CN105296435A

CN105296435A - Hybridoma cell strain, foot-and-mouth disease resistant O-type (O/GX/09-7) virus specificity monoclonal antibody secreted by hybridoma cell strain and application of hybridoma cell strain and antibody to detection of foot-and-mouth disease O-type viruses

Info

Publication number: CN105296435A
Application number: CN201510753950.6A
Authority: CN
Inventors: 刘国英; 郑金来; 范秀丽; 吴园园; 任旭荣; 李蓉; 张燕红; 郝金宝; 魏学峰; 商晓桂
Original assignee: Beijing Sanlian Boyue Biotechnology Co ltd; Spirit Jinyu Biological Pharmaceutical Co ltd
Current assignee: Beijing standard Zai Hui Bio Technology Co., Ltd.; Jinyu Baoling Bio-pharmaceutical Co., Ltd.
Priority date: 2015-11-06
Filing date: 2015-11-06
Publication date: 2016-02-03
Anticipated expiration: 2035-11-06
Also published as: CN105296435B

Abstract

The invention discloses a hybridoma cell strain 6D6 of a specificity monoclonal antibody capable of continuously and stably secreting foot-and-mouth disease resistant O-type (O/GX/09-7) viruses, and the specificity monoclonal antibody secreted by the hybridoma cell strain. The 6D6anti can recognize the foot-and-mouth disease O-type (O/GX/09-7) viruses in a specificity mode, the 6D6anti is used as a coating and an enzyme labeled antibody in ELISA detection and can be used for detecting the foot-and-mouth disease O-type (O/GX/09-7) viruses, and the advantages of high specificity and high sensitivity are achieved; the hybridoma cell strain, the antibody and the application can play an important role in detection of the foot-and-mouth disease O-type (O/GX/09-7) viruses, vaccine production and epidemiologic study.

Description

The monoclonal antibody specific of resistant to foot and mouth disease O type (O/GX/09-7) virus of hybridoma cell strain and secretion thereof and application

Technical field

The invention belongs to biological technical field, relate to the monoclonal antibody of hybridoma cell strain and secretion thereof, particularly the monoclonal antibody specific of foot and mouth disease O type (O/GX/09-7) virus is detecting the application in foot and mouth disease O type (O/GX/09-7) virus with the hybridoma cell strain of this antibody of secretion and this antibody.

Background technology

Foot and mouth disease (Foot-and-mouthDisease, FMD) a class transmissible disease is belonged to, popular name " aphtha ", " warding off Huang ", by foot and mouth disease virus (Foot-and-mouthDiseaseVirus, FMDV) acute, hot, the contagious disease that the artiodactyl that infection causes suffers from altogether, its Clinical symptoms is oral mucosa, hoof and skin of breast generation blister.Foot and mouth disease virus mainly encroaches on artiodactyl beast, occasionally in people and other animal, its main host comprises the Some Livestocks such as pig, ox, sheep and other is domestic, wild artiodactyl etc., susceptible animal reaches kind more than 70, the most susceptible animal is ox, pig, camel, sheep, deer etc., and the wildlife such as wild boar, wild ox is this disease of easy infection also.Foot and mouth disease route of transmission is many, speed is fast, once repeatedly worldwide outbreak of epidemic, causes huge politics, financial loss.Foot and mouth disease in Asia, Africa and the Middle East and South America all has popular, also has Sporadic cases in non-Endemic Area.Given this, OIE (French: Officeinternationaldes é pizooties, OIE, also claim " International Office of Epizootics OIE) be classified as first of category-A transmissible disease.At present, have the popular foot and mouth disease of OIE member states of 2/3rds, the moment threatens safety of livestock without foot and mouth disease countries and regions and Livestock Product Trade.

General not lethal after foot and mouth disease morbidity, but the mouth of disease beast can be made, there is a large amount of blister, high fever in hoof, actual animal yield is fallen sharply.In addition, the mutation of indivedual foot and mouth disease virus is had to be transmitted to people.Therefore, can only butcher after breaking out at every turn and destroy the livestock that catches an illness by fire with trouble without offspring with collective.Because foot and mouth disease propagation is rapid, be difficult to control, remedial measures is few, be called as " number one killer " of livestock industry.The prevention of China to foot and mouth disease is inoculated mainly through vaccination, and then catching and killing of foot and mouth disease occurs.

Foot and mouth disease virus (FMDV) is the cause of disease of artiodactyls hyperinfection disease (foot and mouth disease), belong to micro ribonucleic acid (tiny RNA) Viraceae (Picornaviridae) Hostis (Aphthovirus), its largest particle diameter is 23 nanometers, smallest particles diameter is 7-8 nanometer, at the positive chain RNA that the center of virus is a strand, by about 8000 based compositions, infect and hereditary basis, the protein be around wrapped in determines the antigenicity of virus, immunity and serological reaction ability, virus coat is 20 symmetrical bodies.

At present, foot and mouth disease virus mainly contains O, A, C, SAT1, SAT2, SAT3 (i.e. South Africa foot and mouth disease virus 1,2,3 type) and Asia1 (Type Asia 1) 7 serotypes in the whole world, and more than 65 hypotypes.Almost do not have immune protective efficiency between various, the animal having infected a type foot and mouth disease still can infect another type foot and mouth disease virus and fall ill, and therefore usually reduces with polyvalent vaccine the risk catched a foot and mouth disease.O type foot and mouth disease is the popular the widest serotype in the whole world, and the popular foot and mouth disease of China is mainly O, A, C tri-type and ZB type (Baoshan, Yunnan type).Foot and mouth disease O type comprises multiple strain: O/GX/09-7, O/Mya98/XJ/2010, O/Mya98/BY/2010, O/JMS/2000 etc.Wherein, foot and mouth disease O type (O/GX/09-7) virus is provided by China Veterinery Drug Inspection Office, and belong to the new pig poison of Cathay topological type, this strain strain is better to the adaptability of suckling mouse, and virulence is stronger; On BHK-21 cell, adaptability is good, inheritance stability; Stronger to pig virulence; Strain has wider spectrotype; 1 milliliter of antigen presentation amount can reach more than 3 micrograms, every milliliter of TCID ₅₀can 10 be reached ^7.5above.

At present, inactivated foot-and-mouth disease vaccine be prevention this pathogenetic main effective means, mainly through by after foot and mouth disease virus vitro culture, deactivation, be then mixed and made into immune vaccine with emulsifying agent.

The immunity of FMDV is the B cell response relying on T cell, and the generation of neutralizing antibody is mainly induced in vaccine inoculation.Vaccine inoculation is the reliable and effective means of specificity prevention FMDV, and vaccine is the prerequisite of successfully preventing, controlling and even finally eliminate FMDV safely and effectively.The conventional vaccines such as FMDV attenuated vaccine and inactivated vaccine all have good immunogenicity, play an important role in the process of prevention and corntrol FMDV.Along with the develop rapidly of Protocols in Molecular Biology, FMDV recombinant vaccine such as subunit vaccine, edible vaccine, synthetic peptide vaccine, protein carrier vaccine, gene-deleted vaccine, live vector vaccine, nucleic acid vaccine etc. continue to bring out.At present, the commonly foot and mouth disease multiple strain polyvalent vaccine of preparing, as two ingredient vaccines that foot and mouth disease O type (O/GX/09-7) is mixed with foot and mouth disease O type (O/Mya98/BY/2010).Production of vaccine enterprise often adopts the content of sucrose density gradient centrifugation gauge hatch aphtovirus at present, but cannot carry out separately quantitatively certain strain of polyvalent vaccine.

At present, to the diagnosis and detection of foot and mouth disease, mainly based on clinical judgment and PCR Molecular Detection.PCR is a high-sensitive method, but specificity Shortcomings, also need to coordinate nucleic acid sequencing to making a definite diagnosis of foot and mouth disease.Therefore, high, the complicated operation of this method cost, also high to the requirement of personnel.

Based on the immunology detection of antigen-antibody, because simple to operate, highly sensitive, high specificity, plant and instrument be cheap etc., advantage has been widely used in clinical detection.At present, the accidental enzyme-linked immunologic detecting kit that can detect foot-and-mouth disease antigen on market, but mostly have employed polyclonal antibody to make, such as Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences's exploitation, the O type foot and mouth disease 146S antigen quantify ELISA detection kit of producing, submit patent application (CN103076451A) to, this test kit have employed the rabbit anti-serum of O type foot and mouth disease and guinea pig antiserum two kinds of polyclonal antibodies are made, but the different strains of O type cannot be distinguished, such as Xinjiang Strain (O/Mya98/XJ/2010 strain) and Guangxi Strain (O/GX/09-7 strain), thus just cannot carry out separately quantitatively to often kind of O type foot and mouth disease 146S antigen in many components O type vaccine, its chief reason is just that polyclonal antibody is for plurality of antigens epi-position, and identical epitope may be there is between the different strains of the especially same serotype of different serotype.Therefore polyclonal antibody is adopted to go the different strain of specific detection foot and mouth disease to be impossible successfully, the strain that especially same serotype is different.

Monoclonal antibody because of its for antigen site single, specificity is better, and the repeatability of producing is better than polyclonal antibody, is used at present in a lot of clinical detection.The domestic accidental research having preparation foot and mouth disease monoclonal antibody, but but be still not enough to be applied to the detection of foot and mouth disease O type (O/GX/09-7) virus, the monoclonal antibody of specific detection foot and mouth disease O type (O/GX/09-7) virus can be still research direction.

Summary of the invention

First object of the present invention is to provide the monoclonal antibody specific for the hybridoma cell strain and generation thereof detecting foot and mouth disease O type (O/GX/09-7) virus.

Provided by the present invention with foot and mouth disease O type (O/GX/09-7) virus for immunogen immune mouse obtain continue, the hybridoma cell strain of monoclonal antibody specific that stably excreting resistant to foot and mouth disease O type (O/GX/09-7) is viral, name is called 6D6, this cell strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 16th, 2015, and deposit number is CGMCCNo.11196.Depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.

The monoclonal antibody specific called after 6D6anti secreted by hybridoma cell strain 6D6, derives from Mus mouse (Musmusculus), also belongs to the present invention.

The variable region of heavy chain of 6D6anti have the SEQIDNo:1 in sequence table amino acid residue sequence or by SEQ ID No: the amino acid residue sequence of 1 is through the replacement of one to ten amino-acid residue, disappearance or add and can with the polypeptide of foot and mouth disease O type (O/GX/09-7) viral specific combination, variable region of light chain have the SEQIDNo:2 in sequence table amino acid residue sequence or by SEQ ID No: the amino acid residue sequence of 2 is through the replacement of one to ten amino-acid residue, disappearance or add and can with the polypeptide of foot and mouth disease O type (O/GX/09-7) viral specific combination.

SEQIDNo:1 in sequence table is made up of 113 amino-acid residues, and the SEQIDNo:2 in sequence table is made up of 99 amino-acid residues.

The gene (6D6anti) of encodes monoclonal antibody 6D6anti, its variable region of heavy chain encoding gene has SEQ ID No: in the DNA sequence dna of 3 or polynucleotide SEQIDNo:1 DNA sequence dna or under high high stringency conditions can with SEQ ID No: the nucleotide sequence that 3 DNA sequence dnas limited are hybridized, its variable region of light chain encoding gene has SEQ ID No: in the DNA sequence dna of 4 or polynucleotide SEQIDNo:2 DNA sequence dna or under high high stringency conditions can with SEQ ID No: the nucleotide sequence that 4 DNA sequence dnas limited are hybridized;

Described high high stringency conditions is in the solution of 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS, hybridizes and wash film under 65 DEG C of conditions.

SEQIDNo:3 in sequence table is by 339 based compositions, coding has SEQ ID No: the protein of the amino acid residue sequence of 1, SEQIDNo:4 in sequence table is by 297 based compositions, and coding has SEQ ID No: the protein of the amino acid residue sequence of 2.

Expression vector containing gene 6D6anti of the present invention, transgenic cell line or Host Strains all belong to the present invention.

Obtain the method for hybridoma cell strain 6D6 of the present invention, can comprise the following steps:

1) viral as immunogen immune animal with foot and mouth disease O type (O/GX/09-7);

2) splenocyte of separating immune animal, by itself and myeloma cell fusion, forms hybridoma;

3) screen hybridoma, obtain hybridoma cell strain 6D6.

Obtaining the method for monoclonal antibody specific 6D6anti, is increase following steps on the basis of above-mentioned steps:

4) be separated from the ascites fluid of the animal of the nutrient solution of hybridoma cell strain 6D6 or inoculation hybridoma cell strain 6D6 and be purified into monoclonal antibody.

In the above-mentioned methods, step 1) in foot and mouth disease O type (O/GX/09-7) virus can be live virus or inactivation of viruses, be preferably inactivation of viruses, concentration is 10-1000 μ g/mL; Immune animal for the preparation of monoclonal antibody can be the Mammalss such as mouse, rat, rabbit, goat, sheep, pig, donkey, horse, is preferably mouse.

Step 2) in when the antibody level of serum of immunized animal reaches peak value, the splenocyte of separable animal is also prepared into single cell suspension.If desired, immunosorption method can be used to screen splenocyte, and merge to form hybridoma with myeloma cell's (being preferably murine myeloma cell SP2/0) under the induction of suitable fusogen (as polyoxyethylene glycol).

Step 3) in can cultivate screen the hybridoma merged in the selective medium (as HAT substratum), and can use further the methods such as flow cytometry, western blot method, immuno-precipitation identify needed for positive resistant cell strain.

Step 4) within external (as in tissue culture flasks or multiporous fiber reactor) or body, (mouse ascites) the hybridoma cell strain 6D6 of secretion foot and mouth disease O type (O/GX/09-7) viral monoclonal antibodies 6D6anti can be cultivated, and collect from cell culture fluid or mouse ascites liquid and be purified into monoclonal antibody specific 6D6anti.

The present invention also provides the application of monoclonal antibody specific 6D6anti in the ELISA of foot and mouth disease O type (O/GX/09-7) virus detects, be with the monoclonal antibody specific 6D6anti of foot and mouth disease O type (O/GX/09-7) virus as the double crush syndrome detection method of coated antibody and enzyme labelled antibody, can comprise the following steps:

1) wrap by elisa plate with monoclonal antibody specific 6D6anti, wash plate;

2) close the microwell plate through bag quilt, wash plate;

3) add testing sample, wash plate;

4) add the monoclonal antibody 6D6anti that horseradish peroxidase (HRP) or alkaline phosphatase (ALP) mark, wash plate;

5) substrate colour developing is added;

6) termination reaction;

7) OD is measured ₄₅₀value.

Another object of the present invention is to provide a kind of ELISA kit detecting foot and mouth disease O type (O/GX/09-7) virus.

The ELISA kit of detection foot and mouth disease O type (O/GX/09-7) provided by the present invention virus, comprises with 6D6anti as the microwell plate of coated antibody bag quilt with the enzymic-labelled antibody working fluid of 6D6anti as enzyme labelled antibody.

Described 6D6anti refers to as the microwell plate of coated antibody bag quilt, with 10mMpH7.0-7.4PBS, 6D6anti is diluted to 0.5-10 μ g/mL, and add in microwell plate, 110 μ l/ holes, are placed in 2-8 DEG C and spend the night.After patting dry, add the 10mMpH7.0-7.4PBS containing 1%BSA, 300 μ l/ holes.Be placed in 2-8 DEG C to spend the night.Pat dry, dry final vacuum loads in aluminium foil bag for subsequent use.

Described enzyme labelled antibody can use the marker enzyme traget antibody such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP), and (formula is: Na then to use enzymic-labelled antibody diluent ₂hPO ₄12H ₂o, 2.9g; NaH ₂pO ₄2H ₂o, 0.296g; NaCl, 8.5g; Proclin300,0.6mL; BSA, 10g; Foetal calf serum, 150mL; Enzyme stabilizers, 5g; Tween-200.25mL; Distilled water, is dissolved to 1000mL; Adjustment pH to 7.6-7.8) be diluted to working fluid, the concentration of working fluid is 0.1-1.0 μ g/mL.

Described test kit also can comprise nitrite ion A liquid, nitrite ion B liquid and stop buffer, and described nitrite ion A liquid is hydrogen peroxide or urea peroxide solution, and described nitrite ion B liquid is O-Phenylene Diamine or tetramethyl biphenyl amine aqueous solution, and described stop buffer is H ₂sO ₄solution.

In addition, for convenience of detecting, described test kit also can comprise: diluent, as pH7.0-7.410mMPBS buffered soln; Washings, as the washing reagent of the routines such as PBST.

For ease of observed result, described test kit also can comprise standard foot and mouth disease O type (O/GX/09-7) virus-positive serum (positive control) and standard foot and mouth disease O type (O/GX/09-7) viral negative serum (negative control), and available diluent uses as negative control.

Still a further object of the present invention is to provide a kind of golden millimeter paper chromatographic test paper detecting foot and mouth disease O type (O/GX/09-7) virus.

Golden millimeter paper chromatographic test paper for detecting foot and mouth disease O type (O/GX/09-7) virus is made up of glass fibre membrane sample pad, binding substances release pad, absorbent pad, nitrocellulose filter (NC film), and nitrocellulose filter is provided with detection line (T line) and nature controlling line (C line).

The preparation method of gold millimeter paper chromatographic test paper comprises the following steps:

1) bag is by nitrocellulose filter (NC film)

Be that the monoclonal antibody 2H10anti of 1-5mg/mL or 6D6anti wrap tested survey line (T line) by concentration; Wrap by nature controlling line (C line) with the goat anti-mouse immunoglobulin (purchased from Beijing Bo Erxi Science and Technology Ltd.) that concentration is 1-5mg/mL, concrete operations: monoclonal antibody 2H10anti or 6D6anti, goat anti-mouse immunoglobulin be sprayed on respectively on the nitrocellulose filter that 300mm is long, 25mm is wide with BIODOT company XYZ3000 Membrane jetter, form the detection line and nature controlling line that are separated from each other, detection line and nature controlling line spacing are 0.3-1.0cm, usually select 0.5cm.37 DEG C of dryings are for subsequent use after 1 hour;

2) preparation of binding substances release pad

2.1 use colloid gold label monoclonal antibody 6D6anti, and method is: under magnetic stirring, adds 155mL purified water, boil in triangular flask.Add 1% 4 chlorogold solution 5mL, boil.Add 1% citric acid three sodium solution 7mL again, boil 5 minutes.2-8 DEG C is stored in after cooling.Get 1 milliliter of colloidal gold solution in centrifuge tube.Add 15 μ l0.2M solution of potassium carbonate, the static 5min of room temperature.Add 10 μ l antibody, after mixing, leave standstill 30min.Add 10 μ l20%BSA solution, balance 5min.Add 10 μ l20%PEG20000 solution, balance 30min; Use whizzer 10000rpm, centrifugal 10min, removes supernatant liquor.Add 100 μ l gold mark redissolution liquid (borate buffer containing 2% sucrose, 1% casein, 0.5%BSA, 0.1TritonX100,0.1%SDS), for subsequent use after redissolving.

2.2 prepare binding substances release pad: the material of binding substances release pad is glass fibre membrane, it is coated with the monoclonal antibody specific 6D6anti of colloid gold label, according to the speed spray film of 8 μ l/cm after being diluted by gold 1:4 after redissolution, at 37 DEG C, place drying in 2 hours, for subsequent use.

3) golden millimeter paper chromatographic test paper is prepared

PVC backboard first pastes nitrocellulose filter, absorbent pad is pasted in the one end near nitrocellulose filter nature controlling line, binding substances release pad and sample pad is being pasted near testing wire one end, obtain for detecting the viral golden millimeter paper chromatographic test paper of foot and mouth disease O type (O/GX/09-7), then can cut by required size, obtain for detecting the viral golden millimeter paper chromatographic test strips of foot and mouth disease O type (O/GX/09-7), after adding siccative, sealing is preserved.

Test strip as above-mentioned steps prepared loads in plastic clip, makes test card, and is assembled into test kit, and the position that this test kit corresponds to sample pad is provided with point sample mouth, and the position corresponding to detection line and nature controlling line is provided with observation window.

The present invention's use can be brought out body and produce immunoreactive foot and mouth disease O type (O/GX/09-7) virus as immunogen, adopt conventional hybridoma technology through cytogamy and the hybridoma cell strain 6D6 of the monoclonal antibody specific that screening obtains continuing, stably excreting resistant to foot and mouth disease O type (O/GX/09-7) is viral, and secreted the monoclonal antibody specific 6D6anti obtained by cell strain 6D6.Monoclonal antibody 6D6anti of the present invention can identify foot and mouth disease O type (O/GX/09-7) virus specifically, with foot and mouth disease O type (O/Mya98/XJ/2010), foot and mouth disease O type (O/Mya98/BY/2010), foot and mouth disease O type (O/JMS/2000), Type Asia 1 (JSL/06) and A type (Re-A/WH/09) pathogenic agent no cross reaction, therefore, can be used for using 6D6anti as coated antibody detecting foot and mouth disease O type (O/GX/09-7) virus, and there is high specific, highly sensitive advantage.Experiment proves, monoclonal antibody specific of the present invention accurately can detect the level of foot and mouth disease O type (O/GX/09-7) virus in sample, and with foot and mouth disease O type (O/Mya98/BY/2010), Type Asia 1 (JSL/06), A type (Re-A/WH/09) virus, cross reaction does not occur.The present invention by foot and mouth disease O type (O/GX/09-7) virus detection, production of vaccine, play a significant role in epidemiological study, have a extensive future.

Below in conjunction with specific embodiment, the present invention is described in further details.

Accompanying drawing explanation

Fig. 1 is the monoclonal antibody specific 6D6anti of resistant to foot and mouth disease O type (O/GX/09-7) virus and the SDS-PAGE electrophoresis detection result of non-specific MAbs 2H10anti purity 8%;

Fig. 2 is the monoclonal antibody specific 6D6anti of resistant to foot and mouth disease O type (O/GX/09-7) virus and the WesternBlot detected result of non-specific MAbs 2H10anti conformational epitope;

Fig. 3 is the monoclonal antibody specific 6D6anti of resistant to foot and mouth disease O type (O/GX/09-7) virus and the hypotype qualification result of non-specific MAbs 2H10anti;

Fig. 4 is for detecting the matched curve of the sensitivity of foot and mouth disease O type (O/GX/09-7) virus with monoclonal antibody specific 6D6anti and non-specific MAbs 2H10anti and ELISA double antibody sandwich method;

Fig. 5 is Radioactive colloidal gold paper chromatography test strip Facad structure figure;

Fig. 6 is colloidal gold strip assembling schematic diagram;

Fig. 7 is the sensitivity technique result detecting foot and mouth disease O type (O/GX/09-70) virus with golden millimeter paper chromatographic test paper box prepared by monoclonal antibody specific 6D6anti and non-specific MAbs 2H10anti.

Embodiment

In following embodiment, method therefor is ordinary method if no special instructions, concrete steps can be see: " MolecularCloning:ALaboratoryManual " (Sambrook, J., Russell, DavidW., MolecularCloning:ALaboratoryManual, 3rdedition, 2001, NY, ColdSpringHarbor).

Described percentage concentration is mass/mass (W/W if no special instructions, unit g/100g) percentage concentration, mass/volume (W/V, unit g/100mL) percentage concentration or volume/volume (V/V, Unit/mL/100mL) percentage concentration.

The approach that obtains of the various biomaterials be described in embodiment is only to provide a kind of approach of testing acquisition to reach concrete disclosed object, should not become the restriction to biological material source of the present invention.In fact, the source of used biomaterial is widely, and any biomaterial that can obtain with moral ethics that keeps on the right side of the law can replace use according to the prompting in embodiment.

The primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..

Embodiment is implemented under premised on technical solution of the present invention, gives detailed embodiment and concrete operating process, and embodiment will contribute to understanding the present invention, but protection scope of the present invention is not limited to following embodiment.

Embodiment 1, acquisition continue, the hybridoma of the monoclonal antibody of stably excreting resistant to foot and mouth disease O type (O/GX/09-7) virus

The present embodiment with foot and mouth disease O type (O/GX/09-7) virus for immunogen immune mouse obtain continue, the hybridoma cell strain of monoclonal antibody that stably excreting resistant to foot and mouth disease O type (O/GX/09-7) is viral.

The acquisition of hybridoma cell strain comprises the following steps:

1, animal immune

1) fundamental immunity: using foot and mouth disease (O/GX/09-7) virus (being provided by Jinyu Baoling Biology Drugs Co., Ltd) as immunogen (antigen), employing sucrose density gradient centrifugation measures, purity >=85%, concentration 10-(1000 (g/mL.If desired, using ultrafiltration pipe concentrating virus antigen is to improve viral level, after concentrated, antigen mixes with Freund's complete adjuvant (being purchased from Sigma company) equal-volume and fully emulsified, multiple spot subcutaneous injection emulsion, every Balb/c mouse (8-12 age in week, female, SPF level animal is cultivated, purchased from Military Medical Science Institute's Experimental Animal Center) per injection amount is 150 (g is by concentrated 10 times of foot and mouth disease (O/GX/09-7) virus antigen super filter tube, after concentrated, antigen mixes with Freund's complete adjuvant (being purchased from Sigma company) equal-volume and fully emulsified, multiple spot subcutaneous injection emulsion, every Balb/c mouse (8-12 age in week, female, SPF level animal is cultivated, purchased from Military Medical Science Institute's Experimental Animal Center) per injection amount is 150 μ g.

2) booster immunization: after concentrating, antigen mixes with freund 's incomplete adjuvant (being purchased from Sigma company) equal-volume and fully emulsified, and multiple spot subcutaneous injection emulsion, every Balb/c mouse per injection amount is 200 μ g.Carrying out cytogamy first 3 days, abdominal injection contains the normal saline solution of 200 μ g antigens, to strengthen immune effect further.

2, the preparation of hybridoma and screening

Collect the splenocyte of mouse by ordinary method, splenocyte and murine myeloma cell SP2/0 cell are merged in the ratio of 10:1 under the induction of 500g/LPEG4000 (fusogen is purchased from Sigma company).Cultivate with HAT (purchased from raw emerging biotechnology (Nanjing) company limited) selectivity nutrient solution, culture condition be 5% carbonic acid gas, 37 DEG C.10-15 days after merging, get the hybridoma cell strain that supernatant adopts indirect elisa method screening secretion resistant to foot and mouth disease O type (O/GX/09-7) virus, the operation steps of indirect elisa method is: foot and mouth disease O type (O/GX/09-7) the viral wrapper sheet by 110 μ L concentration being 4 μ g/mL, after patting dry, close with 1%BSA (being purchased from Sigma company) confining liquid, using immune serum 1:2000 as positive control, without Normal Mouse Serum cleer and peaceful on the substratum of clonal growth as negative control, every hole adds 1:2000HRP-goat anti-mouse IgG (purchased from American ABCAM company) 100 μ L, finally measure 450nmOD value, with OD ₄₅₀value is greater than negative control 2 times for positive basis for estimation.

Adopt limiting dilution assay to carry out subclone to gained positive clone strain, concrete operations are:

1) take out antibody positive porocyte, make cell suspension with HT nutrient solution.And sampling is carried out platform and is expected blue dyeing, counting.

2) with HT nutrient solution cell dilution become 200/mL, 40/mL, 20/mL and suspension.

3) plant cell suspension into microtest plate respectively with suction pipe, every hole 0.05mL, cell content is respectively 10/hole, 2/hole, 1/hole and 0.5/hole.

4) 5%CO2 saturated humidity, 37 DEG C of cultivations.

5) observe clonal growth situation with inverted microscope every day, select the hole only having a colony growth, discard two or more and there is no the hole of Growth of Cells.

6) after cloning amount reproduction, when being covered with the 1/3-1/2 at the bottom of hole, surveying nutrient solution supernatant antibody by indirect elisa method, select positive colony, and pass 4-6 for just building up clone strain.

3, the acquisition of hybridoma

Repeating step 2, carry out 2 cytogamy, through 4 subclones and indirect ELISA screening, obtain the hybridoma of 4 strains for the viral Absorbable organic halogens secrete monoclonal antibody of foot and mouth disease O type (O/GX/09-7), respectively called after 2H10,6D6,3E7,3A11.

4, the bioactivity of hybridoma gained monoclonal antibody

1) cell culture supernatant titration: detect tiring of above-mentioned Hybridoma Cell Culture supernatant with indirect elisa method (method is shown in step 2), result is as shown in table 1, tire as 1:10-1:100, show all there is target antibody in supernatant to be measured, the monoclonal antibody (called after 3E7anti) that wherein cell strain 3E7 secretes is tired lower slightly.

Tiring of table 1 Hybridoma Cell Culture supernatant

2) mouse ascites titration: detect titer of ascites prepared by above-mentioned hybridoma with indirect elisa method (method is shown in step 2), result is as shown in table 2, tire as 1:1000-1:1000000, show all can target antibody be detected in ascites, wherein lower slightly in 3E7, its excess-three kind is tired all at more than 1:8000, has using value.

Tiring of table 2 Hybridoma Cell Culture supernatant

	Cell strain 2H10	Cell strain 6D6	Cell strain 3E7	Cell strain 3A11
					Mouse ascites is tired	1:32768	1:16384	1:1024	1:8192

3) mouse ascites antibodies specific checking: the specificity detecting ascites prepared by above-mentioned hybridoma with indirect elisa method (method is shown in step 2), namely adopt the strain bag that foot and mouth disease is different by microwell plate respectively, detect after ascites pH7.410mMPBS being diluted 100 times.Result is as shown in table 3, wherein the monoclonal antibody (called after 6D6anti) of cell strain 6D6 secretion and the monoclonal antibody (called after 3E7anti) of cell strain 3E7 secretion are specific antibody, and the monoclonal antibody (called after 2H10anti) that cell strain 2H10 secretes and the monoclonal antibody (called after 3A11anti) two kinds that cell strain 3A11 secretes are non-specific antibody.Comparatively speaking, 3E7anti and 3A11anti antibody titer is lower, therefore the present invention selects 6D6anti and 2H10anti for testing the exploitation with late detection reagent.

The specific detection of table 3 Hybridoma Cell Culture supernatant

Therefore, in reagent performance history, following matching method can be formed:

1. in ELISA: 6D6anti (bag quilt)-6D6anti (mark); 6D6anti (bag quilt)-2H10anti (mark)

2. Radioactive colloidal gold: 6D6anti (mark)-6D6anti (bag quilt); 6D6anti (mark)-2H10anti (bag quilt), by above-mentioned Antibody Combination mode, can realize the object of specific detection target antigen.

5, the Secondary Culture of hybridoma

6D6 or the 2H10 hybridoma chosen above is proceeded to cultivate, go down to posterity in containing the RPMI-1640 (containing 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates) of 10% foetal calf serum, cultivate after the 10th generation, hybridoma cell strain still can well-grown, stable to go down to posterity, nutrient solution supernatant is tired still can reach more than 1:50, show to obtain to stablize and go down to posterity, and can continue, the hybridoma of monoclonal antibody of stably excreting resistant to foot and mouth disease O type (O/GX/09-7) virus.

6, hybridoma is preserved

After obtaining the hybridoma of lasting, that stably excreting resistant to foot and mouth disease O type (O/GX/09-7) is viral monoclonal antibody, a part of hybridoma must be preserved, this is because in the process of continuous passage, sudden change may be produced or chromosomal drift motion produces the characteristic of antibody to losing natural characteristics or losing; In addition, in long-term culturing process, do not occur unavoidably to pollute to such an extent as to destroy.

Store method comprises the following steps:

1) remove nutrient solution old in Tissue Culture Flask, add the RPM1640 substratum containing 10% foetal calf serum, make cell suspension.

2) the centrifugal 10min of 1000r/min, removes supernatant.Cell precipitation cells frozen storing liquid (dimethyl sulfoxide (DMSO): foetal calf serum: RPM1640=1:2:7) redissolves makes suspension, concentration 5.0 × 10 ⁵cell/mL.

3) sample, platform expects blue dyeing, and living cell counting, should more than 95%.Particularly:

Take 4g trypan blue, add the grinding of a small amount of distilled water, add distilled water to 100mL, with filter paper filtering, 4 degree of preservations.During use, be diluted to 0.4% with pH7.410mMPBS.Prepare single cell suspension, and do suitably dilution.Cell suspension and 0.4% trypan blue solution are mixed with 9:1 and mixes (final concentration 0.04%).In three minutes, Microscopic observation, dead cell is dyed to obvious blueness, and viable cell refuses dye in water white transparency shape.Calculate living cell rate (%)=total viable cell/(total viable cell+dead cell sum) × 100%.

4) by aseptic subpackaged for cell to 1.8mL cell cryopreservation tube (purchased from Zhejiang Gongdong Medical Technology Co., Ltd.), every bottle of 0.5mL-1.0mL, tightens bottle cap.

5) cell cryopreservation: place 2 hours, then place 2 hours again for-20 DEG C for 4 DEG C, liquid nitrogen container gaseous parts (-70 DEG C) is placed 2 hours afterwards, finally proceeds to liquid nitrogen and preserves for a long time.

By above method with foot and mouth disease O type (O/GX/09-7) virus for immunogen immune mouse obtain continue, the hybridoma cell strain of monoclonal antibody that stably excreting resistant to foot and mouth disease O type (O/GX/09-7) is viral, name is called 6D6 and 2H10.Wherein, the cell strain 6D6 that can produce monoclonal antibody specific is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 16th, 2015, and deposit number is CGMCCNo.11196; The cell strain 2H10 producing non-specific MAbs is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 16th, 2015, and deposit number is CGMCCNo.11195.

The monoclonal antibody of embodiment 2, a large amount of preparation resistant to foot and mouth disease O type (O/GX/09-7) virus

One, a large amount of preparation of monoclonal antibody and purifying

1, induce monoclonal antibody method in employing animal body and prepare monoclonal antibody in a large number: select BALB/c mouse of growing up, intraperitoneal inoculation pristane (immunosuppressor, can cause inflammation reaction), every mouse 0.5mL.7-10 days pneumoretroperitoneum inoculation the 16th generation hybridoma 6D6 or 2H10, every mouse 2 × 10 ⁶-3 × 10 ⁶individual.Interval, after 5 days, treats that belly obviously expands, and when touching with hand, skin has tension, and namely available No. 9 syringe needles gather ascites.

2, antibody purification: by centrifugal for ascites (10000r/min30 minute), removing cellular constituent and other throw out, collect supernatant.By the pH7.0-7.410mMPBS (formula: NaH of supernatant with 15-30 times of volume ₂pO ₄2H ₂o0.296g, Na ₂hPO ₄12H ₂o2.9g, distilled water is dissolved to 1000mL, mensuration pH value is 7.0-7.4) dilution, with ProteinA affinity column (GE company, article No. is 29-0491-04) carry out affinity purification, upper prop liquid is the PBS damping fluid of pH7.0-7.620mM, column chromatography elutriant is the citrate buffer solution (formula: monohydrate potassium 21g adds in 1000mL deionized water of pH3.5100mM, by 5MNaOH or 4MHCl adjusted to ph to 3.5), obtain the monoclonal antibody of resistant to foot and mouth disease O type (O/GX/09-7) virus, called after 6D6anti or 2H10anti.

Two, Identification of the antibodies

1, antibody purity qualification

The monoclonal antibody 6D6anti viral to resistant to foot and mouth disease O type (O/GX/09-7) or 2H10anti carries out reductibility 8%SDS-PAGE electroresis appraisal.

Result is as Fig. 1, swimming lane M is Protein Marker (kDa), swimming lane 6D6 is monoclonal antibody 6D6anti, swimming lane 2H10 is for shown in monoclonal antibody 2H10anti, the purity of monoclonal antibody 6D6anti and 2H10anti, more than 85%, shows that the purity of antibody purification can meet actual needs.

2, antibody conformational epitope checking

Conventional WesternBlot detection method detect the monoclonal antibody 6D6anti of resistant to foot and mouth disease O type (O/GX/09-7) virus and 2H10anti institute for epi-position be conformation type (represent this antibody for antigen site be space structure) or linear (this antibody of expression for antigen site be linear structure), method is: use 150 μ g deactivation foot and mouth disease O type (O/GX/09-7) viruses to carry out reductibility 8%SDS-PAGE electrophoresis, wet robin goes on pvdf membrane, the monoclonal antibody 6D6anti of purifying and 2H10anti is used to carry out WesternBlot hybridization check.

Result as shown in Figure 2, wherein M is Protein Marker (kDa), swimming lane 1 is monoclonal antibody 6D6anti, swimming lane 2 is monoclonal antibody 2H10anti, swimming lane 3 is that positive control (is specially the positive tail blood producing immune mouse in monoclonal antibody preparation process, belong to polyclonal antibody, see embodiment 1).Showing monoclonal antibody 6D6anti and 2H10anti according to this experimental result is conformational epitope, points out this antibody to antigen-reactive that is only possible and native state, as antigen generation cracking, protein denaturation may not react.Application value is had on vaccine quality controls.

3, antibody class and subgroup identification

The monoclonal antibody 6D6anti of resistant to foot and mouth disease O type (O/GX/09-7) virus that the Radioactive colloidal gold hypotype characterization test card using Beijing Wu Kang emerging technology company limited to produce detects, qualification hybridoma 6D6 and 2H10 secretes and the antibody subtype of 2H10anti.

Result is as shown in Fig. 3 (order is 6D6anti and 2H10anti from top to bottom), and two kinds of antibody are IgG type, and wherein monoclonal antibody 6D6anti is IgG1; Monoclonal antibody 2H10anti is IgG2a.

Three, the variable region sequences of monoclonal antibody 6D6anti and 2H10anti measures

Extract the mRNA of hybridoma 6D6 and 2H10, reverse transcription is cDNA, uses variable region universal primer to carry out high-fidelity PCR amplification, is inserted into by pcr amplified fragment in carrier T and carries out determined dna sequence.Determined dna sequence result: the gene (6D6anti) of encodes monoclonal antibody 6D6anti, its variable region of heavy chain encoding gene DNA sequence dna is as SEQ ID No: shown in 3, and its variable region of light chain encoding gene DNA sequence dna is as SEQ ID No: shown in 4; The gene (2H10anti) of encodes monoclonal antibody 2H10anti, its variable region of heavy chain encoding gene DNA sequence dna is as SEQ ID No: shown in 7, and its variable region of light chain encoding gene DNA sequence dna is as SEQ ID No: shown in 8.The DNA sequence dna of acquisition is translated into the aminoacid sequence of protein.The heavy chain variable amino acid residue sequence of 6D6anti is as shown in the SEQIDNo:1 in sequence table, and chain variable region amino acid residue sequence is as shown in the SEQIDNo:2 in sequence table; The heavy chain variable amino acid residue sequence of 2H10anti is as shown in the SEQIDNo:5 in sequence table, and the amino acid residue sequence of variable region of light chain is as shown in the SEQIDNo:6 in sequence table.Above-mentioned sequence does not show identical sequence after ncbi database is compared.

Embodiment 3, use monoclonal antibody 6D6anti, 2H10anti and ELISA double antibody sandwich method detect foot and mouth disease O type (O/GX/09-7) virus

One, foot and mouth disease O type (O/GX/09-7) virus of different concns is detected with monoclonal antibody 6D6anti and ELISA double antibody sandwich method

1,6D6anti had not only been coated antibody but also had detected foot and mouth disease O type (O/GX/09-7) virus of different concns for traget antibody---6D6anti (bag quilt)-6D6anti (mark) matches test kit

There is document (NumberandmolecularweightsofFood-and-MouthDiseaseVirusCap sidProteinsandtheEffectsofMaleylation, JournalofVirology, 1971, Vol7, No.2, P250-259) record, the membranin (VP1, VP2, VP3 and VP4) of foot and mouth disease virus has multiple copy, therefore be not only coated antibody but also for traget antibody, realize specific detection foot and mouth disease O type (O/GX/09-7) virus with monoclonal antibody 6D6anti.

Detect foot and mouth disease O type (O/GX/09-7) virus with monoclonal antibody 6D6anti and ELISA double antibody sandwich method, detection method comprises the following steps:

1) monoclonal antibody 6D6anti pH7.0-7.410mMPBS buffered soln is diluted to 4 μ g/mL, the every hole to microwell plate (purchased from Xiamen experiment equipment company limited of happy Jiamei) adds 110 μ L, wraps and spent the night at 4 DEG C;

2) incline coating buffer, pats dry, in every hole, then add 300 μ L1%BSA (in pH7.0-7.410mMPBS buffered soln), put into 4 DEG C close and spend the night after, to pat dry, dry, preserve;

3) adopt Over-voltage protection and horseradish peroxidase (HRP) labeled monoclonal antibody 6D6anti, obtain 6D6anti-HRP, be diluted to working fluid according to a certain percentage with enzymic-labelled antibody diluent for subsequent use.HRP labelling method is specially:

Take 3mgHRP to be dissolved in 1mL distilled water.The 0.1MNaIO that 0.12mL newly joins is added in upper liquid ₄solution, under room temperature, lucifuge stirs 20 minutes.Loaded in dialysis tubing by above-mentioned solution, dialyse to the sodium-acetate buffer of 1mMpH4.4,4 DEG C are spent the night.Add 30 μ l0.2MpH9.5 carbonate buffer solutions, make the pH of the HRP after dialysis be elevated to 9.0-9.5, then add the 4mgIgG be dissolved in 1mL0.01M carbonate buffer solution immediately, room temperature lucifuge stirs 2 hours gently.Add the 5mg/mLNaBH that 0.12mL newly joins ₄solution, mixing, then put 4 DEG C 2 hours.Loaded by above-mentioned solution in dialysis tubing, to 0.01MpH7.2PBS dialysis, 4 DEG C are spent the night.Add equal-volume high-quality glycerine after taking-up, packing ,-20 DEG C of preservations, are 1F2-HRP.Dilute according to a certain percentage with enzymic-labelled antibody diluent and be enzymic-labelled antibody working fluid.The formula of enzymic-labelled antibody diluent is: Na ₂hPO ₄12H ₂o, 2.9g; NaH ₂pO ₄2H ₂o, 0.296g; NaCl, 8.5g; Proclin300,0.6mL; BSA, 10g; Foetal calf serum, 150mL; Enzyme stabilizers, 5g; Tween-200.25mL; Distilled water, is dissolved to 1000mL; Adjustment pH to 7.6-7.8.

4) in microwell plate, add foot and mouth disease O type (O/GX/09-7) viral gradient dilution liquid 100 μ L/ hole respectively, virus concentration in table 4,37 DEG C of incubations 1 hour;

5) with PBST washing lotion (formula: Na ₂hPO ₄12H ₂o58g, NaH ₂pO ₄2H ₂o5.92g, NaCl170g, Tween-205.0mL, Proclin3000.6mL, be dissolved to 1000mL with distilled water, and adjusted to ph is to 7.2-7.4; Use front distilled water to dilute 20 times) wash plate 5 times after, then add 6D6anti-HRP (1:1000-1:8000 dilution) 100 μ L/ hole, 37 DEG C of incubations 0.5 hour;

6) (formula is: anhydrous sodium acetate 4.5g, Glacial acetic acid 1.2mL, and urea peroxide 0.8g, is dissolved to 1000mL with distilled water to add nitrite ion A after washing plate 5 times with PBST.), (formula is nitrite ion B: citric acid 1.62g, EDTA-2Na0.372g, glycerine 100mL, and tetramethyl biphenyl amine hydrochlorate 0.50g, is dissolved to 1000mL with distilled water.) each 50 μ L/ holes develop the color, 37 DEG C of incubation 15min;

7) (formula is: containing the sulfuric acid 27.8mL of 98% in 1000mL distilled water to add stop buffer.), 50 μ L/ holes, reading OD ₄₅₀.

Detected result is as shown in table 4, can find out that this 6D6anti (bag quilt)-6D6anti (mark) pairing test kit can detect the antigen of 1.875-60ng/mL scope, satisfy the demand in this interval internal linear, show that this test kit has good linear, and sensitivity is very high.

Table 4 monoclonal antibody 6D6anti and ELISA double-antibody sandwich two-step approach detect the result of foot and mouth disease O type (O/GX/09-7) virus

2, to take 6D6anti as coated antibody with 2H10anti be, and traget antibody detects foot and mouth disease O type (O/GX/09-7) virus of different concns---and 6D6anti (bag quilt)-2H10anti (mark) matches test kit

With specificity 6D6anti for coated antibody; With non-specific 2H10anti for traget antibody, adopt following method equally also can realize the object of specific detection target antigen.Detection method comprises the following steps:

1) monoclonal antibody 6D6anti pH7.0-7.410mMPBS buffered soln is diluted to 4 μ g/mL, thinks that every hole of microwell plate adds 110 μ L, wrap at 4 DEG C and spent the night;

3) adopt Over-voltage protection and horseradish peroxidase (HRP) labeled monoclonal antibody 2H10anti, obtain 2H10anti-HRP, be diluted to working fluid according to a certain percentage with enzymic-labelled antibody diluent for subsequent use.HRP labelling method is specially:

5) with PBST washing lotion (formula: Na ₂hPO ₄12H ₂o58g, NaH ₂pO ₄2H ₂o5.92g, NaCl170g, Tween-205.0mL, Proclin3000.6mL, be dissolved to 1000mL with distilled water, and adjusted to ph is to 7.2-7.4; Use front distilled water to dilute 20 times) wash plate 5 times after, then add 2H10anti (1:1000-1:8000 dilution) 100 μ L/ hole, 37 DEG C of incubations 0.5 hour;

7) stop buffer (formula is: containing the sulfuric acid 27.8mL of 98% in 1000mL distilled water) is added, 50 μ L/ holes, reading OD ₄₅₀.

Detected result is as shown in table 5, can find out that 6D6anti (bag quilt)-2H10anti (mark) pairing test kit can detect the antigen of 1.875-60ng/mL scope, satisfy the demand in this interval internal linear, show that this test kit has good linear, and sensitivity is very high.

Table 5 monoclonal antibody 6D6anti, 2H10 and ELISA double-antibody sandwich two-step approach detect the result of foot and mouth disease O type (O/GX/09-7) virus

Two, detect foot and mouth disease O type (O/GX/09-7) virus of different concns with monoclonal antibody 6D6anti, 2H10anti and ELISA double antibody sandwich method, to determine the sensitivity of detection method, detection method is identical with step one

The detected result not only having detected different concns foot and mouth disease O type (O/GX/09-7) viral for coated antibody but also for traget antibody (i.e. 6D6anti (bag quilt)-6D6anti (mark) pairing test kit) with 6D6anti is as shown in table 4, in this detection, (X-coordinate represents with the logarithm of 10 concentration that are the end matched curve such as Fig. 4, ordinate zou represents with the logarithm of the 10 OD values that are the end) shown in the A width of left side, linearly dependent coefficient >=0.99 can be found out, detection sensitivity can reach 1.875ng/mL, and now OD value is 2 times of negative control.Show that the test kit that this Antibody Combination becomes has good sensitivity.

The detected result that to take 6D6anti as coated antibody with 2H10anti be traget antibody (i.e. 6D6anti (bag quilt)-2H10anti (mark) match test kit) detects different concns foot and mouth disease O type (O/GX/09-7) virus is as shown in table 5, in this detection, (X-coordinate represents with the logarithm of 10 concentration that are the end matched curve such as Fig. 4, ordinate zou represents with the logarithm of the 10 OD values that are the end) shown in the B width of right side, linearly dependent coefficient >=0.99 can be found out, detection sensitivity is also no more than 1.875ng/mL, and now OD value is 2 times of negative control.Show that the test kit that this Antibody Combination becomes has good sensitivity.

In a word, take 6D6anti as coated antibody, with 2H10anti or 6D6anti for traget antibody detects foot and mouth disease O type (O/GX/09-7), all can realize high-sensitivity detection, detection sensitivity is near 2ng/mL.

Three, the specific detection of foot and mouth disease O type (O/GX/09-7) virus is detected with monoclonal antibody 6D6anti, 2H10anti and ELISA double antibody sandwich method

By monoclonal antibody 6D6anti, 2H10anti and ELISA double antibody sandwich method detection foot and mouth disease O type (O/Mya98/BY/2010) virus, (virus is all diluted to 1000ng/mL with sample diluting liquid to foot and mouth disease O type (O/GX/09-7), Type Asia 1 (JSL/06) and A type (Re-A/WH/09) virus, diluted sample liquid formula: Na ₂hPO ₄12H ₂o, 2.9g; NaH ₂pO ₄2H ₂o, 0.296g; NaCl, 8.5g; Proclin300,0.6mL; BSA, 10g; Foetal calf serum, 150mL; Gentamicin sulphate, 2 (80,000 units /); Distilled water, is dissolved to 1000mL; Adjustment pH to 7.6-7.8.Filtration sterilization, 2-8 DEG C of preservation), to determine the specificity of detection method, detection method is shown in step one, two.

Result: with monoclonal antibody 6D6anti for coated antibody, with 2H10anti or 6D6anti for traget antibody detects, foot and mouth disease O type (O/Mya98/BY/2010), Type Asia 1 (JSL/06) and A type (Re-A/WH/09) Viral diagnosis result are negative (2 times of OD value≤negative control), no cross reaction, and foot and mouth disease O type (O/GX/09-7) Viral diagnosis result is positive (2 times of OD value >=negative control), illustrate with monoclonal antibody 6D6anti as coated antibody (2H10anti or 6D6anti is for traget antibody), adopt ELISA double-antibody sandwich two-step approach can identify foot and mouth disease O type (O/GX/09-7) virus specifically, with foot and mouth disease O type (O/Mya98/BY/2010), other viruses such as Type Asia 1 (JSL/06) and A type (Re-A/WH/09) virus are without any reaction.

And with monoclonal antibody 2H10anti coated antibody, be that traget antibody detects with 2H10anti, result shows above-mentioned hoof-and-mouth disease poison strain and all reacts, and shows monoclonal antibody 2H10anti to foot and mouth disease O type (O/GX/09-7) for nonspecific.With monoclonal antibody 2H10anti coated antibody, be that traget antibody detects with 6D6anti, although only have O/GX/09-7 to have positive findings, all the other do not react; But when multiple strain hybrid detection, nonspecific strain meeting Interference Detection, affects the accuracy of detected result.

Consider above result and application, determination monoclonal antibody 2H10anti or 6D6anti of the present invention matches as the traget antibody in ELISA double antibody sandwich method detection foot and mouth disease O type (O/GX/09-7) and monoclonal antibody specific 6D6anti and uses, or Radioactive colloidal gold double antibody sandwich method detects in foot and mouth disease O type (O/GX/09-7), monoclonal antibody 2H10anti or 6D6anti matches as coated antibody and monoclonal antibody specific 6D6anti and uses specific detection foot and mouth disease O type (O/GX/09-7) virus.

Four, preparation detects the test kit of foot and mouth disease O type (O/GX/09-7) virus with ELISA double antibody sandwich method

The invention provides a kind of test kit detecting foot and mouth disease O type (O/GX/09-7) virus, comprising the monoclonal antibody specific 6D6anti of foot and mouth disease O type (O/GX/09-7) virus.

Concrete, can be the ELISA kit detecting foot and mouth disease O type (O/GX/09-7) virus with ELISA double antibody sandwich method, wherein use the monoclonal antibody specific 6D6anti of foot and mouth disease O type (O/GX/09-7) virus as the microwell plate of coated antibody bag quilt, and with the enzymic-labelled antibody working fluid of 6D6anti as enzyme labelled antibody.

More specifically, using monoclonal antibody 6D6anti not only as coated antibody but also as traget antibody, the test kit detecting foot and mouth disease O type (O/GX/09-7) virus by ELISA double-antibody sandwich two-step approach comprises following reagent:

1) pre-coated microwell plate: in advance with monoclonal antibody 6D6anti according to the concentration of 4 μ g/mL wrap in advance by, close, and sealing be kept in vacuum aluminium foil bag, the microwell plate in each test kit one piece of 96 hole;

2) enzymic-labelled antibody working fluid: use the 6D6anti antibody that horseradish peroxidase (HRP) or alkaline phosphatase (ALP) mark in advance, and become suitable concn as working fluid by enzymic-labelled antibody diluted, be generally 0.1-1.0 μ g/mL, each test kit 10.0mL.

Enzymic-labelled antibody diluent formula is: Na ₂hPO ₄12H ₂o, 2.9g; NaH ₂pO ₄2H ₂o, 0.296g; NaCl, 8.5g; Proclin300,0.6mL; BSA, 10g; Foetal calf serum, 150mL; Enzyme stabilizers (purchased from Xi Bao bio tech ltd, Shanghai), 5g; Tween-200.25mL; Distilled water, is dissolved to 1000mL; Adjustment pH to 7.6-7.8.

3) diluent: for the dilution of standard substance and testing sample, its formula is Na ₂hPO ₄12H ₂o, 2.9g; NaH ₂pO ₄2H ₂o, 0.296g; NaCl, 8.5g; Proclin300,0.6mL; BSA, 10g; Foetal calf serum, 150mL; Gentamicin sulphate, 2 (80,000 units /); Distilled water, is dissolved to 1000mL; Adjustment pH to 7.6-7.8.Filtration sterilization, 2-8 DEG C of preservation.Each test kit 1 bottle, 25mL/ bottle.

4) washings: the concentrated washing lotion being 20 times, uses front dilution.Its formula is: Na ₂hPO ₄12H ₂o58g, NaH ₂pO ₄2H ₂o5.92g, NaCl170g, Tween-205.0mL, Proclin3000.6mL, be dissolved to 1000mL with distilled water, and adjusted to ph is to 7.2-7.4.Each test kit 1 bottle, 25mL/ bottle.

5) nitrite ion A: each test kit 1 bottle, 7mL/ bottle.Formula is: anhydrous sodium acetate 4.5g, Glacial acetic acid 1.2mL, and urea peroxide 0.8g, is dissolved to 1000mL with distilled water.

6) nitrite ion B: each test kit 1 bottle, 7mL/ bottle.Nitrite ion B formula is: citric acid 1.62g, EDTA-2Na0.372g, glycerine 100mL, tetramethyl biphenyl amine hydrochlorate 0.50g, is dissolved to 1000mL with distilled water.

8) stop buffer: each test kit 1 bottle, 7mL/ bottle.Formula is: containing 98% sulfuric acid 27.8mL in 1000mL distilled water.

9) standard foot and mouth disease O type (O/GX/09-7) virus-positive serum: each test kit 1 bottle, 1mL/ bottle.

10) standard foot and mouth disease O type (O/GX/09-7) viral negative serum: sample diluting liquid can be used as negative serum and uses.

Embodiment 4, detect foot and mouth disease O type (O/GX/09-7) virus with monoclonal antibody 6D6anti, 2H10anti and golden millimeter paper chromatographic test paper (card, box)

1, golden millimeter paper chromatographic test paper is prepared

(Fig. 5 is the Facad structure figure of golden millimeter paper chromatographic test paper as shown in Figure 5, Figure 6, Fig. 6 is the vertical section structure figure of golden millimeter paper chromatographic test paper), golden millimeter paper chromatographic test paper for detecting foot and mouth disease O type (O/GX/09-7) virus is discharged pad 4 formed by absorbent pad 1, nitrocellulose filter (NC film) 2, glass fibre membrane sample pad 3, glass fibre element film binding substances, and nitrocellulose filter 2 is provided with detection line (T line) 6 and nature controlling line (C line) 5.

1) bag is by nitrocellulose filter (NC film) 2

Being that the monoclonal antibody 6D6anti of 1-5mg/mL or 2H10anti wrap tested survey line (T line) by concentration, is that goat anti-mouse immunoglobulin (purchased from the Jing Boerxi Science and Technology Ltd.) bag of 1-5mg/mL is by nature controlling line (C line) by concentration.Concrete operations are: with BIODOT company XYZ3000 Membrane jetter by being used as the monoclonal antibody 6D6anti (or 2H10anti) of coated antibody, goat anti-mouse immunoglobulin is sprayed on the nitrocellulose filter (purchased from Millipore company) 2 that 300mm is long, 25mm is wide respectively, form the detection line 6 and nature controlling line 5 that are separated from each other, nature controlling line and testing wire spacing are generally 0.3-1.0cm, preferred 0.5cm, 37 DEG C of dryings 1 hour are for subsequent use, 37 DEG C of dryings 1 hour.

2) preparation of binding substances release pad 4

2.1 use colloid gold label monoclonal antibody 6D6anti, and method is: under magnetic force heated and stirred, adds 155mL purified water, boil in triangular flask.Add 1% 4 chlorogold solution 5mL, boil.Add 1% citric acid three sodium solution 7mL again, boil 5 minutes, be colloidal gold solution, after cooling, be stored in 2-8 DEG C.Get 1 milliliter of colloidal gold solution in centrifuge tube.Add 15 μ l0.2M solution of potassium carbonate, the static 5min of room temperature.Add 10 μ l antibody, after mixing, leave standstill 30min.Add 10 μ l20%BSA solution, balance 5min.Add 10 μ l20%PEG20000 solution, balance 30min; Use whizzer 10000rpm, centrifugal 10min, removes supernatant liquor.Add 100 μ l gold mark redissolution liquid (borate buffer containing 2% sucrose, 1% casein, 0.5%BSA, 0.1TritonX100,0.1%SDS), for subsequent use after redissolving.

2.2 prepare binding substances release pad: the material of binding substances release pad is glass fibre membrane, it is marked with the monoclonal antibody specific 6D6anti of colloid gold label, according to the speed spray film of 8 μ l/cm after gold after redissolution is diluted with gold mark redissolution liquid 1:4, drying in 2 hours is placed at 37 DEG C, for subsequent use.

3) golden millimeter paper chromatographic test paper is prepared

Nitrocellulose filter 2 first pasted by PVC backboard 7, absorbent pad 1 is pasted in the one end near nitrocellulose filter nature controlling line, binding substances release pad 4 and sample pad 3 is being pasted near testing wire one end, obtain for detecting the viral golden millimeter paper chromatographic test paper of foot and mouth disease O type (O/GX/09-7), can cut by required size, after adding siccative, sealing is preserved.

Test strip as above-mentioned steps prepared loads in plastic clip, make test card, and being assembled into test kit, the position that this test kit corresponds to sample pad is provided with point sample mouth (in Fig. 7 S place), and the position corresponding to detection line and nature controlling line is provided with observation window (in Fig. 7 C/T place).

2, the use of golden millimeter paper chromatographic test paper (card, box)

The using method of gold millimeter paper chromatograph test strip: sample pad end is immersed in sample, sample pad 3 i.e. imbitition moves to upper end, when flowing through binding substances release pad 4, the colloid gold label monoclonal antibody 6D6anti on dry plate is redissolved, and drive it to ooze to nitrocellulose membrane 2 to move.If there is specific antigen to be measured (positive sample) in sample, it can be combined with colloid gold label monoclonal antibody 6D6anti, this immune complex flow to detection line 6 namely obtain by insolubilized antibody 6D6anti or 2H10anti, film shows and red detects lines (T line).Superfluous colloid gold label monoclonal antibody 6D6anti continues to move ahead, and combines, and show red Quality Control lines (C line) to nature controlling line 5 and goat anti-mouse immunoglobulin (solid phase two resists).Otherwise, if without specific antigen to be measured (negative sample) in sample, then without detection lines (T line), and only show Quality Control lines (C line).If detection line (T line) and nature controlling line (C line) all do not develop the color or only detection line (T line) develops the color, then represent that golden millimeter paper chromatographic test paper (card) lost efficacy (Fig. 5).

The using method of gold millimeter paper chromatography test card and test kit: during detection, get measuring samples 1-2 to drip, drop in the point sample mouth of paper box, whether occur that colour band is determined whether to there is foot and mouth disease O type (O/GX/09-7) virus in sample according to the detection line (T line) in observation window and nature controlling line (C line).

3, foot and mouth disease O type (O/GX/09-7) virus of different concns is detected with golden millimeter paper chromatography test kit

(combined thing release pad is wrapped with the monoclonal antibody specific 6D6anti of colloid gold label with golden millimeter paper chromatography test kit, wrap tested survey line (T line) with monoclonal antibody 2H10anti) detect different concns (concentration is respectively 120,60,30,15,5ng/mL) foot and mouth disease O type (O/GX/09-7) virus, with the sensitivity of detection kit.

Detected result display detection sensitivity can reach 15ng/mL (see Fig. 7), shows that this product has good sensitivity, has actual application value.

Claims

1. with foot and mouth disease O type (O/GX/09-7) virus for immunogen immune mouse obtain continue, the hybridoma cell strain of monoclonal antibody specific that stably excreting resistant to foot and mouth disease O type (O/GX/09-7) is viral, name is called 6D6, and its deposit number is CGMCCNo.11196.

2. the monoclonal antibody specific 6D6anti secreted by hybridoma cell strain 6D6 described in claim 1, its variable region of heavy chain has SEQ ID No: the amino acid residue sequence of 1 or by SEQ ID No: the amino acid residue sequence of 1 is through the replacement of one to ten amino-acid residue, disappearance or add and can with the polypeptide of foot and mouth disease O type (O/GX/09-7) viral specific combination, variable region of light chain has SEQ ID No: the amino acid residue sequence of 2 or by SEQ ID No: the amino acid residue sequence of 2 is through the replacement of one to ten amino-acid residue, disappearance or add and can with the polypeptide of foot and mouth disease O type (O/GX/09-7) viral specific combination.

3. monoclonal antibody according to claim 2, it is characterized in that: the gene (6D6anti) of encodes monoclonal antibody 6D6anti, its variable region of heavy chain encoding gene has SEQ ID No: in the DNA sequence dna of 3 or polynucleotide SEQIDNo:1 DNA sequence dna or under high high stringency conditions can with SEQ ID No: the nucleotide sequence that 3 DNA sequence dnas limited are hybridized, its variable region of light chain encoding gene has SEQ ID No: in the DNA sequence dna of 4 or polynucleotide SEQIDNo:2 DNA sequence dna or under high high stringency conditions can with SEQ ID No: the nucleotide sequence that 4 DNA sequence dnas limited are hybridized.

4. obtain the method for hybridoma cell strain 6D6 according to claim 1, comprise the following steps:

3) screen hybridoma, obtain hybridoma cell strain 6D6CGMCCNo.11196.

5. obtain the method for the monoclonal antibody 6D6anti that hybridoma cell strain 6D6 according to claim 2 secretes, on the basis of claim 4 step, continue to comprise the following steps:

4) be separated from the ascites fluid of the animal of the nutrient solution of hybridoma cell strain 6D6CGMCCNo.11196 or inoculation hybridoma cell strain 6D6CGMCCNo.11196 and be purified into described monoclonal antibody.

6. detect an ELISA kit for foot and mouth disease O type (O/GX/09-7) virus, comprise the monoclonal antibody specific 6D6anti of foot and mouth disease O type (O/GX/09-7) virus.

7. ELISA kit according to claim 6, it is characterized in that, comprise: with the monoclonal antibody specific 6D6anti of foot and mouth disease O type (O/GX/09-7) virus as the microwell plate of coated antibody bag quilt, and with the enzymic-labelled antibody working fluid of 6D6anti as enzyme labelled antibody.

8. ELISA kit according to claim 6, it is characterized in that, described 6D6anti refers to as the microwell plate of coated antibody bag quilt, with 10mMpH7.0-7.4PBS, 6D6anti is diluted to 0.5-10 μ g/mL, adds in microwell plate, 110 μ l/ holes, are placed in 2-8 DEG C and spend the night; After patting dry, add the 10mMpH7.0-7.4PBS containing 1%BSA, 300 μ l/ holes, are placed in 2-8 DEG C and spend the night; Pat dry, dry final vacuum loads in aluminium foil bag for subsequent use;

Described enzyme labelled antibody is for using the marker enzyme traget antibodies such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP), and (formula is: Na then to use enzymic-labelled antibody diluent ₂hPO ₄12H ₂o, 2.9g; NaH ₂pO ₄2H ₂o, 0.296g; NaCl, 8.5g; Proclin300,0.6mL; BSA, 10g; Foetal calf serum, 150mL; Enzyme stabilizers, 5g; Tween-200.25mL; Distilled water, is dissolved to 1000mL; Adjustment pH to 7.6-7.8) be diluted to working fluid, the concentration of working fluid is 0.1-1.0 μ g/mL.

9. the using method of the ELISA kit of foot and mouth disease O type (O/GX/09-7) virus, for the detection to foot and mouth disease O type (O/GX/09-7) virus, comprises the following steps:

1) wrap by microwell plate with monoclonal antibody 6D6anti, wash plate;

2) close the elisa plate through bag quilt, wash plate;

3) add testing sample, wash plate;

5) substrate colour developing is added;

6) termination reaction;

7) OD is measured ₄₅₀value.

10. one kind is detected the golden millimeter paper chromatographic test paper of foot and mouth disease O type (O/GX/09-7) virus, be made up of glass fibre membrane sample pad, binding substances release pad, absorbent pad, nitrocellulose filter (NC film), nitrocellulose filter is provided with detection line (T line) and nature controlling line (C line), it is characterized in that:

By concentration be 1-5mg/mL claim 2 described in monoclonal antibody 6D6anti wrap tested survey line (T line), be that the goat anti-mouse immunoglobulin bag of 1-5mg/mL is by nature controlling line (C line) by concentration;

Binding substances release pad is coated with the monoclonal antibody specific 6D6anti of colloid gold label.