CN101363866B - Test paper strip for detecting encephalitis virus IgM antibody colloidal gold, method for making same and applications - Google Patents
Test paper strip for detecting encephalitis virus IgM antibody colloidal gold, method for making same and applications Download PDFInfo
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Abstract
The invention provides a test strip for rapid detection of Japanese encephalitis virus IgM antibody. A Japanese encephalitis virus E gene antigen domain III and a double-antibody IgM III coat a nitrate cellulose film (NC film), and a membrane chromatography indirect sandwich method is adopted to detect the Japanese encephalitis virus specific IgM antibody in an infected human body specimen in combination with a colloidal gold labeled antihuman IgM monoclonal antibody. The test strip is simple in operation, convenient, and fast, and has the advantages of no requirements of special instruments and special training, clear and identified result, and easy popularization. The test strip is suitable for base course, site detection and epidemiological investigation, and has auxiliary effect on the diagnosis of Japanese encephalitis virus infection.
Description
Technical field
The present invention relates to a kind of test paper strip for detecting encephalitis virus IgM antibody colloidal gold, its preparation method and application thereof, belong to field of biological detection.
Background technology
(Japanese encephalitis JE) is called for short encephalitis to Japanese Type-B encephalitis, is to belong to enveloped virus section Flavivirus by central nervous system sexually transmitted disease [1] .JEV due to the encephalitis B virus (JEV) of having a liking for nerve; Be ball-type, diameter 20～30nm, core contains single-stranded RNA; Capsid is arranged, three structural proteins are arranged, promptly E albumen, nucleoprotein C and memebrane protein M.E albumen are glycoprotein; Be wrapped in the surface of virus; It is determining the virulence and the host range thereof of virus, not only in the film fusion that combines with host cell receptor to reach subsequently, plays a significant role, and can stimulate the generation neutralizing antibody aspect immunoprotection, to play main effect.Its characteristic feature is high heat headache, occurs a series of brain symptoms and meningismus subsequently.Case fatality rate generally between 10-30%, has quite a few to leave sequelae.
Result of study shows that E albumen is the major protein of encephalitis patients serum IgG identification.Think that at present E albumen space structure is divided into three parts, wherein domain II I is folding at virus surface independently, exists in encephalitis B virus main and epitope.Therefore obtain this regional expressing protein through genetic engineering, and with its specific antibody as the Detection of antigen encephalitis B virus.
The encephalitis traditional detection method is that virus is separated at present, with C6/36 cell separation virus; The mouse intracranial inoculation.Identify in the serological method of encephalitis B virus, neutralization test, complement fixation test (CFT) and to coagulate the enzyme inhibition test be first generation method, the second generation has immunofluorescence method.
Mainly contain following several kinds in the antibody test: (one) specificity lgM detects: the infection and encephalitis B virus infection morbidity promptly produces specificity lgM in early days, and 2～3 weeks peaked after being ill, so single sera can be made early diagnosis.Can use: 1. lgM catches the ELISA method; 2. 2ME-NI method, hemagglutination inhibition antibody is present among lgM and the lgG, and early stage single sera is divided into two parts; A with 2 mercaptoethanols (2ME) processing, to destroy lgM, another part do not handled; Doing HI test then simultaneously, if handle serum antibody titer than untreated decline >=4 times, then be the lgM positive; 3. micro-IIF with fluorescein-labeled anti-u chain serum, detects the lgM that has combined with the cellular antigens sheet, according to the specificity fluorescent particle, judges that the lgM in the serum specimen exists.
(2) conventional serological test: 1. hemagglutination-inhibition test is as far as the encephalitis diagnosis, and this law specificity is lower, but susceptibility is high, simple and easy to do.2. the complement fixation test (CFT) complement fixation antibody exists only among the lgG, occurs after being ill late, and it is fast to disappear, and is applicable to the diagnosis recent infection.3. neutralization test antibody is present among lgG, the lgM, occurs early keeping for a long time.Neutralization test specificity and susceptibility are all high, but trivial operations, need with a large amount of animals or tissue culture tube, take more for a long time, are inappropriate for that clinical diagnosis is conventional to be used, and valuable in serology epidemiology survey and virus evaluation.
Colloidal gold immunity chromatography (Immunochromatography Assay) starts from the mid-90 in last century, is on the basis of immunity percolation method, to grow up.It is the combination of immune affine technology, engram technology, immunolabelling technique and chromatographic technique.With envelope antigen, colloid gold label antibody immobilization, combine with sample sorbing material etc., be prepared as immunochromatography diagnostic test/plate, only need be during use inserting sample solution under the test strips, several minutes just can judged result.With the immunity percolation method relatively, good stability operate easylier, quick, and owing to be strip/test plate (panel) form, need not cryopreservation, accumulating makes things convenient for.
To the popular present situation and control requirement of encephalitis, for the diagnosis of encephalitis, the reagent of not only demanding specificity and susceptibility also needs quick, easy, as to be easy to primary care and health unit use method.
Summary of the invention
The purpose of this invention is to provide a kind of easy to usely, quick, be used to detect the test strips of encephalitis virus specificity antibody, detect encephalitis virus specificity IgM antibody in the infected patient sample, be used for the auxiliary diagnosis of people's infection and encephalitis B virus infection.
Another object of the present invention provides a kind of preparation method of test paper strip for detecting encephalitis virus IgM antibody colloidal gold.
A purpose more of the present invention provides the application of a kind of test paper strip for detecting encephalitis virus IgM antibody colloidal gold in detecting encephalitis virus IgM antibody.
A kind of test paper strip for detecting encephalitis virus IgM antibody colloidal gold of the present invention, it comprises: the nitrocellulose membrane that encapsulates encephalitis B virus E gene antigen territory III and two bands of two anti-IgG; And contain the glass fibre membrane of the anti-people IgM monoclonal antibody of colloid gold label.
Described two anti-IgG are anti-mouse IgG antibody.
The concentration of said anti-mouse IgG antibody is 2mg/ml.
The concentration 3.2mg/ml of encephalitis B virus E gene antigen territory III in the said nitrocellulose membrane.
The optimum pH of the anti-people IgM of colloid gold label is 7.6～8.0 in the said glass fibre membrane,, the proportioning of collaurum and antibody is with 20～24 μ g/ml collaurums.
Test paper strip for detecting encephalitis virus IgM antibody colloidal gold of the present invention; Also comprise reaction holder, golden labeling antibody diaphragm and adsorptive pads, overlap golden labeling antibody diaphragm, glass fibre membrane, nitrocellulose membrane and the adsorptive pads of pasting on the said reaction holder successively each other.
The preferred PVC plate of reaction holder; The preferred filter paper for oil of adsorptive pads; Gold labeling antibody diaphragm has absorbent function simultaneously, preferably uses polyester film, spun glass or filter paper fibre.
The preparation method of test paper strip for detecting encephalitis virus IgM antibody colloidal gold of the present invention may further comprise the steps:
1) preparation encephalitis B virus E gene antigen territory III;
2) preparation of nitrocellulose membrane: territory III is diluted to 3.2mg/ml with encephalitis B virus E gene antigen, will resist mouse IgG antibody dilution to become 2mg/ml, is sprayed on the nitrocellulose membrane, forms detection line and control line respectively, and in 37 ℃ dry 2 hours, subsequent use;
3) preparation of glass fibre membrane: the pH value of the antibody of colloid gold label is 7.6～8.0; The proportioning of collaurum and antibody is 20～24 μ g/ml collaurums, (contains 0.05%BSA, pH8.0 through stabilizing agent; 0.01MTris damping fluid) after the processing; Amount by every square centimeter 65 μ l evenly is adsorbed on the glass fibre membrane, and freeze drying is subsequent use;
4) on the reaction holder, overlap the golden labeling antibody diaphragm of stickup, glass fibre membrane, nitrocellulose membrane and adsorptive pads at last successively each other.
The application of test paper strip for detecting encephalitis virus IgM antibody colloidal gold according to the invention in detecting encephalitis virus IgM antibody.
The present invention adopts the encephalitis B virus E gene antigen territory III and the anti-mouse IgG difference solid phase (NC film) on nitrocellulose membrane of purifying; The anti-people IgM monoclonal antibody of association colloid gold mark is used rete and is analysed the encephalitis virus IgM antibody in the principle detection people sample of indirect sandwich assay.
Test strips of the present invention utilizes colloidal gold-labeled method and rete to analyse technology, is used for the encephalitis virus specificity IgM antibody that fast qualitative half-quantitative detection sample possibly exist, and reaches quick screening patient, the purpose of diagnoses and treatment in time.Can save a large amount of manpower and materials, easily and fast, simple and direct, not need special instruments and equipment, not need professional training; Clear being prone to of result distinguish, and be simple to operate, is easy to promote; Be fit to basic unit, be suitable for on-the-spot the detection and early diagnosis, the Infect And Diagnose of encephalitis B virus is played booster action.
Description of drawings
Figure 1A is the front schematic view of test paper strip for detecting encephalitis virus IgM antibody colloidal gold according to the invention;
Figure 1B is the side schematic view of test paper strip for detecting encephalitis virus IgM antibody colloidal gold according to the invention.
Wherein: 1 adsorptive pads; 2 nitrocellulose membranes (T: encephalitis B virus E gene antigen territory III; C: the Quality Control band that encapsulates anti-encephalitis B virus polyclonal antibody); 3 contain the glass fibre membrane of the anti-people IgM monoclonal antibody of colloid gold label; 4 gold medal labeling antibody diaphragms; 5 reaction holders.
Fig. 2 is a test strip testing result synoptic diagram.
Be followed successively by from left to right: T, two line positives of C; Line feminine gender of C; T, two line feminine genders of C are invalid.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
If do not specialize the conventional means that used technological means is well known to those skilled in the art among the embodiment.
The preparation of embodiment 1 encephalitis B virus E gene antigen territory III
(1) acquisition of genes of interest
The primer that contains the restricted interior enzyme BamH1 of association, HindIII restriction enzyme site according to the characteristics design two ends of target gene fragment sequence and pGEX-4T-1 expression vector:
5’TAAGGATCCCACCTGAAATGTAGGCTG 3’
5’CGGGAAGCTTGAAGACCCCTCCAATAGA 3’
Propose virus total RNA with conventional method, then, carry out RT-PCR immediately, reverse transcription product is identified with 1% agarose gel electrophoresis and is reclaimed.
(2) screening of the clone of genes of interest and positive recombinant
With pcr amplification product that reclaims and PMD-18T cloning vector spend the night for 16 ℃ is connected after, be transformed in the DH5a competent cell, picking monoclonal bacterial strain, 37 ℃ of incubated overnight, extract plasmid after, be that template is carried out PCR evaluation positive colony bacterial strain, mensuration sequence with the plasmid.
(3) structure of fusion expression vector
With restriction enzyme BamH1, HindIII respectively enzyme cut T/EIII and pGEX-4T-1; 1% agarose electrophoresis is cut the big fragment after glue reclaims purpose fragment and pGEX-4T-1 double digestion,, connects product and changes the BL21 competent cell over to both 16 ℃ of connections of spending the night with the T4 ligase; The LB solid medium was cultivated 10～12 hours; Extract plasmid after the picking list bacterium colony overnight incubation, identify respectively that with PCR and restriction enzymes double zyme cutting PCR product and enzyme are cut product and analyzed with 1% agarose electrophoresis; Screening positive clone is with the plasmid order-checking of reorganization.
(4) abduction delivering of pGEX-EIII fusion
The positive colony bacterium that filters out shakes the bacterium overnight incubation in the LB nutrient culture media after, the bacterium that will spend the night is seeded in the 1000mlLB fluid nutrient medium according to 1: 100 ratio, and 37 ℃ of shaking tables are cultivated.The genetic transformation bacterium is induced opportunity (OD600nm 0.5) back 2 hours of inoculation for the best, and IPTG concentration 0.6mmol/L induced 8 hours for 29 ℃, and destination protein is present in the cell pyrolysis liquid supernatant.
(5) purifying of pGEX-EIII fusion, evaluation
1000ml bacterium liquid 12000r/m in (4), 4 ℃ is centrifugal; Abandon supernatant; Thalline is used the cell pyrolysis liquid cracking, use the affinity chromatography pillar purified fusion protein that contains the GST label with GST-fusion purification kit (B-PER bacterium GST tag fusion protein pillar purifying Kit article No.: the silent generation that science and technology that flies of 78200 matches), carry out purifying by kit recommendation step and system; Purified product carries out the SDS-PAGE electrophoresis to judge purification effect, records productive rate more than 95% through the ultraviolet thin layer.
The Western-Blot of fusion identifies
Cutting half glue changes on nitrocellulose filter with the half-dried electroporation that BioRad company produces, and carries out the trace test, and one anti-ly is encephalitis B virus monoclonal antibody (Beijing Bo Aosen Bioisystech Co., Ltd), and after the TMB colour developing, the result has the specific proteins band to occur.
(1) preparation of collaurum-antibody conjugates:
Confirm that through experiment its best combination pH value of anti-people IgM monoclonal antibody colloid mark is 8.0, the proportioning of collaurum and antibody is 18 μ g/ml collaurums.The mark collaurum by the amount of every square centimeter 65 μ l, is got collaurum-antibody conjugates solution after stabilizing agent (containing 0.5%BSA, pH8.0,0.01MTris damping fluid) is handled, evenly is adsorbed on the spun glass, and freeze drying, and in dry environment, preserve.
(2) envelope antigen is in nitrocellulose membrane:
Encephalitis B virus E gene antigen territory III albumen is diluted to 3.2 ± 0.1mg/ml with 0.01MPBS.To resist mouse IgG polyclonal antibody to be diluted to 2 ± 0.1mg/ml with 0.01MPBS.With Membrane jetter the two speed with 1 μ l/cm is sprayed on the nitrocellulose membrane, forms detection line and control line respectively.Article two, be spaced apart 0.5cm between the line.
Put the cellulose nitrate that is fixed with antibody in 37 ℃ of baking boxs dry 2 hours.Preserve subsequent use in the dry environment.
(3) test paper strip for detecting encephalitis virus IgM antibody colloidal gold
(4) test paper strip for detecting encephalitis virus IgM antibody colloidal gold specificity and susceptibility
Specific detection: detect 5 parts of IgM positive serums (comprising influenza, mycoplasma pneumoniae, measles, rubella, Respiratory Syncytial Virus(RSV)) with the encephalitis virus IgM antibody test strip; 5 parts of healthy subjects serum; 5 parts of hepatitis B, third liver and infection of the upper respiratory tract patients' blood serum sample; Testing result is all negative, shows that this product is special.
Susceptibility detects: with the positive encephalitis patients serum of encephalitis totivirus ELISA ELISA Plate screening IgM, with the neutralization experiment IgM titre of serum is demarcated then.With physiological saline the serum of 10 parts of different titers is carried out doubling dilution; Detect with the encephalitis virus IgM antibody test strip then; The result shows, different patients' serum is can detected minimum dilutability different, and the minimum titre of its IgM antibody is 1: 10～20.
Patient's whole blood, blood plasma or serum specimen 100-150 μ l directly are added dropwise to embodiment 2 test strips " 4 " locate, sample liquid is up along film, 10～15 minutes sentence read result.
The result:
As detect and contain encephalitis virus IgM antibody in the sample; Then with test strips on the anti-people IgM monoclonal antibody of colloid gold label form corresponding compound; Up be coated on nitrocellulose membrane on encephalitis virus specificity antigen combine, form red lines, promptly form red stripes at the T place.
No matter whether contain corresponding antibody in the sample, the anti-people IgM monoclonal antibody of colloid gold label continues upwards to creep and the anti-mouse IgG that is coated on the film forms the red precipitate line, promptly locates to form red stripes at " C ".This line is a nature controlling line, loses efficacy like collaurum, and this line just can not occur, and explains that test strips lost efficacy.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Sequence table
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< 120>a kind of test paper strip for detecting encephalitis virus IgM antibody colloidal gold, its preparation method and application thereof
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Claims (3)
1. a test paper strip for detecting encephalitis virus IgM antibody colloidal gold is characterized in that it comprises: the nitrocellulose membrane (2) that encapsulates encephalitis B virus E gene antigen territory III and two bands of anti-mouse IgG antibody; And contain the glass fibre membrane (3) of the anti-people IgM monoclonal antibody of colloid gold label;
The concentration of said anti-mouse IgG antibody is 2mg/ml, and the concentration of encephalitis B virus E gene antigen territory III is 3.2mg/ml;
The pH value of the anti-people IgM of colloid gold label is 7.6～8.0 in the glass fibre membrane, and the proportioning of collaurum and antibody is 20～24 μ g/ml collaurums.
2. test paper strip for detecting encephalitis virus IgM antibody colloidal gold according to claim 1; It is characterized in that; Also comprise reaction holder (5), golden labeling antibody diaphragm (4) and adsorptive pads (1), overlap golden labeling antibody diaphragm (4), glass fibre membrane (3), nitrocellulose membrane (2) and the adsorptive pads of pasting (1) on the said reaction holder (5) successively each other.
3. method for preparing the described test paper strip for detecting encephalitis virus IgM antibody colloidal gold of claim 2 may further comprise the steps:
1) preparation encephalitis B virus E gene antigen territory III;
2) preparation of nitrocellulose membrane: territory III is diluted to 3.2mg/ml with encephalitis B virus E gene antigen, will resist mouse IgG antibody dilution to become 2mg/ml, is sprayed on the nitrocellulose membrane, forms detection line and control line respectively, and in 37 ℃ dry 2 hours, subsequent use;
3) preparation of glass fibre membrane: the pH value of the antibody of colloid gold label is 7.6～8.0, and the proportioning of collaurum and antibody is 20～24 μ g albumen/ml collaurums, evenly be adsorbed on the glass fibre membrane by the amount of every square centimeter 65 μ l, and freeze drying, subsequent use;
4) on the reaction holder, overlap the golden labeling antibody diaphragm of stickup, glass fibre membrane, nitrocellulose membrane and adsorptive pads at last successively each other.
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| CN102262156B (en) * | 2010-06-04 | 2014-01-29 | 北京庄笛浩禾生物医学科技有限公司 | Detection test strip for coxsackie virus A16 type (CA16) IgM (Immune Globulin M) antibody |
| CN106442980A (en) * | 2016-08-28 | 2017-02-22 | 张金凤 | Test paper for quickly detecting human epidemic encephalitis B virus IgM antibody |
| CN113640514A (en) * | 2021-06-09 | 2021-11-12 | 上海农林职业技术学院 | Colloidal gold test strip for detecting canine encephalitis B virus antibody and preparation method thereof |
Citations (2)
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| CN1834650A (en) * | 2006-04-18 | 2006-09-20 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immunity chromatography test paper for detecting farcina Boeck Hold's bacteria infection and prepn. method thereof |
| CN201053964Y (en) * | 2007-05-21 | 2008-04-30 | 四川省迈克科技有限责任公司 | Vaccine protective antibody quick detection reagent kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1834650A (en) * | 2006-04-18 | 2006-09-20 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immunity chromatography test paper for detecting farcina Boeck Hold's bacteria infection and prepn. method thereof |
| CN201053964Y (en) * | 2007-05-21 | 2008-04-30 | 四川省迈克科技有限责任公司 | Vaccine protective antibody quick detection reagent kit |
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| 王祥等.猪乙型脑炎血清抗体DIGFA检测方法的建立与应用.《畜牧兽医学报》.2002,第33卷(第2期),第200-204页. * |
| 谢荣辉等.流行性乙型脑炎病毒囊膜蛋白截片段的克隆及表达.《中国计划免疫》.2007,第13卷(第6期),第533-535页. * |
| 赵中夫等.滴金免疫法快速检测流行性乙型脑炎血清特异性IgM.《中国人兽共患病杂志》.1998,第14卷(第6期),第44-45页. * |
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Effective date of registration: 20161205 Address after: 125208 Suizhong Binhai Economic Zone, Liaoning, No. 25 1-10 Patentee after: Liaoning Di Hao Biotechnology Co., Ltd. Address before: 100043 West Street, Shijingshan District, Beijing, No. 33 Patentee before: Beijing Zhuangdi Haohe Biomedicine Science and Technology Co., Ltd. |
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Inventor after: Liu Ming Inventor after: Zhang Lili Inventor after: Li Minggang Inventor before: Liu Ming |