CN101266248A - HCV IgM antibody rapid detection test paper - Google Patents
HCV IgM antibody rapid detection test paper Download PDFInfo
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- CN101266248A CN101266248A CNA2008100529611A CN200810052961A CN101266248A CN 101266248 A CN101266248 A CN 101266248A CN A2008100529611 A CNA2008100529611 A CN A2008100529611A CN 200810052961 A CN200810052961 A CN 200810052961A CN 101266248 A CN101266248 A CN 101266248A
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Abstract
The invention relates to a biology applied technique field, especially relates to a hepatitis C virus antibody quick detection test paper, comprising a sampling pad (1); a colloidal gold pad (2) closely connected with one end of the sampling pad and containing HCV labelled mixed antigen; a cellulose nitrate (NC) membrane closely connected with another end of the sampling pad and a sucking sample pad (4) closely connected with another end of the NC membrane; a test T line (5) and quality control C line (6) mutually separated with each other and coated by NC membrane, wherein the T line is anti human IgM antibody coated on the NC membrane and the C line is anti HCV antibody coated on the NC membrane. During detecting, the detected sample is added on the sampling pad of the test paper and the immunoreaction result is directly observed to accomplish detection. The invention has features: high sensitivity, specificity, quickspeed, convenience and is suitable for detection on site and real time recording and storing the result.
Description
Technical field
The present invention relates to the biologic applications technical field, particularly relates to a kind of with colloidal gold immunity chromatography fast detecting hepatitis C virus (HCV) IgM antibody fast test.
Background technology
HCV was found from infected chimpanzee blood preparation by U.S. Choo etc. in 1989 at first, mainly by blood, body fluid communication, accounted for 70% of post-transfusion hepatitis.According to World Health Organization's statistics, about 1.7 hundred million people's HCV infection are estimated in the whole world.Hepatitis C chronicity rate is 50%-85%, wherein has 20% can develop into liver fibrosis again, endangers very serious.China HCV antibody the carrier reach about 4,000 ten thousand.Up to the present hepatitis C had not both had effective vaccine, did not also have effective methods of treatment, and it is unique effective method that prevention is infected.HCV antibody is checked and hepatitis B surface antigen (HBsAg), human immunodeficiency virus (HIV) antibody and the inspection of syphilis (TP) antibody are four legal haematogenous examination projects of China, and meaning is very important.
HCV antibody test reagent mainly detects HCV IgG at present.IgM detects has only research report few in number, concentrates on enzyme linked immunosorbent assay analysis method (ELISA) and detects.Detection can not illustrate that the patient is that existing disease is infected or previous infection to IgG, though the recall rate of IgM is the 50%-80% of IgG, it is the infection of existing disease that IgM detects positive basic explanation patient.In addition, IgM antibody is that HCV RNA recall rate is relevant with the body viral replication in; Relevant with hepatitis development degree in the body endosome, necrosis of liver cells; In to HCV patient process, the quality of result of treatment is also pointed out in the variation of IgM; The infection of the different subtype of the appearance of IgM and HCV is relevant in addition, thereby detection has positive reference significance to HCV IgM.
Utilization immunochromatography colloidal gold technique detects HCV IgM is not still had report.The immunochromatography colloidal gold technique is novel diagnostic techniques, obtained comparatively extensively using, ultimate principle is as follows: utilize a kind of antigen of colloid gold label or antibody, bag is matched antigen or antibody accordingly on the NC of reagent film, during detection when containing corresponding specific antibody or antigen in the sample, the part formation compound that combines in colloid gold label particle and the sample, chromatography on the NC film then, coated again antigen or antibody capture, form macroscopic detection (T) line, have or not the judgement of realization the result by detection line.Have easy and simple to handle, reaction fast, high, the high specificity of susceptibility, be fit to on-the-spot the detection and advantage such as economical and practical.
Summary of the invention
The purpose of this invention is to provide a kind of HCV IgM antibody fast test, have easy and simple to handle, reaction fast, high, the high specificity of susceptibility, be fit to on-the-spot the detection and advantage such as economical and practical.
The invention provides a kind of HCV IgM antibody fast test.Comprise sample pad (1), closely be connected in the collaurum pad (2) that contains underlined HCV hybrid antigen of sample pad one end, with the close-connected cellulose nitrate of the other end of collaurum pad (NC) film (3) with closely be connected in the suction sample pad (4) of the NC film other end, the NC film is coated with detection (T) line (5) and Quality Control (C) line (6) that is separated from each other, last sample pad, the collaurum pad, NC film and suction sample pad paste plastic support board (7) and go up the formation test strips, test strips can also be packed into and be formed the cassette packing in the plastic clip, it is characterized in that the T line is the anti-human IgM antibody that is coated on the NC film, the collaurum pad is that the HCV hybrid antigen of mark constitutes.
Described HCV IgM antibody fast test is characterized in that cAg (Core) and non-structural protein (NS) 3 antigens of described HCV hybrid antigen for the reorganization expression of HCV, also alternative NS4, NS5, E1 and the E2 district antigen that adds HCV again.
Described HCV IgM antibody fast test is characterized in that described upward sample pad is glass fibre membrane or nonwoven fabrics, inhales the sample pad and is made of absorbent filter.
Described HCV IgM antibody fast test, it is characterized in that, the method for coating of described antigen is: will resist human IgM antibody to be mixed with the solution of 1mg/ml with 0.01M pH 7.2 phosphate buffers (PBS), rule with the parameter of 1ul/cm in NC film bottom with spray film instrument, bag is wrapped by HCV antigen/antibody combination as the C line on NC film top simultaneously by the T line.After the line with the NC film drying room (temperature 20-25 ℃, humidity is less than 30%) dry 8-10 hour.
Described HCV IgM antibody fast test, it is characterized in that, the method of described hybrid antigen mark colloid gold particle is: prepare the colloidal gold solution that diameter is 30-50nm with gold chloride-trisodium citrate reduction method, get 100ml collaurum liquid after preparation is finished and be placed in the beaker, use 0.2M K
2CO
3Transfer to pH8.0, press the 100ml colloidal gold solution and add 1mg HCV reorganization hybrid antigen, stirring at room 2 hours, add 1% bovine serum albumin(BSA) (BSA), 0.1% polyglycol (PEG), 20000 sealing 20min, centrifugal 30 minutes of 12000r/m abandons supernatant, redissolve to 100ml with the collaurum working fluid, press 1ml solution shop 22cm
2Ratio be layered on equably on glass fibre membrane or the nonwoven fabrics, put drying room (temperature 20-25 ℃, humidity is less than 30%) drier 2-4 hour, make the collaurum pad.
Described HCV IgM antibody fast test, the assembly method that it is characterized in that described test paper is: in hothouse (temperature 20-25 ℃, humidity is less than 30%), get the plastic support board plate, paste at the middle part that the NC film that wraps quilt is placed on plastic support board, paste at NC film T line one side overlap joint collaurum pad (take collaurum pad 1/3), paste the sample pad at collaurum pad opposite side overlap joint and (take glue
Described HCV IgM antibody fast test, it is characterized in that, detection method is: with tested serum or blood plasma balance to the greenhouse, test strips or reagent card are kept flat, on last sample pad, add the 50-100ul test sample, the sample dissolution collaurum and on the NC film chromatography, the appearance situation of Direct observation C, T line in 30 minutes with the naked eye then, and judge testing result
The invention has the beneficial effects as follows: a kind of novel test paper that utilizes the immunochromatography colloidal gold technique to detect HCV IgM is provided.Adopt colloid gold label HCV antigen, bag is by the detection of anti-people IgM realization to HCV IgM in the sample.Have easy and simple to handle, reaction fast, high, the high specificity of susceptibility, be fit to on-the-spot the detection and advantage such as economical and practical.
Description of drawings:
Fig. 1 HCV antibody fast test structural representation.
The reference numeral explanation:
1: go up sample pad 2: the collaurum pad
3:NC film 4: inhale the sample pad
5: detect (T) line 6: Quality Control (C) line
7: plastic support board
Embodiment
Embodiment: preparation HCV IgM antibody fast test
1 main material
1.1HCV CORE, NS3 fused antigen: U.S. Biotech Atlantic Inc (BAI.) product is used for colloid gold label; Mouse-anti people IgM, HCV antigen/antibody combination: Military Medical Science Institute's product is used for NC film bag quilt; Gold chloride: Sigma company product; Cellulose nitrate (NC) film: Millipore company product; Bovine serum albumin(BSA) (BSA), polyglycol (PEG) 20000, caseinhydrolysate: Sigma product.Other common agents is analytical reagent.
1.2 clinical serum obtains 80 parts of HCV IgG antibody positive serum, 300 parts of HCV negative antibody serum by company in relevant hospital or blood station.
1.3HCV IgM antibody test ELISA kit, U.S. Abbott product.
2 methods
2.1HCV the colloid gold label gold chloride-trisodium citrate reduction method of recombinant antigen prepares diameter
Be the colloidal gold solution of 40nm, get three parts of collaurums after preparation is finished, use 0.2M K respectively
2CO
3Solution is transferred to pH7.0, pH8.0 and pH9.0.Then solution is placed on the magnetic stirring apparatus and slowly stir, slowly be added drop-wise to albumen in the colloidal gold solution recombinant antigen by the every adding of 100ml solution 0.5mg, 1mg, 1.5mg, continue to stir 2 hours, be added dropwise to final concentration again and be 0.1% PEG2000 and 1% BSA and seal 20min, it is centrifugal with 12000r/m that mark finishes the back, abandon supernatant, precipitation is pressed original volume and is redissolved to the collaurum working fluid (borate buffer solution of different proportionings, pH8.0, contain BSA, sheep blood serum, sucrose and surfactant) in.Then the mark colloidal gold solution is pressed 1ml solution shop 22cm
2The ratio application of sample on nonwoven fabrics, at temperature 20-25 ℃, relative humidity is made the collaurum pad dry 2-4 hour of＜30% drying room.
2.2 the bag of mouse-anti people IgM is diluted to 0.5mg/ml, 1mg/ml, 2mg/ml with 0.01M pH7.2PBS respectively with mouse-anti people IgM, then with spray film instrument in NC film bottom by the 1ul/cm bag quilt of ruling, wrap on NC film top simultaneously by HCV antigen/antibody combination, be used for the Quality Control of product, after bag is done with the NC film at temperature 20-25 ℃, relative humidity was dry 8-10 hour of＜30% drying room.
2.3HCV the IgM antibody fast test be assembled in the hothouse (temperature 20-25 ℃, relative humidity＜30%) gets plastic support board, paste at the middle part that the NC film that wraps quilt is placed on plastic support board, paste at NC film T line one side overlap joint collaurum pad (take collaurum pad 1/3), paste sample pad (take collaurum pad 1/5) at collaurum pad opposite side overlap joint; Inhale sample pad (take inhale sample pad 1/10) at NC film C line one side overlap joint; Plastic plate be will post with cutter then and 3mm or the wide test strips of 4mm will be cut into.The test strips that cuts can reinstall in the plastic clip, forms HCV IgM antibody test reagent card.
2.4 detection method with tested serum or blood plasma balance to the greenhouse, the test strips or the reagent card that prepare are kept flat, on last sample pad, add the 50-100ul test sample, if contain anti-HCV IgM antibody in the sample, then with sample pad on the collaurum combination of mark HCV antigen, form compound, and be diffused on the NC film further chromatography, when running into the mouse-anti people IgM that is coated on T line place on the NC film, compound then again with the bag by anti-IgM antibodies, be trapped in bag and located, when captive colloidal gold composite reaches some, then form a macroscopic T line; If do not contain specific antibody in the serum, then can not form immune complex, also can not form the T line, the C line is as the quality control standard of reagent, and positive and negative sample all can occur when detecting.The appearance situation of Direct observation C, T line in 15,20,30 and 45 minutes with the naked eye, and judgement testing result.
2.5 test paper technological parameter debugging with concentration not the reagent of isolabeling, bag quilt make up pairing, the preparation sample utilizes quality controlled serum that reagent is tested, and finds best of breed.
2.6 clinical serum detects and all HCV IgG positive serums, negative serum detected by detection method with this reagent, positive serum carries out control test with Abbott IgM antibody test ELISA reagent simultaneously.
3 results
3.1 the test paper parameter is determined the testing result according to sample, the optimum mark pH value of having determined test paper is 8.0; The optimum mark amount of reorganization hybrid antigen is the 1mg/100ml colloidal gold solution; Best collaurum working fluid is a 10mM borosilicate damping fluid, and pH8.0 contains 0.5%BSA, 10% sheep blood serum, 2% sucrose, and 0.2%Tween 20; Best bag mouse-anti people IgM bag is 1mg/ml by concentration.The optimal decision time of testing result is 5-30 minute.
3.2 it is contrast that clinical serum detects with ELISA reagent, ELISA detects 54 parts of IgM positive serums in 80 parts of HCV IgG positive serums, and it is 52 parts that this reagent detects the positive, sensitivity=52/54=96.3%; This reagent detects 5 parts of false positives, specificity=295/300=98.3% to 300 parts of negative serums.The analytical reagent performance is compared there was no significant difference with ELISA reagent.
Claims (7)
1, a kind of hepatitis C virus (HCV) IgM antibody fast test, comprise sample pad (1), closely be connected in the collaurum pad (2) that contains underlined HCV hybrid antigen of sample pad one end, with the close-connected cellulose nitrate of the other end of collaurum pad (NC) film (3) with closely be connected in the suction sample pad (4) of the NC film other end, the NC film is coated with detection (T) line (5) and Quality Control (C) line (6) that is separated from each other, last sample pad, the collaurum pad, NC film and suction sample pad paste plastic support board (7) and go up the formation test strips, test strips can also be packed into and be formed the cassette packing in the plastic clip, it is characterized in that the T line is the anti-human IgM antibody that is coated on the NC film, the collaurum pad is that the HCV hybrid antigen of mark constitutes.
2, HCV IgM antibody fast test according to claim 1, it is characterized in that cAg (Core) and non-structural protein (NS) 3 antigens of described HCV hybrid antigen, also alternative NS4, NS5, E1 and the E2 district antigen that adds HCV again for the reorganization expression of HCV.
3, HCV IgM antibody fast test according to claim 1 is characterized in that described upward sample pad is glass fibre membrane or nonwoven fabrics, inhales the sample pad and is made of absorbent filter.
4, according to claim 1 and 2 described HCV IgM antibody fast tests, it is characterized in that, the method for coating of described antigen is: will resist human IgM antibody to be mixed with the solution of 1mg/ml with 0.01M pH 7.2 phosphate buffers (PBS), rule with the parameter of 1ul/cm in NC film bottom with spray film instrument, bag is wrapped by HCV antigen/antibody combination on NC film top as the C line simultaneously by the T line, after the line with the NC film at drying room, temperature 20-25 ℃, humidity is less than 30%, dry 8-10 hour.
5, according to claim 1 and 2 described HCV IgM antibody fast tests, it is characterized in that, the method of described hybrid antigen mark colloid gold particle is: prepare the colloidal gold solution that diameter is 30-50nm with gold chloride-trisodium citrate reduction method, get 100ml collaurum liquid after preparation is finished and be placed in the beaker, use 0.2M K
2CO
3Transfer to pH8.0, press the 100ml colloidal gold solution and add 1mg HCV reorganization hybrid antigen, stirring at room 2 hours, add 1% bovine serum albumin(BSA) (BSA), 0.1% polyglycol (PEG), 20000 sealing 20min, centrifugal 30 minutes of 12000r/m abandons supernatant, redissolve to 100ml with the collaurum working fluid, press 1ml solution shop 22cm
2Ratio be layered on equably on glass fibre membrane or the nonwoven fabrics, put drying room again, temperature 20-25 ℃, humidity dry 2-4 hour, is made the collaurum pad less than 30%.
6, according to claim 1 and 3 described HCV IgM antibody fast tests, the assembly method that it is characterized in that described test paper is: in hothouse, temperature 20-25 ℃, humidity is got the plastic support board plate less than 30%, and paste at the middle part that the NC film that wraps quilt is placed on plastic support board, at NC film T line one side overlap joint collaurum pad, take 1/3 of collaurum pad and paste, paste the sample pad, take 1/5 of collaurum pad at collaurum pad opposite side overlap joint; Inhale the sample pad at NC film C line one side overlap joint, take and inhale 1/10 of sample pad; To post plastic plate with cutter at last and be cut into 3mm or the wide test strips of 4mm, the test strips that cuts can reinstall in the plastic clip, forms test card.
7, HCV IgM antibody fast test according to claim 1, it is characterized in that, detection method is: with tested serum or blood plasma balance to the greenhouse, test strips or reagent card are kept flat, on last sample pad, add the 50-100ul test sample, the sample dissolution collaurum and on the NC film chromatography, the appearance situation of Direct observation C, T line in 30 minutes with the naked eye then, and judge testing result.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100529611A CN101266248A (en) | 2008-04-30 | 2008-04-30 | HCV IgM antibody rapid detection test paper |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100529611A CN101266248A (en) | 2008-04-30 | 2008-04-30 | HCV IgM antibody rapid detection test paper |
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| CN101266248A true CN101266248A (en) | 2008-09-17 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102109519A (en) * | 2009-12-29 | 2011-06-29 | 北京库尔科技有限公司 | Rubella virus IgG and IgM antibody joint inspection kit and preparation method thereof |
| CN102109523A (en) * | 2009-12-29 | 2011-06-29 | 北京库尔科技有限公司 | Hepatitis C virus IgG and IgM antibody joint detection kit and preparation method thereof |
| CN102707055A (en) * | 2012-06-11 | 2012-10-03 | 郑州安图绿科生物工程有限公司 | Kit for joint or single detection of autoimmune liver disease related antibody and detection method of kit |
| CN102955027A (en) * | 2012-06-11 | 2013-03-06 | 郑州安图绿科生物工程有限公司 | Kit for detecting LC-1 antibody relative to autoimmune liver diseases, and detection method with kit |
| CN103163298A (en) * | 2011-12-19 | 2013-06-19 | 天津中新科炬生物制药有限公司 | Rapid detective reagent strip of treponema pallidum immunoglobulin m (IgM) antibody and preparation method thereof |
| CN103207271A (en) * | 2012-04-23 | 2013-07-17 | 齐明山 | Hepatitis C antibody confirmation reagent |
| CN103575884A (en) * | 2012-07-25 | 2014-02-12 | 上海纽卓生物科技有限公司 | Colloidal gold method-based hepatitis c virus (HCV) fast-diagnosis kit |
| CN104407143A (en) * | 2014-11-28 | 2015-03-11 | 山东博科生物产业有限公司 | Hepatitis c virus antigen-antibody joint detection kit |
| CN112816700A (en) * | 2020-12-28 | 2021-05-18 | 杭州新脉生物科技有限公司 | Colloidal gold test strip for detecting HIV antibody in urine |
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2008
- 2008-04-30 CN CNA2008100529611A patent/CN101266248A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102109519A (en) * | 2009-12-29 | 2011-06-29 | 北京库尔科技有限公司 | Rubella virus IgG and IgM antibody joint inspection kit and preparation method thereof |
| CN102109523A (en) * | 2009-12-29 | 2011-06-29 | 北京库尔科技有限公司 | Hepatitis C virus IgG and IgM antibody joint detection kit and preparation method thereof |
| CN103163298A (en) * | 2011-12-19 | 2013-06-19 | 天津中新科炬生物制药有限公司 | Rapid detective reagent strip of treponema pallidum immunoglobulin m (IgM) antibody and preparation method thereof |
| CN103163298B (en) * | 2011-12-19 | 2015-04-08 | 天津中新科炬生物制药有限公司 | Rapid detective reagent strip of treponema pallidum immunoglobulin m (IgM) antibody and preparation method thereof |
| CN103207271A (en) * | 2012-04-23 | 2013-07-17 | 齐明山 | Hepatitis C antibody confirmation reagent |
| CN102707055A (en) * | 2012-06-11 | 2012-10-03 | 郑州安图绿科生物工程有限公司 | Kit for joint or single detection of autoimmune liver disease related antibody and detection method of kit |
| CN102955027A (en) * | 2012-06-11 | 2013-03-06 | 郑州安图绿科生物工程有限公司 | Kit for detecting LC-1 antibody relative to autoimmune liver diseases, and detection method with kit |
| CN103575884A (en) * | 2012-07-25 | 2014-02-12 | 上海纽卓生物科技有限公司 | Colloidal gold method-based hepatitis c virus (HCV) fast-diagnosis kit |
| CN104407143A (en) * | 2014-11-28 | 2015-03-11 | 山东博科生物产业有限公司 | Hepatitis c virus antigen-antibody joint detection kit |
| CN104407143B (en) * | 2014-11-28 | 2016-04-27 | 山东博科生物产业有限公司 | A kind of C hepatitis virus antigen-antibody combined detection kit |
| CN112816700A (en) * | 2020-12-28 | 2021-05-18 | 杭州新脉生物科技有限公司 | Colloidal gold test strip for detecting HIV antibody in urine |
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Application publication date: 20080917 |