CN102633878B - H1-subtype influenza A virus double-antibody sandwich ELISA kit and application - Google Patents
H1-subtype influenza A virus double-antibody sandwich ELISA kit and application Download PDFInfo
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Abstract
The invention belongs to the fields of detection technology of animal virology and epizootiology. The invention particularly relates to an H1-subtype influenza A virus double-antibody sandwich ELISA kit and an application. The kit of the invention comprises an enzyme label plate which contains anti-H1-subtype influenza A virus hemagglutinin, has a preservation number of CCTCC NO: C201106, and is coated by a monoclonal antibody, and a horseradish peroxidase labeled H1-subtype influenza A virus hemagglutinin monoclonal antibody is used as a second antibody. The invention discloses separation, amplification, inactivation, and purification methods of the H1-subtype influenza A virus, and preparation and purification methods of the H1-subtype influenza A virus hemagglutinin monoclonal antibody. The invention also discloses an H1-subtype influenza A virus double-antibody sandwich ELISA detection method.
Description
Technical field
The present invention relates to animal virology and epizootiology detection technique field.Specifically, the present invention relates to a kind of A type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection kit and the application of A type H1 subtype influenza virus antigen detection in swinery.
Background technology
1975, Kohler G and Milstein C delivered cytogamy method and have set up hybridoma technology on Nature magazine, had created the authentic monoclonal antibody technology being with historically new significance.After clone, produce structure and the identical high purity antibody of various characteristics, be called monoclonal antibody (Monoclonal Antibody, McAb), be called for short monoclonal antibody.The discovery of monoclonal antibody technique and use, played huge pushing effect to the development of modern life science research, become an importance of biological technical field.So far, the application of this technology has been widely used in the aspects such as fundamental research, medical diagnosis on disease, treatment, prevention.
In June, 2009, the World Health Organization announced the warning rank of A type (H1N1) influenza to rise to 6 grades, meaned that worldwide being very popular of A type (H1N1) influenza forms.Worldwide being very popular of the last influenza originated from Mexico, be called as at the beginning porcine influenza (Swine flu), afterwards according to viral analysis, rename as pig source property influenza infection (swine origin influenza virus infection, be called for short S-OIV infection), finally, for the seasonal influenza A with people (H1N1) difference, rename as new influenza A (H1N1), in China, be called Influenza A H1N1.Influenza A H1N1 causes tremendous influence to the health of people and animal, and economy has been caused to huge loss.
The people such as Engvall of Sweden in 1971 are usingd respectively Mierocrystalline cellulose and poly-the third ethene test tube as solid phase carrier adsorption antigen/antibody, have set up enzyme-linked immunosorbent assay (Enzyme Linked ImmunosrbentAssay is called for short ELISA method).The people such as Voller in 1974 use polystyrene micro-reaction plate instead as solid-phase immunity absorption carrier, ELISA method is applied, make to develop into for the enzyme-labelled antibody technique of Antigen Location the measuring method of liquid sample micro substance, and become gradually a kind of method the most conventional in antigen and antibody.It combines the immune response of enzyme labelling thing synantigen antibody complex with the catalytic amplification of enzyme, both kept the susceptibility of enzymic catalytic reaction, has kept again the specificity of antigen antibody reaction, thereby has improved greatly sensitivity.It is again a kind of heterogeneous immune analysis method simultaneously, and each step in reaction has washing process, thereby has removed unreacted reactant and interfering substance.Due to ELISA method have highly sensitive, high specificity, easy and simple to handle, detect rapidly and on-radiation and the plurality of advantages such as can measure in batches, make ELISA method obtain application more and more widely.(Jiao Kui etc. enzyme immunoassay application [M]. Beijing: Chemical Industry Press, 2004,84～141; Li Wenmin. technical progress and the application [J] of Enzyme-linked Immunosorbent Assay reaction. Hubei Vocationl Technical College journal, 2003,4 (6): 65～69)
Enzyme-labelled antibody technique is the gordian technique of ELISA detection method, in the present invention, enzymic-labelled antibody used is that this laboratory produces anti-A type H1 subtype influenza viral hemagglutinin monoclonal antibody voluntarily, through horseradish peroxidase (being called for short HRP) mark, have very high enzymic activity, output is high and cost is low.
Summary of the invention
This main purpose be to overcome the defect of prior art, provide a kind of A type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection kit, to solve the detection of A type H1 subtype influenza virus antigen in swinery.
First object of the present invention is the monoclonal antibody of anti-A type H1 subtype influenza virus hemagglutinin of core reagent that can be used for assembling ELISA test kit that obtains a kind of high specificity.
Second object of the present invention is to set up a kind of double-antibody sandwich elisa detection method of A type H1 subtype influenza virus
The 3rd object of the present invention is the double-antibody sandwich elisa detection kit of a kind of A type H1 subtype influenza virus of assembling.
The 4th object of the present invention is to utilize the application of this test kit in swinery A type H1 subtype influenza virus antigen detection.
The present invention is achieved in that
Agricultural microorganism National Key Laboratory of the Hua Zhong Agriculture University Viral Laboratory at applicant place is the separated former strain of a strain A type H1 subtype influenza virus A-Influ/JML-F9 obtaining from swinery, this strain is delivered Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on January 27th, 2011, and deposit number is CCTCC NO:V201105.
Applicant has prepared a kind of monoclonal antibody of high specificity, it is the monoclonal antibody of anti-A type H1 subtype influenza viral hemagglutinin (HA) albumen, secrete the hybridoma cell strain 5F7 of this monoclonal antibody, on January 27th, 2011, deliver Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province, deposit number is CCTCC NO:C201106.
Applicant utilizes described monoclonal antibody to be prepared into double-antibody sandwich elisa core reagent, has assembled a kind of double-antibody sandwich elisa test kit that is applicable to the A type H1 subtype influenza virus of swinery for rapid detection.Test kit of the present invention is by box body and is included in sample diluting liquid, washings, substrate A liquid, substrate B liquid, stop buffer, positive control sample and the negative control sample composition in box body.
More detailed technical scheme is as described below.
The preparation of the monoclonal antibody of anti-A type H1 subtype influenza virus hemagglutinin, it comprises the following steps:
1) take from swinery the A type H1 subtype influenza virus A-Influ/JML-F9 that the separated strain deposit number obtaining is CCTCC NO:V201105 is antigen, through amplification, deactivation and purifying, 5-8 week BALB/c mouse in age (purchased from Animal Experimental Study center, Hubei Province) is carried out to immunity.
2) cytogamy, the spleen of the BALB/c mouse (purchased from Animal Experimental Study center, Hubei Province) after booster immunization of learning from else's experience, merges with SP2/0 myeloma cell's (purchased from Ministry of Health's Wuhan institute of Biological Products) of fresh preparation.
3) utilize blood clotting suppress method (HI) (comparison [J] of the .RT-PCR method such as Xiao Jinhui and blood clotting inhibition method evaluation influenza virus. Tropical China medical science, 2005,5:401～402), filter out the positive hole of secretion anti-A type H1 hypotype subtype influenza viral hemagglutinin (HA) antibody.To the positive hole screening use at once limiting dilution assay (but the Chinese wait preparation and the evaluation [J] of .H5 subtype avian influenza virus hemagglutinin monoclonal antibody. animal medicine progress, 2006,27 (8): 67～69) clone, screen.Through 3～5 time cloning purifying, finishing screen is selected the monoclonal hybridoma strain (deposit number is CCTCC NO:C201106) of secretion anti-A type H1 subtype influenza viral hemagglutinin (HA) protein antibodies.
4) preparation of ascites, only gets female 5-6 week BABL/c mouse in age (purchased from Animal Experimental Study center, Hubei Province) abdominal injection Freund's complete adjuvant 0.5ml/, pneumoretroperitoneum injection 1 * 10 in 5 days
6individual hybridoma/only, collect ascites after 9 days, blood clotting suppresses (HI) method and detects titer of ascites ,-70 ℃ of preservations.
5) by deposit number, be that CCTCC NO:C201106 monoclonal antibody is carried out purifying and mark.
Above-mentioned antibody (deposit number is CCTCC NO:C201106) can be used for the double-antibody sandwich elisa test kit that preparation detects A type H1 subtype influenza virus after purifying and mark.
Compared with prior art the present invention has following remarkable advantage:
1, test kit of the present invention can directly detect A type H1 subtype influenza virus, has high specificity, and highly sensitive, detection time is short, detects the feature of sample wide ranges (chick embryo allantoic liquid, viscera tissue homogenate).
2, test kit of the present invention is applicable to A type H1 hypotype swine influenza virus to detect, and classical H1 hypotype swine influenza virus is not reacted, and has specificity.
3, test kit of the present invention is applicable to A type H1 subtype influenza virus to detect, and influenza virus to other hypotype, as: the influenza virus of classical H1 hypotype, H3 hypotype, H5 hypotype, H9 hypotype, do not react, there is good specificity.
4, the present invention is assembled into test kit by required all ingredients, and operation is simple, need not operated by professional.Test kit good stability of the present invention, long preservative period are placed and can not affected its susceptibility under 4 ℃ of conditions half a year.
5, the present invention once can process a plurality of samples simultaneously, is applicable to very much the clinical extensive detection of A type H1 hypotype swine influenza virus.And can meet test requirements document, also can be used as scientific research and use.
6, also do not detect in the market the test kit of A type H1 hypotype swine influenza virus antigen (North America influenza).Also without the differential diagnosis kit of distinguishing A type H1 hypotype swine influenza virus (North America influenza) and classical H1 subtype influenza virus.
Accompanying drawing explanation
Fig. 1: be general technical route map of the present invention.
Fig. 2: be SP2/0 cell chromosome counting.
Fig. 3: 5F7 hybridoma chromosome counting.
Fig. 4: anti-A type H1 subtype influenza viral hemagglutinin (HA) protein monoclonal antibody indirect immunofluorescene assay figure.Fig. 4 A wherein: be anti-A type H1 subtype influenza viral hemagglutinin (HA) protein monoclonal antibody indirect immunofluorescene assay figure; The negative contrast of Fig. 4 B
Embodiment
Embodiment 1 Preparation Example
One, the separation of A type H1 subtype influenza virus
1, sepn process: gather disease pig throat sample with the throat swab of sterilizing, be placed in ditalimfos phthalate buffer and (be called for short PBS, formula: Na
2cO
31.59g, NaHCO
32.93g, uses ddH
2o is settled to 1000ml) in, subsequently throat swab sample liquid 0.2ml is inoculated to 9 age in days no-special pathogens (Specific pathogen Free, SPF) chicken embryo (purchased from the logical laboratory animal technology of Beijing Cimmeria dimension company limited), put at 35 ℃ and hatch 72h, the allantoic fluid of receiving is done blood clotting qualitative test, the sample that hemagglutination test is positive, then through RT-PCR test sizing.
2, appraisal basis: the sample of RT-PCR test positive, amplification HA, serves Hai Shenggong biotechnology company limited order-checking, to gained DNA sequence dna and the online sequence of announcing (
http:// blast.ncbi.nlm.nih.gov/) compare, to determine virus subtype.
3, the virological Features of A type H1 subtype influenza virus strain: the HA-DNA sequence alignment of process, draw with North America influenza HA-DNA nucleic acid homology and reach 99.2%, amino acid homology reaches 99.0%, and therefore certain influenza virus that we obtain is A type H1 subtype influenza virus.
4, the preservation of patented procedure: the A type H1 subtype influenza virus A-Influ/JML-F9 that obtains obtaining after identifying is delivered to Chinese Typical Representative culture collection center (CCTCC) preservation that is positioned at Wuhan City, Hubei Province Wuhan University, and its deposit number is CCTCC NO:V201105.
Two, amplification, deactivation and the purifying of A type H1 subtype influenza virus (deposit number is CCTCC NO:V201105)
1, by deposit number, be that the A type H1 subtype influenza virus A-Influ/JML-F9 of CCTCC NO:V201105 increases; (1) strictly screen 9～10 age in days no-special pathogens (Specific pathogen Free, SPF) chicken embryo (purchased from the logical laboratory animal technology of Beijing Cimmeria dimension company limited)
Every batch of chicken embryo of bringing will be through strict screening.Totally 4 of outer inspections: white shell, size, breakage and dirty embryo, totally 9 of interior inspections: remove husky shell, biasing gas chamber, faint breath chamber, inversion embryo, infertile egg, termination embryo, weak embryo, pollute embryo, crack embryo.
(2) virus inoculation (A-Influ/JML-F9) is in chick embryo allantoic cavity
Select 9～10 age in days no-special pathogens (Specific pathogen Free, SPF) chicken embryo (purchased from the logical laboratory animal technology of Beijing Cimmeria dimension company limited), draw air chamber and embryo position, at air chamber, approach that the tincture of iodine is smeared at embryo position place and alcohol carries out disinfection, peg and wear an aperture, subsequently 1ml syringe needle is inserted to (avoiding blood vessel) along this aperture, inoculation A type H1 subtype influenza virus (A-Influ/JML-F9).Finally use paraffin sealing, at 35 ℃ of juxtapositions, hatch 72h.Check chicken embryonic development situation every day, every 12h turns over ovum ovoscopy once.Dead chicken embryo in 24h, thinks nonspecific death and discards, 72h collects chicken embryo, places at 4 ℃ and spends the night.
(3) contain the results of A type H1 subtype influenza virus (A-Influ/JML-F9) allantoic fluid
Alcohol disinfecting chick embryo air sac end by 75% concentration, tears chick embryo air sac eggshell with aseptic nipper, at chorioallantoic membrane, without great vessels place, wears out.With aseptic pipette, extract chick embryo allantoic liquid, be placed in corresponding collection tube.The centrifugal 5min of chicken embryo harvest liquid 3000r/min is removed to blood and cell.Then carry out red cell agglutination experiment, determine chick embryo allantoic liquid hemagglutinative titer.
2, the deactivation of A type H1 subtype influenza virus A-Influ/JML-F9
To by the formaldehyde of final concentration 0.8%, slowly add formaldehyde solution containing after viral A-Influ/JML-F9 allantoic fluid freeze thawing 3 times, and fully mix, 37 ℃ of deactivations 24 hours, every jolting in 6 hours 1 time.Each inactivation of virus container should sample immediately, carries out respectively Validation of Virus Inactivation in Human test.The allantoic fluid of the A type H1 subtype influenza virus of deactivation container is through assay approval.8000r/min, centrifugal 10min.Discard precipitation, supernatant is standby.
3, A type H1 subtype influenza virus A-Influ/JML-F9 concentrates and purifying
(1) A type H1 subtype influenza virus A-Influ/JML-F9 is concentrated, method is: by A type H1 subtype influenza virus chick embryo allantoic liquid in 27000r/min, centrifugal 2h, supernatant discarded, precipitation is resuspended with phosphate buffered saline buffer (PBS), fully mixes standby.
(2) A type H1 subtype influenza virus A-Influ/JML-F9 purifying
In ultracentrifugation pipe, add successively 60%, 45%, 30%, 20% concentration sucrose solution to form density gradient.Resuspended good viral A-Influ/JML-F9 sample is laid in to the superiors, in 32000r/min, centrifugal 1h.Aspect sample between centrifugal rear taking-up 45%, 30%, takes out sample with aspect sample between PBS resuspended 30%, 45%, 32000r/min, and centrifugal 1h, gets precipitation (obtaining described viral A-Influ/JML-F9) and carries out protein content determination.Using this as immunogen.
The preparation of the anti-A type H1 subtype influenza of embodiment 2 hemagglutinin monoclonal antibody
1, the former strain of A type H1 subtype influenza virus A-Influ/JML-F9 with deactivation and purifying (is deposited in the Chinese Typical Representative culture collection center in the Wuhan University of Wuhan City, Hubei Province, deposit number is CCTCC NO:V201105) be antigen, add the immune 5-8 of freund's adjuvant emulsification (head exempts to select Freund's complete adjuvant, and two, three exempt to select Freund's incomplete adjuvant) week BALB/c mouse in age (purchased from Animal Experimental Study center, Hubei Province).It is 20ug/ that head exempts from dosage, every subcutaneous multi-point injection in mouse carotid back.After two weeks with same dose booster immunization once, after two weeks again booster immunization once, dosage be 40ug/ only, after two weeks, docking gathers a small amount of Mouse Blood, collects serum, with blood clotting inhibition method detection mouse antibodies, tires.Treat that mouse antibodies reaches test requirements document, then through the A type H1 of abdominal injection deactivation and purifying subtype influenza virus A-Influ/JML-F9, injected dose be 40ug/ immunity once.After three days, desirable immune spleen cell and SP2/0 myeloma cell (purchased from Ministry of Health's Wuhan institute of Biological Products) are merged by laboratory ordinary method.
2, cytogamy, one of the BALB/c mouse of the reinforced immunological of learning from else's experience (purchased from Animal Experimental Study center, Hubei Province), eye socket sacrificed by exsanguination (collect serum, be positive serum) is soaked 10min sterilization in 75% concentration alcohol.Aseptic taking-up mouse spleen, isolates splenocyte, presses 1 * 10 with SP2/0 myeloma cell's (purchased from Ministry of Health's Wuhan institute of Biological Products) of fresh preparation
7individual SP2/0 and 10
8the ratio of individual immunocyte (number was than 1: 10) mixes in 50ml centrifuge tube, 1500r/min, centrifugal 10min.Supernatant discarded, tube wall blots with the filter paper of sterilizing, the light shake pipe end, makes cell precipitation slightly loosening.The centrifuge tube that cell mixture is housed is positioned in 37 ℃ of waters bath with thermostatic control.Then in 1min, slowly splash into 50% polyoxyethylene glycol (PEG) 0.8ml (purchased from sigma company) of pre-temperature to 37 ℃, limit edged stirs with pipette tip gently, continues to stir 1min.Then 1640 (purchased from sigma company) the cell culture fluid 40ml that slowly adds pre-temperature to 37 ℃.
Merge concrete steps as follows:
Within first minute, dropwise splash into 50% polyoxyethylene glycol (PEG) 0.8ml, standing 0.5min; Within second minute, add 1640 cell culture fluid 1ml, standing 0.5min (repeating once); Within the 4th minute, add 1.5ml, standing 0.5min (repeating once); Within the 6th minute, add 5ml, standing 0.5min (repeating once); Within the 8th minute, add 10ml, standing 0.5min (repeating once); Each added-time need slowly add, and constantly stirs lightly.Standing 1min, the centrifugal 10min of 1000r/min, abandons supernatant, in 37 ℃ of environment, places 8min.With HAT substratum (purchased from Sigma company), suspend, simultaneously also with HAT substratum suspend the raising splenocyte for preparing and with merge after cytomixis, add as required appropriate HAT substratum, minute plant in 96 well culture plates approximately 200 μ l/ holes.Single cell fusion can be inoculated 4～8 96 orifice plates.Also can plant less as required, generally press the cell count of SP2/0 and calculate, every hole inoculum size is approximately containing 10
4a left and right SP2/0 cell.In 37 ℃, in 5%CO2 incubator, cultivate.After merging, second day starts to observe 96 orifice plate inner cells has pollution-freely, in the 4th day, with HT substratum, changes HAT substratum 100 μ l.Treat that fused cell colony grows to culture hole 1/4, when substratum omits flavescence, carry out antibody titer detection.Adopt deactivation A type H1 subtype influenza virus (A-Influ/JML-F9) as screening antigen, utilize blood clotting suppress method (comparison [J] of the .RT-PCR method such as Xiao Jinhui and blood clotting inhibition method evaluation influenza virus. Tropical China medical science, 2005,5:401～402) filter out the positive hole of secretion anti-A type H1 hypotype subtype influenza viral hemagglutinin (HA) antibody.To the positive hole screening use at once limiting dilution assay (but the Chinese wait preparation and the evaluation [J] of .H5 subtype avian influenza virus hemagglutinin monoclonal antibody. animal medicine progress, 2006,27 (8): 67～69) clone, screen.Through 3～5 time cloning purifying, finishing screen is selected the monoclonal hybridoma strain 5F7 of secretion anti-A type H1 hypotype subtype influenza viral hemagglutinin (HA) antibody, on January 27th, 2011, deliver Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province, deposit number is CCTCC NO:C201106.
3, the preparation of ascites, only selects 5-6 week female BABL/c mouse in age (purchased from Animal Experimental Study center, Hubei Province) abdominal injection Freund's complete adjuvant 0.5ml/, pneumoretroperitoneum injection 1 * 10 in 5 days
6individual hybridoma/only, collect ascites after 9 days, blood clotting suppresses method (HI) and detects titer of ascites ,-70 ℃ of preservations.
4, the purifying of ascites antibody: caprylic acid-ammonium
Concrete steps are as follows:
(1) by the centrifugal 10min of ascites 1000r/min of above-mentioned steps 3 preparations, with 0.45 μ m membrane filtration, filtrate adds while stirring with 4 times of volume 60mmol/L acetate buffer solutions (pH=4.5);
(2) dropwise adding while stirring n-caprylic acid to final concentration is 33 μ l/ml, under room temperature condition, stirs 30min, and the centrifugal 30min of 8000r/min abandons precipitation and collects supernatant;
(3) by the supernatant liquor of step (2) through filter paper filtering once, filtrate pH modulation 7.4;
(4) filtrate to step (3) gained adds saturated ammonium sulphate solution (ammonium sulfate volume/cumulative volume≤45%) while stirring, when filtrate journey white opacity liquid, continues to stir 30min, then in 4 ℃ of standing 5h.Centrifugal 30min under 4 ℃ of condition 12000r/min again;
(5) abandon supernatant, precipitation is resuspended with 10mmol pH=9.0Tris-HCl; In the Tris-HCl of the 10mmol of 100 times of volumes pH=9.0, in 4 ℃ of condition lower magnetic forces, stir dialysis, dialysis dialysis 36h, during 3 times (12h/ time) change fresh Tris-HCl liquid.Dialysis finishes, and gets supernatant, identifies the purity of antibody by the method for SDS-PAGE electrophoresis, measures antibody concentration, packing, frozen with ultraviolet spectrophotometer.
In the test of above-mentioned antibody purification, agents useful for same is according to following formulated:
Acetate buffer solution (60mmol/L): C
2h
3naO
22.463g, use ddH
2o is settled to 500ml (pH=4.5).
Saturated ammonium sulphate solution: every 100ml ddH
2in O, add (NH
4)
2sO
490g, is heated to 80 ℃ of dissolvings, and filter paper filtering, is down to the existing crystallization of room temperature while hot, and gained is saturated ammonium sulphate solution with crystallization filtrate.With 25% ammoniacal liquor adjust pH to 7.2, standby.
5, horseradish peroxidase (HRP) labeled monoclonal antibody (deposit number is CCTCC NO:C201106)
(1) horseradish peroxidase (HRP) 5.0mg that gets 5.0mg is dissolved in 5ml ddH
2o, solution is red-brown; Drip subsequently NaIO
4solution (60mmol/L) 0.5ml, makes solution be grass green, and 4 ℃ of conditions are placed 30min; Drip ethylene glycol solution (160mmol/L) 0.5ml, stop oxidizing reaction, under room temperature, lucifuge condition is placed 30min, and solution is brown color;
(2) get the liquid mixing that monoclonal antibody (deposit number is CCTCC NO:C201106) 5ml (1mg/ml) prepared by the present invention and above-mentioned steps (1) are handled well, 10mmol pH=9.5 carbonate buffer solution, 4 ℃ of condition lower magnetic forces stir dialysed overnight.
(3) to completing, in dialysis liquid, add the NaBH that freshly prepared concentration is 5mg/ml
4solution 0.2ml places 2h at 4 ℃; Then equal-volume adds saturated ammonium sulphate solution, standing 30min at 4 ℃, the centrifugal 10min of 7000r/min, supernatant discarded.3ml is resuspended for PB solution (20mmol/L pH=7.4), and in the PB of 1000 times of volumes (20mmol/L pH=7.4), 4 ℃ of condition lower magnetic forces stir dialysis, dialysis 36h, during 3 times (12h/ time) change liquid.
(4) traget antibody (deposit number is CCTCC NO:C201106) completes after dialysis, adds PB (20mmol/L pH=7.4), glycerine (final concentration 30%) is settled to 5ml, in-20 ℃ of preservations.
In labelled antibody test, agents useful for same is pressed following formulated:
NaIO4 solution (60mmol/L): NaIO
41.283g, use ddH
2o is settled to 100ml;
Ethylene glycol solution (160mmol/L): ethylene glycol 13.4 μ l, use ddH
2o is diluted to 1.5ml;
Carbonate buffer solution 10mmol pH=9.5:Na
2cO
31.59g, NaHCO
32.93g, uses ddH
2o is settled to 1000ml (pH=9.5);
NaBH
4solution (5mg/ml): NaBH
40.05g, uses ddH
2o is settled to 10ml, now with the current;
Saturated ammonium sulphate solution: every 100ml ddH
2in O, add (NH
4)
2sO
490g, is heated to 80 ℃ of dissolvings, and filter paper filtering, is down to the existing crystallization of room temperature while hot, and gained is saturated ammonium sulphate solution with crystallization filtrate.With 25% ammoniacal liquor adjust pH to 7.2, standby.
PB solution (20mmol/L pH=7.4): Na
2hPO
412H
2o 3.58g, NaH
2pO
41.56g, uses ddH
2o is settled to 1000ml, pH=7.4.
The evaluation of the anti-A type H1 subtype influenza of embodiment 3 hemagglutinin monoclonal antibody (deposit number is CCTCC NO:C201106)
1, the evaluation that HI tires
Adopt the A type H1 subtype influenza virus (deposit number is CCTCCNO:V201105) that separation of the present invention obtains by blood clotting inhibition method, to measure tiring of Hybridoma Cell Culture supernatant and mouse ascites as antigen.The results are shown in Table 1.
Table 1: utilize blood clotting to suppress method (HI) and measure tiring of Hybridoma Cell Culture supernatant and mouse ascites
2, the evaluation of the Ig subclass (type) of monoclonal antibody (deposit number is CCTCC NO:C201106)
With mouse source monoclonal antibody hypotype identification kit (Mouse Mab Isotyping Test Kit, purchased from ROCKLAND company), the resulting monoclonal antibody of the present invention is identified, determined that monoclonal antibody antibody prepared by the present invention is IgG
1subclass.
3, the specificity identification of monoclonal antibody (deposit number is CCTCC NO:C201106)
Adopt blood clotting to suppress the influenza virus that method (HI) is measured respectively anti-A type H1 subtype influenza hemagglutinin (HA) monoclonal antibody and classical H1 hypotype, H3 hypotype, H5 hypotype, H9 hypotype, result is all shown as feminine gender, illustrates that anti-A type H1 subtype influenza hemagglutinin (HA) monoclonal antibody obtaining has good specificity.The results are shown in Table 2.
Table 2: the influenza virus result of blood clotting inhibition method mensuration monoclonal antibody and classical H1 hypotype, H3 hypotype, H5 hypotype, H9 hypotype, H10 hypotype
4, chromosome counting
Carry out according to a conventional method hybridoma chromosome counting (Zhang Gusheng etc. monoclonal antibody is in application [M] medically. Shanghai science tech publishing house, 1987:281～406), between 85-97, (SP2/0 myeloma cell's modal number is 70 to the hybridoma modal number of all acquisitions, BALB/c mice spleen cell chromosome number is 40), all, higher than the chromosome number of two parental cells, the hybridoma that proves all acquisitions is really the heterozygote of SP2/0 cell and immune spleen cell.
Embodiment 4 A type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection methods
1, core reagent preparation:
Coating buffer (25mmol/L carbonate buffer solution): Na
2cO
31.59g, NaHCO
32.93g, uses ddH
2o is settled to 1000ml (pH9.6).
10 times of washingss: NaCl 80g, KCl 2g, Na
2hPO
412H
2o 29g, KH
2pO
42g, Tween-20 5ml, uses ddH
2o is settled to 1000ml (pH=7.4).
Confining liquid: 5g skimming milk is dissolved in 100ml washings.
Substrate solution: be divided into substrate A liquid and substrate B liquid.Specifically composed as follows:
Substrate A liquid: the H of 0.006% concentration
2o
2damping fluid.
Substrate B liquid: get Na
2hPO
412H
2o14.2g, citric acid 10.5g, uses ddH
2o is settled to 500m and is made into 0.1ml phosphoric acid salt citrate buffer solution (pH=5.0), by final concentration, is then that 20mg/L adds benzidine (TMB) (during use, A liquid and B liquid equal-volume are mixed, mix in latter 5 minutes and use, be now with the current).
Stop buffer (final concentration 0.025% hydrofluoric acid solution): getting concentration is 40% hydrofluoric acid solution 625 μ L, is settled to 100mL with ddH2O.
2, ELISA detection method step:
(1) the best coated concentration of coated antibody (deposit number is CCTCC NO:C201106) and the best weaker concn of Horseradish Peroxidase Conjugates (deposit number is CCTCC NO:C201106) than adopt square formation volumetry (Li Haiyan etc. the research of avian influenza virus recombinant nucleocapsid protein ELISA diagnostic techniques, 2000,22 (3): 182～185) determine.Detectable antigens is A type H1 subtype influenza virus (deposit number is CCTCCNO:V201105) allantoic fluid.Test-results shows that the best coated concentration of coated antibody (deposit number is CCTCC NO:C201106) is 2 μ g/ml, and the best weaker concn ratio of Horseradish Peroxidase Conjugates (deposit number is CCTCC NO:C201106) is 1: 1000.
(2) preparation of enzyme reaction plate: the dilution proportion that antibody purification (deposit number is CCTCC NO:C201106) is 1: 1000 with coating buffer according to volume ratio, 100 μ l/ holes join in enzyme plate, place the rearmounted Cool Room 4℃ of 1h for 37 ℃ and spend the night; Dry coating buffer, with washings washing 1 time, each 5min, dries washings; Add confining liquid 200 μ l/ holes in enzyme plate, in 37 ℃ of sealing 2h, dry confining liquid.In 4 ℃ of refrigerations, spend the night, seasoning.
3, determining of result decision content:
By the A type H1 subtype influenza virus negative sample to 89 parts of known background, detect, obtain result, ask its mean value
standard deviation S D=0.03; Determine that yin and yang attribute stagnation point is
be the OD of testing sample
630value≤0.23 item is judged to be feminine gender, OD
6300.23 of value > is judged to be the positive.
4, the preparation of yin and yang attribute contrast agents:
To by the formaldehyde of final concentration 0.8%, slowly add formaldehyde solution containing after viral allantoic fluid freeze thawing 3 times, and fully mix, 37 ℃ of deactivations 24 hours, every jolting in 6 hours 1 time.Add 0.02%NaN
3anticorrosion, and as 4 ℃ of preservations of positive control sample.
Blank chick embryo allantoic liquid adds 0.02%NaN
3anticorrosion conduct is as 4 ℃ of preservations of negative control sample.
5, the use step of A type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection method:
(1) coated: the best coated concentration coated elisa plate of the antibody purification of take is 100 μ l/ holes, hatches rearmounted 4 ℃ of 1h for 37 ℃ and spends the night.
(2) wash plate: abandon coating buffer, PBST washings, 200 μ l/ holes, wash 3 times, each 3min.
(3) sealing: pat dry enzyme plate, add confining liquid, 200 μ l/ holes, hatch 2h for 37 ℃, sealing nonspecific binding site.
(4) wash plate: abandon confining liquid, same step (2).
(5) add sample to be checked: pat dry enzyme plate, add sample to be checked, 100 μ l/ holes, establish negative control, hatch 30min for 37 ℃.
(6) wash plate: abandon sample liquid to be checked, same step (2).
(7) add enzyme labelled antibody: pat dry the anti-monoclonal antibody (preserving number is CCTCC NO:C201106) that adds HRP mark after enzyme plate, volume ratio is 1: 1000 times of dilution, 100 μ l/ holes, place 30min for 37 ℃.
(8) wash plate: abandon enzyme labelled antibody, same step (2).
(9) colour developing: every hole adds substrate A liquid, each the 50 μ l of substrate B liquid that newly join, room temperature lucifuge colour developing 10min.
(10) termination reaction: every hole adds 50 μ l stop buffer termination reactions;
(11) measure OD
630value: OD value when microplate reader mensuration wavelength is 630nm.
Sensitivity test and the specific test of embodiment 5 A type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection methods
1, the sensitivity test of A type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection method
By sensitivity test of the present invention, show that the minimum detectable viral level of ELISA method of the present invention is TCID
50=10
-2.33(approximately 215 TCID
50) (Yin Zhen etc. animal virology (the 2nd edition) [M]. Beijing: Science Press, 1997).The results are shown in Table 3.
Table 3: sensitivity test of the present invention
2, the specific test of A type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection method
(1) influenza virus of classical H1 hypotype, H3 hypotype, H5 hypotype, H9 hypotype is detected respectively to OD by method of the present invention
630end value all≤0.23, is judged to be feminine gender, illustrates that the present invention has good specificity to each hypotype of influenza.It the results are shown in Table described in 4.
Table 4: the detected result of the present invention to the influenza virus of classical H1 hypotype, H3 hypotype, H5 hypotype, H9 hypotype, H10 hypotype
(2) by the main virus of fowl poultry kind as: Pestivirus suis (CSFV), pig reproduction are detected respectively to OD by method of the present invention with respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), foot and mouth disease virus (FMDV), pig encephalitis b virus, newcastle disease virus (NDV), infectious bursal disease virus (IBDV), chicken egg-decreasing syndrome viral (EDSV), SPF chick embryo allantoic liquid equal samples
630end value all≤0.23, is judged to be feminine gender, illustrates that the present invention has good specificity to above various virus.It the results are shown in Table described in 5.
Table 5: the detection effect of the present invention to the main virus of fowl poultry kind
The comparison of embodiment 6 A type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection methods and PCR method
1, detect allantoic fluid sample: 16 strain A type H1 subtype influenza virus chick embryo allantoic liquids are got in this laboratory and by the inventive method and RT-PCR method, detect and compare simultaneously.It the results are shown in Table described in 6.
Table 6 shows: the inventive method detects 15 parts of positive sample altogether, 1 part of negative sample, recall rate 93.75% (15/16); RT-PCR method detects 16 parts of positive altogether, 0 part of negative sample, recall rate 100% (16/16); The inventive method and RT-PCR method coincidence rate 93.75% (15/16).
Table 6: the result comparison of ELISA detection method of the present invention and RT-PCR method
2, detect tissue samples: by the positive sample of known background 17 parts (comprising the heart, liver, spleen, lung, kidney, tonsilla, tracheae and tracheae washing fluid), negative sample 16 parts (comprising the heart, liver, spleen, lung, kidney, tonsilla, tracheae and tracheae washing fluid) totally 33 increments by the inventive method and RT-PCR method, detect and compare simultaneously.It the results are shown in Table described in 7.
Table 7 shows: the present invention detects 16 increments altogether, and this is positive, and 17 increments are originally negative.By RT-PCR method detect altogether 17 parts positive, 16 parts are negative.The present invention is 94.1% (16/17) to the recall rate of positive sample, the coincidence rate 94.1% (16/17) of two kinds of methods; The present invention is 100% (16/16) to the recall rate of negative sample, the coincidence rate 100% (16/16) of two kinds of methods.
Table 7: the comparison of the recall rate of the present invention and RT-PCR method
3, detect clinical sample: by totally 284 this use of the increment double-antibody sandwich elisa detection method tests of various places institute collecting sample.Result shows: detection method of the present invention detects 0 part of positive sample, and positive rate is 0% (0/284).
The assembling of embodiment 7 A type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection kit
1, A type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection kit comprises:
1) 96 hole enzyme plates of Sheet clonal antibody (CCTCC NO:C201106)
(8 hole * 12 * 2)
2) (concrete formula refers to: 2, the preparation of related reagent) in positive and negative contrast
Each 1 pipe (0.5ml/ pipe)
3) preserving number of horseradish peroxidase-labeled is the monoclonal antibody of CCTCC NO:C201106
1 bottle (25ml/ bottle)
4) 10 times of 1 bottle of washings concentrated solutions (50ml/ bottle)
5) substrate A liquid, substrate B liquid each 1 bottle (12ml/ bottle)
6) 1 bottle of stop buffer (12ml/ bottle)
7) 1 bottle of sample preparation A liquid (internal organs are organized in processing) (50ml/ bottle)
8) sample preparation B liquid (processing larynx, nose swab) 1 bottle (50ml/ bottle)
2, the preparation of related reagent
Coating buffer (25mmol/L carbonate buffer solution): Na
2cO
31.59g, NaHCO
32.93g, uses ddH
2o is settled to 1000mL (pH9.6).
10 times of washingss: NaCl 80g, KCl 2g, Na
2hPO
412H
2o 29g, KH
2pO
42g, Tween-20 5ml, uses ddH
2o is settled to 1000ml (pH=7.4).
Confining liquid: 5g skimming milk is dissolved in 100ml washings.
Substrate solution: substrate A liquid: 0.006%H
2o
2damping fluid; Substrate B liquid: get Na
2hPO
412H
2o14.2g, citric acid 10.5g, uses ddH
2o is settled to 500ml, is made into 0.1ml phosphoric acid salt citrate buffer solution (pH=5.0), then adds benzidine (TMB).During use, substrate A liquid and substrate B liquid equal-volume are mixed, mix in latter 5 minutes and use, now with the current.
Stop buffer (final concentration 0.025% hydrofluoric acid solution): getting concentration is 40% hydrofluoric acid solution 625 μ L, is settled to 100mL with ddH2O.
The preparation of sample preparation A liquid: take Na
2b
4o
710H
2o 13.3g, H
3bO
316.08g, NaCl 8.5g, ddH
2o 800ml, adjust pH is 8.4 with distilled water, to be settled to 1000ml afterwards.At 121 ℃, high pressure steam sterilization packing after 30 minutes, puts 4 ℃ of storages, for the treatment of viscera tissue.
The preparation of sample preparation B liquid: take Na
2b
4o
710H
2o 13.3g, H
3bO
316.08g, NaCl 8.5g, ddH
2o 800ml, adjust pH is 8.4 with distilled water, to be settled to 1000ml afterwards.Under 121 ℃ of high pressure steam, sterilizing adds 5g N-acetyl-L-cysteine after 30 minutes, and 5mL NP-40, mixes rear packing, puts 4 ℃ of storages, for the treatment of larynx swab.
The preparation of positive control: will slowly add formaldehyde solution by the formaldehyde of final concentration 0.8% containing after viral allantoic fluid freeze thawing 3 times, and fully mix, 37 ℃ of deactivations 24 hours, every jolting in 6 hours 1 time.Add 0.02%NaN
3anticorrosion, and be distributed into 0.5ml/ pipe as positive control sample, put at 4 ℃ and preserve.
The preparation of negative control: the aseptic SPF chick embryo allantoic liquid of collecting,, adds 0.02%NaN after the centrifugal 30min of 12000r/min by 4 ℃
3anticorrosion.Be distributed into 0.5ml/ pipe, put at 4 ℃ and preserve.
The operation steps of embodiment 8 A type H1 type influenza virus double-antibody sandwich elisa detection kit
1) get enzyme plate (how many removable gradation are used per sample), 10 times of concentrated cleaning solutions, with after distilled water diluting, are washed to plate and once after (200 μ l/ hole), patted dry;
2) add sample to be checked, 100 μ l/ holes, establish negative control, hatch 30min for 37 ℃;
3) abandon sample liquid to be checked,
4) add HRP traget antibody after patting dry enzyme plate, 1: 1000 times of dilution, 100 μ l/ holes, place 30min for 37 ℃;
5) abandon enzyme labelled antibody, add washings, 200 μ l/ holes, wash 3 times, each 3min;
6) every hole adds substrate A, each the 50 μ l of B liquid that newly join, room temperature lucifuge colour developing 10min;
7) termination reaction: every hole adds 50 μ l stop buffer termination reactions;
8) measure OD630 value: OD value when microplate reader mensuration wavelength is 630nm.
9) result is judged: the OD value of positive and negative contrast is the important symbol of reaction kit quality and test operation standard, and we select the A type H1 subtype influenza virus of deactivation as positive control, and the blank allantoic fluid of SPF chicken embryo is as negative control.The condition that ELISA test is set up is that positive control OD value is greater than 0.8, and negative control OD value is less than 0.2.Positive control, negative control must be controlled within the scope of this, otherwise detected result is invalid.
Although content of the present invention is to describe in conjunction with the present embodiment, can not think limitation of the scope of the invention, scope of the present invention is limited by appended claims.In addition, in the scope that those skilled in the art limits at appended claims, the present invention is carried out to various changes or modification, these changes or modified forms drop in protection scope of the present invention equally.
Claims (1)
1. the hybridoma cell strain 5F7 of the monoclonal antibody of anti-A type H1 subtype influenza viral hemagglutinin is secreted in a strain, it is characterized in that: this hybridoma cell strain is deposited in Chinese Typical Representative culture collection center, preserving number is CCTCC NO:C201106, and it is that the H1N1virus A-Influ/JML-F9 that is CCTCC NO:V201105 by preserving number is prepared from.
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