CN101187665A - Enterovirus immunofluorescence chromatographic assay test paper and its preparation method - Google Patents
Enterovirus immunofluorescence chromatographic assay test paper and its preparation method Download PDFInfo
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- CN101187665A CN101187665A CNA2007103044196A CN200710304419A CN101187665A CN 101187665 A CN101187665 A CN 101187665A CN A2007103044196 A CNA2007103044196 A CN A2007103044196A CN 200710304419 A CN200710304419 A CN 200710304419A CN 101187665 A CN101187665 A CN 101187665A
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Abstract
The invention relates to a detecting testing paper for enterovirus fluorescence immune chromatographies, which comprises a base plate, a sampling absorbing mat, a marking antibody mat, a reaction film and a water absorbing mat. The sampling absorbing mat, the marking antibody mat, the reaction film and the water absorbing mat are connected closely in turn and are adhered on the base plate. The invention is characterized in that one or a plurality of enterovirus fluorescence marking monoclonal antibodies are wrapped by the marking antibody mat, one or a plurality of testing belts and a quality control belt are arranged on the reaction film, an enterovirus monoclonal antibody is fixed on each testing belt, and a sheep anti mouse IgC antibody is arranged on the quality control belt. Enteroviruses which are tested include one or a plurality of Norwalk virus, enteric adenovirus, human astrovirus and rotavirus. The purpose of qualitative or quantitative tests can be realized through a specific reaction of fluorescein or fluorescence microspheres marking antibody and applying special instrument tests.
Description
Technical field
The present invention relates to a kind of immunofluorescence chromatographic assay test paper, specifically a kind of immunofluorescence chromatographic assay test paper bar that is used to detect the enteron aisle common virus, detectable enterovirus comprises norwalk virus, EAd, astrovirus and rotavirus, and this test paper can be used for association areas such as epidemic disease monitoring, clinical examination.
Background technology
Immune chromatography method is with things that serves as a mark such as collaurum, fluorescein, enzymes, and utilizes the characteristic of specific antigen-antibody reaction and label, to antigen or antibody materials position, the labelling technique of qualitative and even quantitative examination.Since founding colloidal gold-labeled method in 1971, Geoghegan_1 uses immune technology for gold and detects the bone-marrow-derived lymphocyte surface antigen from Faulk and Taylor, has set up the immune technology for gold (IGS) of light microscopic level.Danscher improves on this basis and has developed the immunogold staining (IGSS) that strengthens light microscopic gold grain observability with silver-colored developer solution.Holgate also improves this method.In recent years, colloidal gold technique also is introduced into immunochemistry check field, be called golden labelled immune adsorption test (gold labeledimmunosorbent assay, GLISA).Because collaurum is when serving as a mark thing, its test strips easy and simple to handle need be by complicated superiority such as monitoring instrument, thereby have been subjected to paying attention to widely and using in immunohistochemistry detects, but its detection sensitivity is lower generally speaking.When serving as a mark thing, relate to a plurality of steps such as application of sample, warm bath, washing, colour developing, termination with enzyme (biotin enzyme, alkaline phosphatase etc.), consuming time longer, higher to operating personnel and environment requirement; When serving as a mark thing, can improve the sensitivity of detection to a certain extent by fluorescence detector with fluorescein.
In immune chromatography method, be further to improve sensitivity, existing with the serve as a mark report of thing of fluorescein.Use the fluorescein thing that serves as a mark, as detecting instrument, can improve the sensitivity of its detection in theory with fluorescence detector.
Diarrhoea is human especially children, old man and immunodeficiency person's a kind of common disease and frequently-occurring disease, and wherein virus diarrhea accounts for 70%～80%, is the major reason that causes death of child.Rotavirus (Rotavirus wherein, RV), astrovirus (Astrovirus, AstV), norwalk virus (Norwalk virus, NV) and EAd (Enteric Adenovirus, EAdV) etc. four kinds of enteroviruses are the most common, and nearly all children live through the infection of above-mentioned diarrhea virus before 3 years old.
The diarrhoea that rotavirus causes is the principal element of virus diarrhea, the diarrhoea of 40%-60% is caused by rotavirus, the whole world has 1.4 hundred million people that rotavirus diarrhea takes place every year approximately, its incidence of disease and the order of severity occupy the first place of diarrhoea autumn and winter, and causing about 800,000 death of child, serious rotavirus infection is mainly seen in 6～24 months infants.EAd also is the important cause of disease of virus diarrhea, and cardinal symptom is diarrhoea, watery stool or just rare, and the diarrhoea number of times is more, the course of disease 7～14 days, 10～14 days toxin expelling time, can be with heating, vomiting and respiratory symptom, the state of an illness is lighter relatively, and minority also can cause serious dehydration to cause death.Astrovirus is similar to rotavirus, with the pilosity of children below 2 years old, can distribute, also can outbreak of epidemic.Norwalk virus is widely distributed, can cause being grown up and the virus of children's acute diarrhea, and be the topmost virus causing disease that except that rotavirus, causes diarrhoea, the epidemiology survey of Japan shows that the norwalk virus infection rate has surpassed rotavirus.In the U.S., have 42% to be caused that by this viroid China also lacks epidemiology survey in the outburst of adult's nonbacterial gastroenteritis.
Still needleless is to the specific treatment medicine of these four kinds of enteroviruses clinically at present, though the report of Rotavirus Vaccine development is arranged, its effect also needs to observe, and seems particularly important so prevent, control viral communication.Reasons such as bacterium, parasite and food poisoning also can cause diarrhoea, and clinical symptoms is similar, but different with the therapeutic scheme of virus diarrhea.Have report to find the diarrhoea of dual virus or multiple virus infections, its symptom is more serious, and disease progression is fast, causes dead risk higher.
Because the clinical manifestation of enteric infectious disease and auxiliary examination commonly used lack tangible specificity, therefore exist in the indefinite hazards of the early stage pathogen diagnosis of disease, thereby exist the early stage abuse of antibiotics of disease, the sick phase prolongs and increase the objective factor of patient suffering and financial burden.Therefore,, develop diagnostic techniques fast and accurately,, instruct clinical symptomatic treatment and avoid the microbiotic abuse significant prophylactic generation and minimizing mortality ratio to the further investigation of this type of disease.
Use the etiological diagnosis that highly sensitive, quick and special immunofluorescence chromatography method carries out enteric infectious disease, help the early stage pathogen of just determining to cause enteric infectious disease that occurs at disease symptoms.Help carrying out effective antibiotic therapy, shorten the course of disease of disease, alleviate patient's misery and financial burden in the early stage specific aim of disease.To the timely and accurately etiological diagnosis of enterovirus, help the epidemiology of enterovirus is studied, guidance prevented at the initial stage of disease popularity, thereby the popular scope and the degree of the reduction enteric infectious disease of the very big degree of energy reduce enteric infectious disease to social harm.
At present adopt immunology detection for the detection of enterovirus more, traditional immune detection has latex agglutination test (LA), immunolabelling technique [comprises enzyme-linked immunoassay (EIA), radiommunoassay (RIA), immunogold staining (immunogold staining, technology such as IGS)], along with the application of novel marker and the development of solid phase carrier technology and antibody production techniques, immune quantitative technology such as time-resolved fluorescent immunoassay (time-resolvedfluoro-immunoassay, TR-IFMA or TrFIA) and quantitative latex agglutination test (quantitative latex agglutination technique, QLAT) etc. the immunology new technology is used for the detection of pathogen in succession, simplify the detection step, also improved specificity and sensitivity that immunological method detects.Can be used for the monoclonal antibody specific of the immunofluorescence chromatographic assay test paper bar method of the many inspections of enterovirus by different pathogens, come enterovirus is carried out etiological diagnosis, just can diagnose at the early stage of disease development, thereby have conspicuous clinical diagnosis meaning.
Summary of the invention
The object of the present invention is to provide the immunofluorescence chromatography quick detection test paper of a kind of common enterovirus, it has quick, effective and easy characteristics, and the pathogen that relates to is one or more in rotavirus (RV), astrovirus (AstV), norwalk virus (NV) and the EAd (EAdV).
Another object of the present invention is to provide the method for the above-mentioned detection test paper of preparation.
Immunofluorescence chromatography quick detection test paper of the present invention comprises base plate, attached to the absorption of sample pad, labelled antibody pad, reaction film and the adsorptive pads that closely link to each other successively on the base plate.Wherein said labelled antibody pad is embedded with the fluorescence labeling monoclonal antibody of one or more enteroviruses; Have one or more on the described reaction film and detect band and a quality control band, every detection with on be fixed with the monoclonal antibody of a kind of enterovirus, detect the bar number of band and fixing enterovirus monoclonal antibody and the labelled antibody of labelled antibody pad embedding and adapt, quality control band be fixed with can with the antiantibody of fluorescence labeling monoclonal antibody specific bond.
Wherein, described enterovirus monoclonal antibody is one or more in rotavirus monoclonal antibody, enteron aisle adenopathy monoclonal antibody, astrovirus monoclonal antibody or the norwalk virus monoclonal antibody.Wherein, the immunogene of preparation rotavirus monoclonal antibody is preferably rotavirus vp 6 albumen; The immunogene of preparation EAd monoclonal antibody is preferably adenovirus 40/41 type hexon (hexon); The immunogene of preparation astrovirus monoclonal antibody is preferably I type astrovirus CP albumen; The immunogene of preparation norwalk virus monoclonal antibody is preferably norwalk virus VP1 albumen.Above-mentioned immunogene is to adopt the escherichia coli prokaryotic expression system expression, utilize Protocols in Molecular Biology to amplify required nucleotide sequence, nucleic acid is connected on the expression plasmid carrier, and transfection abduction delivering in the Escherichia coli is purified into destination protein by the nickel affinity column again.Adopt Fu Shi immunologic adjuvant and purifying destination protein emulsion immunization experiment animal Balb/c mouse, every injection volume is 50 μ g albumen, the immunity position is groin and abdominal cavity, be 14 days immune interval, immunity 4 times (adjuvant of selecting for use for the first time is a Freund's complete adjuvant, and back three times is incomplete Freund); Take mice serum test Elisa antibody titer, choose the mouse of tiring greater than 1: 5000 and carry out booster immunization, the protein immunization amount is 100 μ g/, and immune position is a spleen, can carry out test for fusion after 3 days.Asepticly get its spleen cell and hybridize fusion with SP2/0-Ag14 myeloma cell, fusion agent is PEG3350.Use the indirect ELISA method screening positive clone, filter out the monoclonal cell strain of special secretion enterovirus recombinant protein by limiting dilution assay.Carry out the ascites preparation to induce method in the body, with Protein-A, protein-G or sad-ammonium sulfate method purification monoclonal antibody.The monoclonal antibody purifying thing of preparation is kept in-20 ℃ of refrigerators.Cell strain of monoclonal antibody adds cryopreserving liquid and is kept in the liquid nitrogen through enlarged culture.
What wherein, described quality control band was fixing can be sheep anti-mouse igg antibody with the antiantibody of fluorescence labeling monoclonal antibody specific bond.
Wherein, described labelled antibody is with fluorescein or the fluorescent microsphere thing that serves as a mark, and carries out crosslinked acquisition with specific enterovirus monoclonal antibody.Fluorescein is Alexa Fluor series fluorescein (the Alexa Fluor@350 of MolecularProbes company, AlexaFluor@405, Alexa Fluor@488, Alexa Fluor@555, Alexa Fluor@568, Alexa Fluor@594, Alexa Fluor@647, Alexa Fluor@660, AlexaFluor@680, Alexa Fluor@750 etc.) or the Cy series fluorescein (Cy3, Cy3.5, Cy5 etc.) of peace agate West Asia company.
Wherein, described reaction film is a nitrocellulose filter.
The present invention also provides a kind of method for preparing above-mentioned detection test paper, and it comprises the steps:
1) on the diverse location of reaction film according to the kind of antibody fixedly enterovirus monoclonal antibody and antiantibody respectively, form and detect band and quality control band;
2) preparation labelled antibody, and in sample labelled antibody pad the embedding labelled antibody;
3) assembling plate, absorption of sample pad, labelled antibody pad, reaction film and adsorptive pads;
4) cutting, packing.
In use, on the sample pad of chromatographic film, add sample liquid (supposition contains viral antigen), add dilution in case of necessity, under capillary action, sample liquid forms immune complex at labelled antibody pad place and labelled antibody, more further swimming to adsorptive pads one end swimming, different immune complexs combines the immune complex that forms similar double-antibody sandwich respectively with the monoclonal antibody at relevant detection band place, unconjugated labelled antibody then can combine with the quality control band antiantibody.
Labelled antibody is with a kind of fluorescein or fluorescent microsphere as if what adopt, then should be quality control band is separated from one another according to different coated antibodies, when detecting,, illustrate that then test paper is out of date if band does not appear in the quality control band place; If band appears in the quality control band place, band does not appear and detect the band place, then do not contain virus in the interpret sample; Band occurs if detect the band place, then have corresponding virus in the interpret sample liquid certain or some.
Fixedly being meant of described detection antibody, Quality Control antibody (how anti-sheep anti-mouse igg is) according to common collaurum manufacture craft, a utilization point film machine will detect antibody, Quality Control antibody is scoring to nitrocellulose filter (solid phase carrier) and goes up and solid phase carrier is sealed, and its objective is when detecting to be with and quality control band as detecting.
Described antibody labeling is to instigate monoclonal antibody and fluorescein or fluorescent microsphere to form compound by chemical crosslink technique.Fluorescein-labelled monoclonal antibody: the active ester of fluorescein and the carboxyl reaction on the antibody form covalent bond; The fluorescent microsphere labeled monoclonal antibody: fluorescent particle adopts macromolecule polysterol material parcel fluorescein(e) dye to be prepared from, and diameter is 200～300nm, and there is the functional group carboxyl on the fluorescent microsphere surface.Under bi-functional cross-linking agent (EDAC and NHS) effect, fluorescent microsphere combines with monoclonal antibody.
Described labelled antibody pad preparation is the antibody dilution (0.5%BSA+3% trehalose+0.1%Tween-20 with fluorescein-labeled antibody or fluorescent microsphere mark, 10mMPH 7.5Tris-HCl) dilutes, then antibody labeling pad (glass fibre membrane, polyester film) is immersed in the dilution, after oven dry or vacuum freeze drying, refill and be fitted on the test strips end liner.
Described test strips assembling and cutting are to stick sample pad and labelled antibody pad in a side of the nitrocellulose filter that is coated with capture antibody and Quality Control antibody, and opposite side sticks high-intensity water absorbent paper, utilizes cutting machine to carry out the cutting of test strips.
Described immunochromatography reaction comprises double antibody sandwich method and two kinds of immunological methods of indirect method.Double antibody sandwich method is the reaction method that detects band, determined antigen forms immune complex at labelled antibody pad place and fluorescein-labeled antibody, this compound forms the immune complex that solidifies carrying out swimming on the nitrocellulose filter and combining with detection band antibody on the film; Indirect method is the reaction method of quality control band, and fluorescein-labeled antibody combines in swimming on the film and with antiantibody on the quality control band, forms the immune complex that solidifies.
It is that the special-purpose fluorescence detector of utilization detects quality control band and detection band that the result detects, and the content of specific antigen is directly proportional in the fluorescence intensity of detection band and the testing sample.
Compare with existing enterovirus detection method, the present invention has following advantage:
1) fluorescein or fluorescent microsphere are combined with traditional colloidal gold immunochromatographimethod technology, the variation by fluorescence intensity comes the antigenic content in the response sample, can realize the detection by quantitative of pathogen qualitatively simultaneously.
2) result is objective and highly sensitive.Come comparison with traditional colloidal gold strip detection method, the collection of fluorescein intensity is finished by fluorescence detector, and the result is objective and sensitive, and sensitivity can reach the several more than 10 times of traditional colloidal gold strip.
3) can carry out the detection of multiple common enterovirus simultaneously.
Description of drawings
Fig. 1 is the structural representation of the immunofluorescence chromatography quick detection test paper bar of enterovirus 4 joint inspections;
Fig. 2 is all positive result schematic diagram of norwalk virus, EAd, astrovirus, rotavirus;
Fig. 3 is the positive result schematic diagram of EAd, rotavirus;
Fig. 4 is the structural representation that single virus detects test paper.
Among the figure, 1 is that base plate, 2 is that reaction film, 3 is that thieving paper, 4 is that labelled antibody pad, 5 is that absorption of sample pad, T1～T4 are that detection is with, C is a quality control band.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Should be appreciated that these embodiment only are used to illustrate the present invention, and can not limit protection scope of the present invention.
The preparation of four joint inspection test paper of embodiment 1 enterovirus
With reference to accompanying drawing 1, the enterovirus of present embodiment is inspected paper bag more and draws together base plate 1, adheres to being embedded with quality control band (C band) and detecting nitrocellulose filter 2, the high-intensity water absorbent paper 3 that is covered in nitrocellulose filter one side, the labelled antibody pad 4 that is covered in the opposite side of nitrocellulose filter, the absorption of sample pad 5 of being with (T1 band, T2 band, T3 band, T4 band) on the base plate.
The C region of described nitrocellulose filter is embedded with sheep anti-mouse igg antibody, the T1 band is embedded with the monoclonal antibody of norwalk virus, the T2 band is embedded with the monoclonal antibody of EAd, and the T3 band is embedded with the monoclonal antibody of astrovirus, and the T4 band is embedded with the monoclonal antibody of rotavirus.And the immunogene of preparation rotavirus monoclonal antibody is rotavirus vp 6 albumen; The immunogene of preparation EAd monoclonal antibody is adenovirus 40/41 a type hexon; The immunogene of preparation astrovirus monoclonal antibody is an I type astrovirus antigen; The immunogene of preparation norwalk virus monoclonal antibody is a norwalk virus VP1 albumen.
1. MONOCLONAL ANTIBODIES SPECIFIC FOR
1.1, being prepared as follows of rotavirus vp 6 protein monoclonal antibodies:
The purpose fragment derives from clinical separation strain, and going up sequence number according to GENEBANK is the VP6 gene order of AF260931, and is as follows according to its sequence and pQE-30 prokaryotic expression carrier design primer:
Upstream primer is 5 '-TTGGCATGCATGGAGGTTCTGTACTCATTGTC
Downstream primer is 5 '-GGGGTCGACACTTAATCAACATGCTTCTAATGG
Carry out PCR (PCR), according to the archaeal dna polymerase that is adopted reaction system is set, wherein the addition of each primer is 0.6pmol/ μ L, and response procedures is provided with as follows:
The first step: 95 ℃ of 2min; Second step: 95 ℃ of 30sec, 57 ℃ of 30sec, 72 ℃ of 1min, 36 circulations; The 3rd step: 72 ℃ of 5min.
The PCR product is checked order, sequencing result shown in sequence table SEQ ID NO:1, with the homology of AF260931 be 100%.
The PCR product of the pQE-30 plasmid that extracts and purifying is carried out the double digestion of SalI and SphI restriction enzyme, and the enzyme tangent condition is set to 37 ℃ of 3hr; Enzyme is cut product also to be reclaimed with 1% agarose gel electrophoresis; With the T4 dna ligase carrier is connected with genes of interest.Connect product transformed into escherichia coli M15 competent cell.Choose single bacterium colony cultivate increase bacterium after, extract plasmid and carry out double digestion and identify.
Correct recombinant plasmid that has the VP6 protein gene (called after pQE-30/VP6) and the transformed bacteria of empty plasmid pQE-30 are identified in order-checking, used 37 ℃ of cultivations of LB fluid nutrient medium (Amp that adds 50 μ g/mL) 250rpm; When bacteria concentration was OD=0.5-0.6, the IPTG that adds final concentration and be 1mmol/L carried out abduction delivering 6hr.Carry out protein s DS-PAGE electrophoresis observation behind the centrifugation thalline.Adopt the fusion of the method acquisition purifying of nickel column, carry out the purity checking of purifying protein, require purity greater than 99% by protein s DS-PAGE electrophoresis.
Adopt Fu Shi immunologic adjuvant and purifying protein emulsion immunization experiment animal Balb/c mouse, every injection volume is 50 μ g albumen, the immunity position is groin and abdominal cavity, be 14 days immune interval, immunity 4 times (adjuvant of selecting for use for the first time is a Freund's complete adjuvant, and back three times is incomplete Freund); Take mice serum test Elisa antibody titer, choose the mouse of tiring greater than 1: 5000 and carry out booster immunization, the protein immunization amount is 100 μ g/, and immune position is a spleen, can carry out test for fusion after 3 days.Asepticly get its spleen cell and hybridize fusion with SP2/0-Ag14 myeloma cell, fusion agent is PEG3350.Use the indirect ELISA method screening positive clone, filter out the monoclonal cell strain of special secretion anti-rotavirus VP6 albumen by limiting dilution assay.Carry out the ascites preparation to induce method in the body, with Protein-A, protein-G or sad-ammonium sulfate method purification monoclonal antibody.The monoclonal antibody purifying thing of preparation is kept in-20 ℃ of refrigerators.Cell strain of monoclonal antibody adds cryopreserving liquid and is kept in the liquid nitrogen through enlarged culture.
1.2, the Monoclonal Antibody of adenovirus 40/41 type hexon is as follows:
The purpose fragment derives from clinical separation strain, and going up sequence number according to GENEBANK is the hexon gene order of D13781, and conservative region and the pQE-30 prokaryotic expression carrier design primer chosen wherein are as follows:
Upstream primer is 5 '-GGGGCATGCGTGTACAAGCCAGATGTAG
Downstream primer is 5 '-AAAGTCGACGCAGGTCGTTACCCAGACTG
Carry out PCR (PCR), according to the archaeal dna polymerase that is adopted reaction system is set, wherein the addition of each primer is 0.6pmol/ μ L, and response procedures is provided with as follows:
The first step: 95 ℃ of 2min; Second step: 95 ℃ of 30sec, 57 ℃ of 30sec, 72 ℃ of 1min, 36 circulations; The 3rd step: 72 ℃ of 5min.
The PCR product is checked order, sequencing result shown in sequence table SEQ ID NO:2, with the homology of D13781 be 100%.
The PCR product of the pQE-30 plasmid that extracts and purifying is carried out the double digestion of SalI and SphI restriction enzyme, and the enzyme tangent condition is set to 37 ℃ of 3hr; Enzyme is cut product also to be reclaimed with 1% agarose gel electrophoresis; With the T4 dna ligase carrier is connected with genes of interest.Connect product transformed into escherichia coli M15 competent cell.Choose single bacterium colony cultivate increase bacterium after, extract plasmid and carry out double digestion and identify.
Correct recombinant plasmid that has the hexon protein gene (called after pQE-30/hexon) and the transformed bacteria of empty plasmid pQE-30 are identified in order-checking, used 37 ℃ of cultivations of LB fluid nutrient medium (Amp that adds 50 μ g/mL) 250rpm; When bacteria concentration was OD=0.5-0.6, the IPTG that adds final concentration and be 1mmol/L carried out abduction delivering 6hr.Carry out protein s DS-PAGE electrophoresis observation behind the centrifugation thalline.Adopt the fusion of the method acquisition purifying of nickel column, carry out the purity checking of purifying protein, require purity greater than 99% by protein s DS-PAGE electrophoresis.
Follow-up Monoclonal Antibody step is with the Monoclonal Antibody of rotavirus vp 6 albumen.
1.3, astrovirus CP protein monoclonal antibody is prepared as follows:
The purpose fragment derives from clinical separation strain, and going up sequence number according to GENEBANK is the CP gene order of AB009985, and conservative region and the pQE-30 prokaryotic expression carrier design primer chosen wherein are as follows:
Upstream primer is 5 '-GGGGCATGCAACGGACGCAACAAATATCAATCT;
Downstream primer is 5 '-AAAGTCGACGCGCACCAGTGCGAGC;
Carry out PCR (PCR), according to the archaeal dna polymerase that is adopted reaction system is set, wherein the addition of each primer is 0.6pmol/ μ L, and response procedures is provided with as follows:
The first step: 95 ℃ of 2min; Second step: 95 ℃ of 30sec, 57 ℃ of 30sec, 72 ℃ of 1min, 36 circulations; The 3rd step: 72 ℃ of 5min.
The PCR product is checked order, sequencing result shown in sequence table SEQ ID NO:3, with the homology of AB009985 be 100%.
The PCR product of the pQE-30 plasmid that extracts and purifying is carried out the double digestion of SalI and SphI restriction enzyme, and the enzyme tangent condition is set to 37 ℃ of 3hr; Enzyme is cut product also to be reclaimed with 1% agarose gel electrophoresis; With the T4 dna ligase carrier is connected with genes of interest.Connect product transformed into escherichia coli M15 competent cell.Choose single bacterium colony cultivate increase bacterium after, extract plasmid and carry out double digestion and identify.
Correct recombinant plasmid that has the CP protein gene (called after pQE-30/CP) and the transformed bacteria of empty plasmid pQE-30 are identified in order-checking, used 37 ℃ of cultivations of LB fluid nutrient medium (Amp that adds 50 μ g/mL) 250rpm; When bacteria concentration was OD=0.5-0.6, the IPTG that adds final concentration and be 1mmol/L carried out abduction delivering 6hr.Carry out protein s DS-PAGE electrophoresis observation behind the centrifugation thalline.Adopt the fusion of the method acquisition purifying of nickel column, carry out the purity checking of purifying protein, require purity greater than 99% by protein s DS-PAGE electrophoresis.
Follow-up step is with the Monoclonal Antibody of rotavirus vp 6 albumen.
1.4, being prepared as follows of norwalk virus VP1 protein monoclonal antibody:
The purpose fragment derives from clinical separation strain, and going up sequence number according to GENEBANK is the VP1 protein gene sequence of EF535854, and is as follows according to its sequence and pET-28a prokaryotic expression carrier design primer:
Upstream primer is 5 '-CGCGGATCCTCGAATGACGCCGCTCCATC
Downstream primer is 5 '-TCCGAGCTCTCTTCTACGCCCATTGCCAG
Carry out PCR (PCR), according to the archaeal dna polymerase that is adopted reaction system is set, wherein the addition of each primer is 0.6pmol/ μ L, and response procedures is provided with as follows:
The first step: 95 ℃ of 2min; Second step: 95 ℃ of 45sec, 56 ℃ of 45sec, 72 ℃ of 80sec, 36 circulations; The 3rd step: 72 ℃ of 8min.
The PCR product is checked order, sequencing result shown in sequence table SEQ ID NO:4, with the homology of EF535854 be 100%.
The PCR product of the pET-28a plasmid that extracts and purifying is carried out the double digestion of SalI and BamHI restriction enzyme, and the enzyme tangent condition is set to 37 ℃ of 3hr; Enzyme is cut product also to be reclaimed with 1% agarose gel electrophoresis; With the T4 dna ligase carrier is connected with genes of interest.Connect product transformed into escherichia coli BL21 competent cell.Choose single bacterium colony cultivate increase bacterium after, extract plasmid and carry out double digestion and identify.
Correct recombinant plasmid that has the VP1 protein gene (called after pET-28a/VP1) and the transformed bacteria of empty plasmid pET-28a are identified in order-checking, used 37 ℃ of cultivations of LB fluid nutrient medium (Amp that adds 50 μ g/mL) 250rpm; When bacteria concentration was OD=0.5-0.6, the IPTG that adds final concentration and be 1mmol/L carried out abduction delivering 6hr.Carry out protein s DS-PAGE electrophoresis observation behind the centrifugation thalline.Adopt the fusion of the method acquisition purifying of nickel column, carry out the purity checking of purifying protein, require purity greater than 99% by protein s DS-PAGE electrophoresis.
Follow-up step is with the Monoclonal Antibody of rotavirus vp 6 albumen.
Pathogen to be checked is the norwalk virus of common enterovirus, EAd, four kinds of astrovirus and rotaviruss.
2, the preparation of test strips
2.1, be embedded with the monoclonal antibodies of four kinds of enteroviruses of fluorescein or fluorescent microsphere mark on the labelled antibody pad.
Fluorescein-labelled enterovirus monoclonal antibody: with 4 ℃ of dialysed overnight of enterovirus monoclonal anti body and function 0.1M sodium bicarbonate solution of purifying, antibody concentration is controlled at 5-20mg/ml; Fluorescein (there is the group of activation on the surface: for example tetrafluoro phenyl ester, sulphonic acid chloride phenolic ester, amber platinum imide ester etc.) is with dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO) (DMSO) dissolving, and concentration is 10mg/ml; With the monoclonal antibody after the adding of the fluorescein after the dissolving dialysis, magnetic agitation is 1 hour at ambient temperature, and the mass ratio of fluorescein and monoclonal antibody is 1: 10～20; Reaction back 1.5M azanol chloride cessation reaction, the fluorescein-labelled process of antibody is finished; With Sephadex G25 gel column the fluorescein-labelled thing of antibody is carried out purifying, obtain the fluorescein-labelled thing of the high monoclonal antibody of purity.Configuration dilution buffer liquid: 0.5%BSA+3% trehalose+0.1%Tween-20,10mM pH 7.5Tris-HCl.Four kinds of fluorescein-labelled things of antibody are diluted (antibody concentration remains on 0.01 μ g/ml～0.1 μ g/ml), again antibody labeling pad (glass fibre membrane or polyester film) is soaked wherein, time was controlled at 2～5 minutes, take out the back and adopt 37 ℃ of air-casing formula oven for drying or vacuum freeze drying, under 45% humidity, preserve standby.
Fluorescent microsphere mark enterovirus monoclonal antibody: fluorescent particle adopts macromolecule polysterol material parcel fluorescein(e) dye to be prepared from, and diameter is 200～300nm, and there is the functional group carboxyl on the fluorescent microsphere surface; With fluorescent microsphere 50mM MES (2-(N-morpholino) ethane sulfonicacid), pH6.0) damping fluid dialysis, concentration is adjusted to 10mg/ml, volume 20ml; Preparation 9.38% carbodiimide (N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, the MES damping fluid of MES damping fluid EDC) and 10% NHS (N-Hydroxysuccinimide); The 20ml particulate adds NHS solution 7.2ml, and EDAC solution 1.6ml replenishes volume to 40ml (fluorescent microsphere concentration 5mg/ml) with the MES damping fluid, 37 ℃ of continuous revolving reactions 1 hour; Get above-mentioned activation fluorescent particle 20mg (4ml),, and be condensed into 1ml (20mg/ml) with 50mM pH8.0 phosphate buffer centrifuge washing; Add enterovirus monoclonal antibody 4mg (50mM pH 8.0 phosphate buffer dialysis treatment), and additional volume makes end reaction concentration be to 2ml: fluorescent microsphere 10mg/ml, antibody 2mg/ml, 37 ℃ of revolving reactions 4 hours; Stop reaction, will react 4 ℃ of back fluorescent microsphere 12000rpm centrifugal 30 minutes, supernatant discarded, sediment dilutes with the 50mg/ml bovine serum albumin(BSA), ultrasonic Treatment.Configuration dilution buffer liquid: 0.5%BSA+3% trehalose+0.1%Tween-20,10mM pH7.5Tris-HCl.Four kinds of antibody fluorescent microsphere labels are diluted (microballoon concentration remains on 0.05mg/ml～0.2mg/ml), again antibody labeling pad (glass fibre membrane or polyester film) is soaked wherein, time was controlled at 2～5 minutes, take out the back and adopt 37 ℃ of air-casing formula oven for drying or vacuum freeze drying, under 45% humidity, preserve standby.
2.2, the bag quilt of four kinds of enterovirus monoclonal antibodies
Four kinds of enteroviruses (rotavirus, EAd, astrovirus, norwalk virus) monoclonal anti body and function 50mM pH7.2 phosphate buffer is diluted to 1mg/ml.On nitrocellulose membrane, draw rotavirus monoclonal antibody, EAd monoclonal antibody, astrovirus monoclonal antibody, the norwalk virus monoclonal antibody of dilution in order with spray film instrument, the spacing of every line is 0.3cm, and package amount is 1.0～2.0 μ l/cm.
2.3, quality control band preparation
Be diluted to 2mg/ml with 50mM pH7.2 phosphate buffer with sheep anti-mouse igg is how anti-, detect below the band quality control band in the 0.5cm place stroke at nitrocellulose filter, package amount is 1.0～2.0 μ l/cm.
2.4, the equipment and the cutting of test strips
Stick the nitrocellulose filter of coated antibody (how anti-4 kinds of enterovirus monoclonal antibodies and sheep anti-mouse igg be) in the centre position of offset plate; Glass fibre membrane is sticked in left side at offset plate, and spreads the antibody labeling pad at handing-over one end of nitrocellulose filter, and it is overlapping that antibody labeling pad and cellulose nitrate have 1～2mm position; Thieving paper is sticked on right side at offset plate, and it is overlapping that thieving paper and cellulose nitrate have 1～2mm position.Utilize cutting cutter to be cut into the test strip of 3～5mm width.
2.5, the detection of sample: during use test strips is tiled on the monitor station, gets the sample to be checked of 50 μ L, splash on the sample pad, react and should carry out the fluorescence signal collection with fluorescence detector after 5 minutes, can be with the relevant position collect fluoroscopic image at C band and T.If C takes existing band out of, it is positive that T1-T4 takes the different enterovirus of the representative of existing fluoroscopic image out of, and its antigenic content can be illustrated by fluorescence detector; If band does not appear in the C band, illustrate that test strips lost efficacy.
With reference to accompanying drawing 2, fluoroscopic image all appears in C band, T1 band, T2 band, T3 band, T4 band, shows that norwalk virus, EAd, astrovirus, rotavirus are all positive, and its antigenic content can be illustrated by fluorescence detector.
With reference to accompanying drawing 3, C band, T2 band, T4 take existing fluoroscopic image out of, show that EAd, rotavirus are positive, and norwalk virus, astrovirus are negative.EAd, wheel virus antigen content can be illustrated by fluorescence detector.
1, four joint inspection paper slips of enterovirus specificity test
Choose 5 parts of samples (sample 1 infects stool sample for single EAd infection stool sample, sample 4 single astroviruss infection stool samples, samples 5 for single norwalk virus for single rotavirus infection stool sample, sample 3 for salmonella infection stool sample, sample 2), with sample diluent (0.5%Triton-X100+2%NaCl+0.1%Casein, 10mM pH7.2PB) dilution in 1: 10, each is drawn 50 μ l and is added on the sample pad of four joint inspection paper slips of enterovirus, placed 15 minutes last fluorescence detector testing result under the room temperature.
Four joint inspection test strips of table 1 enterovirus test value
2, four joint inspection sensitivity tests of enterovirus
10 times of dilutions of wheel virus antigen (concentration 3 μ g/ml) gradient, concentration (ng/ml) is respectively 300,30,3,0.3,0, each is drawn 50 μ l and is added on the sample pad of four joint inspection paper slips of enterovirus, places 15 minutes last fluorescence detector testing result under the room temperature.10 times of dilutions of EAd antigen (concentration 3 μ g/ml) gradient, concentration (ng/ml) is respectively 300,30,3,0.3,0, each is drawn 50 μ l and is added on the sample pad of four joint inspection paper slips of enterovirus, places 15 minutes last fluorescence detector testing result under the room temperature.10 times of dilutions of astrovirus antigen (concentration 3 μ g/ml) gradient, concentration (ng/ml) is respectively 300,30,3,0.3,0, each is drawn 50 μ l and is added on the sample pad of four joint inspection paper slips of enterovirus, places 15 minutes last fluorescence detector testing result under the room temperature.10 times of dilutions of norwalk virus antigen (concentration 3 μ g/ml) gradient, concentration (ng/ml) is respectively 300,30,3,0.3,0, each is drawn 50 μ l and is added on the sample pad of four joint inspection paper slips of enterovirus, places 15 minutes last fluorescence detector testing result under the room temperature.Four kinds of enterovirus detection sensitivities are all below 1.0ng/ml.
Four joint inspection sensitivity tests of table 2 enterovirus value of reading
Annotate: the critical value that fluorescence value of reading 1 is judged for yin and yang attribute.
The preparation of enterovirus individual event test strip
With reference to accompanying drawing 4, the enterovirus of present embodiment is inspected paper slip more and comprises base plate 1, adheres to being embedded with quality control band (C band) and detecting nitrocellulose filter 2, the thieving paper 3 that is covered in nitrocellulose filter one side, the labelled antibody pad 4 that is covered in the opposite side of nitrocellulose filter, the absorption of sample pad 5 of being with (T band) on the base plate.
The C region of described nitrocellulose filter is embedded with sheep anti-mouse igg antibody, detects band and is embedded with rotavirus monoclonal antibody or EAd monoclonal antibody or astrovirus monoclonal antibody or norwalk virus monoclonal antibody.
Pathogen to be checked is rotavirus or EAd or astrovirus or norwalk virus.
1, is embedded with the monoclonal antibody of the enterovirus of fluorescein or fluorescent microsphere mark on the labelled antibody pad.
Fluorescein-labelled enterovirus monoclonal antibody: with 4 ℃ of dialysed overnight of enterovirus monoclonal anti body and function 0.1M sodium bicarbonate solution of purifying, antibody concentration is controlled at 5-20mg/ml; Fluorescein (there is the group of activation on the surface: for example tetrafluoro phenyl ester, sulphonic acid chloride phenolic ester, amber platinum imide ester etc.) is with dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO) (DMSO) dissolving, and concentration is 10mg/ml; With the monoclonal antibody after the adding of the fluorescein after the dissolving dialysis, magnetic agitation is 1 hour at ambient temperature, and the mass ratio of fluorescein and monoclonal antibody is 1: 10～20; Reaction back 1.5M azanol chloride cessation reaction, the fluorescein-labelled process of antibody is finished; With Sephadex G25 gel column the fluorescein-labelled thing of antibody is carried out purifying, obtain the fluorescein-labelled thing of the high monoclonal antibody of purity.Configuration dilution buffer liquid: 0.5%BSA+3% trehalose+0.1%Tween-20,10mM pH 7.5Tris-HCl.The fluorescein-labelled thing of antibody is diluted (antibody concentration remains on 0.01 μ g/ml～0.1 μ g/ml), again antibody labeling pad (glass fibre membrane or polyester film) is soaked wherein, time was controlled at 2～5 minutes, take out the back and adopt 37 ℃ of air-casing formula oven for drying or vacuum freeze drying, under 45% humidity, preserve standby.
Fluorescent microsphere mark enterovirus monoclonal antibody: fluorescent particle adopts macromolecule polysterol material parcel fluorescein(e) dye to be prepared from, and diameter is 200～300nm, and there is the functional group carboxyl on the fluorescent microsphere surface; With fluorescent microsphere 50mM MES (2-(N-morpholino) ethane sulfonicacid), pH6.0) damping fluid dialysis, concentration is adjusted to 10mg/ml, volume 20ml; Preparation 9.38% carbodiimide (N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, the MES damping fluid of MES damping fluid EDC) and 10% NHS (N-Hydroxysuccinimide); The 20ml particulate adds NHS solution 7.2ml, and EDAC solution 1.6ml replenishes volume to 40ml (fluorescent microsphere concentration 5mg/ml) with the MES damping fluid, 37 ℃ of continuous revolving reactions 1 hour; Get above-mentioned activation fluorescent particle 20mg (4ml),, and be condensed into 1ml (20mg/ml) with 50mM pH8.0 phosphate buffer centrifuge washing; Add enterovirus monoclonal antibody 4mg (50mM pH 8.0 phosphate buffer dialysis treatment), and additional volume makes end reaction concentration be to 2ml: fluorescent microsphere 10mg/ml, antibody 2mg/ml, 37 ℃ of revolving reactions 4 hours; Stop reaction, will react 4 ℃ of back fluorescent microsphere 12000rpm centrifugal 30 minutes, supernatant discarded, sediment dilutes with the 50mg/ml bovine serum albumin(BSA), ultrasonic Treatment.Configuration dilution buffer liquid: 0.5%BSA+3% trehalose+0.1%Tween-20,10mM pH 7.5Tris-HCl.Antibody fluorescent microsphere label is diluted (microballoon concentration remains on 0.05mg/ml～0.2mg/ml), again antibody labeling pad (glass fibre membrane or polyester film) is soaked wherein, time was controlled at 2～5 minutes, take out the back and adopt 37 ℃ of air-casing formula oven for drying or vacuum freeze drying, under 45% humidity, preserve standby.
2, the bag quilt of enterovirus monoclonal antibody
Enterovirus (rotavirus or EAd or astrovirus or norwalk virus) monoclonal anti body and function 50mM pH7.2 phosphate buffer is diluted to 1mg/ml.On nitrocellulose membrane, draw rotavirus monoclonal antibody or EAd monoclonal antibody or the astrovirus monoclonal antibody or the norwalk virus monoclonal antibody of diluting with spray film instrument, package amount is 1.0～2.0 μ l/cm.
3, quality control band preparation
Be diluted to 2mg/ml with 50mM pH7.2 phosphate buffer with sheep anti-mouse igg is how anti-, detect below the band quality control band in the 0.5cm place stroke at nitrocellulose filter, package amount is 1.0～2.0 μ l/cm.
4, the equipment of test strips and cutting
Stick the nitrocellulose filter of coated antibody (how anti-enterovirus monoclonal antibody and sheep anti-mouse igg be) in the centre position of offset plate; Glass fibre membrane is sticked in left side at offset plate, and spreads the antibody labeling pad at handing-over one end of nitrocellulose filter, and it is overlapping that antibody labeling pad and cellulose nitrate have 1～2mm position; Thieving paper is sticked on right side at offset plate, and it is overlapping that thieving paper and cellulose nitrate have 1～2mm position.Utilize cutting cutter to be cut into the test strip of 3～5mm width.
5, the detection of sample: during use test strips is tiled on the monitor station, gets the sample to be checked of 50 μ L, splash on the sample pad, react and to carry out the fluorescence signal collection with fluorescence detector after 15 minutes, can collect fluoroscopic image at C band and T band relevant position.
Sequence table
Claims (9)
1. enterovirus immunofluorescence chromatography quick detection test paper, comprise base plate, attached to the absorption of sample pad, labelled antibody pad, reaction film and the adsorptive pads that closely link to each other successively on the base plate, it is characterized in that described labelled antibody pad is embedded with the fluorescence labeling monoclonal antibody of one or more enteroviruses; Have one or more on the described reaction film and detect band and a quality control band, every detection with on be fixed with the monoclonal antibody of a kind of enterovirus, detect the bar number of band and fixing enterovirus monoclonal antibody and the labelled antibody of labelled antibody pad embedding and adapt, quality control band be fixed with can with the antiantibody of fluorescence labeling monoclonal antibody specific bond.
2. detection test paper according to claim 1, it is characterized in that described enterovirus monoclonal antibody is one or more in rotavirus monoclonal antibody, enteron aisle adenopathy monoclonal antibody, astrovirus monoclonal antibody or the norwalk virus monoclonal antibody.
3. detection test paper as claimed in claim 1 is characterized in that, described antiantibody is a sheep anti-mouse igg antibody.
4. detection test paper as claimed in claim 1 is characterized in that, described reaction film is a nitrocellulose filter.
5. as described in the claim 1, it is characterized in that the mark fluorescent element of described fluorescence labeling monoclonal antibody is the Alexa Fluor series fluorescein of Molecular Probes company or the anthocyanidin Cy series fluorescein of peace agate West Asia company.
6. as each described detection test paper of claim 2～5, it is characterized in that the immunogene of preparation rotavirus monoclonal antibody is rotavirus vp 6 albumen; The immunogene of preparation EAd monoclonal antibody is adenovirus 40/41 a type hexon; The immunogene of preparation astrovirus monoclonal antibody is an I type astrovirus CP albumen; The immunogene of preparation norwalk virus monoclonal antibody is a norwalk virus VP1 albumen.
7. method for preparing each described test paper of claim 1～6, it comprises the steps:
1) on the diverse location of reaction film according to the kind of antibody fixedly enterovirus monoclonal antibody and antiantibody respectively, form and detect band and quality control band;
2) preparation labelled antibody;
3) assembling plate, absorption of sample pad, labelled antibody pad, reaction film and adsorptive pads, and in the labelled antibody pad embedding labelled antibody.
8. method as claimed in claim 7 is characterized in that, the preparation method of labeled monoclonal antibody is: with 4 ℃ of dialysed overnight of enterovirus monoclonal anti body and function 0.1M sodium bicarbonate solution of purifying, antibody concentration is controlled at 5-20mg/ml; Fluorescein is 10mg/ml with dimethyl formamide or dmso solution, concentration; With the monoclonal antibody after the adding of the fluorescein after the dissolving dialysis, magnetic agitation is 1 hour at ambient temperature, and the mass ratio of fluorescein and monoclonal antibody is 1: 10～20; Reaction back 1.5M azanol chloride cessation reaction, the fluorescein-labelled process of antibody is finished; With Sephadex G25 gel column the fluorescein-labelled thing of antibody is carried out purifying, obtain the fluorescein-labelled thing of the high monoclonal antibody of purity.
9. method as claimed in claim 7 is characterized in that, the preparation method of labeled monoclonal antibody is: fluorescent microsphere is dialysed with 50mM pH6.0MES damping fluid, and concentration is adjusted to 10mg/ml, volume 20ml; The MES damping fluid of the MES damping fluid of the carbodiimide of preparation 9.38% and 10% NHS; The 20ml particulate adds NHS solution 7.2ml, and EDAC solution 1.6ml replenishes volume to 40ml with the MES damping fluid, 37 ℃ of continuous revolving reactions 1 hour; Get above-mentioned activation fluorescent particle 20mg,, and be condensed into 1ml with 50mM pH 8.0 phosphate buffer centrifuge washings; Add enterovirus monoclonal antibody 4mg, and additional volume makes end reaction concentration be to 2ml: fluorescent microsphere 10mg/ml, antibody 2mg/ml, 37 ℃ of revolving reactions 4 hours; Stop reaction, will react 4 ℃ of back fluorescent microsphere 12000rpm centrifugal 30 minutes, supernatant discarded, sediment dilutes with the 50mg/ml bovine serum albumin(BSA), ultrasonic Treatment.
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