CN202794177U - Kit for enzyme-linked immune-chromatography - Google Patents
Kit for enzyme-linked immune-chromatography Download PDFInfo
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- CN202794177U CN202794177U CN 201220299634 CN201220299634U CN202794177U CN 202794177 U CN202794177 U CN 202794177U CN 201220299634 CN201220299634 CN 201220299634 CN 201220299634 U CN201220299634 U CN 201220299634U CN 202794177 U CN202794177 U CN 202794177U
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Abstract
The utility model discloses a kit for enzyme-linked immune-chromatography. The kit comprises a box body, an enzyme-linked immune-chromatography test paper strip in the box body, a reagent bottle and a sunken bottle position for placing the reagent bottle, wherein the test paper strip comprises a sample pad (1), a combined pad (2), a chromatography membrane (3), a water absorbent pad (4) and a backing pad (7), wherein the sample pad (1), the combined pad (2), the chromatography membrane (3) and the water absorbent pad (4) are arranged above the backing pad (7); the sample pad (1) is connected to the combined pad (2); the combined pad (2) is connected to the chromatography membrane (3); a quality control band (5) and a detecting band (6) are arranged on the chromatography membrane (3); the chromatography membrane (3) is connected to the water absorbent pad (4); and the reagent bottle is respectively provided with a standard product to be detected, a sample dilution solution, a substrate developing solution and a cleaning solution which are of series of concentrations. The kit provided by the utility model has the advantages of high flexibility, strong specificity, capacity of accurately determining quantity by combining with a reading device, and the like.
Description
Technical field
The utility model relates to technical field of biological, particularly a kind of enzyme linked immunological chromatography kit.
Background technology
The immunology detection technology is widely used in medical science and field of biology.The immunology detection technology that wherein is most widely used at present is immunolabelling technique, namely uses labelled antibody or the antigens such as collaurum, fluorescein, enzyme or radioactive nuclide, carries out antigen-antibody reaction.Label and the immunological characteristic that does not change the latter after antibody or antigen are connected, have highly sensitive, quick, can be qualitative, quantitative, the advantage such as location.
Traditionally multiplex fluorescent immune method, chemoluminescence method, enzyme linked immunosorbent assay (ELISA) and colloidal gold immunity chromatography etc.But fluorescent immune method and chemoluminescence method are high to technical requirement, and must be equipped with expensive task equipment use, therefore are only applicable to some large hospitals and quarantine departments etc., are difficult for carrying out routine to carry out; The enzyme linked immunosorbent assay complicated operation, the use procedure reaction time is longer, need to carry out to sample the processing such as enzymic digestion, concussion, and it is not very convenient using; Although it is few that colloidal gold immunity chromatography has a sample consumption, easy fast, cheap advantage, yet when antigen or antibody content are extremely low in running into some sample, the color of collaurum will be very shallow, be difficult to the naked eye judged result, erroneous judgement occurs easily, sensitivity is lower.
Summary of the invention
For the defective that above-mentioned prior art exists, the technical problems to be solved in the utility model is compared with existing method for a kind of enzyme linked immunological chromatography kit is provided, the utility model kit detection sensitivity height, good resolution, high specificity.
Particularly, the utility model provides a kind of enzyme linked immunological chromatography kit, the recessed bottle position that comprises enzyme linked immunological chromatograph test strip, the reagent bottle in box body and the box and put reagent bottle; Described test strips comprises sample pad (1), pad (2), chromatographic film (3), adsorptive pads (4) and backing pad (7), wherein sample pad (1), pad (2), chromatographic film (3) and adsorptive pads (4) are equipped on the backing pad (7), sample pad (1) is connected in pad (2), pad (2) is connected in chromatographic film (3), be provided with quality control band (5) on the chromatographic film (3) and detect band (6), chromatographic film (3) is connected in adsorptive pads (4);
Determinand standard items, sample diluting liquid, substrate nitrite ion, the cleansing solution of series concentration are housed respectively in the described reagent bottle.
In the mentioned reagent box, be fixed with enzyme labeling or biotin labeled specific antibody on the described pad, but described specific antibody is the antibody of specific binding determinand; Being coated with two of anti-described enzyme labeling or biotin labeled specific antibody on the described quality control band resists; Described detection with on be fixed with can with the antigen of determinand competitive binding specific antibody.
Described enzyme is horseradish peroxidase, alkaline phosphatase, glucose oxidase or beta galactosidase.
Described can be determinand molecule haptens-BSA conjugate with the antigen of determinand competitive binding specific antibody.
In the mentioned reagent box, described chromatographic film (3) is the hybrid films of nitrocellulose filter or nitrocellulose filter and cellulose acetate membrane composition.
Described sample diluting liquid is phosphate buffer or borate buffer solution.
Described substrate nitrite ion is comprised of A liquid and B liquid, and described A liquid is H
2O
2Solution, described B liquid comprises the hydrogen donor of sedimentation type, and described hydrogen donor is OPD, TMB, DAB, biphenylamine, 5-aminosalicylic acid or 4-chloro-1-naphthols, and described A liquid and B liquid are loaded on respectively in two reagent bottles.
The qualitative detection principle of the utility model kit:
Detect the coated thing to be checked of band-BSA conjugate, two of the coated antienzyme mark of quality control band or biotin labeled antibody resists.On the pad in conjunction with biotin labeled monoclonal antibody.In the testing process, get a certain amount of sample solution and add sample pad, sample and dissolved enzyme labelled antibody move at filter together.If sample solution contains corresponding thing to be checked, then thing to be checked will with in advance reaction of antibody, therefore detect when being with when diffusing to, the avtive spot of antibody will because of occupied by the thing to be checked in the sample solution can't with detection with on thing antigen to be checked be combined; The Avidin that adds afterwards the HRP mark is combined with the biotin that is fixed on the film by monoclonal antibody; Add cleansing solution, wash the peroxidase that does not have combination off, add at last substrate and use liquid, when the thing content to be checked in the sample surpasses the agent plate detection limit, the detection band on the agent plate will than quality control band develop the color light or even without colour developing, be judged to be the positive.Otherwise when thing content to be checked in the sample below the agent plate detection limit or during noresidue, the detection band colour developing on the agent plate is close with quality control band or partially dark, is judged to be feminine gender.
When with the utility model kit when supporting quantitative analysis instrument is combined with, can draw the concrete content of thing to be checked, realize the purpose that quantitatively detects.
The major advantage of the utility model kit is as follows:
1) compares detection sensitivity height, good resolution, high specificity with existing method;
2) easy and simple to handle, need not specialized instrument and equipment, visual result, safety is cheap, is fit to field quick detection.
3) when readout instrument is combined, content that can the Accurate Determining determinand.
Description of drawings
Fig. 1 is enzyme linked immunological chromatograph test strip assembling synoptic diagram, and wherein 1 is sample pad, and 2 is pad, and 3 is chromatographic film, and 4 is adsorptive pads, and 5 is quality control band, and 6 for detecting band, and 7 is the backing pad.
Embodiment
In order to make those skilled in the art understand better the technical solution of the utility model, the utility model is described in further detail below in conjunction with specific embodiment.
The preparation of embodiment 1 the utility model organophosphorus immune chromatography reagent kit
(1) the standby enzyme labelled antibody of improvement sodium periodate legal system
Get HRP 5mg and be dissolved in 0.2mol/L, among the pH5.6 acetate buffer 1ml, add 1%DNFB ethanol solution 0.1ml, gentle agitation 1h under the room temperature; The 0.1mol/L NaIO that adds fresh configuration
40.5ml, put 4 ℃ and place 30min, add 2.5% ethylene glycol 1ml, gentle agitation 1h under the room temperature, cessation reaction; Add anti-organophosphorus monoclonal antibody 5 ~ 10mg to be marked, use 1.0mol/L, pH9.5CBS regulates pH value to 9.0; Mixing is put 4 ℃ and is spent the night; Add sodium borohydride solution 0.1ml, mixing is placed 3h for 4 ℃; To 0.01mol/L, pH7.4PBS, 4 ℃ of dialysed overnight are changed liquid 3 times; The centrifugal 30min of 3000r/min removes sediment; Collect supernatant and be enzymic-labelled antibody.
(2) test strips assembling
Enzymic-labelled antibody dilutes with dilution, wherein dilution contains 10% ~ 20% sucrose, 1% ~ 5% trehalose, 0.5% ~ 1% bovine serum albumin(BSA) (BSA), 0.5% ~ 1% Tween-20, then with BIO-DOT metal spraying lining instrument enzymic-labelled antibody is sprayed on the glass fibre membrane, obtain pad, seal 4 ℃ of lower preservations behind 37 ℃ of dry 4h.
Draw two rule parallel bands with BIO-DOT metal spraying lining instrument at nitrocellulose filter, band interval 5mm, the bar bandwidth all is about 1mm.Wherein article one is quality control band, and second is quantitatively to be with.Spraying sheep anti mouse immunoglobulin (Ig) (IgG) on the quality control band, concentration is 0.5 ~ 1mg/ml; The second band is coated with machine phosphorus coating antigen, and concentration is 0.2 ~ 1mg/ml.Get chromatographic film, seal behind 37 ℃ of dry 4h, 4 ℃ of lower preservations.
On the PVC plate, paste successively upper chromatographic film (long 30cm, wide 2.5cm), pad (long 30cm, wide 8mm), sample pad (long 30cm, wide 17mm) and adsorptive pads (long 30cm, wide 17mm).With cutting cutter the plastic plate that pastes vertically is cut into the wide test strips of 3.8mm, puts into the button card and be assembled into immune chromatography reagent kit, dry rear sealing, 4 ℃ keep in Dark Place.
(3) reagent preparation:
The sodium tetraborate solution of sample diluting liquid: 0.2mol/L need not transfer pH(to be about 9.2), contain 3% acetone and 0.5% methyl alcohol;
Standard items: high-purity thing to be checked;
The substrate nitrite ion: 100mL B liquid+20 μ L A liquid, wherein A liquid is 33% H
2O
2Solution, B liquid are the 40%(quality percent by volume with substrate buffer solution preparation) TMB solution; Described substrate buffer solution is phosphoric acid-citrate buffer of pH5.0, and it specifically is formulated as follows:
0.1mol/L citric acid (19.2g/L) 24.3mL
0.2mol/L Na
2HPO
4（28.4g/L） 25.7mL
Ultrapure water is settled to 100mL
Cleansing solution: 0.01mol/L, the PBS+0.05%(v/v of pH7.4) Tween-20
The preparation of embodiment 2 the utility model organophosphorus immune chromatography reagent kits
(1) prepares biotin labeled antibody.
With 4 ℃ of 50mM carbonate buffer solutions (pH9.6) dialysis antibody, go out sodium azide (NaN3), and will treat that biotinylated anti-organophosphorus monoclonal antibody is diluted to 1mg/ml with this carbonate buffer solution.Biotin ester (BNHS) is dissolved in dimethyl formamide (DMF), and concentration is 20mg/mL.Ratio in the every 1mg antibody of 100 μ gBNHS adds BNHS, mixing, and reaction is 4 hours under the room temperature.Removed educt in three days with phosphate buffer dialysis under 4 ℃, obtain biotinylated monoclonal antibody, save backup under 4 ℃, long preservation needs-20 ℃ of preservations.
(2) assembling of test strips
Method assembling test strips according to embodiment 1.
(3) preparation of reagent
With embodiment 1
The qualitative detection organophosphorus residue of embodiment 3 the utility model kits
Get a certain amount of sample solution and add sample pad, sample and dissolved enzyme labelled antibody move at filter together.If sample solution contains the residual of relative medicine, medicine will react with antibody in advance, and therefore when diffusing to the detection band, the avtive spot of antibody will can't be with upper medicine antigen to be combined with detection because being occupied by the medicine in the sample solution; Add afterwards substrate and use liquid.When the thing content to be checked in the sample surpasses the agent plate detection limit, the detection band on the agent plate will than quality control band develop the color light or even without colour developing, be judged to be the positive.Otherwise when the thing content to be checked in the sample below the agent plate detection limit or during noresidue, the detection band colour developing on the agent plate is close with quality control band or partially dark, is judged to be feminine gender; Do not develop the color with quality control band if detect band, illustrate that detection is invalid.
Embodiment 4 the utility model kits quantitatively detect the drafting of organophosphorus typical curve
With embodiment 1 or 2 described sample diluted thing standard items to be checked, configuration series concentration standard items are as follows: 10 μ g/mL, 1 μ g/mL, 0.1 μ g/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL, 1ng/mL, 0ng/mL.
Vertically drip to negative sample dilution or above-mentioned series concentration standard items 70 μ L on the sample pad during detection, chromatography, after 1 minute, 2 ~ 5 μ L embodiment 1 or embodiment 2 described substrate nitrite ions are dripped on the chromatographic film, treat quantitatively band and all obviously demonstrations of quality control band behind the 2min, 5 ~ 10 μ L sample cleansing solutions are dripped on the chromatographic film, (each sample is measured 3 times with 3 test strips respectively to observe background area, quality control band and quantitative band signal intensity behind the 1min, average), after quantitatively band is deducted the background area signal, the drawing standard curve.Wherein quality control band is proofreaied and correct and work is corresponding for affected by environment to quantitative band signal.
The above only is preferred implementation of the present utility model; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the utility model principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection domain of the present utility model.
Claims (6)
1. enzyme linked immunological chromatography kit, the recessed bottle position that comprises enzyme linked immunological chromatograph test strip, the reagent bottle in box body and the box and put reagent bottle; Described test strips comprises sample pad (1), pad (2), chromatographic film (3), adsorptive pads (4) and backing pad (7), wherein sample pad (1), pad (2), chromatographic film (3) and adsorptive pads (4) are equipped on the backing pad (7), sample pad (1) is connected in pad (2), pad (2) is connected in chromatographic film (3), be provided with quality control band (5) on the chromatographic film (3) and detect band (6), chromatographic film (3) is connected in adsorptive pads (4); Determinand standard items, sample diluting liquid, substrate nitrite ion, the cleansing solution of series concentration are housed respectively in the described reagent bottle.
2. kit as claimed in claim 1 is characterized in that, is fixed with enzyme labeling or biotin labeled specific antibody on the described pad, but described specific antibody is the antibody of specific binding determinand; Being coated with two of anti-described enzyme labeling or biotin labeled specific antibody on the described quality control band resists; Described detection with on be fixed with can with the antigen of determinand competitive binding specific antibody.
3. kit as claimed in claim 2 is characterized in that, described enzyme is horseradish peroxidase, alkaline phosphatase, glucose oxidase or beta galactosidase.
4. kit as claimed in claim 2 is characterized in that, described can be determinand molecule haptens-BSA conjugate with the antigen of determinand competitive binding specific antibody.
5. kit as claimed in claim 1 is characterized in that, described chromatographic film (3) is nitrocellulose filter.
6. kit as claimed in claim 1 is characterized in that, described sample diluting liquid is phosphate buffer or borate buffer solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201220299634 CN202794177U (en) | 2012-06-20 | 2012-06-20 | Kit for enzyme-linked immune-chromatography |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201220299634 CN202794177U (en) | 2012-06-20 | 2012-06-20 | Kit for enzyme-linked immune-chromatography |
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| Publication Number | Publication Date |
|---|---|
| CN202794177U true CN202794177U (en) | 2013-03-13 |
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ID=47821420
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201220299634 Expired - Lifetime CN202794177U (en) | 2012-06-20 | 2012-06-20 | Kit for enzyme-linked immune-chromatography |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104730237A (en) * | 2013-12-23 | 2015-06-24 | 国家纳米科学中心 | Test paper, application and method for alpha-fetoprotein antigen, hepatitis B surface antigen or HIV gp41 antibody |
| CN105067584A (en) * | 2015-08-11 | 2015-11-18 | 郑州安图生物工程股份有限公司 | Immunochromatographic kit for quantitative detection of RV (rubella virus) IgG (immunoglobulin G) antibody through quantum dots |
| CN107110860A (en) * | 2014-08-30 | 2017-08-29 | 新加坡科技研究局 | The test-strips determined for paper substrate |
| CN108802389A (en) * | 2018-06-04 | 2018-11-13 | 郭伟 | A kind of kit for Early stage NSCLC diagnosis |
| CN111830252A (en) * | 2020-06-30 | 2020-10-27 | 广州佰芮慷生物科技有限公司 | Enzyme-linked immunosorbent assay sensor based on intelligent mobile terminal and use method |
| CN111855648A (en) * | 2020-03-05 | 2020-10-30 | 美康生物科技股份有限公司 | Dry type homocysteine test card and application thereof |
-
2012
- 2012-06-20 CN CN 201220299634 patent/CN202794177U/en not_active Expired - Lifetime
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104730237A (en) * | 2013-12-23 | 2015-06-24 | 国家纳米科学中心 | Test paper, application and method for alpha-fetoprotein antigen, hepatitis B surface antigen or HIV gp41 antibody |
| CN107110860A (en) * | 2014-08-30 | 2017-08-29 | 新加坡科技研究局 | The test-strips determined for paper substrate |
| CN105067584A (en) * | 2015-08-11 | 2015-11-18 | 郑州安图生物工程股份有限公司 | Immunochromatographic kit for quantitative detection of RV (rubella virus) IgG (immunoglobulin G) antibody through quantum dots |
| CN108802389A (en) * | 2018-06-04 | 2018-11-13 | 郭伟 | A kind of kit for Early stage NSCLC diagnosis |
| CN111855648A (en) * | 2020-03-05 | 2020-10-30 | 美康生物科技股份有限公司 | Dry type homocysteine test card and application thereof |
| CN111830252A (en) * | 2020-06-30 | 2020-10-27 | 广州佰芮慷生物科技有限公司 | Enzyme-linked immunosorbent assay sensor based on intelligent mobile terminal and use method |
| CN111830252B (en) * | 2020-06-30 | 2023-02-28 | 广州佰芮慷生物科技有限公司 | Enzyme-linked immunosorbent assay sensor based on intelligent mobile terminal and use method |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term |
Granted publication date: 20130313 |
|
| CX01 | Expiry of patent term |