CN202149901U - Hapatitis A virus IgM and IgM antibody combined test paper - Google Patents

Hapatitis A virus IgM and IgM antibody combined test paper Download PDF

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Publication number
CN202149901U
CN202149901U CN201120186081U CN201120186081U CN202149901U CN 202149901 U CN202149901 U CN 202149901U CN 201120186081 U CN201120186081 U CN 201120186081U CN 201120186081 U CN201120186081 U CN 201120186081U CN 202149901 U CN202149901 U CN 202149901U
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virus
igm
hepatitis
pad
line
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霍五奎
岳晓燕
马雪明
刘寅
王晶
罗云萍
张有青
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JOYSBIO (TIANJIN) BIOTECHNOLOGY CO Ltd
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JOYSBIO (TIANJIN) BIOTECHNOLOGY CO Ltd
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Abstract

The utility model discloses hapatitis A virus IgM and IgM antibody combined test paper, which is characterized in that a sample loading pad (1) is adhered to one end of a plastic supporting board (8), one end of the sample loading pad (1) is tightly connected with a colloidal gold pad (2) containing marked hapatitis A virus antigen VP1 or VP3 in a pressing mode, and one end of the colloidal gold pad (2) is tightly connected with a nitric acid cellulose nitro cellulose (NC) film (3). A test line T1 (5), a test line T2 (6) and a quality control C line which are separated from each other are coated on the film. The test line T1 (5) is anti-human IgM antibodies, the test line T2 (6) is anti-human IgG antibodies, and the quality control C line (7) is anti-hapatitis A virus VP or VP1 antibodies. The other end of the nitric acid cellulose film is connected with a sample absorption pad (4) to form the test paper. During test, products to be sampled are added on the sample loading pad of the test paper, the samples can be viewed directly by naked eyes in 15 minutes to enable users to judge test results. The hapatitis A virus IgM and IgM antibody combined test paper has the advantages of being convenient and quick to operate, suitable for on-site test, economical and practical and the like.

Description

The antibody combined detection test paper of hepatitis A virus IgM and IgG
Technical field
The utility model relates to the biologic applications technical field, particularly relates to a kind of with colloidal gold immunity chromatography fast detecting hepatitis A virus IgM and the antibody combined detection test paper of IgG
Background technology
Viral hepatitis type A (abbreviation hepatitis A) is a kind of virus hepatitis that is caused by hepatitis A virus (HAV).Hepatitis A virus belongs to picornavirus, and various extraneous factors are had stronger resistibility and can in external environment, survive for a long time, can pass through all contaminations article and water and food transmission, also can carry and propagate through fly.Behind human infection's hepatitis A virus, show as subclinical or subclinical infection mostly, only acute hepatitis A is asked in the minority performance.Generally can recover fully, not transfer chronic hepatitis to.
Hepatitis A is mainly propagated through fecal oral route, and in 15-45 days latent period, virus is everlasting in the blood and ight soil that just were present in the patient in 5-6 days of patient's transaminase before raising.Anti--HAV IgM antibody occurs very soon in the serum of infection back, about two weeks, reaches the peak, descends gradually then, within 8 weeks, disappear, and be the serological evidence of HAV acute infection and recent infection; Anti--HAV IgG antibody produces later; Occur in early days (when IgM begins to descend in later stage acute stage and convalescence; IgG rises gradually) belong to protection antibody, reach the peak in convalescence, can exist lastingly; Can protect no longer subinfection of human body, significant to hepatitis A epidemiology survey, health examination, understanding Susceptible population.
The main method that at present hepatitis A virus is detected is that the hepatitis A virus specific antibody detects.Detect hepatitis A virus IgM and take a decision as to whether the infection of existing disease, detect IgG and judge whether once infected, avoid subinfection again.Detecting two kinds of antibody helps the carrying out of checked object comprehensively judged.The detection method of anti-HAV mainly is detection methods such as RIA, ELISA at present.In the detection method of collaurum, the report of independent detection hepatitis A virus IgM or IgG is arranged, but detect hepatitis A virus IgM simultaneously and IgG detection of antibodies test paper does not have relevant report as yet with collaurum para-immunity chromatographic technique.With detect IgM respectively with two kinds of reagent and compare with IgG antibody, detect the workload that IgM and IgG antibody capable reduce the testing staff simultaneously, practice thrift cost; Reduce blood sampling volume simultaneously, reduce patient's misery.
The immunochromatography colloidal gold technique is novel diagnostic techniques; Aspect antibody test, obtained comparatively extensively using, ultimate principle is following: utilize a kind of antigen of colloid gold label or antibody, on the NC of reagent film, encapsulate corresponding pairing antigen or antibody; 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 through 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 the utility model provides a kind of hepatitis A virus IgM and IgG antibody test test paper, 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 utility model provides a kind of hepatitis A virus IgM and the antibody combined detection test paper of IgG; Appearance is filled up (1) to it is characterized in that pasting upward by plastic support board (8) one ends; The tight crimping of one end of last appearance pad (1) contains the collaurum pad (2) of specific antigen VP1 of underlined hepatitis A virus or VP3; Collaurum pad (2) one ends tight crimping cellulose nitrate NC films (3); Nitrocellulose filter is coated with detection line T1 (5), detection line T2 (6) and the Quality Control C line that is separated from each other, and detection line T1 (5) is for being coated on the anti-human IgM antibody on the NC film, and detection line T2 (6) is for being coated on the anti-human IgG antibody on the NC film; Quality Control C line (7) is for being coated on anti-hepatitis A virus VP1 or the VP3 antibody on the NC film, and the other end of nitrocellulose filter connects suction appearance pad (4) and forms test paper.
The antibody combined detection test paper of described hepatitis A virus IgM and IgG is characterized in that specific major antigen VP1 of described antigen hepatitis A virus or VP3 antigen, antigen can utilize genetic engineering recombinant expressed or in cultivating virus purifying.
The antibody combined detection test paper of described hepatitis A virus IgM and IgG is characterized in that the described appearance pad (1) of going up is glass fibre membrane or nonwoven fabrics, inhales appearance pad (4) and is made up of absorbent filter.
The antibody combined detection test paper of described hepatitis A virus IgM and IgG; The method for coating of described antigen is: will resist human IgM antibody to be mixed with the solution of 1-2mg/ml with 0.01M pH 7.2 phosphate buffers (PBS), will resist the human IgG antibody to be mixed with the solution of 1-2mg/ml with 0.01M pH 7.2 phosphate buffers (PBS), and rule with the parameter of 1-1.5ul/cm in NC film bottom with spray film appearance; Encapsulate T1, T2 line; Simultaneously encapsulate anti-hepatitis A virus VP1 antibody as the C line on NC film top, after the line with the NC film at drying room, temperature 20-25 ℃; Humidity is less than 30%, dry 2-5 hour.
The antibody combined detection test paper of described hepatitis A virus IgM and IgG; The method of described antigenic 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 accomplished and be placed in the beaker, use 0.2M K 2CO 3Transfer to pH8.5, press the 100ml colloidal gold solution add 0.5-2mg hepatitis A virus VP1 with (or) VP3 antigen, stirring at room 2 hours; Add 1% bovine serum albumin(BSA) BSA; 1% polyglycol PEG20000 seals 20min, and centrifugal 30 minutes of 12000r/m abandons supernatant; Redissolve to 100ml with the collaurum working fluid, press 1ml solution shop 20cm 2Ratio be layered on equably on glass fibre membrane or the nonwoven fabrics, put drying room again, temperature 20-25 ℃, humidity dry 2-5 hour, is processed the collaurum pad less than 30%.
The antibody combined detection test paper of described hepatitis A virus IgM and IgG, the assembly method of described test paper is: in hothouse, temperature 20-25 ℃; Humidity is less than 40%; Get the plastic support board plate, paste at the middle part that the NC film that has encapsulated is placed on plastic support board, pastes at NC film T line one side overlap joint collaurum pad (take collaurum pad 1/3); Paste the appearance pad at collaurum pad opposite side overlap joint, take 1/5 of collaurum pad; Inhale the appearance pad at NC film C line one side overlap joint, take and inhale 1/10 of appearance pad; To post plastic plate with cutter at last and be cut into the wide test strips of 2-5mm, the test strips that cuts can reinstall in the plastic clip, forms test card.
The antibody combined detection test paper of described hepatitis A virus IgM and IgG; Detection method is: seized serum or blood plasma balance to the greenhouse, are kept flat test strips or reagent card, on last appearance pad, add the 5-20ul test sample; The sample diluting liquid that adds 50-100ul again; Make collaurum dissolving and on the NC film chromatography, the appearance situation of Direct observation C, T1, T2 line in 15 minutes with the naked eye then, and judge testing result.
The beneficial effect of the utility model is: a kind of novel test paper that utilizes the immunochromatography colloidal gold technique to detect hepatitis A virus IgM and IgG antibody is provided.Adopt colloid gold label hepatitis A virus specific antigen, encapsulate anti-people IgM and IgG antibody and realize the detection to anti-HAV in the blood, one-time detection obtains two kinds and detects index, saves time and cost, and alleviates patient's misery.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:
The antibody combined detection test paper of Fig. 1 hepatitis A virus IgM and IgG structural representation.
The reference numeral explanation:
1: go up the appearance pad; 2: the collaurum pad; The 3:NC film; 4: inhale the appearance pad
5: detection line T1; 6: detection line T2; 7: Quality Control C line; 8: plastic support board
Embodiment
Embodiment: preparation hepatitis A virus IgM and the antibody combined detection test paper of IgG
1 main material
1.1 hepatitis A virus specificity VP1 antigen, VP1 antibody: the Shenzhen City Fapon Biotech Co., Ltd product, be recombinant antigen, VP1 antigen is used for colloid gold label, and VP1 antibody is used for NC film nature controlling line and encapsulates; Mouse-anti people IgM and IgG antibody: the Arista Company products is used for the NC film and encapsulates; Gold chloride: Sigma Company products; Cellulose nitrate (NC) film: Millipore Company products; Bovine serum albumin(BSA) (BSA), polyglycol PEG20000, caseinhydrolysate: Sigma product.Other common agents is AR.
1.2 clinical serum is obtained in relevant hospital by company, wherein hepatitis A virus IgG antibody positive sample is 50 parts, and 15 parts in IgM antibody positive sample is comprising 8 parts of two kinds of equal positive sample of antibody.60 parts in two kinds of complete negative normal person's samples of antibody.
1.3 antihepatitis A virus IgM detection kit (ELISA method), hepatitis A virus IgG antibody assay kit (ELISA method): domestic registered commercial product.
2 methods
2.1 the colloid gold label gold chloride-trisodium citrate reduction method of hepatitis A virus VP1 recombinant antigen prepares the colloidal gold solution that diameter is 40nm, preparation is got three parts of collaurums after accomplishing, and uses 0.2M K respectively 2CO 3Solution is transferred to pH7.5, pH8.5 and pH9.5.Then solution is placed on the magnetic stirring apparatus and slowly stir; Add 0.5mg, 1mg, 1.5mg by every 100ml solution recombinant antigen slowly is added drop-wise to albumen in the colloidal gold solution, 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, deposition is pressed original volume and is redissolved to the collaurum working fluid borate buffer solution of different proportionings, pH8.5; Contain BSA, sheep blood serum, in sucrose and the surfactant.Then the mark colloidal gold solution is pressed 1ml solution shop 20cm 2The ratio application of sample on nonwoven fabrics, at temperature 20-25 ℃, relative humidity is processed the collaurum pad dry 2-4 hour of<30% drying room.
2.2NC film encapsulates with 0.01M pH7.2PBS mouse-anti people IgM is diluted to 0.5mg/ml, 1mg/ml, 1.5mg/ml, 2mg/ml respectively; The mouse-anti human IgG is diluted to 0.5mg/ml, 1mg/ml, 1.5mg/ml, 2mg/ml respectively; Ruling respectively by 1ul/cm in NC film bottom with spray film appearance then encapsulates, and encapsulates anti-VP1 antibody on NC film top simultaneously, is used for the Quality Control of product; Encapsulate after the completion the NC film at temperature 20-25 ℃, relative humidity was dry 2-5 hour of<30% drying room.
2.3 the antibody combined detection test paper of hepatitis A virus IgM and IgG 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 has encapsulated is placed on plastic support board; Paste at NC film T line one side overlap joint collaurum pad (take collaurum pad 1/3), paste appearance pad (take collaurum pad 1/5) at collaurum pad opposite side overlap joint; Inhale appearance pad (take inhale appearance 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 hepatitis A virus IgM and the antibody combined detectable card of IgG.
2.4 detection method to the greenhouse, keeps flat seized serum or blood plasma balance with test strips for preparing or reagent card, on last appearance pad, add the 10ul test sample; If contain anti-hepatitis A virus IgM or IgG antibody in the sample, then with sample pad on the collaurum of mark VP1 antigen combine, form compound; And be diffused on the NC film further chromatography; When running into the mouse-anti people IgM that is coated on T1 line place on the NC film, compound then again with encapsulate anti-IgM antibodies, be trapped in and encapsulate the place; When running into when being coated on the NC film T2 line IgG of place, compound then combines with coated antibody again, is trapped in and encapsulates line T2 place; When captive colloidal gold composite reaches some, then form a macroscopic T1 or T2 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 5,10,15,20 and 30 minutes with the naked eye, and judgement testing result.
2.5 test paper technological parameter debugging with concentration not isolabeling, the reagent that encapsulates 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 clinical serum is detected by detection method with this reagent, carries out control test with the commercial ELISA reagent that detects simultaneously.
3 results
3.1 the test paper parameter is confirmed the testing result according to sample, the optimum mark pH value of having confirmed test paper is 8.5; 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 pH8.5 contains 0.5%BSA, 10% sheep blood serum, 2% sucrose, and 0.2%Tween 20; Best bag mouse-anti people IgM and IgG encapsulate concentration and are 1.5mg/ml.The optimal decision time of testing result is 5-15 minute.But above parameter possibly need suitably adjustment when preparation different batches product.
3.2 it is contrast that clinical serum detects with ELISA reagent, and IgG antibody is detected, this reagent IgG detects positive 48 parts, and ELISA detects positive 50 parts of IgG, sensitivity=48/50=96%; This reagent detects 59 parts of negative sample numbers, relative specificity=59/60=98.3% to the IgG negatives.P>0.05。With ELISA reagent is contrast, and IgM antibody is detected, and this reagent IgM detects positive 10 parts, and ELISA detects positive 10 parts of IgM, sensitivity=10/10=100.0%; This reagent detects all negative to the IgM negatives, relative specificity 100%.P>0.05。Simultaneously positive this reagent of sample of IgM and IgG detect and ELISA reagent in full accord.Clinical detection shows that the performance of this reagent compares there was no significant difference with ELISA reagent, is suitable for Clinical detection.

Claims (3)

1. hepatitis A virus IgM and the antibody combined detection test paper of IgG; Appearance is filled up (1) to it is characterized in that pasting upward by plastic support board (8) one ends; The tight crimping of one end of last appearance pad (1) contains the collaurum pad (2) of specific antigen VP1 of underlined hepatitis A virus or VP3; Collaurum pad (2) one ends tight crimping cellulose nitrate NC films (3); Nitrocellulose filter is coated with detection line T1 (5), detection line T2 (6) and the Quality Control C line that is separated from each other, and detection line T1 (5) is for being coated on the anti-human IgM antibody on the NC film, and detection line T2 (6) is for being coated on the anti-human IgG antibody on the NC film; Quality Control C line (7) is for being coated on anti-hepatitis A virus VP1 or the VP3 antibody on the NC film, and the other end of nitrocellulose filter connects suction appearance pad (4) and forms test paper.
2. the antibody combined detection test paper of hepatitis A virus IgM according to claim 1 and IgG; It is characterized in that described antigen is specific major antigen VP1 of hepatitis A virus or VP3 antigen, antigen can utilize genetic engineering recombinant expressed or in cultivating virus purifying.
3. the antibody combined detection test paper of hepatitis A virus IgM according to claim 1 and IgG is characterized in that the described appearance pad (1) of going up is glass fibre membrane or nonwoven fabrics, inhales appearance pad (4) and is made up of absorbent filter.
CN201120186081U 2011-06-03 2011-06-03 Hapatitis A virus IgM and IgM antibody combined test paper Expired - Lifetime CN202149901U (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102692502A (en) * 2012-04-26 2012-09-26 北京北方生物技术研究所 Rapid hepatitis A virus IgM antibody detection kit and preparation method thereof
CN103018446A (en) * 2012-12-24 2013-04-03 青岛汉唐生物科技有限公司 Method for detecting hepatitis A virus antibodies, kit for detection through method and preparation method for kit
CN104087609A (en) * 2014-07-10 2014-10-08 山东省农业科学院家禽研究所 General type duck hepatitis A virus antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit
CN111381049A (en) * 2020-03-30 2020-07-07 天津纽赛生物技术有限公司 Serology type detection test paper and detection method for rapid new coronavirus antibody

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102692502A (en) * 2012-04-26 2012-09-26 北京北方生物技术研究所 Rapid hepatitis A virus IgM antibody detection kit and preparation method thereof
CN102692502B (en) * 2012-04-26 2015-02-04 北京北方生物技术研究所 Rapid hepatitis A virus IgM antibody detection kit and preparation method thereof
CN103018446A (en) * 2012-12-24 2013-04-03 青岛汉唐生物科技有限公司 Method for detecting hepatitis A virus antibodies, kit for detection through method and preparation method for kit
CN103018446B (en) * 2012-12-24 2014-08-20 青岛汉唐生物科技有限公司 Method for detecting hepatitis A virus antibodies, kit for detection through method and preparation method for kit
CN104087609A (en) * 2014-07-10 2014-10-08 山东省农业科学院家禽研究所 General type duck hepatitis A virus antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit
CN104087609B (en) * 2014-07-10 2016-08-24 山东省农业科学院家禽研究所 A kind of universal DHAV antibody ELISA detection kit
CN111381049A (en) * 2020-03-30 2020-07-07 天津纽赛生物技术有限公司 Serology type detection test paper and detection method for rapid new coronavirus antibody

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