CN102426231A - Immunochromatography reagent strip and sealing agent composition used for same - Google Patents
Immunochromatography reagent strip and sealing agent composition used for same Download PDFInfo
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- CN102426231A CN102426231A CN2011102729313A CN201110272931A CN102426231A CN 102426231 A CN102426231 A CN 102426231A CN 2011102729313 A CN2011102729313 A CN 2011102729313A CN 201110272931 A CN201110272931 A CN 201110272931A CN 102426231 A CN102426231 A CN 102426231A
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Abstract
The invention provides a sealing agent composition used for an immunochromatography reagent strip, which comprises protein, protein protective agent, surface active agent and first buffer solution, wherein the protein is casein, casein treatment solution, methylation bovine serum albumin (BSA) and human serum albumin; and the protein protective agent is saccharide. The sealing agent composition provided by the invention is capable of effectively reducing the background of the immunochromatography reagent strip, and is long in service life and high in detection sensitivity. The invention also provides the immunochromatography reagent strip.
Description
Technical field
The present invention relates to immunochromatography technique, be specifically related to a kind of immunity-chromatography test strip with sealant compositions and a kind of immunity-chromatography test strip.
Background technology
Along with the development of immune diagnostic technique, the integrated application of various starting material and various detection meanss, technology more and more develops to the direction small-sized, simple to operate, quick, that accuracy is high.Immunochromatography technique is a very important part in the middle of this development; It from the space, the time changed this technological developing direction, spatially, it from before the laboratory move family to; In time, it shortens to a few minutes and goes out testing result going out testing result in several days.Thereby promoted the fast development of immune diagnostic technique greatly.
Immunochromatography technique is a reaction principle of utilizing antigen-antibody high specific, high sensitivity, detects antigen (or antigen) through efficient signal tracer labelled antibody (or antigen).At the central immobilization material that very important technological difficulties are exactly antigen (or antibody) of technology; Situation from present solution; Several kinds of immobilization materials below general selection the: nitrocellulose filter (NC film), nylon film, polyvinylidene fluoride film (PVDF); Because these films can both be firm conjugated protein, and binding ability is very high.Wherein modal is the NC film, because its conjugated protein ability is strong, price is relatively cheap.
Though the NC film can strong adsorption antigen or antibody; But in the detection of biological sample processes; Owing to have the big molecule of a large amount of other biologicals in the biological specimen; These biomacromolecules also can non-specific adsorption to the NC film, thereby seriously disturb the recognition capability of antigen-antibody, lower detection sensitivity.Therefore, need find a kind of technology solve antigen (or antibody) fixing after, other biomacromolecule can not disturb the reagent of antigen-antibody reaction
At present, have a lot of a large amount of patent documentations and academic documents to report the problem of the background interference that lowers the NC film, but their overwhelming majority is to adopt following two kinds of methods to handle:
Adopt elder generation at the big molecule of NC film fixed biologically, use organic polymer material (like PVP, PEG, PVP) to combine to seal processing with surfactant then.Adopt earlier at the big molecule of NC film fixed biologically, use BSA, skimmed milk power, casein or various haemocyanins (like sheep blood serum etc.) individual processing then or the processing that combines with surfactant.
After the NC film is handled through above-mentioned several method; Making originally, hydrophobic film becomes hydrophilic; Simultaneously the adsorptive power of biomacromolecule on the NC film reduces greatly, but the NC film through more than after a series of processing, also brought a lot of problems: as changing because the adding of surfactant produces the protein position that originally is fixed on the NC film; Disperse appears in the protein line, and the albumen quality that originally is fixed on the NC film reduces.Secondly because certain variation takes place in immersion, dry run the NC film the whole performance of strip is changed, once more for the strip production technology, these several kinds of treatment process bother very much, are unfavorable for the amplification of production technology.
Therefore, the NC film after sealing is handled is mostly selected to buy by at present general strip manufacturer, when producing strip, need not carry out extra NC membrane closure and handle.But this processing has generally only solved the hydrophilicity of NC film; The performance of NC film adsorbed proteins does not almost change; The ability of NC film adsorbed proteins is also very strong, also has a large amount of protein binding sites on the film, and therefore a lot of strip manufacturers just add BSA, surfactant and seal processing once more in the chromatographic flow component; To reach the detection background that reduces the NC film, still this disposal route is very desirable.
The chromatography strip generally is made up of these several parts: sample pad, NC film, adsorptive pads.When adding detected material in the sample pad, detected material is along with solution flows and the immune particle reaction, and along with the flowing of liquid, immune particle reacts with the antigen or the antibody that are fixed on the NC film more then, and responseless particle is collected by adsorptive pads at last.Most of bibliographical informations are in sample pad, to add BSA and surfactant to solve the high problem of reduction NC film background; Like the inventor Zhang Tao (patent No.: 01126931.6; Proprietary term: " the immune golden detection kit of using flow sealing technique ") reported a kind of sealing treatment technology, he adds sealer (the sealer prescription is: skimmed milk power, BSA, PVP, polysorbas20) in sample pad, when adding sample; Sealer in the sample pad discharges, and plays sealing effect.This sealing disposal route has solved the closed question of NC film on the colloidal gold chromatographic strip really; But this method is to seal the non-specific adsorption problem of NC film to latex particle for the chromatography strip of latex particle; Possibly be because the latex particle hydrophobicity is stronger than colloid gold particle hydrophobicity, grain diameter is bigger; Thereby cause latex particle " dead gold " phenomenon to occur through the NC film front end of being everlasting, it is unsatisfactory therefore to continue to use collaurum sealing disposal route.Need finding more suitably, closed reagent component and method solve this problem.While was not also reported the sealer protection problem after the strip longer-term storage at present, because sealer biologically active, inactivation easily; Therefore need to add some protein protectants, thereby reach the long preservation of strip performance.
Summary of the invention
The technical matters that the present invention will solve is to provide a kind of immunity-chromatography test strip that can reduce the strip background with sealant compositions and the lower immunity-chromatography test strip of a kind of background.
In order to solve the prior art problem, the invention provides a kind of immunity-chromatography test strip and use sealant compositions, comprising:
Albumen, protein protective agent, surfactant and first buffer solution;
Wherein, said albumen is casein, casein treating fluid, MBSA, human serum albumins; Said protein protective agent is a carbohydrate.
Preferably, said surfactant is polyoxyethylene 20 sorbitan monolaurate, polyoxyethylene sorbitan monooleate, NONIN HS 240 or polyoxyethylene lauric acid ether.
Preferably; Said first buffer solution is phosphate buffer solution, tromethamine-hydrochloride buffer, borate buffer, glycocoll-hydrochloride buffer, citrate buffer solution, veronal buffer, malonic acid-sodium hydrate buffer solution, maleic acid-sodium hydrate buffer solution; Succinic acid-sodium hydrate buffer solution; 4-HEPES damping fluid, 3-(N-morpholinyl) propane sulfonic acid damping fluid, 2-morpholino b acid damping fluid.
Preferably, said casein treating fluid comprises: the treating fluid that casein and the mixed solution of second buffer solution obtain behind autoclaving.
Preferably, said second buffer solution is the organic acid damping fluid.
Preferably, said carbohydrate is: sucrose, trehalose, sweet mellow wine, sorbierite, glycerine, maltose.
Preferably, said albumen, protein protective agent, the mass concentration ratio of surfactant in said buffer solution are: (0.01～5): (0.1～20): (0.05～5).
Preferably, the ion concentration of said buffer solution is that 0.01-1mol/L, pH value are 4～9.
The present invention also provides a kind of immunity-chromatography test strip, comprising: plastic plate; Be arranged on the adsorptive pads on the said plastic plate; Be arranged on the nitrocellulose filter on the said adsorptive pads; Be arranged on the sample pad on the said nitrocellulose filter;
Wherein, said sample pad comprises: albumen, protein protective agent, surfactant and buffering solution; Wherein, said albumen is casein treating fluid, MBSA, human serum albumins, and said protein protective agent is a carbohydrate.
Preferably, said casein treating fluid comprises: the treating fluid that casein and the mixed solution of buffering solution obtain behind autoclaving.
The invention provides a kind of immunity-chromatography test strip and use sealant compositions, comprising: albumen, protein protective agent, surfactant and first buffer solution; Wherein, said albumen is casein, casein treating fluid, MBSA, human serum albumins; Said protein protective agent is a carbohydrate.Casein, casein treating fluid, MBSA, human serum albumins have been used in the sealant compositions provided by the invention; These albumen have good hydrophobic nature; With respect to prior art, can in colloidal sol, better use, in emulsion, can not make albumen soluble in water with flowing of solution; Firm is fixed on the NC film, has reduced the background that causes owing to environment for use and has raise.In addition, in the composition provided by the invention, also comprise protein protective agent, make the albumen in the composition can keep more permanent use, improved the serviceable life of composition.
The invention provides a kind of immunity-chromatography test strip, comprising: plastic plate; Be arranged on the adsorptive pads on the said plastic plate; Be arranged on the nitrocellulose filter on the said adsorptive pads; Be arranged on the sample pad on the said nitrocellulose filter; Wherein, said sample pad comprises: albumen, protein protective agent, surfactant and buffering solution; Wherein, said albumen is casein treating fluid, MBSA, human serum albumins, and said protein protective agent is a carbohydrate.The prior art of comparing contains sealer in the immunity-chromatography test strip sample pad provided by the invention, and said sealer comprises albumen, protein protective agent, surfactant and buffering solution; Said albumen is casein treating fluid, MBSA, human serum albumins, and said protein protective agent is a carbohydrate, makes that reaction site does not change in the strip, and sealing effect is good, and background is low, long service life.
Description of drawings
Fig. 1, immunity-chromatography test strip structural representation provided by the invention.
Embodiment
In order further to understand the present invention, below in conjunction with embodiment the preferred embodiments of the invention are described, but should be appreciated that these just restriction for further specifying feature and advantage of the present invention rather than patent of the present invention being required is described.
The invention provides a kind of immunity-chromatography test strip and use sealant compositions, comprising: comprising: albumen, protein protective agent, surfactant and first buffer solution; Wherein, said albumen is casein, casein treating fluid, MBSA, human serum albumins; Said protein protective agent is a carbohydrate.
The prior art of comparing; Albumen provided by the invention is hydrophobicity albumen preferably, can be firm in emulsion on the strip site, play sealing process, can site change in location or site not take place along with flowing of water increase; Said albumen is selected casein, casein treating fluid, MBSA for use; Human serum albumins is preferably casein or casein treating fluid, most preferably is the casein treating fluid.The preparation method of said casein treating fluid does, is that second buffer solution of 6～7 0.1mol/L mixes with casein and pH, and dissolving is handled through autoclaving then and being obtained the casein treating fluid.According to the present invention, the mass concentration of said albumen in first buffer solution is preferably 0.01%-5%, and more preferably 0.04%-1% most preferably is 0.2%.Said second buffer solution is preferably glycocoll-hydrochloric acid buffer solution, and the pH value is preferably 6.5.
According to the present invention; In order to make albumen have longer serviceable life; Make said composition after long-time the placement, still have good sealing effect, and low background, protein protective agent increased in the composition provided by the invention; Said protein protective agent is a carbohydrate, is preferably sucrose, trehalose, sweet mellow wine, sorbierite, glycerine, maltose.More preferably sucrose, trehalose, sorbierite.Most preferably be sucrose.According to the present invention, the mass concentration of said protein protective agent in first buffer solution is preferably 0.1%-20%, and more preferably 0.5%-10% most preferably is 5%.Carbohydrate all is a polyol, and high concentration sugar can form glassy state in the high temperature drying process with materials such as protein, enzymes, thereby makes protein active be able to protection.
According to the present invention, also comprise in the said composition increasing albumen surfactant that mixes with emulsion and first buffer solution of regulating sealant compositions pH value.According to the present invention; Said surfactant is preferably non-ionics, is preferably polyoxyethylene 20 sorbitan monolaurate (Tween20), polyoxyethylene sorbitan monooleate (Tween80), NONIN HS 240 (Triton x 100) or polyoxyethylene lauric acid ether (Brij 35).More preferably polyoxyethylene 20 sorbitan monolaurate or NONIN HS 240 most preferably are polyoxyethylene 20 sorbitan monolaurate.Mass concentration in first buffer solution is preferably 0.05%-5%, and more preferably 0.1%-2% most preferably is 0.5%
Said first buffer solution is preferably phosphate buffer solution, tromethamine-hydrochloride buffer, borate buffer, glycocoll-hydrochloride buffer, citrate buffer solution, veronal buffer, malonic acid-sodium hydrate buffer solution, maleic acid-sodium hydrate buffer solution; Succinic acid-sodium hydrate buffer solution; 4-HEPES damping fluid; 3-(N-morpholinyl) propane sulfonic acid damping fluid, 2-morpholino b acid damping fluid.Phosphate buffer more preferably, veronal buffer, 4-HEPES damping fluid, optimal selection phosphate buffer.The pH value of said first buffer solution is preferably between the 5-8 between 4-9, most preferably is 6.5, and ionic strength is 0.005-1mol/L, is preferably 0.05-0.5mol/L, and optimum is 0.1mol/L,
According to the present invention, said albumen is mixed with protein protective agent, surfactant is blended in first buffer solution, then albumen and protein protective agent evenly are blended in first buffer solution, obtain sealant compositions.
Sealant compositions provided by the invention can use in the strip sample pad, also can mix use with latex particle, is used to reduce the background of immunochromatography.
According to the present invention; Sealer is added in the sample pad, then the NC film is attached on the plastic plate, to Membrane jetter, rule then; NC film dried overnight behind the spray film; Second day assembling strip, strip is pasted on the plastic plate according to the order of sample pad, NC film, thieving paper, to cutting machine, is cut into strip to strip at last.
The present invention also provides a kind of immunity-chromatography test strip, comprising: plastic plate; Be arranged on the adsorptive pads on the said plastic plate; Be arranged on the nitrocellulose filter on the said adsorptive pads; Be arranged on the sample pad on the said nitrocellulose filter; Wherein, said sample pad comprises: albumen, protein protective agent, surfactant and buffering solution; Wherein, said albumen is casein treating fluid, MBSA, human serum albumins, and said protein protective agent is a carbohydrate.
According to the present invention; As shown in Figure 1, said immunity-chromatography test strip comprises: sample pad 2, nitrocellulose filter 3, adsorptive pads 4, plastic plate 5 add sample solution 1 on the strip; Obtain the result after 5～10 minutes, that T and C line all develop the color is negative, C colour developing and T do not develop the color is positive.
Below be specific embodiment:
1, the comparison of protein sealing effect
The particle mark: experiment is at first active through EDC and NHS to the fluorescence latex particle; The albuminous monoclonal antibody reactive of particle and AHS then; Behind centrifugal, wash-out; The particle suspending that is marked with antibody in following 5 kinds of different protein confining liquids: 1, blank control group, the confining liquid of 2,0.5% gelatin; 3, the confining liquid of 0.5%B SA; 4,0.5% caseic confining liquid; 5, the confining liquid of 0.5% skimmed milk power; 6, casein treating fluid; 7,1% MBSA.
The compound method of casein treating fluid: glycocoll-hydrochloride buffer of the 0.1mol/l of secure ph=6.5 at first, be dissolved in casein in this damping fluid then, after handling, autoclaving obtains the casein treating fluid.
Strip is made: earlier the NC film is attached on the plastic plate; To Membrane jetter, rule NC film dried overnight behind the spray film, second day assembling strip then; Strip is pasted on the plastic plate according to the order of sample pad, NC film, thieving paper, to cutting machine, is cut into the wide strip of 4mm to strip at last.
Strip detects: strip is divided into 7 groups, and every group is divided into 2 parts again, and portion is negatives (this test item is a microdose urine protein, and the cutoff value is 20mg/l, and the albumin concentration of negatives is 5mg/l.); Another part is positive sample (being that albumin concentration is 25mg/l). every group of 40 strips, 20 of positive sample, 20 of negatives.When strip detects, will be stored in particle solution and the mixed of positive perhaps negatives according to 10: 1 in the different proteins confining liquid earlier, it is 0.05% that promptly final particle contains concentration admittedly.Join then in the sample pad, every strip solution addition is 80ul.The experiment test result is following:
| Protein sealer type | Positive | Negative | Positive coincidence rate (%) | Negative match-rate (%) |
| Blank | 0 | 7 | 0 | 35 |
| Gelatin | 0 | 6 | 0 | 30 |
| BSA | 8 | 9 | 40 | 45 |
| Casein | 16 | 16 | 80 | 80 |
| Skimmed milk power | 16 | 15 | 80 | 75 |
| The casein treating fluid | 17 | 17 | 85 | 85 |
| MBSA | 17 | 16 | 85 | 80 |
2, the comparison of protein protectant
Protein protectant generally adopts polyalcohols such as carbohydrate, glycerine to protect; So experimental design is following: at first with the latex particle activation; Then to the particle labeled monoclonal antibody, with the particle suspending of labeled monoclonal antibody in following several solns: 1, blank control group; 2, add 1% sucrose group; 3, add 1% trehalose group; 4, add the glycerine group; 5, add 1% sweet mellow wine group; 6, add 1% maltose group; All added the casein sealer in the above solution, in 37 ℃ baking oven, worn out 20 days then, worn out and mix with the latex particle that is marked with monoclonal antibody after 20 days.
Strip detects: strip is divided into 6 groups, and every group is divided into 2 parts again, and portion is negatives (this test item is a microdose urine protein, and the cutoff value is 20mg/l, and the albumin concentration of negatives is 5mg/l.); Portion is positive sample (being that albumin concentration is 25mg/l). every group of 40 strips, 20 of positive sample, 20 of negatives.When strip detects, will be stored in particle solution and the mixed of positive perhaps negatives according to 10: 1 in the different proteins confining liquid earlier, it is 0.05% that promptly final particle contains concentration admittedly.Join then in the sample pad, every strip solution addition is 80ul.The experiment test result is following:
| The protein protectant type | Positive | Negative | Positive coincidence rate (%) | Negative match-rate (%) |
| Blank control group | 0 | 3 | 0 | 15 |
| The sucrose group | 14 | 15 | 70 | 75 |
| The trehalose group | 15 | 16 | 75 | 80 |
| The glycerine group | 10 | 12 | 50 | 60 |
| The sweet mellow wine group | 13 | 14 | 65 | 70 |
| The maltose group | 9 | 13 | 45 | 65 |
3, the comparison of surfactant
In above-mentioned casein, trehalose confining liquid, add different surface active agents and compare, adding surfactivity mainly is to make sample pad wetting evenly, also guarantees the consistance that latex particle flows on the NC film simultaneously.Experimental design is following: at first also be with the latex particle activation, then to the particle labeled monoclonal antibody, with the particle suspending of labeled monoclonal antibody in following several solns: 1, blank control group; 2, add 20 groups of 0.5%Tween; 3, add the 0.5%Tween80 group; 4, add 0.5%triton x-100 group; 5, add 35 groups of 0.5%Brij; All added casein, trehalose sealer in the above solution, mixed with the latex particle that is marked with monoclonal antibody then.
Strip detects: strip is divided into 5 groups, and every group is divided into 2 parts again, and portion is a negatives; Part is every group of 40 strips of positive sample in addition, 20 of positive sample, 20 of negatives.When strip detects, will be stored in particle solution and the mixed of positive perhaps negatives according to 10: 1 in the different proteins confining liquid earlier, it is 0.05% that promptly final particle contains concentration admittedly.Join then in the sample pad, every strip solution addition is 80ul.The experiment test result is following:
| Surfactant types | Positive | Negative | Positive coincidence rate (%) | Negative match-rate (%) |
| Blank control group | 16 | 16 | 80 | 80 |
| 20 groups of Tween | 18 | 19 | 90 | 95 |
| 80 groups of Tween | 15 | 14 | 75 | 70 |
| Triton x-100 group | 17 | 18 | 85 | 90 |
| 35 groups of Brij | 17 | 17 | 85 | 85 |
4, the comparison of buffer system
Through to protein sealer, carbohydrate and surface-active comparison, need to add casein in the component of confining liquid, trehalose and Tween 20, but these confining liquids also need can be more stable in certain damping fluid.Therefore to human body microdose urine protein project, experimental selection following several kinds of buffer solution carried out comparative study, damping fluid is following: 1, blank control group (water); 2, add the phosphate buffer group; 3, add the citrate buffer solution group; 4, add glycocoll-hydrochloride buffer group; 5, add tris-hydrochloride buffer group; All added 0.5% casein, 1% trehalose, 0.5%Tween 20 confining liquid components in the above solution, mixed with the latex particle that is marked with monoclonal antibody then.The strip detection method is consistent with top method of testing, and the experiment test result is following:
| Buffer type | Positive | Negative | Positive coincidence rate (%) | Negative match-rate (%) |
| Blank control group | 18 | 19 | 90 | 95 |
| The phosphate buffer group | 20 | 20 | 100 | 100 |
| The citrate buffer solution group | 18 | 19 | 90 | 95 |
| Glycocoll-hydrochloride buffer group | 19 | 18 | 95 | 90 |
| Tris-hydrochloride buffer group | 17 | 19 | 85 | 95 |
More than a kind of immunity-chromatography test strip provided by the invention has been carried out detailed introduction with sealant compositions and a kind of immunity-chromatography test strip; Having used concrete example among this paper sets forth principle of the present invention and embodiment; The explanation of above embodiment just is used for helping to understand method of the present invention and core concept thereof; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of claim of the present invention.
Claims (10)
1. an immunity-chromatography test strip is used sealant compositions, it is characterized in that, comprising:
Albumen, protein protective agent, surfactant and first buffer solution;
Wherein, said albumen is casein, casein treating fluid, MBSA, human serum albumins; Said protein protective agent is a carbohydrate.
2. composition according to claim 1 is characterized in that, said surfactant is polyoxyethylene 20 sorbitan monolaurate, polyoxyethylene sorbitan monooleate, NONIN HS 240 or polyoxyethylene lauric acid ether.
3. composition according to claim 1; It is characterized in that; Said first buffer solution is phosphate buffer solution, tromethamine-hydrochloride buffer, borate buffer, glycocoll-hydrochloride buffer, citrate buffer solution, veronal buffer, malonic acid-sodium hydrate buffer solution, maleic acid-sodium hydrate buffer solution, succinic acid-sodium hydrate buffer solution, 4-HEPES damping fluid; 3-(N-morpholinyl) propane sulfonic acid damping fluid, 2-morpholino b acid damping fluid.
4. composition according to claim 1 is characterized in that, said casein treating fluid comprises: the treating fluid that casein and the mixed solution of second buffer solution obtain behind autoclaving.
5. composition according to claim 4 is characterized in that, said second buffer solution is the organic acid damping fluid.
6. composition according to claim 1 is characterized in that, said carbohydrate is: sucrose, trehalose, sweet mellow wine, sorbierite, glycerine, maltose.
7. composition according to claim 1 is characterized in that, said albumen, protein protective agent, the mass concentration ratio of surfactant in said buffer solution are: (0.01～5): (0.1～20): (0.05～5).
8. according to claim 1,3,5 any described compositions, it is characterized in that the ion concentration of said buffer solution is that 0.01-1mol/L, pH value are 4～9.
9. an immunity-chromatography test strip is characterized in that, comprising: plastic plate; Be arranged on the adsorptive pads on the said plastic plate; Be arranged on the nitrocellulose filter on the said adsorptive pads; Be arranged on the sample pad on the said nitrocellulose filter;
Wherein, said sample pad comprises: albumen, protein protective agent, surfactant and buffering solution; Wherein, said albumen is casein treating fluid, MBSA, human serum albumins, and said protein protective agent is a carbohydrate.
10. immunity-chromatography test strip according to claim 9 is characterized in that, said casein treating fluid comprises: the treating fluid that casein and the mixed solution of buffering solution obtain behind autoclaving.
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