CN101769919B - Immuno-chromatography detection device and detection method thereof - Google Patents
Immuno-chromatography detection device and detection method thereof Download PDFInfo
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Abstract
The invention discloses an immuno-chromatography detection device and a detection method thereof, which comprise a membrane matrix. The membrane matrix is provided with a sample pad, a marker pad carrying a marker which consists of reactive substance and indicative substance, a detection belt coated or chemically linked crosswise with the reactive substance and an absorbent pad in turn. The marker is multi-functional polymer formed by the coupling of the reactive substance and the indicative substance on a polymer framework. The detection belt is the reactive substance coated or chemically linked crosswise with the membrane matrix or reactive polymer formed by the coupling of the reactive substance on the polymer framework. The detection method is that the sample swims on a membrane and the object to be tested reacts with the marker on the marker pad through the marker pad, then swims to the detection belt to react with the reactive substance under the capillary action, and is developed or checked out through the indicative substance. The invention provides the immuno-chromatography device with high sensitivity and specificity, and the immuno-chromatography detection method with high sensitivity, quickness and reliability.
Description
Technical field
The invention belongs to technical field of medical detection, relate to a kind of immuno-chromatography detection device and detection method thereof.
Background technology
Immunochromatography (Immunochromatography, IC) is a kind of tachysynthesis analytical technology that last century, early eighties grew up.Principle is by capillary action, sample swimming on the fibre strip film, the respective ligand that test substance is wherein preset on film is combined, and obtains intuitively result by chromogenic reaction (or fluorescence or directly use colored marker) within the short time (20min).Immunochromatography is different because of usage flag thing difference title, such as enzyme immunochromatography, emulsion particle immunochromatography, colloidal gold immunochromatographimethod, electroselenium immunochromatography, CI immunochromatography etc.Immunochromatography technique the widest in current application and that technology is the most ripe is colloidal gold immunochromatographimethod.Only at home on the market, just can find easily hundreds of colloidal gold immuno-chromatography test paper strip, they play an important role at aspects such as early pregnancy detection, communicate illness detection, illicit drugs inspection, food hygiene detection (such as residue of veterinary drug etc.) and genetic tests.Yet, the major limitation of colloidal gold immunochromatographimethod chromatography detection technique is that sensitivity and accuracy all are difficult to reach the level that ELASA detects, in addition, collaurum easily produces cohesion, and the label that forms is unstable, poor for little molecular labeling, the mark specificity is difficult to be held, and this technical bottleneck has greatly limited further development and the application of immunochromatography technique.
In immunochromatography detects, the reaction of determinand and its reactive materials (antibody, nucleic acid or acceptor etc. have the material of specific bond ability) is not balanced reaction, determinand in the sample all will obtain enrichment by the detection region that is comprised of reactive materials, so that rete is analysed the efficient of reaction is higher.But the sensitivity of collaurum indicant commonly used is on the low side, has caused present immunochromatography technique sensitivity on the low side, and this also is one of slow most important reason that does not substitute ELISA of colloidal gold immunochromatographimethod technology.Therefore, people improve rete and analyse the effort of sensitivity and never stopped, weeding out the old and bring forth the new of label then is to improve rete to analyse the most important approach of sensitivity, the label that once used has collaurum, electroselenium, CI, latex particle and has also used in recent years liposome, and its purpose all is to wish the sensitivity that chromatography detects is reached a new high.Recently, the researcher is arranged with paramagnetic particle mark, the technology such as quantum dot-labeled, or immune response signal is converted into the electronic signal pattern to be improved sensitivity and has also all obtained good effect.But these new technologies are quite high for the requirement of equipment and technology, have got long long way to go apart from practical application.
Summary of the invention
The technical problem to be solved in the present invention is, for immunity testing equipment sensitivity is on the low side in the prior art, accuracy is bad, equipment and the higher problem of technical requirement thereof, provides the device for immunochromatography of a kind of high sensitivity, high specific.
The technical matters that the present invention further will solve is, and is on the low side for prior art medium sensitivity, accuracy is bad, equipment and the higher problem of technical requirement thereof, and a kind of immunochromatography detection method of highly sensitive, fast and reliable is provided.
The technical solution adopted for the present invention to solve the technical problems is: a kind of immuno-chromatography detection device, comprise the film matrix, be disposed with sample pad at the film matrix, the labeling pad of the label that carrying is comprised of reactive materials and indicative material, the detection band of coated or responding property of chemical crosslinking material, adsorptive pads, described label is that reactive materials and indicative material are coupled at the multifunctional polymer that forms on the polymer backbone, and described detection band is coupled at the reactive polymer that polymer backbone forms for coated or reactive materials or the responding property material of chemical crosslinking on the film matrix.
Described polymer backbone be with-CHO ,-NH
2,-OH ,-COOH in the polymkeric substance of at least one functional group.
Described polymer backbone is glucosan, poly-D-lysine, tygon or polystyrene.
The immunochromatography detection method is: sample swimming on the film that fiber or gel are made, determinand in the sample is at first by labeling pad and the reaction of the label on it, then under capillary action swimming to detecting band and reactive materials reaction, colour developing or be detected by indicator substance; Described label is that reactive materials and indicative material are coupled at the multifunctional polymer that forms on the polymer backbone, and described detection band is coated or chemical crosslinking reactive materials or responding property material is coupled at the reactive polymer that polymer backbone forms on the film matrix.
In the immunochromatography detection method, described polymer backbone be with-CHO ,-NH
2,-OH ,-COOH in the polymkeric substance of at least one functional group.
In the immunochromatography detection method, described polymer backbone can also be glucosan, poly-D-lysine, tygon or polystyrene.
In the immunochromatography detection method, reactive materials, indicative material are coupled on the polymer backbone by chemical modification or biological crosslinked method.
In the immunochromatography detection method, described reactive polymer is at least one reactive materials of coupling on the polymer backbone.
In the immunochromatography detection method, described multifunctional polymer is at least one reactive materials of coupling and at least one indicative material on the polymer backbone.
The present invention, is coupled to identical function or difference in functionality material on the privileged site of polymer backbone by chemical modification or biological crosslinked take polymkeric substance as skeleton, gives its multi-biological active.These materials that are connected on the polymer backbone can be divided into two kinds according to function: the one, and reactive materials, the 2nd, indicative material.Reactive materials refers to carry out the material of idiosyncrasy function such as antigen, antibody, enzyme, part, acceptor, nucleic acid etc.Indicative material refers to that naked eyes are visible or the semiochemicals that can be detected by instrument such as pigment, fluorescent material, enzyme quantum dot etc.Analyse in the detection at rete, the polymkeric substance with reactive materials is only called reactive polymer, coated or detect the band position by chemical crosslinking to cellulose membrane; The 2nd, not only having carried reactive materials but also the multifunctional polymer of the indicative material thing that serves as a mark being arranged, join on the labeling pad of rete analysis apparatus.Such multifunctional polymer is applied in rete and analyses when detecting, when passing through labeling pad, the test substance in the sample just generates compound with corresponding multifunctional polymer reaction, arrive when being coated with in advance the detection band that reacts with this determinand, this compound is just assembled with detecting the band reaction, and its aggregation extent can judge to reflect the result by enzymatic colour developing, fluoroscopic examination or color.Because polymer backbone is inert substance to film, adopt simultaneously the special processing process that reduces non-specific absorption, all the other unreacted multifunctional polymers can not be adsorbed on the film, continue swimming in absorbent material.
Owing to reactive materials or indicative material covalent coupling can be changed surface nature and the dissolubility of reactive materials or indicative material to the polymer backbone, participating in various water soluble liquid phase reactions for the not good material of dissolubility and have great meaning.And aspect check analysis, play the immunoreactive effect of amplification as common reagent.Concrete principle is: antigen or antibody isoreactivity material and the indicative materials such as pigment, fluorescent material, horseradish peroxidase (HRP) are coupled to by chemical modification or the crosslinked mode of biology are prepared into compound on the polymer backbone, the existing multiple reaction group of this compound, can need to add upper a large amount of indicative material by difference again, therefore can increase sensitivity and the specificity of immunochromatography reaction, improve the accuracy of reaction.Kept again simultaneously rete analyse technology fast, convenient, economic advantage, forms a kind of brand-new immunochromatography detection new technology that integrates high sensitivity, high specific, fast and reliable.
The present invention substitutes the collaurum thing that serves as a mark with multifunctional polymer, has obtained ideal result, is not reducing in the specific situation, and sensitivity is greatly improved.Multifunctional polymer not only can avoid owing to the collaurum poor stability, being difficult to the shortcoming of the little molecule of mark and mark poor specificity, and can also improve the stability of protein (polypeptide), so that it is longer to detect the term of validity of reagent, testing result is more accurate and reliable.Solved well the fast detecting that exists in the modern detecting and the contradiction between the high sensitivity, and, by multiple labelling, can carry out simultaneously the multiple term purpose and detect.Meet the developing direction of modern experiment detection technique, have outstanding Social benefit and economic benefit.
Description of drawings
The invention will be further described below in conjunction with drawings and Examples, in the accompanying drawing:
Fig. 1 is preparation technology's process flow diagram of the embodiment of the invention 1 multifunctional polymer.
Fig. 2 is the structural representation of the embodiment of the invention 2 immunoassay devices.
Embodiment
Embodiment 1, and the immunochromatography detection method is: sample swimming on the film that fiber is made, and the determinand elder generation in the sample reacts with the label on the film, and swimming is reacted colour developing to detecting to be with reactive materials under capillary action; Described label is that reactive materials and indicative material are coupled at the multifunctional polymer that forms on the polymer backbone, and described detection band is coated with or responding property of chemical crosslinking material is coupled at the reactive polymer that forms on the polymer backbone.
One, the preparation of multifunctional polymer:
Be preparation technology's process flow diagram of the multifunctional polymer of the embodiment of the invention as shown in Figure 1, take antibody as reactive materials, illustrate take horseradish peroxidase (HRP) as indicative material.
1, the preparation of HRP-glucosan: at first under neutrallty condition, use the sodium periodate method to activate as the glucosan of polymer backbone.Take by weighing the 10mg glucosan, be dissolved in the phosphate buffer (PBS) of 0.01mol/L pH7.2, add the sodium periodate solution 0.5ml of 0.15mol/L, 25 ℃ of reaction 60min.After desalting column is removed unreacted sodium periodate, add again the hydrazine (NH of 10mg
2-NH
2-), 25 ℃ of lucifuge reaction 30min.Phosphate buffer to 0.01mol/L is fully dialysed, and forms the amination glucosan; The same sodium periodate method activation HRP that adopts takes by weighing the NaHCO that 10mg HRP is dissolved in the freshly prepared 0.1mol/L of 1.0ml
3In the solution, add the sodium periodate solution 0.5ml of 0.15mol/L, 25 ℃ of lucifuge reaction 30min after desalting column is removed unreacted sodium periodate, form the HRP of aldehyde radical; The color liquid that has of collecting is all joined in the above-mentioned amidized glucosan, 25 ℃ of lucifuge reaction 120min, the sodium borohydride 0.4ml of adding 4.0mg/ml, 25 ℃ of lucifuges reaction 60min, phosphate buffer to 0.01mol/L is fully dialysed, and just forms the HRP-glucosan.
Because the dextran molecule amount is larger, the site of activation is many, make it with upper a large amount of amino, and because dextran molecule is the long-chain shape, so horseradish peroxidase (HRP) more than 10 on each molecule of the skeleton energy coupling, the all right coupling fluorescent material of HRP-glucosan or dye molecule, but fluorescent material or the dye molecule of coupling more than 100.The use monoethanolamine sealed reactive group after coupling was finished.
2, antibody uses protease digestion and reduction: get 10mg antibody, adjust about pH4.2 with the acetate buffer of 70mmol/L, the pepsin that adds 1.0mg, 37 ℃ of water-baths 16 hours, adjust about pH8.0 with the Tris damping fluid of 1.0mol/L, on use in advance Superdex 75 molecular sieve columns of 0.01mol/L phosphate buffer balance, collect the first eluting peak, be concentrated to 5mg/ml, namely obtain antibody fragment F (ab ')
2Then according to every milliliter of above-mentioned F (ab ')
2The ratio mixing F of DTT (DTT) 0.01ml of adding 0.1mol/L (ab ')
2And DTT, 37 ℃ of water-bath 90min, just form after the desalting column desalination antibody fragment that contains sulfydryl (Fab '-SH).This process also can be used crosslinking chemical method activation antibody fragment, formation F (ab ')
2-SH.
3, the HRP-glucosan uses the crosslinking chemical activation: the HRP-glucosan of preparation in the step 1 is condensed into the concentration that is about 5mg/ml, the concentration that every milliliter of adding prepares is isodigeranyl functional cross-link agent GMBS (N-γ-Maleimidobutyryloxy-oxysuccinimide ester) 0.1ml of 10mg/ml, 25 ℃ of lucifuge reaction 120min, after the desalting column desalination, the collection first peak is the HRP-glucosan with the maleimide reactive group.
4, the HRP-glucosan of the product of the product Fab ' of step 2-SH and step 3 with the maleimide reactive group mixed according to mass ratio at 1: 1,25 ℃ of lucifuge reaction 120min add 50mg25 ℃ of lucifuge reaction of glycocoll 60min with the sealing reactive group.That finally finishes reactive materials and polymkeric substance is connected the multifunctional polymer that obtains.
The multifunctional polymer periphery of preparation is with reactive materials like this, and the inboard is conducive to multifunctional polymer and participates in various biologicallies with indicative material.For containing-NH
2Polymer backbone can directly use difunctional or three functional cross-link agents and indicative material and reactive materials coupling, form multifunctional polymer.
Two, the preparation of reactive polymer:
Take antibody as reactive materials, poly-D-lysine is polymer backbone, adopts the standby reactive polymer of crosslinking chemical legal system.
1, the reduction of antibody: antibody is placed the PBS of 0.1mol/L pH7.2, adjustment concentration is 10mg/ml, adds the EDTA of final concentration 10mmol/L; Every milliliter of antibody adds the Mercamine Cysteamine of 6mg, stirring and dissolving, 37 ℃ of insulation 90min.Cross Sephadex G-25 or similar desalting column, the absorption peak that is determined at 280nm determines that the position of antibody, the antibody of collection namely are the antibody fragments with-SH.
2, the activation of poly-D-lysine: the concentration that poly-D-lysine is mixed with 10mg/ml, adding the concentration for preparing in every milliliter of such poly-D-lysine is isodigeranyl functional cross-link agent GMBS (N-γ-Maleimidobutyryloxy-oxysuccinimide ester) 0.1ml of 10mg/ml, 25 ℃ of lucifuge reaction 120min, after the desalting column desalination, the collection first peak is the poly-D-lysine with the maleimide reactive group.
3, the generation of reactive polymer: mixing according to 4: 1 ratio of mass ratio with the poly-D-lysine that the product of step 2 activates with-the antibody fragment of SH that step 1 produces, 25 ℃ of lucifuge reaction 120min, cross Superdex 200 molecular sieve and remove unconjugated antibody, collect the first eluting peak and be reactive polymer.
The noun explanation
The sodium periodate method: being a kind of chemical modification method ,-OH is oxidized to-CHO, generate the method for attachment of amido link with-NH2 reaction and reduction again under alkali condition, is traditional carbohydrate and protein interconnection technique, does not repeat them here.
Crosslinking chemical method: be a kind of chemical modification method, crosslinking chemical is connected different material as the bridge chain and generates the extending arm that length differs.Be prior art, do not repeat them here.
The biological crosslinked method that in bacterium or cell, two class materials linked together by means such as genetic engineerings of mainly referring to.Biology is crosslinked to be prior art, does not repeat them here.
Chemical modification or biological cross-linking method also comprise the EDC method, and the EDC method is to utilize carbodiimide (EDC) or derivatives thereof activating carboxy acid, enable the reaction with-NH2, are prior art, do not repeat them here.
Coated: having reactive protein or nucleic acid point sample in detecting the band position, after the drying, use albumin or casein solution to seal not binding site, this process is referred to as to be coated with.Coated principle generally is physical adsorption process, also can use Chemical Crosslinking Methods.
The present invention's immunity rete analysis method can improve sensitivity from two aspects, the reactive materials that at first a plurality of participations of coupling are reacted on the polymer backbone, increased the relative concentration of reactive materials, and rete is analysed the chance that can improve the reactive materials contact, so that reaction sensitivity increases, in addition, each polymer backbone can with the indicative material more than 10 (enzyme, fluorescent material or more dye molecule), improve the sensitivity that detects.Film during rete is analysed can guarantee therein normal swimming of multifunctional polymer, and is the chromatography inert substance the polymer backbone (such as glucosan), can not be adsorbed onto on the film, has guaranteed the specificity of reaction.Being coated with reactive polymer has increased package amount, has further increased sensitivity.Multiple reactive materials and indicative material on can coupling on the polymer backbone can be coated with the multiple reaction band on the film, carry out multiple reaction, and diverse location position demonstration on film, therefore can detect simultaneously multiple determinand.
As shown in Figure 2, hepatitis C and HCV infect detection kit and comprise the film matrix, the film matrix is comprised of the polyester film 10 of bottom and the chromatographic film 11 on top, from chromatographic film 11 1 ends of film matrix be disposed with the label that sample pad 1, carrying be comprised of reactive materials and indicative material labeling pad 2, be coated with or the detection of responding property of chemical crosslinking material is with 3, adsorptive pads 4.
Label is for using HCV antigen fragment or HCV core antibody as reactive materials, with horseradish peroxidase as indicative material, be coupled at polymer backbone--the multifunctional polymer that forms on the glucosan, described detection band is many, is coated with respectively HCV antigen or the HCV core antibody of different fragments.
So according to detect result that band shows just can draw contain in the sample antibody for clip types or the no cAg that contains.By the multifunctional polymer immunochromatographic method being detected synchronously the detection of HCV antigen-antibody kit technical indicator, and make comparisons with ELISA and colloid gold reagent, the results are shown in following table 1:
Table 1: the multifunctional polymer immunochromatographic method detects the comparison of HCV antigen-antibody and ELISA and collaurum synchronously
In the table 1, the antigen detection sensitivity that the present invention detects HCV antigen-antibody kit synchronously is 10pg, reaches the level of ELISA, and far above the detection level of collaurum, antibody test sensitivity is because less demanding, and three kinds of kits all are 2.0ncu; The specificity aspect, the present invention detects synchronously HCV antigen-antibody kit and also reaches 100%; Detection for clinical samples, (the RIBA kit of U.S. Chiron company is used in antibody test with the import reagent box, antigen detects and is the cAg detection kit of U.S. Ortho company) relatively, the coincidence rate of this method is respectively up to 99.2% and 100%; Be higher than ELISA kit (it is 91.7% that antigen detects, and antibody test is 85%) and colloidal gold kit (it is 79.2% that antigen detects, and antibody test is 81.7%).Simultaneously, the ELISA kit can only carry out single detectable antigens or antibody in a test, can not divide segment to detect even detect simultaneously, although and collaurum can divide the segment detection, can not detect synchronously.Aspect detection time and ease-to-operate, the present invention detects synchronously HCV antigen-antibody kit and also has obvious technical advantage in addition.
In addition, the present invention can also carry out the associating fast detecting that different virus infects, such as hepatitis type B virus (HBV), hepatitis C virus (HCV), AIDS virus (HIV), the associating fast detecting of the blood transfusion such as syphilis test item, only need respectively the HBV surface antibody, HCV-cAg, HIV antigen, syphilis antigen is coupled at respectively on the different polymer backbones, and the HBV surface antibody that on zones of different endoperidium on the chromatographic film, matches with marker antibody (former), HCV-cAg, HIV antigen, syphilis antigen etc. form different detection bands, will detect at the corresponding band that detects when containing material to be checked in the sample.
Claims (1)
1. immuno-chromatography detection device, comprise the film matrix, the film matrix be disposed with the label that sample pad, carrying be comprised of reactive materials and indicative material labeling pad, be coated with or detection band, the adsorptive pads of responding property of chemical crosslinking material, it is characterized in that, described label is that reactive materials and indicative material are coupled at the multifunctional polymer that forms on the polymer backbone, and described detection band is that reactive materials is coupled at the reactive polymer that forms on the polymer backbone;
Wherein, described reactive materials is the antibody fragment that contains sulfydryl;
The preparation of described multifunctional polymer may further comprise the steps:
S1. prepare horseradish peroxidase-glucosan: take by weighing in the phosphate buffer that the 10mg glucosan is dissolved in 0.01mol/LpH7.2, add the sodium periodate solution 0.5ml of 0.15mol/L, 25 ℃ of reaction 60min; After desalting column is removed unreacted sodium periodate, add again the hydrazine of 10mg, 25 ℃ of lucifuge reaction 30min; Phosphate buffer to 0.01mol/L is fully dialysed, and forms the amination glucosan; Take by weighing the NaHCO that the 10mg horseradish peroxidase is dissolved in the freshly prepared 0.1mol/L of 1.0ml
3In the solution, add the sodium periodate solution 0.5ml of 0.15mol/L, 25 ℃ of lucifuge reaction 30min after desalting column is removed unreacted sodium periodate, form the HRP of aldehyde radical; The color liquid that has of collecting is all joined in the described amidized glucosan, 25 ℃ of lucifuge reaction 120min, the sodium borohydride 0.4ml of adding 4.0mg/ml, 25 ℃ of lucifuges reaction 60min, phosphate buffer to 0.01mol/L is fully dialysed, and just forms the HRP-glucosan;
S2. preparation contains the antibody fragment of sulfydryl: get 10mg antibody, adjust about pH4.2 with the acetate buffer of 70mmol/L, the pepsin that adds 1.0mg, 37 ℃ of water-baths 16 hours, adjust about pH8.0 with the Tris damping fluid of 1.0mol/L, on use in advance Superdex 75 molecular sieve columns of 0.01mol/L phosphate buffer balance, collect the first eluting peak, be concentrated to 5mg/ml, obtain antibody fragment F (ab ')
2Then according to every milliliter of above-mentioned F (ab ')
2The ratio mixing F of the DTT 0.01ml of adding 0.1mol/L (ab ')
2And DTT, 37 ℃ of water-bath 90min form the antibody passage Fab that contains sulfydryl after the desalting column desalination '-SH;
S3. use crosslinking chemical activation horseradish peroxidase-glucosan: with the described horseradish peroxidase that makes among the described step S1-glucosan simmer down to 5mg/mL concentration, every milliliter adds concentration is the isodigeranyl functional cross-link agent GMBS0.1mL of 10mg/mL, 25 ℃ of lucifuge reaction 120min, first peak is collected in the desalting column desalination, obtains the horseradish peroxidase-glucosan with the maleimide reactive group;
S4. the generation of multifunctional polymer: with the antibody passage Fab that contains sulfydryl that makes among the described step S2 '-horseradish peroxidase-glucosan with the maleimide reactive group that makes among SH and the described step S3 mixes according to mass ratio at 1: 1,25 ℃ of lucifuge reaction 120min, add subsequently glycocoll 50mg in 25 ℃ of lucifuge reaction 60min sealing reactive groups, obtain described multifunctional polymer.
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| WO2012003617A1 (en) * | 2010-07-05 | 2012-01-12 | 深圳大学 | Immuno-chromatographic detection device and detection method thereof |
| FR2966248B1 (en) * | 2010-10-18 | 2020-05-01 | Centre National De La Recherche Scientifique (Cnrs) | METHOD FOR FUNCTIONALIZING SURFACES FOR THE DETECTION OF ANALYTES |
| AU2012308326B2 (en) * | 2011-09-16 | 2018-11-22 | Credo Biomedical Pte Ltd | Molecular diagnostic assay device and method of use |
| CN107764992B (en) * | 2017-10-16 | 2018-10-12 | 南京诺唯赞医疗科技有限公司 | A kind of orientation coupling method and the application of microballoon and antibody |
| CN112881691A (en) * | 2020-12-24 | 2021-06-01 | 杭州百殷生物科技有限公司 | Immunocytochemistry labeling developing kit for cervical cancer auxiliary diagnosis |
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