CN104744590A - A monoclonal antibody for H1N1 swine influenza A virus hemagglutinin protein and a double-antibody sandwich ELISA kit - Google Patents
A monoclonal antibody for H1N1 swine influenza A virus hemagglutinin protein and a double-antibody sandwich ELISA kit Download PDFInfo
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Abstract
A monoclonal antibody for H1N1 swine influenza A virus hemagglutinin protein and a double-antibody sandwich ELISA kit are disclosed. The kit comprises a solid phase vector coated with the monoclonal antibody, a rabbit polyclonal antibody labeled by horseradish peroxidase, an H1N1 hemagglutinin protein standard substance, a sample diluting liquid, a washing liquid, a substrate coloring liquid and a reaction terminating liquid. The kit is good in sensitivity, capable of performing quantitative detection for the swine influenza virus hemagglutinin protein, and specifically recognizing main prevalent strains of A(H1N1) influenza outbreak in 2009, and free of cross reactions with other strains of the H1N1 influenza viruses and other influenza virus subtypes. The kit is simple to operate, can be used for supporting fundamental research of the H1N1 swine influenza virus, and has important significance for performing epidemiologic study on the H1N1 influenza viruses in different zones and different years.
Description
Technical field
The invention belongs to technical field of immunoassay, be specifically related to a kind of anti-H 1 N 1 swine influenza virus hemagglutinin monoclonal antibody and double crush syndrome immue quantitative detection reagent box thereof.
Background technology
Influenza virus H1N1 hypotype, also claims H1N1 virus, is a kind of RNA viruses, belongs to Orthomyxoviridae family, the one of influenza A virus, is also one of influenza virus of the most often infecting of the mankind.The strain of some H1N1 can be propagated between the mankind, and comprise the influenza great outburst of 1918, other can propagate between bird and pig.H1N1 is the abbreviation of the important target point protein title of two-strain, and wherein " H " refers to homo agglutinin (Hemagglutinin), and also known as hemagglutinin, " N " refers to neuraminidase (Neuraminidase).Therefore, H1N1 is the influenza virus sub-strain that one has " homo agglutinin 1 type and neuraminidase 1 type " viral protein.
Flu outbreak is one of biggest threat of facing mankind, often brings huge or even catastrophic loss to the whole world in history every many decades flu outbreak once.The mortality ratio of the flu outbreak of 1918 greatly about 2.5%, but still causes the death of about 5,000 ten thousand people in the world.3 to April in 2009, H1N1 porcine influenza epidemic situation, at first in Mexico's outburst, caused hundred people to infect.Epidemic situation bamboo telegraph is thereafter to the whole world.Morning on April 30th, 2009, the World Health Organization brings up to the 5th grade the Global influenza warning level that is very popular, by the end of the accumulative report death in the whole world is routine more than 5000 according to incompletely statistics at present, and the World Health Organization is thought, actual morbidity and death toll are considerably beyond number of reports.By on December 7th, 2009, in China, ground added up report Influenza A H1N1 severe cases 4328 example, dead 326 examples.
Although the lethality rate of Influenza A H1N1 is not very high, but influenza virus often has strong variability, the immune pressure be subject to due to H1N1 is larger, the amino acid change constantly accumulation of 8 gene fragments, its antigenic variation is obvious, once some sudden change causes pathogenic obvious increase, the impact that cannot estimate is produced by the life security of the mankind and social stability, therefore, carry out permanently effective monitoring and research to H1N1 swine influenza virus and other influenza virus, its public health meaning is day by day remarkable.The investigative technique means of current published H1N1 swine influenza virus haemagglutinin antigen mainly rely on chicken embryo and are separated, PCR etc., virus purification length consuming time, PCR needs professional equipment and technician, all should not popularize, and the qualitative porcine influenza detection kit of some ELISA, then cannot carry out accurate quantification detection to viral antigen proteins, and be often difficult to different strain and other influenza virus sub-strain of accurately distinguishing H1N1 influenza virus, be unfavorable for basis and the epidemiological study of supporting influenza virus, simple to operate in the urgent need to one, sensitivity is good, the quantitative detecting reagent that specificity is high.
Summary of the invention
The double crush syndrome immue quantitative detection reagent box that the object of this invention is to provide a kind of anti-H 1 N 1 swine influenza virus hemagglutinin monoclonal antibody and be key ingredient with it.。
Detection kit provided by the present invention comprises the solid phase carrier including Sheet clonal antibody, and enzyme mark detects polyclonal antibody and protein standard substance, sample diluting liquid, washings, substrate nitrite ion and reaction terminating liquid.
Wherein said Sheet clonal antibody is mouse monoclonal antibody, and its light chain and heavy chain amino acid sequence are respectively SEQ ID NO:1 and SEQ ID NO:2; It is the rabbit polyclonal antibody that horseradish peroxidase (Horseradish peroxidase, HRP) marks that described enzyme mark detects polyclonal antibody; Described protein standard substance is restructuring H1N1 porcine influenza hemagglutinin.
Described mouse monoclonal antibody adopts restructuring H1N1 porcine influenza hemagglutinin immune animal, then selects high serum titer mouse to be prepared by classical hybridoma technology; Described enzyme mark rabbit polyclonal antibody adopts H1N1 porcine influenza hemagglutinin immune animal, is prepared the polyclonal antibody of purifying, then conventionally utilize HRP traget antibody to obtain by Protein A purification and antigen affinity purification technology.
Technical scheme of the present invention is preferably have highly sensitive, the monoclonal antibody of high specific and enzyme mark polyclonal antibody combinations of pairs, monoclonal antibody is adsorbed on solid phase carrier as coated antibody, coated antibody specificly can catch H1N1 hemagglutinin in sample and protein standard substance, after adding enzyme mark detection antibody, form coated antibody, antigen, detection antibody complex, stop after corresponding substrate colour developing, read sample light absorption value, by comparing the content that can draw H1N1 porcine influenza hemagglutinin in sample with typical curve.
Hemagglutinin is the key function albumen of influenza infection host cell, simultaneously, also be the important target spot for the treatment of of influenza medicine and vaccine development, the present invention adopts double-antibody method, can H1N1 porcine influenza hemagglutinin level in qualitative or detection by quantitative sample, the method of inspection is convenient and easy, detection sensitivity and accuracy high, high specificity, can the simultaneously large quantities of sample of rapid detection, bag quilt as test kit key components is independent research with detection antibody, therefore cost is low, reliability is strong, easily trace to the source, expectation will play a significant role in H1N1 relevant rudimentary and clinical study.
Accompanying drawing explanation
Fig. 1 .H1N1 porcine influenza hemagglutinin ELISA kit canonical plotting
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described and explanation.
The component preparation of embodiment 1H1N1 porcine influenza hemagglutinin ELISA kit
1. the preparation of protein standard substance
Standard substance in described test kit are restructuring H1N1 porcine influenza hemagglutinin, from Sino Biological Inc. (article No. is: 11055-V08H), this albumen utilizes human archeocyte expression system to express in vitro, then the high purity protein of purifying acquisition, concrete purity data can see the explanation of Yi Qiao Divine Land company's site for this product.
2. the preparation of mouse monoclonal antibody:
1) animal immune
Adopt Balb/c mouse as immune animal, the restructuring H1N1 porcine influenza hemagglutinin produced with Yi Qiao Divine Land Bioisystech Co., Ltd is for immunogen, and immunizing dose is the albumen of every mouse immune 50 μ g at every turn.During first immunisation, the complete Freund's adjuvant of immunogen and equivalent is made emulsifying agent, subcutaneous abdomen multi-point injection, interval gets same dose immunogen after 2 ~ 3 weeks and equivalent incomplete Freund's adjuvant makes emulsifying agent, booster immunization twice, indirect elisa method is used to measure serum titer after three immunity, serum titer reaches after 1: 16000, and once, after 4 days, extracting spleen cell carries out cytogamy to the mouse peritoneal booster immunization that selection is tired the highest.
2) cytogamy and cloning
Get mouse spleen, the rear single splenocyte suspension of centrifugal acquisition is filtered in grinding, after cell counting, ratio in 5: 1 or 10: 1 mixes with the SP2/0 murine myeloma cell being in logarithmic phase, adopts polyoxyethylene glycol (PEG) method to carry out cytogamy, bed board, by the effect of Selective agar medium, myeloma cell and splenocyte etc. do not merge or the cell of fusion not yet in effect cannot grow, and the hybridoma of effective integration will grow, breed in culture hole, and secretory antibody.After changing liquid three times, within 9-12 days, cells and supernatant is got after merging, using H1N1 porcine influenza hemagglutinin of recombinating as envelope antigen, indirect elisa method is utilized to measure supernatant liquor, screen positive hole, and by limiting dilution assay, colonized culture is carried out to positive cell, until obtain the monoclonal hybridoma strain of stably excreting specific antibody.Above cytogamy and cloning method are the conventional classical way in immunology monoclonal antibody technique.
3) production of monoclonal antibody and purifying
Choose hybridoma cell strain, culturing bottle or bio-reactor is utilized to carry out cell cultures, collecting cell culture supernatant, routine protein A affinity column is utilized to carry out purifying, the antibody obtained is by packing after SDS-PAGE electrophoresis and indirect elisa method qualification antibody purity and specificity, for subsequent use in-20 DEG C of cryopreservation.
4) sequencing of monoclonal antibody
Hybridoma is preserved for a long time and positive colony may be caused to lose due to unstable after repeatedly going down to posterity and pollution problem, for solving the problem, in process of the present invention, Protocols in Molecular Biology is utilized to be extracted light chain of antibody and the heavy chain gene of positive colony respectively, carry out sequencing, the plasmid including antibody gene can steady in a long-termly under-20 DEG C of conditions be preserved, simultaneously, according to antibody gene sequences, the technician in this professional domain conveniently can obtain identical monoclonal antibody by molecular biology method clonal expression.
It is as follows that antibody gene extracts concrete grammar: collect the hybridoma that growth conditions is good, hybridoma total serum IgE is extracted by the operation scheme of the classical total RNA isolation kit specification sheets of BBI company, by electrophoresis detection quality, UV measures concentration.Be cDNA by the operation scheme of the MMLV first strand cDNAsynthesis kit specification sheets of BBI company by mRNA reverse transcription ,-20 DEG C frozen for subsequent use.Reverse transcription reaction system is: 11 μ l RNA (2.7 μ g), 5 × reaction buffer4 μ l, RNase inhibitor (20U/ μ l) 1 μ l, dNTP mix (10mmol/L) 2 μ l, M-MuLV reverse transcriptase1 μ l, total reaction volume is 20 μ l.According to reference design primer, take cDNA as light chain and the heavy chain fragment that template difference PCR obtains antibody, reaction system is: 2.0 μ l10 × pyrobest buffer, 1.6 μ l2.5mM dNTP, 1.4 μ l10 μM primer pairs, 0.4 μ l hybridoma cDNA, 0.2 μ l5U/ μ l Pyrobest DNA Polymerase, reaction system is 20 μ l.Amplification condition: sex change 94 DEG C of 4min; Sex change 94 DEG C of 1min, anneal 58 DEG C of 1min, extends 72 DEG C of 1min, 30 circulations; Extend 72 DEG C of 5min.Light chain and heavy chain fragment are inserted on pcDNA3T carrier, build pcDNA3-anti-H1N1-L and pcDNA3-anti-H1N1-H carrier.By vector intestinal bacteria, picking positive colony, carries out order-checking qualification, analyzes sequencing result, obtain correct light chain and heavy chain amino acid sequence.The light-chain amino acid sequence that measurement result and sequential analysis show this antibody is SEQ ID NO:1; Heavy chain amino acid sequence is SEQ ID NO:2, refers to sequence table.Specific experiment operation is with reference to [Sa curtain Brooker J etc., " molecular cloning texts guide ", Beijing Science Press] well known to those skilled in the art.
3. the preparation of rabbit polyclonal antibody:
Choose new zealand rabbit as immune animal, carry out immunity with H1N1 porcine influenza hemagglutinin of recombinating for immunogen, immunizing dose is the albumen of every rabbit immune 500 μ g at every turn.The complete Freund's adjuvant of immunogen and equivalent is made emulsifying agent by first immunisation, neck dorsal sc multi-point injection, interval gets same dose immunogen in 2 ~ 3 weeks and equivalent incomplete Freund's adjuvant makes emulsifying agent, booster immunization, immune 4-5 time altogether, indirect elisa method measures serum titer and reaches after 1: 25000, heart extracting blood, by obtaining the polyclonal antibody of purifying after routine protein A affinity column and hemagglutinin antigen affinity column two-step purifying, packing, is used for the preparation of enzyme labelled antibody in-20 DEG C of cryopreservation.Wherein hemagglutinin antigen affinity purification concrete steps are as follows:
A) sepharose (GE company) of 0.7g cyanogen bromide-activated is claimed, swelling with 1mM HCl, then wash three times with 1mM HCl, for subsequent use;
B) hemagglutinin getting 2mg utilizes hyperfiltration process albumen buffer exchange to be become 0.1M NaHCO3, pH8.3, and to control volume be 1 ~ 2ml;
C) joined by protein solution in the activated sepharose that step a) washed, room temperature shakes 4h;
D) with the Tris buffer blind unreacted group of pH8.0,0.1M;
E) fill gravity post, column volume is about 2ml, rinses, and balance with PBS;
Get Protein A purification how anti-cross post, rinse non-binding antibody with PBS, with the antibody of pH3.0 citrate buffer solution wash-out specific combination, with in Tris damping fluid and elutriant to pH7.0-7.5.
4. the preparation of enzyme labelled antibody:
A) HRP taking 5mg is dissolved in 0.5mL distilled water;
B) NaIO of the 0.1M that 0.5mL newly joins is added
4solution, under room temperature, lucifuge stirs 20 minutes;
C) loaded in dialysis tubing by above-mentioned solution, the sodium-acetate buffer (pH4.4) of 1mM is dialysed 4 DEG C and is spent the night;
D) get the polyclonal antibody of 5mg affinity purification, join in 1mL0.01M carbonate buffer solution, for subsequent use;
E) in c) liquid, add 0.2M carbonate buffer solution (pH9.5), make pH be elevated to 9.0 ~ 9.5, then add immediately d) in liquid, room temperature lucifuge stirs 2 hours gently;
F) the 4mg/mL NaBH that 0.2mL now joins is added
4liquid, mixing, places 2 hours in 4 DEG C;
G) loaded in dialysis tubing by above-mentioned liquid, dialyse in the PBS of pH7.4,0.15M, 4 DEG C are spent the night.
The establishment of embodiment 2H1N1 porcine influenza hemagglutinin ELISA kit
The ELISA kit set up, comprises following reagent:
A) mouse monoclonal coated antibody;
B) rabbit polyclonal antibody of HRP mark;
C) H1N1 porcine influenza hemagglutinin standard substance;
D) bag is buffered liquid: the carbonate buffer solution of pH9.6,0.05mol/L;
E) confining liquid: the Tris damping fluid containing 2% bovine serum albumin;
F) sample diluting liquid: the phosphate buffered saline buffer containing 0.1% bovine serum albumin;
G) washings: the phosphate buffered saline buffer containing 0.1% tween;
H) substrate nitrite ion: be made up of nitrite ion A and nitrite ion B, nitrite ion A is hydrogen peroxide or urea peroxide, and nitrite ion B is tetramethyl biphenyl;
The sulfuric acid of i) stop buffer: 2mol/L
Embodiment 3 detect the ELISA kit of H1N1 porcine influenza hemagglutinin preparation
1. utilize orthogonal test to grope optimum antibody combination and the working concentration of enzyme linked immunological kit
According to ultraviolet spectrophotometer method, measure the concentration of antibody and antigen.Adopt orthogonal test method, grope optimum antibody combination and optimum antibody working concentration, it is 4 μ g/ml, 2 μ g/ml, 1 μ g/ml that anti-for difference H1N1 porcine influenza hemagglutinin monoclonal antibody is diluted to concentration, recombinate hemagglutinin concentration dilution to 1000pg/ml, 100pg/ml, 0pg/ml, rabbit polyclonal antibody weaker concn to 4 μ g/ml, 2 μ g/ml, 1 μ g/ml, the 0.5 μ g/ml of HRP mark.Consider the absorbance value in blank well background and positive test hole, optimize a strain mouse monoclonal antibody as coated antibody, and confirm that its best effort concentration is that to mark the working concentration of polyclonal antibody be 1 μ g/ml for 2 μ g/ml, HRP.
2. the batch preparation of test kit
1) coated elisa plate: be buffered liquid with carbonate bag and (take 3.18g Na
2cO
3, 5.86g NaHCO
3, 1gNa
2n
3, be settled in the deionized water of 2L, adjust pH is 9.6), it is 2 μ g/ml that anti-H1N1 porcine influenza hemagglutinin monoclonal antibody is diluted to concentration, 100 μ L/ holes, coated elisa plate, and 4 DEG C of overnight incubation, wash plate 1 time with washings, 200 μ L/ holes.
2) close: every hole adds 300 μ L confining liquids and closes nonspecific binding site, incubated at room 1 hour; Then plate is washed 2 times with washings, 200 μ L/ holes; Pat dry rear Vacuum Packaging Machine packaging, 4 DEG C save backup.
3) preparation of protein standard substance: with sample diluting liquid dilution H1N1 porcine influenza hemagglutinin, freeze-drying after packing, 4 DEG C save backup.
4) preparation of enzyme labelled antibody: marked by the rabbit polyclonal antibody HRP after affinity purification, add 50% glycerine, after packing ,-20 DEG C save backup.
Embodiment 4ELISA test kit coated antibody is identified
1. antibody test limit measures:
1) H1N1 porcine influenza hemagglutinin carbonate bag is buffered liquid to be diluted to, 0.1 μ g/mL, 0.05 μ g/mL, 0.025 μ g/mL, 0.0125 μ g/mL, 0.00625 μ g/mL, 100 μ l/ holes, coated elisa plate, 4 DEG C of overnight incubation, plate is washed 1 time, 200 μ L/ holes with washings;
2) every hole adds 300 μ L confining liquids and closes nonspecific binding site, incubated at room 1 hour; Then plate is washed 2 times with washings, 200 μ L/ holes;
3) anti-H1N1 porcine influenza hemagglutinin mouse monoclonal antibody Sample dilution is diluted to 1ug/ml, adds bag quilt and the enzyme plate closed in advance, 200 μ L/ holes, incubated at room 1 hour; Then plate is washed 3 times with washings, 200 μ L/ holes;
4) the anti-mouse IgG bis-adding HRP mark resists, 200 μ L/ holes, incubated at room 1 hour; Then plate is washed 3 times with washings, 200 μ L/ holes;
5) add the tmb substrate of the fresh configuration of 200 μ L, room temperature lucifuge develops the color 20 minutes, adds 50 μ L2mol/L sulfuric acid termination reactions, reads value under microplate reader 450nm wavelength.
6) numerical value of 3 times of standard deviations is deducted with the mean value of each sample group light absorption value and blank group (is buffered liquid coated elisa plate with the bag not containing recombinant protein, the same experimental group of other step) light absorption value mean value adds that the numerical value of 3 times of standard deviations is compared, getting the minimum albumen bag that sample values is greater than under blank group value conditions is the detectability of antibody by concentration.The detectability of result display antibody can reach 5ng/ hole (table 1), illustrates that antibody has higher detection sensitivity.
Table 1 coated antibody detectability measures
| OD-1 | OD-2 | OD-3 | OD-4 | OD-5 | AVERAGE | SD | AERAGE+3SD | AERAGE-3SD | Detectability (ng/ hole) | |
| Blank | 0.038 | 0.039 | 0.038 | 0.039 | 0.038 | 0.038 | 0.001 | 0.040 | ||
| 0.00625ug／mL | 0.038 | 0.043 | 0.041 | 0.042 | 0.044 | 0.042 | 0.002 | 0.035 | ||
| 0.0125ug／mL | 0.045 | 0.046 | 0.051 | 0.048 | 0.048 | 0.003 | 0.040 | |||
| 0.025ug／mL | 0.044 | 0.046 | 0.046 | 0.043 | 0.048 | 0.045 | 0.002 | 0.040 | ||
| 0.05ug／mL | 0.051 | 0.057 | 0.053 | 0.052 | 0.056 | 0.054 | 0.003 | 0.046 | 5 | |
| 0.1ug／mL | 0.071 | 0.072 | 0.073 | 0.072 | 0.072 | 0.072 | 0.001 | 0.070 |
2. antibody subtype detects
Adopt the antibody typing test kit of Sino Biological Inc. to detect antibody subtype, detected result is IgG1 type antibody.
The specific assay of embodiment 5H1N1 porcine influenza hemagglutinin ELISA kit
The restructuring hemagglutinin of different influenza virus sub-strain and the different strain of H1N1 hypotype is diluted to 50ng/ml, test by H1N1 porcine influenza hemagglutinin ELISA kit, result visualizingre agent box can specific identification H1N1 swine influenza virus Major Epidemic strain H1N1, A/California/4/2009 and H1N1, A/California/7/2009, BrevigMission/1/1918 is comprised with other strain of H1N1 influenza virus, Solomon Islands/3/2006, Ohio/UR06-0091/2007, NewCaledonia/20/1999, Puerto Rico/8/1934 and WSN/1933 no cross reaction, H1N2 is comprised with other influenza virus sub-strain, swine/Guangxi/13/2006, H1N3, duck/NZL/160/1976, H2N2, Canada/720/2005, H6N1, California/HKWF115/2007 no cross reaction.Above-mentioned experiment proves that this test kit specificity is good, clearly can distinguish H1N1 porcine influenza Major Epidemic strain and other influenza virus sub-strain and strain, may be used for the epidemiology change monitoring H1N1 porcine influenza Major Epidemic strain, and for the fundamental research of carrying out for influenza virus different subtype and the different strain of same hypotype, also there is important actual application value.
The specific assay of table 2H1N1 porcine influenza hemagglutinin ELISA kit
| The hypotype of hemagglutinin and strain | OD450nm value |
| H1N1(A／California／7／2009) | 1.876 |
| H1N1(A／California／4／2009) | 2.013 |
| H1N1(A／Brevig Mission／1／1918) | 0.001 |
| H1N1(A／Solomon Islands／3／2006) | 0.003 |
| H1N1(A／Ohio／UR06-0091／2007) | 0.015 |
| H1N1(A／New Caledonia／20／1999) | 0.001 |
| H1N1(A／Puerto Rico／8／1934) | 0.005 |
| H1N1(A／WSN／1933) | 0.003 |
| H1N2(A／swine／Guangxi／13／2006) | 0.004 |
| H1N3(A／duck／NZL／160／1976) | 0.001 |
| H2N2(A／Canada／720／2005) | 0.002 |
| H6N1(A／Califomia／HKWF115／2007) | 0.003 |
The detecting step of embodiment 6H1N1 porcine influenza hemagglutinin ELISA kit
1. application of sample
1) take out the standard substance wrapped by good enzyme plate and freeze-drying, add 1ml sample diluting liquid and standard substance are dissolved.Ambient temperatare puts 20 minutes.By standard substance from 800pg/ml, do the doubling dilution of 2 times, totally 6 points, diluent is respectively got 100 μ l and add in 96 hole enzyme plates.
2) get sample to be tested, 100 μ l/ holes add in reacting hole, incubated at room temperature 2 hours;
3) washings washes plate 3 times, and 200 μ L/ holes, pat dry enzyme plate.
2. add detection antibody
HRP traget antibody sample diluting liquid is diluted to 1 μ g/mL, adds in reacting hole, 100 μ L/ holes, incubated at room temperature 1 hour, washings washes plate 3 times, and 200 μ L/ holes, pat dry enzyme plate.
3. develop the color
1) add the freshly prepared substrate nitrite ion of 200 μ L, room temperature reaction 20 minutes, then add 50 μ L stop buffer termination reactions;
2) light absorption value is read under microplate reader 450nm wavelength.
4. the foundation of typical curve
With the concentration of standard substance for X-coordinate, OD value is ordinate zou Criterion curve (Fig. 1), can calculate the content of the H1N1 hemagglutinin obtained in sample according to the sample OD value recorded.
Finally, it is also to be noted that enumerate above be only specific embodiments of the invention son.Obviously, the invention is not restricted to above examples of implementation, many modification can also be had.All modification that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
Claims (5)
1. an anti-H 1 N 1 swine influenza virus agglutinin of blood protein monoclonal antibody, is characterized in that, the light chain of described monoclonal antibody and heavy chain amino acid sequence are respectively SEQ ID NO:1:
MDFQVQIFSFLLISASVIMSRGQIVLTQSPAIMSASLGERVTMTCTASSSVSSTYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
With SEQ ID NO:2:
MKWSWVILFLLSGTAGAHSEIQLQQTGPELVKPGAPVKISCKASGYSLTDYI ILWVKQSHGKSLEWIGNINPYFGTTSYNLKFKDKATLTVDKSSSTAYMELNSLTSEDSAVYYCARNDNYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQP IMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK。
2. one kind is detected the double-antibody sandwich elisa test kit of H1N1 swine influenza virus hemagglutinin, it is characterized in that, include the solid phase carrier of Sheet clonal antibody, the rabbit polyclonal antibody of horseradish peroxidase-labeled, H1N1 hemagglutinin standard substance, sample diluting liquid, washings, substrate nitrite ion and reaction terminating liquid; Described monoclonal antibody light chain and heavy chain amino acid sequence are respectively SEQ ID NO:1 and SEQ ID NO:2.
3. test kit as claimed in claim 2, it is characterized in that, the preparation process of the anti-H1N1 swine influenza virus hemagglutinin rabbit polyclonal antibody of described horseradish peroxidase-labeled is as follows: use restructuring H1N1 swine influenza virus hemagglutinin immunize New Zealand rabbit 0.5mg//time, subcutaneous multiple spot immunity, At intervals of two to three weeks, immunity four times altogether, heart extracting blood, after albumin A and H1N1 hemagglutinin antigen affinity purification, obtain the polyclonal antibody of purifying, then carry out mark acquisition with horseradish peroxidase.
4. test kit as claimed in claim 2, is characterized in that, adopts restructuring H1N1 swine influenza virus hemagglutinin as standard substance, can carry out detection by quantitative to H1N1 swine influenza virus hemagglutinin.
5. test kit as claimed in claim 2, it is characterized in that, specific identification H1N1 swine influenza virus Major Epidemic strain H1N1, A/California/4/2009 and H1N1, A/California/7/2009, Brevig Mission/1/1918 is comprised with other strain of H1N1 influenza virus, Solomon Islands/3/2006, Ohio/UR06-0091/2007, New Caledonia/20/1999, Puerto Rico/8/1934 and WSN/1933 no cross reaction, H1N2 is comprised with other influenza virus sub-strain, swine/Guangxi/13/2006, H1N3, duck/NZL/160/1976, H2N2, Canada/720/2005, H6N1, Cal ifornia/HKWF115/2007 no cross reaction.
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