CN103995112B - For the test strips and preparation method thereof of specificity animal wool dandruff allergens IgE examination - Google Patents

For the test strips and preparation method thereof of specificity animal wool dandruff allergens IgE examination Download PDF

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CN103995112B
CN103995112B CN201410231215.4A CN201410231215A CN103995112B CN 103995112 B CN103995112 B CN 103995112B CN 201410231215 A CN201410231215 A CN 201410231215A CN 103995112 B CN103995112 B CN 103995112B
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test strips
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preparation
antigen
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CN103995112A (en
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蒋琳
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Zhongsheng Beikong Biological Science & Technology Co Ltd
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    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Abstract

The invention provides a kind of test strips for specificity animal wool dandruff allergens IgE examination, described test strips is coated with the animal wool dander antigen of colloid gold label.The present invention utilizes colloidal gold-labeled method, use colloid gold label specificity animal wool dandruff allergens first, then the anaphylactogen after mark is evenly sprayed line in cellulose acetate membrane or nitrocellulose filter, be prepared into test strips product and carry out Allergic skin test, easy and simple to handle, result is accurate, with low cost, can realize multinomially freely detecting.

Description

For the test strips and preparation method thereof of specificity animal wool dandruff allergens IgE examination
Technical field
The present invention relates to colloidal gold-labeled method field, specifically, relate to a kind of test strips for specificity animal wool dandruff allergens IgE examination and preparation method thereof.
Background technology
Anaphylactia is modal a kind of disease in contemporary society, it is caused by the anaphylactogen extensively existed, the crowd of China about 5 ~ 10%, US and European about 5 ~ 25% was invaded and harassed by anaphylaxis, wherein child and teenager particularly evident, endanger more serious.Allergic reaction (allergy) is immune body when again contacting identical allergen, and react the excessively violent and physiological dysfunction that causes and tissue damage pathologic immune reaction.Relate to clinical departments, as internal medicine comprises bronchial astehma, anaphylactic shock, serum sickness, connective tissue disease (CTD), glomerulonephritis, antimigraine etc., surgery comprises naltrindole, ear-nose-throat department has allergic rhinitis, exudative otitis media, meniere's disease etc., gynemetrics comprises erythroblastosis fetalis, ophthalmology comprises allergic conjunctivitis, sympathetic ophthalmia, the department of stomatology comprises RAU, dept. of dermatology's disease about more than half is that allergic reaction is sick, also has transfusion reaction, drug allergy, anaphylactic shock etc. in addition.
The investigation statistics data display that in April, 2011, State Statistics Bureau promulgated, National urban population about 5.7 hundred million, wherein have 1.5 hundred million city anaphylactogens morbidity populations, the crowd being throughout the year in anaphylactogen state reaches 5,122 ten thousand people.Estimate accordingly, the Chinese population city allergic disease incidence of disease is 27%, adds non-metropolitan population, and actual Chinese allergic disease number of the infected is about 3.6 hundred million.But nearly ten years, the diagnosis and treatment rate of China anaphylactogen patient is less than 1%, and the anaphylactogen patient of nearly 99% can not get Diagnosis and Treat timely.
According to incompletely statistics, the whole nation 15000 hospitals above county level in introduce UniCAP less than 150, introduction rate only accounts for 1%.This ratio, compared with the introduction rate of other biochemistry detection equipment (about 40 ~ 50%), is enough to illustrate that the in-vitro diagnosis field of anaphylactogen also exists great development space.Meanwhile, Extrinsic allergen checkout equipment and the main dependence on import of reagent, these raw materials all pick up from American-European import anaphylactogen reagent, with the region difference of China patient and ethnic group difference also still to be tested.Based on above-mentioned situation, develop a kind of Allergic skin test system meeting localization requirement, the research and development comprising the Allergic skin test instrument of China's autonomous innovation and reagent are imperative.
The method of Allergic skin test is mainly divided into in-vivo diagnostic and in-vitro diagnosis.In-vivo diagnostic comprises skin diagnosis (qualitative examination) and provocative diagnosis.Common skin diagnosis comprises intracutaneous experiment, pricking method experiment, scratch experiment, patch experiment etc.Provocative diagnosis comprises conjunctiva experiment, nose experiment, bronchus experiment, food experiment, medicine excitation experiment, physical property excitation experiment etc.Because the influence factor of in-vivo diagnostic is complicated, accuracy is low, and result can only be carried out qualitative, and inspection itself brings misery to patient, and to severe allergy, patient even exists the factors such as life danger, limits further developing of in-vivo diagnostic.
The principle of in-vitro diagnosis is based on IgE antibody diagnosis, and diagnostic techniques comprises Diagnosis of Sghistosomiasis notation, radiation allergen absorption diagnosis (RAST), fluorescence enzyme linked immunosorbent assay (FEIA), euzymelinked immunosorbent assay (ELISA) (ELISA) etc.
At present, the method of domestic conventional clinical detection anaphylactogen is based on the In vivo assay Cells of skin puncture test (SPT), namely with the direct pricking method skin of the irritated stoste of dilution, observe check point and whether occur wheal or flare reaction, carry out feminine gender or positive judgement.The advantages such as although SPT has easy, cost is low, patient's visible result.But its result is by many factors, and accuracy is low, inspection itself brings misery to patient especially pediatric patient, and to severe allergy, patient is even in peril of one's life.Domestic in-vitro diagnosis field is in a backward condition, and substantially concentrates on manual operations and sxemiquantitative semi-automated analysis state.There is loaded down with trivial details, the sensitivity of operation and precision poor, the shortcoming that the test duration is grown.
Compared with in vivo studies, only need a small amount of serum can screen and determine hundred kinds of anaphylactogens, have effectiveness parameters reliable, not by drug influence, nonsystemic reaction danger waits remarkable advantage.What Application comparison was outstanding is PHARDIAUNICAP system and SIEMENS diagnostic system.PHARDIAUNICAP system from the seventies come out since, always by the goldstandard as allergen diagnosis.The eighties enters China, is in monopoly position always.And SIEMENS diagnostic system is the representative of anaphylactogen Automation New Technology, is allergen diagnosis system emerging in recent years, not yet enters Chinese market.
In developed country, full-automatic Allergic skin test current is clinically based on euzymelinked immunosorbent assay (ELISA).Conventional two kinds of methods: enzyme linked immunological fluorometry and biotin-avidin system-euzymelinked immunosorbent assay (ELISA).
The classical system of enzyme linked immunological fluorometry is the ImmunoCAP detection system of Pharmacia (Pharmacia) company, and its adopts cellulose solid phase carrier, this solid phase carrier after bromination oxygen activation with anaphylactogen covalent bond.Anaphylactogen is combined on solid phase carrier in advance, hatches for 37 DEG C after adding blood sample, as there being the specific IgE for this anaphylactogen in patients serum, namely forms this anti-allergen antibodies compound anti-.Add the ELIAS secondary antibody of enzyme labeling after wash-out, form the bond of solid phase carrier-anaphylactogen-specific IgE-ELIAS secondary antibody.Again add substrate after wash-out, produce enzyme catalysis fluorescent product.Measure fluorescent value.Specific IgE content is converted into according to the size of fluorescence absorbance.
Biotin-avidin system-euzymelinked immunosorbent assay (ELISA) adopts the ALLERG-O-LIQ Analytical system of Fooke company, and ELISA Plate wraps quilt by antihuman IgE antibody, can be used to detect total IgE and specific IgE.Hatch after adding blood sample and wash, ELISA Plate is in conjunction with all IgE, washing can avoid other immune globulins to disturb (as specific antigen), add biotin labeled anaphylactogen again, then add the Streptavidin bond and chromogenic substrate that combine enzyme, detect the IgE content for specific allergen by the change of absorbance after enzyme-to-substrate reaction.
Summary of the invention
The object of this invention is to provide a kind of test strips for specificity animal wool dandruff allergens IgE examination.
Another object of the present invention is to provide the preparation method of described test strips.
In order to realize the object of the invention, a kind of test strips for specificity animal wool dandruff allergens IgE examination of the present invention, described test strips is coated with the animal wool dander antigen of colloid gold label.
Particularly, test strips of the present invention is nitrocellulose filter or the cellulose acetate membrane of the animal wool dander antigen being coated with colloid gold label.
Wherein, the pH value of described test strips is 8.5-10, and the mol ratio between colloid gold particle and animal wool dander antigen is 5-20:1.Described animal wool scurf includes but not limited to cat fur bits, dog skin bits, wool scurf, camel hair scurf, rabbit hair scurf, horsehair scurf, pig fur bits, ox hair scurf etc.
The preparation method of described cat hair dander antigen is: get cat fur bits 50g, add water 100g, pulverize with tissue pulverizer after freeze-drying, 2000r/min is centrifugal.Taking precipitate, add the analysis of 200mL acetone and purely carry out ungrease treatment, repeat degreasing more than 5 times, carry out centrifugal after each degreasing terminates, the material after centrifugal is placed in fuming cupboard, and by the volatilization of the acetone of remnants completely, degreasing again after pulverizing, obtains cat hair dander antigen.
The present invention also provides the preparation method of described test strips, comprises the following steps:
1) preparation of animal wool scurf antigenic solution: be dissolved in the NaCl solution of 0.9%pH7.0 by animal wool dander antigen to be marked, makes its final concentration be 50-500 μ g/ml, obtains animal wool scurf antigenic solution;
2) preparation of colloidal gold solution: use 0.1MK 2cO 3solution and 0.1MHCl adjust 0.01%HAuCl 4the pH value of colloidal gold solution is to 8.5-10, and make the particle diameter of collaurum reach 40-60nm, concentration reaches 0.5-2%, obtains colloidal gold solution;
3) colloid gold label animal wool dander antigen: use 0.1MK 2cO 3solution adjusts the pH value of the antigenic solution of step 1) to 8.5-10, add step 2 wherein while stirring) colloidal gold solution, the volume ratio of antigenic solution and colloidal gold solution is 1-10:100, add stabilizing agent simultaneously and consider antigen and colloid gold particle polymeric precipitation to be worth doing to prevent animal fur, obtain collaurum-animal wool scurf antigen conjugates; Described stabilizing agent is the polyglycol of BSA (bovine serum albumin(BSA)) and molecular weight 20KD, and the two final concentration is respectively 1% and 0.1%;
4) purifying of the animal wool dander antigen of colloid gold label: by step 3) in preparation collaurum-animal wool scurf antigen conjugates through the centrifugal 20min of 1500r/min; Collecting precipitation is dissolved in stabilizing agent, makes its concentration reach 5-30%; Then add on SephacrylS-400 chromatographic column and carry out purifying, by the animal wool scurf antigenic solution filtration sterilization of the colloid gold label of purifying, 4 DEG C of preservations; Described stabilizing agent is the polyglycol solution of 1%BSA and 0.1% molecular weight 20KD;
5) the bag quilt of test strips: by step 4) in preparation colloid gold label animal wool scurf antigenic solution spray line in test strips, namely obtain the test strips finished product for specificity animal wool dandruff allergens IgE examination after drying.
Aforesaid preparation method, step 4) in application of sample amount be 1/10 of SephacrylS-400 chromatographic column volume, using stabilizing agent as eluent, flow velocity is 5-10ml/h; Described stabilizing agent is the polyglycol solution of 1%BSA and 0.1% molecular weight 20KD.
During use, test strips of the present invention is immersed in human serum, IgE antibody in serum is combined with specific allergen, produce purple band.Classification is carried out according to bar colored depth contrast ratio colour atla,
The present invention utilizes colloidal gold-labeled method, use colloid gold label specificity animal wool dandruff allergens antigen first, then the anaphylactogen after mark is evenly sprayed line in cellulose acetate membrane or nitrocellulose filter, be prepared into test strips product and carry out Allergic skin test, easy and simple to handle, result is accurate, with low cost, can realize multinomially freely detecting.
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.
Embodiment is used for test strips of specificity animal wool dandruff allergens IgE examination and preparation method thereof
Preparation for the test strips of specificity animal wool dandruff allergens IgE examination comprises: the bag quilt of the preparation of antigenic solution to be marked, the preparation waiting to mark colloidal gold solution, colloid gold label cat hair antigen, the purifying of collaurum cat hair antigen, the particle diameter qualification of collaurum protein body and test strips.Concrete steps are as follows:
1, the preparation of antigenic solution to be marked
The preparation of cat hair dander antigen: get cat fur bits 50g, add water 100g, pulverize with tissue pulverizer after freeze-drying, 2000r/min is centrifugal.Taking precipitate, add the analysis of 200mL acetone and purely carry out ungrease treatment, repeat degreasing more than 5 times, carry out centrifugal after each degreasing terminates, the material after centrifugal is placed in fuming cupboard, and by the volatilization of the acetone of remnants completely, degreasing again after pulverizing, obtains cat hair dander antigen.
Cat hair dander antigen to be marked is dissolved in the NaCl solution of 0.9%pH7.0, makes its final concentration be 50-500 μ g/ml, obtain cat fur bits antigenic solution.
2, the preparation of marking colloidal gold solution is waited
Use 0.1MK 2cO 3solution or 0.1MHCl adjust 0.01%HAuCl 4the pH value to 9.0 of colloidal gold solution, make the particle diameter of collaurum reach 50nm, concentration reaches 1%.
3, colloid gold label cat hair dander antigen: collaurum and antigenic solution are used 0.1MK respectively 2cO 3solution adjusts pH to 9.0, and electromagnetic agitation antigenic solution, adds colloidal gold solution, the volume ratio of antigenic solution and colloidal gold solution is 1-10:100, continue to stir 10min, add the polyglycol of stabilizing agent BSA and molecular weight 20KD, obtain the polyglycol solution of 1%BSA and 0.1% molecular weight 20KD.The consumption of this process need adjustment antigen is that grape wine is red for the best with the solution after colloid gold label antigen completes.
4, the purifying of the cat mao mao dander antigen of colloid gold label: by the collaurum-animal wool scurf antigen conjugates of preparation in step 3 through the centrifugal 20min of 1500r/min; Collecting precipitation is dissolved in stabilizing agent (described stabilizing agent is the polyglycol solution of 1%BSA and 0.1% molecular weight 20KD), makes its concentration reach 5-30%; Then add on SephacrylS-400 chromatographic column and carry out purifying, by the cat mao mao dander antigen solution filtration sterilization of the colloid gold label of purifying, 4 DEG C of preservations.
In purge process, application of sample amount is 1/10 of SephacrylS-400 chromatographic column volume, and using stabilizing agent (described stabilizing agent is the polyglycol solution of 1%BSA and 0.1% molecular weight 20KD), as eluent, flow velocity is 5-10ml/h.
5, the particle diameter qualification of collaurum protein body
The measurement of colloid gold particle mean diameter: observing colloid gold grain under transmission electron microscope, the mean diameter calculating 100 gold grains should within the scope of 40 ~ 60nm.
6, the bag quilt of test strips
By negative control solution (PBS solution containing 40-70% bovine serum albumin(BSA)), the antigenic solution of step 4 purifying evenly sprays line.
The spray line of negative control and antigenic solution is carried out on immune chromatography test paper nitrocellulose filter surface.Quantitative work is carried out with pneumatic sprayhead.With the three-dimensional platform reciprocal spray of interval back and forth line.Finally that the nitrocellulose filter sprayed is dry and cut slivering.
7, result judges
Carry out classification according to bar colored depth contrast ratio colour atla, stage division is in table 1:
Table 1 grade scale
IgE concentration in serum Classification Specific IgE content
＜0.35IU/mL 0 Nothing
0.35-0.75IU/mL 1 Low
0.75-3.5IU/mL 2 Increase
3.5-17.5IU/mL 3 Remarkable increase
17.5-50IU/mL 4 Remarkable increase
50-100IU/mL 5 Higher
＞100IU/mL 6 High
In visual colorimetry, the band on colorimetric card is for directly to carry out the red spray line of Grape Skin on immune chromatography test paper nitrocellulose filter surface.The concentration difference red according to spray line Grape Skin forms gradient (table 2):
Table 2 different I gE concentration corresponds to the concentration that on colorimetric card, Grape Skin is red
IgE concentration in serum The red concentration of Grape Skin
＜0.35IU/mL Nothing
0.35-0.75IU/mL 0.25g/L
0.75-3.5IU/mL 0.5g/L
3.5-17.5IU/mL 2g/L
17.5-50IU/mL 5g/L
50-100IU/mL 9g/L
＞100IU/mL 12g/L
Test strips for specificity animal wool dandruff allergens IgE examination provided by the invention is suitable for industrialization large-scale production, reduces production cost, greatly reduces the price that anaphylactogen individual event detects.In addition, to the anaphylactogen raw material with China domestic feature, realize localization, be more suitable for the diagnosis of Chinese population anaphylactogen, improve the accuracy of diagnosis.
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 (3)

1. for a test strips for specificity cat hair dandruff allergens IgE examination, it is characterized in that, described test strips is nitrocellulose filter or the cellulose acetate membrane of the cat hair dander antigen being coated with colloid gold label;
The pH value of described test strips is 8.5-10, and the mol ratio between colloid gold particle and cat hair dander antigen is 5-20:1;
The preparation method of described cat hair dander antigen is: get cat fur bits 50g, add water 100g, pulverize with tissue pulverizer after freeze-drying, 2000r/min is centrifugal, taking precipitate, add the analysis of 200mL acetone and purely carry out ungrease treatment, repeat degreasing more than 5 times, carry out centrifugal after each degreasing terminates, material after centrifugal is placed in fuming cupboard, by the volatilization of the acetone of remnants completely, degreasing again after pulverizing, obtains cat hair dander antigen.
2. the preparation method of test strips described in claim 1, is characterized in that, comprises the following steps:
1) preparation of cat fur bits antigenic solution: be dissolved in the NaCl solution of 0.9%pH7.0 by cat hair dander antigen to be marked, make its final concentration be 50-500 μ g/ml, obtains cat fur bits antigenic solution;
2) preparation of colloidal gold solution: use 0.1MK 2cO 3solution and 0.1MHCl adjust 0.01%HAuCl 4the pH value of colloidal gold solution is to 8.5-10, and make the particle diameter of collaurum reach 40-60nm, concentration reaches 0.5-2%, obtains colloidal gold solution;
3) combination of collaurum and cat hair dander antigen: use 0.1MK 2cO 3solution adjusts the pH value of the antigenic solution of step 1) to 8.5-10, add step 2 wherein while stirring) colloidal gold solution, the volume ratio of antigenic solution and colloidal gold solution is 1-10:100, add stabilizing agent to prevent cat hair dander antigen and colloid gold particle polymeric precipitation simultaneously, obtain collaurum-cat fur bits antigen conjugates; Described stabilizing agent is the polyglycol of BSA and molecular weight 20KD, and the two final concentration is respectively 1% and 0.1%;
4) purifying of the cat hair dander antigen of colloid gold label: by step 3) in the collaurum-cat fur bits antigen conjugates of preparation through the centrifugal 20min of 1500r/min; Collecting precipitation is dissolved in stabilizing agent, makes its concentration reach 5-30%; Then add on SephacrylS-400 chromatographic column and carry out purifying, by the filtration sterilization of the cat fur of the colloid gold label of purifying bits antigenic solution, 4 DEG C of preservations; Described stabilizing agent is the polyglycol solution of 1%BSA and 0.1% molecular weight 20KD;
5) the bag quilt of test strips: by step 4) in preparation colloid gold label cat fur bits antigenic solution spray line in test strips, namely obtain the test strips finished product for specificity cat hair dandruff allergens IgE examination after drying.
3. method according to claim 2, is characterized in that, step 4) in application of sample amount be 1/10 of SephacrylS-400 chromatographic column volume, using stabilizing agent as eluent, flow velocity is 5-10ml/h; Described stabilizing agent is the polyglycol solution of 1%BSA and 0.1% molecular weight 20KD.
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CN105353134A (en) * 2015-11-17 2016-02-24 苏州浩欧博生物医药有限公司 Kit detecting dog-hair allergen specific IgE antibody and method
CN105445257A (en) * 2015-11-17 2016-03-30 苏州浩欧博生物医药有限公司 Kit and method for detecting allergen-specific IgE antibody of cat hair
CN105403692A (en) * 2015-11-17 2016-03-16 苏州浩欧博生物医药有限公司 Kit and method for detecting dermatophagoides pteronyssinus allergen specificity IgE antibody
CN105353133A (en) * 2015-11-17 2016-02-24 苏州浩欧博生物医药有限公司 Kit detecting dermatophagoides-farinae allergen specific IgE antibody and method

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