |
| CN106645710A (en) * | 2016-12-27 | 2017-05-10 | 斯德润(北京)医疗诊断用品有限公司 | Kit for detecting 30 food allergen IgG antibodies and preparation method of kit |
| CN113125739A (en) * | 2021-04-16 | 2021-07-16 | 杭州浙大迪迅生物基因工程有限公司 | Fluorescent chip quantitative detection kit |
| CN113125753A (en) * | 2021-04-16 | 2021-07-16 | 杭州浙大迪迅生物基因工程有限公司 | Kit for detecting specific antibody of dust mite component |
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| CN106596929A (en) * | 2016-12-27 | 2017-04-26 | 斯德润(北京)医疗诊断用品有限公司 | Kit for detecting IgG antibodies of allergens of 30 types of foods and preparation method |
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| CN113125739A (en) * | 2021-04-16 | 2021-07-16 | 杭州浙大迪迅生物基因工程有限公司 | Fluorescent chip quantitative detection kit |
| CN113125753A (en) * | 2021-04-16 | 2021-07-16 | 杭州浙大迪迅生物基因工程有限公司 | Kit for detecting specific antibody of dust mite component |
| CN113125753B (en) * | 2021-04-16 | 2022-07-26 | 杭州浙大迪迅生物基因工程有限公司 | Kit for detecting specific antibody of dust mite component |
| CN113125739B (en) * | 2021-04-16 | 2022-07-26 | 杭州浙大迪迅生物基因工程有限公司 | Fluorescent chip quantitative detection kit |
| WO2022217732A1 (en) * | 2021-04-16 | 2022-10-20 | 杭州浙大迪迅生物基因工程有限公司 | Kit for detecting dust mite component specific antibody |
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