CN102633878A - 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 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 the application of a kind of first type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection kit and first type H1 subtype influenza virus antigen detection in swinery thereof.
Background technology
1975, Kohler G and Milstein C delivered the cytogamy method and have set up hybridoma technology on the Nature magazine, had created the authentic monoclonal antibody technology that is with historically new significance.Behind the clone, produce structure and the identical high purity antibody of various characteristics, (Monoclonal Antibody McAb), is called for short monoclonal antibody to be called monoclonal antibody.The discovery of monoclonal antibody technique and use have been played huge pushing effect to the development of modern life science research, have become an importance of biological technical field.So far, this The Application of Technology has been widely used in aspects such as fundamental research, medical diagnosis on disease, treatment, prevention.
In June, 2009, the World Health Organization announced the warning rank of first type (H1N1) influenza is risen to 6 grades, meaned that worldwide being very popular of first type (H1N1) influenza forms.Worldwide being very popular of the last influenza originated from Mexico; Be called as porcine influenza (Swine flu) at the beginning, the back renames as pig source property influenza infection (swine origin influenza virus infection according to the analysis of virus; Be called for short S-OIV infection); For the seasonal influenza A (H1N1) with the people distinguishes, rename as new influenza A (H1N1) at last, then be called Influenza A H1N1 in China.Influenza A H1N1 causes tremendous influence to the health of people and animal, and economy has been caused tremendous loss.
The people such as Engvall of Sweden in 1971 respectively with Mierocrystalline cellulose with gather the third ethene test tube as solid phase carrier adsorption antigen/antibody, set up enzyme-linked immunosorbent assay (Enzyme Linked ImmunosrbentAssay, be called for short ELISA method).People such as Voller used the PS micro-reaction plate instead as the solid-phase immunity absorption carrier in 1974; Make the ELISA method be able to apply; Make the enzyme-labelled antibody technique that is used for Antigen Location develop into the measuring method of liquid sample micro substance, and become a kind of method the most commonly used in the antigen-antibody detection gradually.It combines the immunoreation of enzyme labelling thing synantigen antibody complex with the catalysis amplification of enzyme, both kept the susceptibility of enzymic catalytic reaction, has kept the specificity of antigen antibody reaction again, thereby has improved sensitivity greatly.It is again a kind of heterogeneous immune analysis method simultaneously, promptly in each step of reaction washing process is arranged all, thereby has removed unreacted reactant and interfering substance.Since the ELISA method have highly sensitive, high specificity, easy and simple to handle, detect rapidly and on-radiation and plurality of advantages such as can measure in batches, make the ELISA method obtain application more and more widely.(Jiao Kui etc. enzyme immunoassay technology and application [M]. Beijing: Chemical Industry Press, 2004,84～141; Li Wenmin. the technical progress of enzyme linked immunological absorption reaction and application [J]. Hubei Vocationl Technical College journal, 2003,4 (6): 65～69)
Enzyme-labelled antibody technique is the gordian technique of ELISA detection method; Used enzymic-labelled antibody is that this laboratory produces anti-first type H1 subtype influenza viral hemagglutinin monoclonal antibody voluntarily among the present invention; Through horseradish peroxidase (being called for short HRP) mark; Have very high enzymic activity, output height and cost are low.
Summary of the invention
This main purpose be to overcome the defective of prior art, a kind of first type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection kit is provided, to solve the detection of first type H1 subtype influenza virus antigen in the swinery.
First purpose of the present invention is the monoclonal antibody of anti-first type H1 subtype influenza virus hemagglutinin of core reagent that can be used for assembling the ELISA test kit that obtains a kind of high specificity.
Second purpose of the present invention is to set up a kind of double-antibody sandwich elisa detection method of first type H1 subtype influenza virus
The 3rd purpose of the present invention is the double-antibody sandwich elisa detection kit of a kind of first type H1 subtype influenza virus of assembling.
The 4th purpose of the present invention is to utilize the application of this test kit in swinery first type H1 subtype influenza virus antigen detects.
The present invention is achieved in that
Agricultural mikrobe National Key Laboratory of the Hua Zhong Agriculture University Viral Laboratory at applicant place separates the former strain of a strain first type H1 subtype influenza virus of A-Influ/JML-F9 that obtains from swinery; This strain is delivered Chinese typical 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.
The applicant has prepared a kind of monoclonal antibody of high specificity; It is the proteic monoclonal antibody of anti-first type H1 subtype influenza viral hemagglutinin (HA); Secrete the hybridoma cell strain 5F7 of this monoclonal antibody; Deliver Chinese typical culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on January 27th, 2011, deposit number is CCTCC NO:C201106.
The applicant utilizes described Monoclonal Antibody to become double-antibody sandwich elisa core reagent, has assembled a kind of double-antibody sandwich elisa test kit that rapid detection is applicable to the first type H1 subtype influenza virus of swinery that is used for.Test kit of the present invention is by box body and is included in the intravital sample diluting liquid of box, washings, substrate A liquid, substrate B liquid, stop buffer that positive control sample and negative control sample are formed.
More detailed technical scheme is described below.
The MONOCLONAL ANTIBODIES SPECIFIC FOR of anti-first type H1 subtype influenza virus hemagglutinin, it comprises the following steps:
1) a strain deposit number that obtains with separation from swinery is that first type H1 subtype influenza virus of A-Influ/JML-F9 of CCTCC NO:V201105 is an antigen; Through amplification, deactivation and purifying, 5-8 week BALB/c mouse in age (available from Animal Experimental Study center, Hubei Province) is carried out immunity.
2) cytogamy, the spleen of the BALB/c mouse behind the booster immunization of learning from else's experience (available from Animal Experimental Study center, Hubei Province) merges with SP2/0 myeloma cell's (available from Ministry of Health's Wuhan institute of Biological Products) of prepared fresh.
3) utilize blood clotting suppress method (HI) (comparison [J] of .RT-PCR method such as Xiao Jinhui and blood clotting inhibition method evaluation influenza virus. Chinese tropical medicine; 2005; 5:401～402), filter out the positive hole of secretion anti-first type H1 hypotype subtype influenza viral hemagglutinin (HA) antibody.To the positive hole that screens use at once limiting dilution assay (but the Chinese and wait .H5 subtype avian influenza virus hemagglutinin MONOCLONAL ANTIBODIES SPECIFIC FOR and evaluation [J]. 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-first type H1 subtype influenza viral hemagglutinin (HA) protein antibodies.
4) preparation of ascites is only got female 5-6 week BABL/c mouse in age (available 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 suppress (HI) method detection ascites and tire-70 ℃ of preservations.
5) be that CCTCC NO:C201106 monoclonal antibody is carried out purifying and mark with deposit number.
Can be used for preparing the double-antibody sandwich elisa test kit that detects first type H1 subtype influenza virus behind above-mentioned antibody (deposit number is CCTCC NO:C201106) process purifying and the mark.
Compared with prior art the present invention has following remarkable advantage:
1, test kit of the present invention can directly detect first type H1 subtype influenza virus, has high specificity, and is highly sensitive, and detection time is short, the characteristics of test sample wide ranges (chick embryo allantoic liquid, viscera tissue homogenate).
2, test kit of the present invention is applicable to first type H1 hypotype swine influenza virus is detected, and classical H1 hypotype swine influenza virus is not reacted, and has specificity.
3, test kit of the present invention is applicable to first type H1 subtype influenza virus is detected, and to the influenza virus of other hypotype, as: the influenza virus of classical H1 hypotype, H3 hypotype, H5 hypotype, H9 hypotype, then do not react, have excellent specificity.
4, the present invention is assembled into test kit with required all ingredients, and operation is simple, does not need to be operated by the professional.Test kit good stability of the present invention, long preservative period can not influence its susceptibility 4 ℃ of condition held half a year.
5, the present invention once can handle a plurality of samples simultaneously, is fit to very much the clinical extensive detection of first type H1 hypotype swine influenza virus.And can satisfy TR, also can be used as scientific research and use.
6, also do not detect the test kit of first type H1 hypotype swine influenza virus antigen (North America influenza) in the market.There is not the differential diagnosis kit of distinguishing first type H1 hypotype swine influenza virus (North America influenza) and classical H1 subtype influenza virus yet.
Description of drawings
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-first type H1 subtype influenza viral hemagglutinin (HA) protein monoclonal antibody indirect immunofluorescence detects figure.Fig. 4 A wherein: be that anti-first type H1 subtype influenza viral hemagglutinin (HA) protein monoclonal antibody indirect immunofluorescence detects figure; The negative contrast of Fig. 4 B
Embodiment
Embodiment 1 preparation embodiment
One, the separation of first type H1 subtype influenza virus
1, sepn process: the throat swab with sterilization is gathered disease pig throat sample, places the ditalimfos phthalate buffer (to be called for short PBS, prescription: Na
2CO
31.59g, NaHCO
32.93g, use ddH
2O is settled to 1000ml) in; Subsequently throat swab sample liquid 0.2ml is inoculated 9 age in days no-special pathogens (Specific pathogen Free; SPF) chicken embryo (available from the logical laboratory animal technology of Beijing Cimmeria dimension ltd) is put under 35 ℃ and is hatched 72h, and the allantoic fluid of receiving is done the blood clotting qualitative test; The sample that hemagglutination test is positive is again through RT-PCR test typing.
2, appraisal basis: the sample of RT-PCR test positive, amplification HA serves the sea and gives birth to the order-checking of worker's biotechnology ltd, to gained dna sequence dna and the online sequence of announcing (
Http:// blast.ncbi.nlm.nih.gov/) compare, to confirm virus subtype.
3, the virusology characteristic of first 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 first type H1 subtype influenza virus.
4, the preservation of patented procedure: the first type H1 subtype influenza virus of A-Influ/JML-F9 that gets that will obtain after will identifying delivers Chinese typical 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 first type H1 subtype influenza virus (deposit number is CCTCC NO:V201105)
1, be that first type H1 subtype influenza virus of A-Influ/JML-F9 of CCTCC NO:V201105 increases with deposit number; (1) strict screening 9～10 age in days no-special pathogens (Specific pathogen Free, SPF) chicken embryo (available from the logical laboratory animal technology of Beijing Cimmeria dimension ltd)
Every batch of chicken embryo of bringing will pass 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 for use; SPF) chicken embryo (available from the logical laboratory animal technology of Beijing Cimmeria dimension ltd); The tincture of iodine is smeared near place, embryo position and alcohol carries out disinfection at air chamber in draw air chamber and embryo position, pegs and wears an aperture; Subsequently the 1ml syringe needle is inserted (avoiding blood vessel) along this aperture, inoculation first type H1 subtype influenza virus (A-Influ/JML-F9).Use paraffin sealing at last, and put under 35 ℃ and hatch 72h.Check chicken embryonic development situation every day, every 12h turns over ovum and ovoscopy once.Dead chicken embryo in the 24h is thought the special death of right and wrong and discarding, and 72h collects the chicken embryo, and 4 ℃ of held are spent the night.
(3) contain the results of first type H1 subtype influenza virus (A-Influ/JML-F9) allantoic fluid
Alcohol disinfecting chick embryo air sac end with 75% concentration tears the chick embryo air sac eggshell with aseptic nipper, does not have the aorta place at chorioallantoic membrane and wears out.Place corresponding collection tube with aseptic pipette, extract chick embryo allantoic liquid.The centrifugal 5min of chicken embryo results liquid 3000r/min is removed blood and cell.Carry out the RCA experiment then, confirm the chick embryo allantoic liquid hemagglutinative titer.
2, the deactivation of first type H1 subtype influenza virus of A-Influ/JML-F9
After will containing virus of A-Influ/JML-F9 allantoic fluid freeze thawing 3 times, slowly add formaldehyde solution by the formaldehyde of final concentration 0.8%, thorough mixing is even, and 37 ℃ of deactivations 24 hours are every at a distance from jolting in 6 hours 1 time.Each inactivation of virus container should be taken a sample immediately, carries out the inactivation of virus proof test respectively.The allantoic fluid of the first type H1 subtype influenza virus of deactivation container is through assay approval.8000r/min, centrifugal 10min.Discard deposition, supernatant is subsequent use.
3, first type H1 subtype influenza virus of A-Influ/JML-F9 concentrates and purifying
(1) first type H1 subtype influenza virus of A-Influ/JML-F9 is concentrated, method is: with first type H1 subtype influenza virus chick embryo allantoic liquid in 27000r/min, centrifugal 2h, supernatant discarded precipitate resuspendedly with phosphate buffered saline buffer (PBS), abundant mixing is subsequent use.
(2) first type H1 subtype influenza virus of A-Influ/JML-F9 purifying
In the ultracentrifugation pipe, add 60%, 45%, 30%, 20% concentration sucrose solution successively and form density gradient.Resuspended good virus of A-Influ/JML-F9 sample is tiled in the superiors, in 32000r/min, centrifugal 1h.Centrifugal back takes out 45%, aspect sample between 30%,, 45% between aspect sample taking-up sample resuspended 30% with PBS, and 32000r/min, centrifugal 1h gets deposition and (promptly gets described virus of A-Influ/JML-F9) and carry out protein content determination.With this as immunogen.
Embodiment 2 anti-first type H1 subtype influenza hemagglutinin MONOCLONAL ANTIBODIES SPECIFIC FOR
1, the former strain of first type H1 subtype influenza virus of A-Influ/JML-F9 with deactivation and purifying (is deposited in the Chinese typical 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 for use Freund's complete adjuvant, and two, three exempt to select for use Freund's incomplete adjuvant) week BALB/c mouse in age (available from Animal Experimental Study center, Hubei Province).It is 20ug/ that head exempts from dosage, every subcutaneous multi-point injection in mouse carotid back.Two week backs with the same dose booster immunization once, after two weeks again booster immunization once, dosage be 40ug/ only, a small amount of mouse blood is gathered in the back docking of two weeks, collects serum, tires with blood clotting inhibition method detection mouse antibodies.Treat that mouse antibodies reaches TR, again through the first type H1 of abdominal injection deactivation and purifying subtype influenza virus of A-Influ/JML-F9, ID be 40ug/ immunity once.After three days, promptly desirable immune spleen cell and SP2/0 myeloma cell (available from Ministry of Health's Wuhan institute of Biological Products) are merged by the laboratory ordinary method.
2, cytogamy, one of the BALB/c mouse of the reinforced immunological of learning from else's experience (available from Animal Experimental Study center, Hubei Province), eye socket sacrificed by exsanguination (collect serum, be positive serum) is soaked the 10min sterilization in 75% concentration alcohol.Aseptic taking-up mouse spleen is isolated splenocyte, with SP2/0 myeloma cell's (available from Ministry of Health's Wuhan institute of Biological Products) of prepared fresh by 1 * 10
7Individual SP2/0 and 10
8The ratio of individual immunocyte (number was than 1: 10) mixing in the 50ml centrifuge tube, 1500r/min, centrifugal 10min.Supernatant discarded, tube wall blots with the filter paper of sterilization, 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.In 1min, slowly splash into 50% polyoxyethylene glycol (PEG) 0.8ml (available from sigma company) of preparatory temperature to 37 ℃ then, the limit edged stirs with pipette tip gently, continues to stir 1min.Slowly add temperature in advance then to 1640 (available from sigma company) cell culture fluid 40ml of 37 ℃.
It is following to merge concrete steps:
Dropwise splash into 50% polyoxyethylene glycol (PEG) 0.8ml in first minute, leave standstill 0.5min; Add 1640 cell culture fluid 1ml in second minute, leave standstill 0.5min (repeating once); Add 1.5ml on the 4th minute, and left standstill 0.5min (repeating once); Add 5ml on the 6th minute, and left standstill 0.5min (repeating once); Add 10ml on the 8th minute, and left standstill 0.5min (repeating once); Each added-time needs slowly to add, and constantly stirs lightly.Leave standstill 1min, the centrifugal 10min of 1000r/min abandons supernatant, in 37 ℃ of environment, places 8min.Suspend with HAT substratum (available from Sigma company), simultaneously also with the HAT substratum suspend the raising splenocyte for preparing and with merge after cytomixis, add an amount of HAT substratum as required, divide and plant in 96 well culture plates, about 200 μ l/ holes.Once merge and to inoculate 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 contains 10 approximately
4About SP2/0 cell.In 37 ℃, cultivate in the 5%CO2 incubator.Merge and began to observe 96 orifice plate inner cells in back second day and have pollution-freely, changed HAT substratum 100 μ l with the HT substratum in the 4th day.Treat that the fused cell colony grows to culture hole 1/4, when substratum omits flavescence, carry out antibody titer and detect.Adopt deactivation first type H1 subtype influenza virus (A-Influ/JML-F9) as screening antigen; Utilize blood clotting suppress method (comparison [J] of .RT-PCR method such as Xiao Jinhui and blood clotting inhibition method evaluation influenza virus. Chinese tropical medicine; 2005,5:401～402) filter out the positive hole of secretion anti-first type H1 hypotype subtype influenza viral hemagglutinin (HA) antibody.To the positive hole that screens use at once limiting dilution assay (but the Chinese and wait .H5 subtype avian influenza virus hemagglutinin MONOCLONAL ANTIBODIES SPECIFIC FOR and evaluation [J]. 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-first type H1 hypotype subtype influenza viral hemagglutinin (HA) antibody; Deliver Chinese typical culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on January 27th, 2011, deposit number is CCTCC NO:C201106.
3, the preparation of ascites is only selected 5-6 week female BABL/c mouse in age (available from Animal Experimental Study center, Hubei Province) abdominal injection Freund's complete adjuvant 0.5ml/ for use, pneumoretroperitoneum injection 1 * 10 in 5 days
6Individual hybridoma/only, collect ascites after 9 days, blood clotting suppress method (HI) detection ascites and tire-70 ℃ of preservations.
4, the purifying of ascites antibody: sad-the ammonium sulfate method
Concrete steps are following:
(1) with the centrifugal 10min of ascites 1000r/min of above-mentioned steps 3 preparations, with 0.45 μ m membrane filtration, filtrating adds with 4 times of volume 60mmol/L acetate buffer solutions (pH=4.5) while stirring;
(2) dropwise adding n-caprylic acid to final concentration while stirring is 33 μ l/ml, and room temperature condition stirs 30min down, and the centrifugal 30min of 8000r/min abandons deposition and collects supernatant;
(3) through filter paper filtering once, filtrating pH modulation 7.4 with the supernatant of step (2);
(4) filtrating to step (3) gained adds saturated ammonium sulphate solution (ammonium sulfate volume/TV≤45%) while stirring, when waiting to filtrate journey white opacity liquid, continues to stir 30min, leaves standstill 5h in 4 ℃ then.Centrifugal 30min under 4 ℃ of condition 12000r/min again;
(5) abandon supernatant, precipitate resuspended with 10mmol pH=9.0Tris-HCl; In the Tris-HCl of the 10mmol of 100 times of volumes pH=9.0, stir dialysis in 4 ℃ of condition lower magnetic forces, dialysis dialysis 36h, during the fresh Tris-HCl liquid of 3 times (12h/ time) replacing.Dialysis finishes, and gets supernatant, with the purity of the electrophoretic method evaluation of SDS-PAGE antibody, measures AC with ultraviolet spectrophotometer, packing, frozen.
Agents useful for same is according to following formulated in the test of above-mentioned antibody purification:
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
2Add (NH among the O
4)
2SO
490g is heated to 80 ℃ of dissolvings, and filter paper filtering is reduced to the existing crystallization of room temperature and separated out while hot, and gained has crystallization filtrating and is saturated ammonium sulphate solution.With 25% ammoniacal liquor adjust pH to 7.2, subsequent use.
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 NaIO subsequently
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, the lucifuge condition is placed 30min under the room temperature, and solution is pale brown look;
(2) get monoclonal antibody (deposit number the is CCTCC NO:C201106) 5ml (1mg/ml) of the present invention's preparation and the liquid mixing that above-mentioned steps (1) is handled well, 10mmol pH=9.5 carbonate buffer solution, 4 ℃ of condition lower magnetic forces stir dialysed overnight.
(3) in accomplishing dialysis liquid, add the NaBH that freshly prepared concentration is 5mg/ml
4Solution 0.2ml is in 4 ℃ of held 2h; Equal-volume adds saturated ammonium sulphate solution then, under 4 ℃, leaves standstill 30min, 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) after traget antibody (deposit number is CCTCC NO:C201106) is accomplished dialysis, add PB (20mmol/L pH=7.4), glycerine (final concentration 30%) is settled to 5ml, in-20 ℃ of preservations.
Agents useful for same is pressed following formulated in the labelled antibody test:
NaIO4 solution (60mmol/L): NaIO
41.283g, use ddH
2O is settled to 100ml;
Ethylene glycol solution (160mmol/L): terepthaloyl moietie 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, use ddH
2O is settled to 1000ml (pH=9.5);
NaBH
4Solution (5mg/ml): NaBH
40.05g, use ddH
2O is settled to 10ml, joins existing usefulness at present;
Saturated ammonium sulphate solution: every 100ml ddH
2Add (NH among the O
4)
2SO
490g is heated to 80 ℃ of dissolvings, and filter paper filtering is reduced to the existing crystallization of room temperature and separated out while hot, and gained has crystallization filtrating and is saturated ammonium sulphate solution.With 25% ammoniacal liquor adjust pH to 7.2, subsequent use.
PB solution (20mmol/L pH=7.4): Na
2HPO
412H
2O 3.58g, NaH
2PO
41.56g, use ddH
2O is settled to 1000ml, pH=7.4.
The evaluation of embodiment 3 anti-first type H1 subtype influenza hemagglutinin monoclonal antibodies (deposit number is CCTCC NO:C201106)
1, the HI evaluation of tiring
The first type H1 subtype influenza virus (deposit number is CCTCCNO:V201105) that adopts the present invention's separation to obtain is measured tiring of Hybridoma Cell Culture supernatant and mouse ascites as antigen with blood clotting inhibition method.The result sees 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)
(Mouse Mab Isotyping Test Kit is available from ROCKLAND company) identifies the resulting monoclonal antibody of the present invention with mouse source monoclonal antibody hypotype identification kit, confirms that the monoclonal antibody antibody that the present invention prepares is IgG
1Subclass.
3, the specificity of monoclonal antibody (deposit number is CCTCC NO:C201106) is identified
Adopt blood clotting to suppress the influenza virus that method (HI) is measured anti-first type H1 subtype influenza hemagglutinin (HA) monoclonal antibody and classical H1 hypotype, H3 hypotype, H5 hypotype, H9 hypotype respectively; The result all is shown as feminine gender, explains that anti-first type H1 subtype influenza hemagglutinin (HA) monoclonal antibody that obtains has excellent specificity.The result sees 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
By ordinary method carry out the hybridoma chromosome counting (Zhang Gusheng etc. monoclonal antibody is in medically application [M]. Shanghai science tech publishing house; 1987:281～406); (SP2/0 myeloma cell's modal number is 70 to the hybridoma modal number of all acquisitions between 85-97; BALB/c mice spleen cell chromosome number is 40), all be higher than the chromosome number of two parent's cells, the hybridoma that proves all acquisitions really is the heterozygote of SP2/0 cell and immune spleen cell.
Embodiment 4 first 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, use 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: the 5g skimming milk is dissolved in the 100ml washings.
Substrate solution: be divided into substrate A liquid and substrate B liquid.The concrete composition as follows:
Substrate A liquid: the H of 0.006% concentration
2O
2Damping fluid.
Substrate B liquid: get Na
2HPO
412H
2O14.2g, Hydrocerol A 10.5g uses ddH
2O is settled to 500m and is made into 0.1ml phosphoric acid salt citrate buffer solution (pH=5.0), is that 20mg/L adds benzidine (TMB) (during use A liquid and B liquid equal-volume are mixed, mix in back 5 minutes and use, join existing usefulness at present) by final concentration then.
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) coated antibody (deposit number is CCTCC NO:C201106) the best encapsulate the best weaker concn of concentration and horseradish peroxidase-labeled antibody (deposit number is CCTCC NO:C201106) than adopt the square formation volumetry (Li Haiyan etc. the research of bird flu virus reorganization nucleoprotein ELISA diagnostic techniques; 2000,22 (3): 182～185) confirm.Detecting antigen is first type H1 subtype influenza virus (deposit number is CCTCCNO:V201105) allantoic fluid.Test-results shows that it is 2 μ g/ml that coated antibody (deposit number is CCTCC NO:C201106) the best encapsulates concentration, and the best weaker concn ratio of horseradish peroxidase-labeled antibody (deposit number is CCTCC NO:C201106) is 1: 1000.
(2) preparation of enzyme reaction plate: it is 1: 1000 dilution proportion according to volume ratio that antibody purification (deposit number is CCTCC NO:C201106) uses coating buffer, and 100 μ l/ holes join in the enzyme plate, place the rearmounted Cool Room 4s 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,, dry confining liquid in 37 ℃ of sealing 2h.Spend the night seasoning in 4 ℃ of refrigerations.
3, confirming of decision content as a result:
First type H1 subtype influenza virus negative sample through to 89 parts of known background detects, and obtains the result, asks its MV
Standard deviation S D=0.03; Confirm that the yin and yang attribute stagnation point does
Be the OD of testing sample
630Value≤0.23 item is judged to be feminine gender, OD
630Value＞0.23 item is judged to be the positive.
4, the preparation of yin and yang attribute contrast agents:
After will containing viral allantoic fluid freeze thawing 3 times, slowly add formaldehyde solution by the formaldehyde of final concentration 0.8%, thorough mixing is even, and 37 ℃ of deactivations 24 hours are every at a distance from 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 first type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection method:
(1) encapsulate: encapsulating the concentration coated elisa plate with antibody purification the best is 100 μ l/ holes, hatches 1h for 37 ℃ and spends the night for rearmounted 4 ℃.
(2) wash plate: abandon coating buffer, the PBST washings, wash 3 times in 200 μ l/ holes, each 3min.
(3) sealing: clap dried enzyme plate, add confining liquid, 2h is hatched for 37 ℃ in 200 μ l/ holes, the sealing nonspecific binding site.
(4) wash plate: abandon confining liquid, same step (2).
(5) add sample to be checked: clap dried enzyme plate, add sample to be checked, negative control is established in 100 μ l/ holes, hatches 30min for 37 ℃.
(6) wash plate: abandon sample liquid to be checked, same step (2).
(7) add enzyme labelled antibody: clap the anti-monoclonal antibody (preserving number is CCTCC NO:C201106) that adds the HRP mark behind the dried enzyme plate, volume ratio is 1: 1000 times of dilution, and 30min are placed for 37 ℃ in 100 μ l/ holes.
(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 ELIASA mensuration wavelength is 630nm.
The sensitivity test of embodiment 5 first type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection methods and specificity test
1, the sensitivity test of first type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection method
Through 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(about 215 TCID
50) (Yin Zhen etc. animal virology (the 2nd edition) [M]. Beijing: Science Press, 1997).The result sees table 3.
Table 3: sensitivity test of the present invention
2, the specificity of first type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection method test
(1) influenza virus with classical H1 hypotype, H3 hypotype, H5 hypotype, H9 hypotype detects OD respectively with method of the present invention
630End value all≤0.23 is judged to be feminine gender, explains that the present invention has excellent specificity to each hypotype of influenza.Its result sees that table 4 is said.
Table 4: the present invention is to the detected result of the influenza virus of classical H1 hypotype, H3 hypotype, H5 hypotype, H9 hypotype, H10 hypotype
(2) with the main virus of fowl poultry kind as: CSFV (CSFV), pig reproduction are detected OD respectively 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 with method of the present invention
630End value all≤0.23 is judged to be feminine gender, explains that the present invention has excellent specificity to above various viruses.Its result sees that table 5 is said.
Table 5: the present invention is to the detection effect of the main virus of fowl poultry kind
The comparison of embodiment 6 first type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection methods and PCR method
1, detects the allantoic fluid sample: 16 strain first type H1 subtype influenzas virus chick embryo allantoic liquid is got in this laboratory detected and compare with the inventive method and RT-PCR method simultaneously.Its result sees that table 6 is said.
Table 6 shows: the inventive method detects 15 parts of positive sample altogether, 1 part of negatives, recall rate 93.75% (15/16); The 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 of ELISA detection method of the present invention and RT-PCR method relatively
2, detect tissue samples: the sample that known background is positive 17 parts (comprising the heart, liver, spleen, lung, kidney, tonsilla, tracheae and tracheae washing fluid), negatives 16 parts (comprising the heart, liver, spleen, lung, kidney, tonsilla, tracheae and tracheae washing fluid) totally 33 increments simultaneously with the inventive method and the detection of RT-PCR method and compare.Its result sees that table 7 is said.
Table 7 shows: the present invention detects 16 increments altogether, and this is positive, and 17 increments are originally negative.Through the 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 negatives, 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: with sample that gather various places totally 284 this usefulness of increment double-antibody sandwich elisa detection methods test.The 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 first type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA detection kit
1, first 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 holes * 12 * 2)
2) (concrete prescription sees for details: 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 liquid concentrators (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 (handling 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, use 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: the 5g skimming milk is dissolved in the 100ml washings.
Substrate solution: substrate A liquid: 0.006%H
2O
2Damping fluid; Substrate B liquid: get Na
2HPO
412H
2O14.2g, Hydrocerol A 10.5g uses ddH
2O is settled to 500ml, is made into 0.1ml phosphoric acid salt citrate buffer solution (pH=5.0), adds benzidine (TMB) then.During use substrate A liquid and substrate B liquid equal-volume are mixed, mix in back 5 minutes and use, join existing usefulness at present.
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 by weighing Na
2B
4O
710H
2O 13.3g, H
3BO
316.08g, NaCl 8.5g, ddH
2O 800ml, adjust pH are that 8.4 backs are settled to 1000ml with zero(ppm) water.At 121 ℃, 4 ℃ of storages are put in high pressure steam sterilization packing after 30 minutes, are used to handle viscera tissue.
The preparation of sample preparation B liquid: take by weighing Na
2B
4O
710H
2O 13.3g, H
3BO
316.08g, NaCl 8.5g, ddH
2O 800ml, adjust pH are that 8.4 backs are settled to 1000ml with zero(ppm) water.Add the 5g N-acetyl-L-cysteine after 30 minutes in sterilization under 121 ℃ of HP steams, 5mL NP-40,4 ℃ of storages are put in packing behind the mixing, are used to handle the larynx swab.
The preparation of positive control: after will containing viral allantoic fluid freeze thawing 3 times, slowly add formaldehyde solution by the formaldehyde of final concentration 0.8%, thorough mixing is even, and 37 ℃ of deactivations 24 hours are every at a distance from jolting in 6 hours 1 time.Add 0.02%NaN
3Anticorrosion, and be distributed into 0.5ml/ pipe as positive control sample, put 4 ℃ and preserve down.
The preparation of negative control: the aseptic SPF chick embryo allantoic liquid of collecting, adds 0.02%NaN behind the centrifugal 30min of 12000r/min by 4 ℃
3Anticorrosion.Be distributed into the 0.5ml/ pipe, put 4 ℃ and preserve down.
The operation steps of embodiment 8 first type H1 type influenza virus double-antibody sandwich elisa detection kit
1) get enzyme plate (per sample what removable gradation use), with 10 times of concentrated cleaning solutions with distilled water diluting after, wash plate and once clap and do in (200 μ l/ hole) back;
2) add sample to be checked, negative control is established in 100 μ l/ holes, hatches 30min for 37 ℃;
3) abandon sample liquid to be checked,
4) add the HRP traget antibody behind the dried enzyme plate of bat, 1: 1000 times of dilution, 30min are placed for 37 ℃ in 100 μ l/ holes;
5) abandon enzyme labelled antibody, add washings, wash 3 times in 200 μ l/ holes, 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 the OD630 value: OD value when ELIASA mensuration wavelength is 630nm.
9) result judges: the OD value of positive and negative contrast is the important symbol of reaction kit quality and test operation standard, and we select the first 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 be positive control OD value greater than 0.8, negative control OD value is less than 0.2.Positive control, negative control must be controlled in this scope, otherwise detected result is invalid.
Although content of the present invention is to combine present embodiment to describe, can not think limitation of the scope of the invention, scope of the present invention is limited appended claims.In addition, those skilled in the art carries out various changes or modification to the present invention in the appended claims restricted portion, and these changes or modified forms drop in protection scope of the present invention equally.
Claims (8)
1. the monoclonal antibody of an anti-first type H1 subtype influenza viral hemagglutinin; It is characterized in that; It is to be that the hybridoma cell strain of H1N1virus A-Influ/JML-F9 preparation of CCTCCNO:V201105 is secreted by preserving number, and described hybridoma cell strain 5F7 preserving number is CCTCC NO:C201106.
2. the hybridoma cell strain 5F7 of the monoclonal antibody of the anti-first type of strain H1 subtype influenza viral hemagglutinin is deposited in Chinese typical culture collection center, and its preserving number is CCTCC NO:C201106.
3. the first type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA antigen detecting agent box that comprises the described monoclonal antibody of claim 1.
4. double-antibody sandwich elisa test kit that the first type H1 subtype influenza virus antigen that is applicable to pig detects; It is characterized in that; This test kit is made up of following part: preserving number is the enzyme plate that the monoclonal antibody of CCTCC NO:C201106 encapsulates; The preserving number of horseradish peroxidase-labeled be the monoclonal antibody of CCTCC NO:C201106 as ELIAS secondary antibody, sample preparation A liquid, sample preparation B liquid, positive control sample, negative control sample, substrate A liquid, substrate B liquid, stop buffer and 10 times of washingss;
Wherein:
Described reagent component and proportioning thereof are following:
(1) preparation of sample preparation A liquid: take by weighing Na
2B
4O
710H
2O 13.3g, H
3BO
316.08g, NaCl 8.5g, ddH
2O 800mL transfers pH to 8.4 back to be settled to 1000mL with zero(ppm) water; In sterilization packing after 30 minutes under 121 ℃ of HP steams, put 4 ℃ of storages;
(2) preparation of sample preparation B liquid: take by weighing Na
2B
4O
710H
2O 13.3g, H
3BO
316.08g, NaCl 8.5g, ddH
2O 800mL, adjust pH are that 8.4 backs are settled to 1000mL with zero(ppm) water; Add the 5g N-acetyl-L-cysteine after 30 minutes in sterilization under 121 ℃ of HP steams, 5mL NP-40,4 ℃ of storages are put in packing behind the mixing;
(3) preparation of positive control: after will containing viral allantoic fluid freeze thawing 3 times, slowly add formaldehyde solution by the formaldehyde of final concentration 0.8%, thorough mixing is even, and 37 ℃ of deactivations 24 hours are every at a distance from jolting in 6 hours 1 time; Add 0.02%NaN
3Anticorrosion, and be distributed into 0.5mL/ pipe as positive control sample, put 4 ℃ and preserve down;
(4) preparation of negative control: collect the SPF chick embryo allantoic liquid, 4 ℃, add 0.02%NaN behind the centrifugal 30min of 12000r/min
3Anticorrosion.Be distributed into the 0.5mL/ pipe, put 4 ℃ and preserve down;
(5) substrate A liquid: 0.006%H
2O
2Damping fluid;
(6) substrate B liquid: get Na
2HPO
412H
2O 14.2g, Hydrocerol A 10.5g uses ddH
2O is settled to 500m and is made into 0.1mL phosphoric acid salt citrate buffer solution, pH5.0; By final concentration is that 20mg/L adds benzidine;
(7) stop buffer: concentration is 40% hydrofluoric acid solution, 625 μ L, is settled to 100mL with ddH2O;
(8) 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, pH7.4.
5. double-antibodies sandwich ELISA that the first type H1 subtype influenza virus antigen that is applicable to pig detects; Comprise that the preparation preserving number is the enzyme plate that the monoclonal antibody of CCTCC NO:C201106 encapsulates; The preserving number of horseradish peroxidase-labeled is the monoclonal antibody of CCTCC NO:C201106; Sample preparation A liquid, sample preparation B liquid, positive control, negative control sample, substrate A liquid, substrate B liquid, stop buffer and 10 times of washingss, its step comprises:
(1) with the preserving number H1N1virus A-Influ/JML-F9 of CCTCC NO:V201105, as immunogen;
(2) immunogen preparing with step (1) obtains the monoclonal antibody that preserving number is CCTCC NO:C201106;
(3) anti-with the described monoclonal antibody coated elisa plate of step (2) as one;
(4) detected sample is handled with sample preparation liquid obtained thing to be detected;
(5) use the preserving number of horseradish peroxidase-labeled anti-as two as the monoclonal antibody of CCTCC NO:C201106;
(6) the described thing to be detected of step (4) is detected, on ELIASA, read the absorption photometric value of 630 nanometers; Wherein the component of each reagent and preparation are as follows:
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;
Sample preparation A liquid: take by weighing Na
2B
4O
710H
2O 13.3g, H
3BO
316.08g, NaCl 8.5g, ddH
2O 800mL, adjust pH are that 8.4 backs are settled to 1000mL with zero(ppm) water; 4 ℃ of storages are put in sterilization back packing under 121 ℃ of HP steams, are used to handle viscera tissue;
Sample preparation B liquid: take by weighing Na
2B
4O
710H
2O 13.3g, H
3BO
316.08g, NaCl 8.5g, ddH
2O 800mL, adjust pH are that 8.4 backs are settled to 1000mL with zero(ppm) water; The sterilization back adds the 5g N-acetyl-L-cysteine under 121 ℃ of HP steams, 5mLNP-40, and 4 ℃ of storages are put in packing behind the mixing, are used to handle the larynx swab.
Substrate A liquid: 0.006%H
2O
2Damping fluid;
Substrate B liquid: get Na
2HPO
412H
2O 14.2g, Hydrocerol A 10.5g uses ddH
2O is settled to 500m and is made into 0.1mL phosphoric acid salt citrate buffer solution, and pH5.0 is that 20mg/L adds benzidine by final concentration;
Stop buffer: concentration is 40% hydrofluoric acid solution, 625 μ L, is settled to 100mL with ddH2O.
6. the described H1N1virus A-Influ/JML-F9 of claim 1 is deposited in Chinese typical culture collection center, and its preserving number is CCTCC NO:V201105.
7. the application of the described monoclonal antibody of claim 1 in preparation first type H1 subtype influenza virus (PRRSV) double-antibody sandwich ELISA antigen detecting agent box.
8. claim 3 or the 4 described test kits application in first type H1 subtype influenza virus antigen vitro detection.
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| CN102796708A (en) * | 2011-05-25 | 2012-11-28 | 华中农业大学 | Method for proliferating influenza A viruses |
| CN104744590A (en) * | 2013-12-30 | 2015-07-01 | 神州细胞工程有限公司 | A monoclonal antibody for H1N1 swine influenza A virus hemagglutinin protein and a double-antibody sandwich ELISA kit |
| CN109142734A (en) * | 2018-10-26 | 2019-01-04 | 北京纳百生物科技有限公司 | H_5 subtype Re-6 strain antibody identification kit |
| CN110272488A (en) * | 2018-03-16 | 2019-09-24 | 洛阳普莱柯万泰生物技术有限公司 | Feline calicivirus monoclonal antibody and its application |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102796708A (en) * | 2011-05-25 | 2012-11-28 | 华中农业大学 | Method for proliferating influenza A viruses |
| CN104744590A (en) * | 2013-12-30 | 2015-07-01 | 神州细胞工程有限公司 | A monoclonal antibody for H1N1 swine influenza A virus hemagglutinin protein and a double-antibody sandwich ELISA kit |
| CN104744590B (en) * | 2013-12-30 | 2018-05-18 | 神州细胞工程有限公司 | Anti-H 1 N 1 swine influenza virus hemagglutinin monoclonal antibody and double crush syndrome kit |
| CN110272488A (en) * | 2018-03-16 | 2019-09-24 | 洛阳普莱柯万泰生物技术有限公司 | Feline calicivirus monoclonal antibody and its application |
| CN109142734A (en) * | 2018-10-26 | 2019-01-04 | 北京纳百生物科技有限公司 | H_5 subtype Re-6 strain antibody identification kit |
| CN109142734B (en) * | 2018-10-26 | 2021-07-16 | 北京纳百生物科技有限公司 | Avian influenza virus H5 subtype Re-6 strain antibody identification kit |
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