CN103995131A - Test strip for screening specific weedy pollen allergen IgE (Immunoglobulin E) and preparation method thereof - Google Patents
Test strip for screening specific weedy pollen allergen IgE (Immunoglobulin E) and preparation method thereof Download PDFInfo
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- CN103995131A CN103995131A CN201410232320.XA CN201410232320A CN103995131A CN 103995131 A CN103995131 A CN 103995131A CN 201410232320 A CN201410232320 A CN 201410232320A CN 103995131 A CN103995131 A CN 103995131A
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Abstract
The invention provides a test strip for screening specific weedy pollen allergen IgE (Immunoglobulin E). The test strip is coated by a weedy pollen antigen labeled by colloidal gold. The colloidal gold labelling technology is utilized in the invention; the specific weedy pollen allergen is labelled by using colloidal gold for the first time; then, the labelled allergen is uniformly sprayed on a cellulose acetate membrane or a nitrocellulose membrane, therefore, the test strip is prepared for detecting allergens; the test strip disclosed by the invention is simple and convenient to operate, accurate in result and low in cost; and a number of free tests can be realized.
Description
Technical field
The present invention relates to colloidal gold-labeled method field, specifically, relate to a kind of test strips for specificity weeds class pollen allergens IgE examination and preparation method thereof.
Background technology
Anaphylactia is modal a kind of disease in contemporary society, it is to be caused by the anaphylactogen extensively existing, the crowd of China approximately 5～10%, US and European approximately 5～25% was invaded and harassed by anaphylaxis, and wherein child and teenager are particularly evident, endangered more serious.Allergic reaction (allergy) is immune body while again contacting identical allergen, the pathologic immune response of the disorderly and tissue damage of the physiological function that is excessively violent and that cause that reacts.Relate to clinical departments, as internal medicine comprises bronchial astehma, anaphylactic shock, serum sickness, connective tissue disease (CTD), glomerulonephritis, antimigraine etc., surgery comprises that naltrindole, ear-nose-throat department have 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 approximately more than half is allergic reaction disease, also has in addition transfusion reaction, drug allergy, anaphylactic shock etc.
The investigation statistics data that in April, 2011, State Statistics Bureau promulgated show, National urban population approximately 5.7 hundred million, wherein has 1.5 hundred million city anaphylactogens morbidity populations, reaches 5,122 ten thousand people in the crowd of anaphylactogen state throughout the year.Estimation accordingly, the Chinese population city allergic disease incidence of disease is 27%, adds non-metropolitan population, actual Chinese allergic disease number of the infected is about 3.6 hundred million.But nearly ten years, China anaphylactogen patient's diagnosis and treatment rate is less than 1%, nearly 99% anaphylactogen patient can not get diagnosing timely and treating.
According to incompletely statistics, 150 of the less thaies of UniCAP are introduced in the whole nation in 15000 hospitals above county level, and introduction rate only accounts for 1%.Compared with the introduction rate of this ratio and other biochemistry detection equipment (approximately 40～50%), be enough to illustrate that the in-vitro diagnosis field of anaphylactogen exists great development space.Meanwhile, the main dependence on import of Extrinsic allergen checkout equipment and reagent, these raw materials all pick up from American-European import anaphylactogen reagent, poor also still to be tested with China patient's region difference and ethnic group.Based on above-mentioned situation, develop a kind of anaphylactogen detection system that meets localization requirement, the research and development including anaphylactogen detector and the reagent of China's autonomous innovation are imperative.
The method that anaphylactogen detects 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.Due to the influence factor complexity of in-vivo diagnostic, accuracy is low, and result can only be carried out qualitative, and inspection itself brings misery to patient, and severe allergy patient is even existed to the factors such as life danger, has limited further developing of in-vivo diagnostic.
The principle of in-vitro diagnosis is based on IgE antibody diagnosis, comprises Diagnosis of Sghistosomiasis notation, radiation allergen absorption diagnosis (RAST), fluorescence enzyme linked immunosorbent assay (FEIA), euzymelinked immunosorbent assay (ELISA) (ELISA) etc. in diagnostic techniques.
At present, the method of domestic conventional clinical detection anaphylactogen is taking skin puncture test (SPT) as main In vivo assay Cells, 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 subject to 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.Exist loaded down with trivial details, the sensitivity of operation and precision poor, the shortcoming that the test duration is long.
Compared with in vivo studies, only need a small amount of serum can screen and determine hundred kinds of anaphylactogens, there is effectiveness parameters reliable, be not subject to drug influence, the dangerous remarkable advantage that waits of nonsystemic reaction.What application was relatively more outstanding is PHARDIA UNICAP system and SIEMENS diagnostic system.PHARDIA UNICAP system, since coming out the seventies, has been used as the goldstandard of allergen diagnosis always.Enter China the eighties, always in monopoly position.And SIEMENS diagnostic system is the representative of anaphylactogen Automation New Technology, be in recent years emerging allergen diagnosis system, not yet enter Chinese market.
In developed country, current full-automatic anaphylactogen detection is based on euzymelinked immunosorbent assay (ELISA) clinically.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 Immuno CAP 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, adds after blood sample 37 DEG C to hatch, and as there being the specific IgE for this anaphylactogen in patients serum, forms anti-this anaphylactogen antibody complex.After wash-out, add the ELIAS secondary antibody of enzyme labeling, form the bond of solid phase carrier-anaphylactogen-specific IgE-ELIAS secondary antibody.Again after wash-out, add substrate, produce enzyme catalysis fluorescent product.Measure fluorescent value.Be converted into specific IgE content according to the size of fluorescence absorbance.
Biotin-avidin system-euzymelinked immunosorbent assay (ELISA) adopts the ALLERG-O-LIQ of Fooke company to measure system, and ELISA Plate is coated with by anti human IgE antibody, can be used to detect total IgE and specific IgE.After adding blood sample, hatch and wash, ELISA Plate is in conjunction with all IgE, washing can avoid other immune globulins to disturb (as specific antigen), add again biotin labeled anaphylactogen, then add the Streptavidin bond and the chromogenic substrate that combine enzyme, after reacting by enzyme-to-substrate, the variation of absorbance detects the IgE content for specific allergen.
Summary of the invention
The object of this invention is to provide a kind of test strips for specificity weeds class pollen 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 weeds class pollen allergens IgE examination of the present invention, described test strips is coated with the weeds class pollen-antigen of colloid gold label.
Particularly, test strips of the present invention is nitrocellulose filter or the cellulose acetate membrane that is coated with the weeds class pollen-antigen of colloid gold label.
Wherein, the pH value of described test strips is 8.5-10, and the mol ratio between colloid gold particle and weeds class pollen-antigen is 10-15:1.Described weeds class includes but not limited to that timothy grass, chrysanthemum thatch, rye grass, reed, bromegrass, humulus grass, argy wormwood, yorkshire fog grass, meadow fescue, hundred acts reach grass, white bent, false Chinese sorghum, rye, oat, wheat, foxtail, barley, corn, salt grass, paspalum notatum etc.
Described weeds class pollen-antigen is timothy grass pollen-antigen; The preparation method of described timothy grass pollen-antigen is: get timothy grass pollen 50g, cross 200 mesh sieves and remove impurity, add that 200mL ethanol analysis is pure carries out ungrease treatment, repeat degreasing more than 10 times, after each degreasing finishes, carry out centrifugal, material after centrifugal is placed in fuming cupboard, by remaining ethanol volatilization completely, obtains timothy grass pollen-antigen.
The present invention also provides the preparation method of described test strips, comprises the following steps:
1) preparation of weeds class pollen-antigen solution: weeds class pollen to be marked is dissolved in the NaCl solution of 0.9%pH7.0, and making its final concentration is 50-500 μ g/ml, obtains weeds class pollen-antigen solution;
2) preparation of colloidal gold solution: use 0.1M K
2cO
3solution and 0.1M HCl adjust 0.01%HAuCl
4the pH value of colloidal gold solution, to 8.5-10, makes the particle diameter of collaurum reach 40-60nm, and concentration reaches 0.5-2%, obtains colloidal gold solution;
3) combination of collaurum and weeds class pollen-antigen: use 0.1M K
2cO
3the pH value of the antigenic solution of solution tune step 1) is to 8.5-10, add wherein while stirring step 2) colloidal gold solution, the volume ratio of antigenic solution and colloidal gold solution is 1-10:100, add stabilizing agent to prevent weeds class pollen-antigen and colloid gold particle polymerization precipitation simultaneously, obtain collaurum-weeds class pollen-antigen bond; 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 weeds class pollen-antigen of colloid gold label: by step 3) in preparation collaurum-weeds class pollen-antigen bond through the centrifugal 20min of 1500r/min; Collecting precipitation is dissolved in stabilizing agent, makes its concentration reach 5-30%; Then add on Sephacryl S-400 chromatographic column and carry out purifying, by the weeds class pollen-antigen solution filter degerming 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) test strips is coated: by step 4) in the weeds class pollen-antigen solution spray line of colloid gold label of preparation in test strips, obtain the test strips finished product for specificity weeds class pollen allergens IgE examination after dry.
Aforesaid preparation method, step 4) in application of sample amount be 1/10 of Sephacryl S-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.
When use, test strips of the present invention is immersed in human serum, IgE antibody in serum is combined with specific allergen, produce purple band.Carry out classification according to the colored depth contrast ratio of bar colour atla,
The present invention utilizes colloidal gold-labeled method, use first colloid gold label specificity weeds class pollen hypersensitivity Proantigen, then the anaphylactogen after mark is evenly sprayed to line on cellulose acetate membrane or nitrocellulose filter, be prepared into test strips product and carry out anaphylactogen detection, easy and simple to handle, result is accurate, with low cost, can realize multinomially freely detecting.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art, the raw materials used commercial goods that is.
Embodiment is used for test strips of specificity weeds class pollen allergens IgE examination and preparation method thereof
The preparation that is used for the test strips of specificity timothy grass pollen allergens IgE examination comprises purifying, the particle diameter qualification of collaurum protein body and being coated with of test strips of the preparation of the preparation of antigenic solution to be marked, colloidal gold solution to be marked, colloid gold label timothy grass pollen-antigen, collaurum timothy grass pollen-antigen.Concrete steps are as follows:
1, the preparation of antigenic solution to be marked
The preparation method of timothy grass pollen-antigen is: get timothy grass pollen 50g, cross 200 mesh sieves and remove impurity, add that 200mL ethanol analysis is pure carries out ungrease treatment, repeat degreasing more than 10 times, after each degreasing finishes, carry out centrifugal, material after centrifugal is placed in fuming cupboard, by remaining ethanol volatilization completely, obtains timothy grass pollen-antigen.
Timothy grass pollen-antigen to be marked is dissolved in the NaCl solution of 0.9%pH7.0, making its final concentration is 50-500 μ g/ml, obtains timothy grass pollen-antigen solution.
2, the preparation of colloidal gold solution to be marked
Use 0.1M K
2cO
3solution or 0.1M HCl adjust the pH value to 9.0 of colloidal gold solution, make the particle diameter of collaurum reach 50nm, and concentration reaches 1%.
3, colloid gold label timothy grass pollen-antigen: collaurum and antigenic solution are used respectively to 0.1MK
2cO
3solution is adjusted 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.This process need is adjusted the consumption of antigen, and the solution after completing taking colloid gold label antigen is grape wine redness as best.
4, the purifying of the timothy grass pollen-antigen of colloid gold label: by step 3 preparation collaurum-timothy grass pollen-antigen bond 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 Sephacryl S-400 chromatographic column and carry out purifying, by the timothy grass pollen-antigen solution filter degerming of the colloid gold label of purifying, 4 DEG C of preservations.
In purge process, application of sample amount is 1/10 of Sephacryl S-400 chromatographic column volume, and using stabilizing agent (described stabilizing agent is as the polyglycol solution of 1%BSA and 0.1% molecular weight 20KD), as eluent, flow velocity is 5-10ml/h.
5, the particle diameter of collaurum protein body qualification
The measurement of colloid gold particle mean diameter: observing colloid gold grain under transmission electron microscope, the mean diameter of calculating 100 gold grains should be within the scope of 40～60nm.
6, test strips is coated
By negative control solution (containing the PBS solution of 40-70% bovine serum albumin(BSA)), the antigenic solution of step 4 purifying evenly sprays line.
Carry out the spray line of negative control and antigenic solution on immune chromatography test paper nitrocellulose filter surface.Carry out quantitative work with pneumatic sprayhead.With back and forth interval spray line back and forth of three-dimensional platform.Finally the nitrocellulose filter having sprayed is dried and cuts slivering.
7, result judgement
Carry out classification according to the colored depth contrast ratio of bar 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 | Significantly increase |
| 17.5-50IU/mL | 4 | Significantly increase |
| 50-100IU/mL | 5 | Higher |
| ＞100IU/mL | 6 | High |
In visual colorimetry, the band on colorimetric card is for directly carrying 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 gradients (table 2):
Table 2 different I gE concentration is corresponding to the red concentration of Grape Skin on colorimetric card
| 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 weeds class pollen allergens IgE examination provided by the invention is suitable for large-scale industrialization produces, and has reduced production cost, greatly reduces the price that anaphylactogen individual event detects.In addition, to thering is the anaphylactogen raw material of China domestic feature, realize localization, be more suitable for the diagnosis of Chinese population anaphylactogen, improved 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 (7)
1. for a test strips for specificity weeds class pollen allergens IgE examination, it is characterized in that, described test strips is coated with the weeds class pollen-antigen of colloid gold label.
2. test strips according to claim 1, is characterized in that, described test strips is nitrocellulose filter or the cellulose acetate membrane that is coated with the weeds class pollen-antigen of colloid gold label.
3. test strips according to claim 1, is characterized in that, the pH value of described test strips is 8.5-10, and the mol ratio between colloid gold particle and weeds class pollen-antigen is 10-15:1.
4. according to the test strips described in claim 1-3 any one, it is characterized in that, weeds class includes but not limited to that timothy grass, chrysanthemum thatch, rye grass, reed, bromegrass, humulus grass, argy wormwood, yorkshire fog grass, meadow fescue, hundred acts reach grass, white bent, false Chinese sorghum, rye, oat, wheat, foxtail, barley, corn, salt grass, paspalum notatum.
5. test strips according to claim 4, is characterized in that, described weeds class pollen-antigen is timothy grass pollen-antigen; The preparation method of described timothy grass pollen-antigen is: get timothy grass pollen 50g, cross 200 mesh sieves, add that 200mL ethanol analysis is pure carries out ungrease treatment, repeat degreasing more than 10 times, after each degreasing finishes, carry out centrifugal, material after centrifugal is placed in fuming cupboard, by remaining ethanol volatilization completely, obtains timothy grass pollen-antigen.
6. the preparation method of test strips described in claim 1-5 any one, is characterized in that, comprises the following steps:
1) preparation of weeds class pollen-antigen solution: weeds class pollen to be marked is dissolved in the NaCl solution of 0.9%pH7.0, and making its final concentration is 50-500 μ g/ml, obtains weeds class pollen-antigen solution;
2) preparation of colloidal gold solution: use 0.1M K
2cO
3solution and 0.1M HCl adjust 0.01%HAuCl
4the pH value of colloidal gold solution, to 8.5-10, makes the particle diameter of collaurum reach 40-60nm, and concentration reaches 0.5-2%, obtains colloidal gold solution;
3) combination of collaurum and weeds class pollen-antigen: use 0.1M K
2cO
3the pH value of the antigenic solution of solution tune step 1) is to 8.5-10, add wherein while stirring step 2) colloidal gold solution, the volume ratio of antigenic solution and colloidal gold solution is 1-10:100, add stabilizing agent to prevent weeds class pollen-antigen and colloid gold particle polymerization precipitation simultaneously, obtain collaurum-weeds class pollen-antigen bond; Described stabilizing agent is the polyglycol of 1%BSA and 0.1% molecular weight 20KD;
4) purifying of the weeds class pollen-antigen of colloid gold label: by step 3) in preparation collaurum-weeds class pollen-antigen bond through the centrifugal 20min of 1500r/min; Collecting precipitation is dissolved in stabilizing agent, makes its concentration reach 5-30%; Then add on Sephacryl S-400 chromatographic column and carry out purifying, by the weeds class pollen-antigen solution filter degerming 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) test strips is coated: by step 4) in the weeds class pollen-antigen solution spray line of colloid gold label of preparation in test strips, obtain the test strips finished product for specificity weeds class pollen allergens IgE examination after dry.
7. method according to claim 6, is characterized in that step 4) in application of sample amount be 1/10 of Sephacryl S-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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Cited By (1)
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| CN105929174A (en) * | 2016-05-01 | 2016-09-07 | 上海大学 | Method for detecting and positioning pollen sensitinogen protein on basis of immunofluorescence technique |
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