CN104744590B - Anti-H 1 N 1 swine influenza virus hemagglutinin monoclonal antibody and double crush syndrome kit - Google Patents
Anti-H 1 N 1 swine influenza virus hemagglutinin monoclonal antibody and double crush syndrome kit Download PDFInfo
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Abstract
The invention discloses a kind of anti-H 1 N 1 swine influenza virus hemagglutinin monoclonal antibody and double-antibody sandwich elisa kits.The kit includes the solid phase carrier of coating monoclonal antibody, rabbit polyclonal antibody, H1N1 hemagglutinins standard items, sample diluting liquid, cleaning solution, substrate developing solution and the reaction terminating liquid of horseradish peroxidase-labeled, not only sensitivity is good, quantitative detection can be carried out to swine influenza virus hemagglutinin, and the Major Epidemic strain of specific recognition Influenza A H1N1 outburst in 2009, other strains and other influenza virus sub-strain no cross reactions with H1N1 influenza viruses.Kit is easy to operate, both can be used for the basic research for supporting H1N1 swine influenza viruses, meanwhile, it is of great significance for the epidemiological study for carrying out different regions, different year H1N1 influenza viruses.
Description
Technical field
The invention belongs to technical field of immunoassay, and in particular to a kind of anti-H 1 N 1 swine influenza virus hemagglutinin
Monoclonal antibody and its double crush syndrome immue quantitative detection reagent box.
Background technology
Influenza virus H1N1 hypotype, also referred to as H1N1 viruses, are a kind of RNA virus, belong to Orthomyxoviridae family, A type
One of influenza virus that one kind of influenza virus and the mankind most often infect.The strain of some H1N1 can be propagated between the mankind,
Including the influenza great outburst of 1918, other can be propagated between bird and pig.H1N1 is two kinds of important target point proteins of virus
The abbreviation of title, wherein " H " refers to hemagglutinin (Hemagglutinin), also known as hemagglutinin and " N " is referred to
Neuraminidase (Neuraminidase).Therefore, H1N1 is a kind of with " 1 type of 1 type of hemagglutinin and neuraminidase " disease
The influenza virus sub-strain of toxalbumin.
Flu outbreak is one of biggest threat of facing mankind, and the flu outbreak every many decades once is past in history
Huge even catastrophic loss is brought toward to the whole world.The death rate of the flu outbreak of 1918 about 2.5%, but
Still the death of about 50,000,000 people is caused in the world.Initially exist to April, H1N1 swine flu epidemic situation within 2009 3
Mexico is broken out, and hundred people was caused to infect.Epidemic situation travels to rapidly thereafter the whole world.Morning on April 30th, 2009, world health
The Global influenza warning level that is very popular is increased to the 5th grade by tissue, and by the end of at present, the whole world add up to report according to incompletely statistics
Death is more than 5000, and the World Health Organization thinks, actual morbidity and death toll are considerably beyond number of reports.By
On December 7th, 2009, ground adds up to report Influenza A H1N1 severe cases 4328, dead 326 in China.
Although the lethality of Influenza A H1N1 is not very high, influenza virus often with there is strong variability, due to H1N1 by
The immune pressure arrived is larger, and the amino acid variation constantly accumulation of 8 genetic fragments, antigenic variation is apparent, once some mutation
Cause pathogenic apparent increase, the influence that will can not be estimated the life security of the mankind and social stability generation, therefore, to A type
H1N1 swine influenza viruses and other influenza viruses carry out permanently effective monitoring and research, public health meaning are increasingly notable.
The investigative technique means of current published H1N1 swine influenza virus haemagglutinin antigen rely primarily on chicken embryo separation, PCR etc.,
Time-consuming for virus purification, and PCR needs professional equipment and technical staff, should not popularize, and the qualitative swine flu detections of some ELISA
Kit then can not carry out accurate quantification detection to viral antigen proteins, and often be difficult to accurately distinguish H1N1 influenza viruses
Different strains and other influenza virus sub-strains, be unfavorable for support influenza virus basis and epidemiological study, compel to be essential
A kind of easy to operate, sensitivity is good, the high quantitative detecting reagent of specificity.
The content of the invention
The object of the present invention is to provide a kind of anti-H 1 N 1 swine influenza virus hemagglutinin monoclonal antibody and with it
For the double crush syndrome immue quantitative detection reagent box of key component..
Detection kit provided by the present invention includes the solid phase carrier of coating monoclonal antibody, and the detection of enzyme mark is more
Clonal antibody and protein standard substance, sample diluting liquid, cleaning solution, substrate developing solution and reaction terminating liquid.
Wherein described coating monoclonal antibody is mouse monoclonal antibody, and light chain and heavy chain amino acid sequence are respectively SEQ
ID NO：1 and SEQ ID NO：2；The enzyme mark detection polyclonal antibody is horseradish peroxidase (Horseradish
Peroxidase, HRP) mark rabbit polyclonal antibody；The protein standard substance is restructuring H1N1 swine flu hemagglutinins.
Animal is immunized using restructuring H1N1 swine flu hemagglutinins in the mouse monoclonal antibody, and high serum is then selected to imitate
Valency mouse is prepared by classical hybridoma technology；The enzyme mark rabbit polyclonal antibody uses H1N1 swine flu hemagglutinins
Immune animal prepares the polyclonal antibody of purifying by Protein A purification and antigen affinity purification technology, then conventionally
It is obtained using HRP labelled antibodies.
The technical scheme is that preferably have the highly sensitive, monoclonal antibody of high specific and enzyme mark Anti-TNF-α
Body combinations of pairs, is adsorbed in monoclonal antibody as coated antibody on solid phase carrier, the capture that coated antibody can be specific
H1N1 hemagglutinins and protein standard substance in sample after adding in enzyme mark detection antibody, form coated antibody, antigen, inspection
Antibody complex is surveyed, is terminated after corresponding substrate colour developing, sample light absorption value is read, by that can draw sample with standard curve comparison
The content of middle H1N1 swine flus hemagglutinin.
Hemagglutinin is the key function albumen of influenza infection host cell, meanwhile and treatment of influenza medicine
The important target spot of object and vaccine development, the present invention can qualitatively or quantitatively detect the A type in sample using double-antibody method
H1N1 swine flus hemagglutinin is horizontal, and the method for inspection is convenient and easy, detection sensitivity and accuracy height, high specificity, can
Large quantities of samples are quickly detected simultaneously, the coating and detection antibody as kit key components are independent research, therefore
It is at low cost, it is highly reliable, it easily traces to the source, it is contemplated that will play a significant role in H1N1 relevant rudimentaries and clinical research.
Description of the drawings
Fig. 1 .H1N1 swine flu hemagglutinin ELISA kit canonical plottings
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be further described and explanation.
It is prepared by the component of embodiment 1H1N1 swine flu hemagglutinin ELISA kits
1. the preparation of protein standard substance
Standard items in the kit are restructuring H1N1 swine flu hemagglutinins, from Yi Qiao Divine Land, Beijing biology skill
(article No. is for art Co., Ltd：11055-V08H), which expressed in vitro using human archeocyte expression system, then
The high purity protein obtained is purified, specific purity data can be found in explanation of Yi Qiao Divine Land company's site for the product.
2. the preparation of mouse monoclonal antibody：
1) animal immune
Using Balb/c mouse as immune animal, the restructuring H1N1 pigs produced with Yi Qiao Divine Land Bioisystech Co., Ltd
Influenza hemagglutinin protein is immunogene, and immunizing dose is the albumen of each every 50 μ g of mouse immune.It will be immune during first immunisation
Emulsifier, subcutaneous abdomen multi-point injection is made in the former complete Freund's adjuvant with equivalent, and interval takes same dose to be immunized after 2~3 weeks
Former that emulsifier is made with equivalent incomplete Freund's adjuvant, booster immunization twice, blood is measured after being immunized three times using indirect elisa method
Clear potency after serum titer reaches 1: 16000, selects the highest mouse peritoneal booster immunization of potency once, takes spleen thin after 4 days
Born of the same parents carry out cell fusion.
2) cell fusion and cloning
Mouse spleen is taken, the single splenocyte suspension of centrifugation acquisition after grinding filtering, after cell count, by 5: 1 or 10: 1
Ratio is mixed with the SP2/0 murine myeloma cells in exponential phase, and carrying out cell using polyethylene glycol (PEG) method melts
It closes, bed board, by the effect of Selective agar medium, myeloma cell and splenocyte etc. are not merged or the cell of fusion not yet in effect is by nothing
Method is grown, and the hybridoma of effective integration will grow in culture hole, be proliferated, and secretory antibody.After changing liquid three times, fusion
Cells and supernatant is taken within 9-12 days afterwards, to recombinate H1N1 swine flus hemagglutinin as envelope antigen, utilize indirect ELISA
Method measures supernatant, screens positive hole, and passes through limiting dilution assay and carry out colonized culture to positive cell, until being stablized
The monoclonal hybridoma strain of secreting specificity antibody.More than cell fusion and cloning method are that immunology monoclonal resists
Common classical way in body technique.
3) production and purifying of monoclonal antibody
Hybridoma cell strain is chosen, cell culture is carried out using blake bottle or bioreactor, collects cells and supernatant,
It is purified using routine protein A affinity columns, the antibody of acquisition is identified by SDS-PAGE electrophoresis and indirect elisa method to be resisted
It is dispensed after body purity and specificity, it is spare in -20 DEG C of Cord bloods.
4) sequencing of monoclonal antibody
Hybridoma for a long time preserve may due to after repeatedly passing on unstable and pollution problem positive colony is caused to be lost
It loses, to solve the above problems, in process of the present invention, the antibody for being extracted positive colony respectively using Protocols in Molecular Biology is light
Chain and heavy chain gene have carried out sequencing, include the plasmid of antibody gene and can steadily in the long term be protected under the conditions of -20 DEG C
It deposits, meanwhile, according to antibody gene sequences, the technical staff in this professional domain can clone according to conventional molecular biological method
Expression obtains identical monoclonal antibody.
Antibody gene extraction specific method is as follows：The good hybridoma of growth conditions is collected, by BBI companies
The operation scheme extraction hybridoma total serum IgE of classical total RNA isolation kit specifications, passes through electrophoresis
Detect quality, UV measured concentrations.By the behaviour of the MMLV first strand cDNA synthesis kit specifications of BBI companies
Make scheme by mRNA reverse transcriptions be cDNA, -20 DEG C freeze it is spare.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 are 20 μ l.Primer is designed according to bibliography, using cDNA as template point
Other PCR obtains the light chain and heavy chain fragment of antibody, and 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 hybridomas cDNA, 0.2 μ l5U/ μ l Pyrobest DNA Polymerase,
Reaction system is 20 μ l.Amplification condition：It is denatured 94 DEG C of 4min；94 DEG C of 1min are denatured, anneal 58 DEG C of 1min, extends 72 DEG C of 1min,
30 Xun Huans；Extend 72 DEG C of 5min.Light chain and heavy chain fragment are inserted on pcDNA3T carriers, build pcDNA3-anti-
H1N1-L and pcDNA3-anti-H1N1-H carriers.Carrier is converted into Escherichia coli, picking positive colony carries out sequencing identification,
Sequencing result is analyzed, obtains correct light chain and heavy chain amino acid sequence.Measurement result and sequence analysis show the light of the antibody
Chain amino acid sequence is SEQ ID NO：1；Heavy chain amino acid sequence is SEQ ID NO：2, refer to sequence table.Specific experiment operates
With reference to well known to those skilled in the art【Sa curtain Brooker J etc.,《Molecular cloning texts guide》, Beijing Science Press】.
3. the preparation of rabbit polyclonal antibody：
New zealand rabbit is chosen as animal is immunized, is immunized using recombinating H1N1 swine flu hemagglutinins as immunogene,
The albumen of 500 μ g is immunized for every rabbit every time for immunizing dose.First immunisation is by immunogene and the complete Freund's adjuvant system of equivalent
Into emulsifier, the subcutaneous multi-point injection of nape part, interval takes same dose immunogene and equivalent incomplete Freund's adjuvant system in 2~3 weeks
Into emulsifier, booster immunization is immunized 4-5 times altogether, and after indirect elisa method measure serum titer reaches 1: 25000, heart takes
Blood passes through the Anti-TNF-α purified after routine protein A affinity columns and hemagglutinin antigen affinity column two-step purifying
Body, packing are used for the preparation of enzyme labelled antibody in -20 DEG C of Cord bloods.Wherein hemagglutinin antigen affinity purification specific steps
It is as follows：
A) claim the Ago-Gel (GE companies) of 0.7g cyanogen bromide-activateds, be swollen with 1mM HCl, then washed with 1mM HCl
Three times, it is spare；
B) hemagglutinin of 2mg is taken to utilize hyperfiltration process by albumen buffer exchange into 0.1M NaHCO3, pH8.3, and
It is 1~2ml to control volume；
C) protein solution is added in the activated sepharose that step a) has been washed, room temperature shakes 4h；
D) with pH8.0, the Tris buffer blind unreacted groups of 0.1M；
E) gravity column is filled, bed volume about 2ml is rinsed with PBS, and balanced；
The how anti-of Protein A purification is taken to cross column, unbonded antibody is rinsed with PBS, is eluted with pH3.0 citrate buffer solutions special
With reference to antibody, in Tris buffer solutions and eluent is to pH7.0-7.5.
4. the preparation of enzyme labelled antibody：
A) HRP for weighing 5mg is dissolved in 0.5mL distilled water；
B) NaIO for the 0.1M that 0.5mL newly matches somebody with somebody is added in4Solution is protected from light stirring 20 minutes at room temperature；
C) above-mentioned solution is fitted into bag filter, the sodium-acetate buffer (pH4.4) of 1mM dialyses 4 DEG C overnight；
D) polyclonal antibody of 5mg affinity purifications is taken, is added in 1mL0.01M carbonate buffer solutions, it is spare；
E) 0.2M carbonate buffer solutions (pH9.5) are added in into c) liquid, pH is made to be increased to 9.0~9.5, is added in immediately after
D) in liquid, room temperature be protected from light be gently mixed 2 it is small when；
F) the 4mg/mL NaBH that 0.2mL is added now to match somebody with somebody4Liquid, mixing, when 4 DEG C of placements 2 are small；
G) above-mentioned liquid is fitted into bag filter, in pH7.4, the PBS dialysis of 0.15M, 4 DEG C overnight.
The establishment of embodiment 2H1N1 swine flu hemagglutinin ELISA kits
The ELISA kit of establishment includes following reagent：
A) mouse monoclonal coated antibody；
B) rabbit polyclonal antibody of HRP marks；
C) H1N1 swine flus hemagglutinin standard items；
D) it is coated with buffer solution：The carbonate buffer solution of pH9.6,0.05mol/L；
E) confining liquid：Tris buffer solutions containing 2% bovine serum albumin(BSA)；
F) sample diluting liquid：Phosphate buffer containing 0.1% bovine serum albumin(BSA)；
G) cleaning solution：Phosphate buffer containing 0.1% tween；
H) substrate developing solution：It is made of developing solution A and developing solution B, developing solution A is hydrogen peroxide or urea peroxide, is developed the color
Liquid B is tetramethyl biphenyl；
I) terminate liquid：The sulfuric acid of 2mol/L
Embodiment 3 detects the preparation of the ELISA kit of H1N1 swine flu hemagglutinins
1. grope optimum antibody combination and the working concentration of enzyme linked immunological kit using orthogonal test
According to ultraviolet spectrophotometer method, the concentration of measure antibody and antigen.Using orthogonal test method, grope most preferably anti-
Body combines and optimum antibody concentration, and different anti-H1N1 swine flus hemagglutinin monoclonal antibodies are diluted to concentration is
4 μ g/ml, 2 μ g/ml, 1 μ g/ml, restructuring hemagglutinin concentration dilution to 1000pg/ml, 100pg/ml, 0pg/ml, HRP marks
The rabbit polyclonal antibody diluted concentration of note is to 4 μ g/ml, 2 μ g/ml, 1 μ g/ml, 0.5 μ g/ml.Consider blank well background and
The absorbance value in positive test hole preferably goes out one plant of mouse monoclonal antibody as coated antibody, and confirms its best effort concentration
Working concentration for 2 μ g/ml, HRP mark polyclonal antibodies is 1 μ g/ml.
2. prepared by the batch of kit
1) coated elisa plate：(3.18g Na are weighed with carbonate coating buffer solution2CO3, 5.86g NaHCO3, 1gNa2N3,
It is settled in the deionized water of 2L, adjusts pH value 9.6), anti-H1N1 swine flus hemagglutinin monoclonal antibody to be diluted to dense
It spends for 2 μ g/ml, 100 μ L/ holes, coated elisa plate, 4 DEG C of overnight incubations, with cleaning solution board-washing 1 time, 200 μ L/ holes.
2) close：300 μ L confining liquids closing nonspecific binding site is added in per hole, when incubation at room temperature 1 is small；Then with washing
Wash liquid board-washing 2 times, 200 μ L/ holes；It is packed after patting dry with vacuum packing machine, 4 DEG C save backup.
3) preparation of protein standard substance：H1N1 swine flu hemagglutinins are diluted with sample diluting liquid, are freezed after packing, 4
It DEG C saves backup.
4) preparation of enzyme labelled antibody：Rabbit polyclonal antibody after affinity purification with HRP is marked, adds in 50% glycerine, point
It is saved backup for -20 DEG C after dress.
Embodiment 4ELISA kits coated antibody is identified
1. antibody test limit measures：
1) H1N1 swine flus hemagglutinin is diluted to carbonate coating buffer solution, 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 are incubated overnight, and use cleaning solution
Board-washing 1 time, 200 μ L/ holes；
2) 300 μ L confining liquids closing nonspecific binding site is added in per hole, when incubation at room temperature 1 is small；Then cleaning solution is used
Board-washing 2 times, 200 μ L/ holes；
3) anti-H1N1 swine flus hemagglutinin mouse monoclonal antibody is diluted to 1ug/ml with Sample dilution, added in pre-
The ELISA Plate for being first coated with and having closed, 200 μ L/ holes, when incubation at room temperature 1 is small；Then cleaning solution board-washing is used 3 times, 200 μ L/ holes；
4) the anti-mouse IgG secondary antibodies of HRP marks, 200 μ L/ holes, when incubation at room temperature 1 is small are added in；Then cleaning solution board-washing 3 is used
It is secondary, 200 μ L/ holes；
5) tmb substrate of the 200 fresh configurations of μ L is added in, room temperature is protected from light colour developing 20 minutes, and it is whole to add in 50 μ L2mol/L sulfuric acid
It only reacts, readings under microplate reader 450nm wavelength.
6) numerical value of 3 times of standard deviations and blank control group are subtracted (with not containing again with the average value of each sample group light absorption value
The coating buffer solution coated elisa plate of histone, other same experimental groups of step) light absorption value average value plus 3 times of standard deviations numerical value
It compares, the minimum albumen coating concentration that sample values are more than under blank control group value conditions is taken to be limited for the detection of antibody.Knot
Fruit shows that the detection limit of antibody can reach 5ng/ holes (table 1), illustrates that antibody has higher detection sensitivity.
1 coated antibody of table detection limit measures
| OD-1 | OD-2 | OD-3 | OD-4 | OD-5 | AVERAGE | SD | AERAGE+3SD | AERAGE-3SD | Detection limit (ng/ holes) | |
| Blank control | 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
Antibody subtype, testing result are detected using the antibody typing kit of Sino Biological Inc.
For IgG1 type antibody.
The specific assay of embodiment 5H1N1 swine flu hemagglutinin ELISA kits
The restructuring hemagglutinin of different influenza virus sub-strains and H1N1 hypotype difference strains is diluted to 50ng/ml, is used
H1N1 swine flu hemagglutinins ELISA kit is tested, and the results show kit is capable of the identification H1N1 pig of specificity
Influenza virus Major Epidemic strain H1N1, A/California/4/2009 and H1N1, A/California/7/2009, with H1N1
Other strains of influenza virus include Brevig Mission/1/1918, Solomon Islands/3/2006, Ohio/UR06-
0091/2007th, New Caledonia/20/1999, Puerto Rico/8/1934 and WSN/1933 no cross reactions, it is and other
Influenza virus sub-strain include H1N2, swine/Guangxi/13/2006, H1N3, duck/NZL/160/1976, H2N2,
Canada/720/2005, H6N1, California/HKWF115/2007 no cross reactions.Above-mentioned experiment proves kit spy
The opposite sex is good, can clearly distinguish H1N1 swine flu Major Epidemic strains and other influenza virus sub-strains and strain, can be used for supervising
The epidemiology variation of H1N1 swine flu Major Epidemic strains is controlled, and for being directed to influenza virus different subtype and same Asia
The development basic research of type difference strain also has important actual application value.
The specific assay of table 2H1N1 swine flu hemagglutinin ELISA kits
| The hypotype and strain of hemagglutinin | OD450nm values |
| 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 swine flu hemagglutinin ELISA kits
1. sample-adding
1) ELISA Plate being coated with and lyophilized standard items are taken out, 1ml sample diluting liquids is added to dissolve standard items.At room temperature
It places 20 minutes.By standard items from 800pg/ml, 2 times of doubling dilution is done, totally 6 points, dilution is respectively taken 100 μ l add in
In 96 hole elisa Plates.
2) sample to be tested is taken, 100 μ l/ holes are added in reacting hole, when incubation 2 is small at room temperature；
3) cleaning solution board-washing 3 times, 200 μ L/ holes, pat dry ELISA Plate.
2. add in detection antibody
HRP labelled antibodies are diluted to 1 μ g/mL with sample diluting liquid, are added in reacting hole, 100 μ L/ holes are incubated at room temperature
Educate 1 it is small when, cleaning solution board-washing 3 times, 200 μ L/ holes pat dry ELISA Plate.
3. colour developing
1) the substrate developing solution of 200 μ L Fresh is added in, is reacted at room temperature 20 minutes, then adds in the termination of 50 μ L terminate liquids
Reaction；
2) light absorption value is read under microplate reader 450nm wavelength.
4. the foundation of standard curve
Using the concentration of standard items as abscissa, OD values establish standard curve (Fig. 1) for ordinate, according to the sample OD measured
Value can calculate the content for obtaining the H1N1 hemagglutinins in sample.
Finally, it should also be noted that it is listed above be only specific embodiments of the present invention son.Obviously, the present invention not
It is limited to above embodiment, it can also be there are many modification.Those of ordinary skill in the art can be straight from present disclosure
Export or all modifications associated are connect, are considered as protection scope of the present invention.
Claims (4)
1. a kind of anti-H 1 N 1 swine influenza virus agglutinin of blood protein monoclonal antibody, which is characterized in that the monoclonal resists
The light chain and heavy chain amino acid sequence of body are respectively SEQ ID NO：1：
MDFQVQIFSFLLISASVIMSRGQIVLTQSPAIMSASLGERVTMTCTASSSVSSTYLHWYQQKPGSSPKLWIYS
TSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTS
GGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSP
IVKSFNRNEC and SEQ ID NO：2：
MKWSWVILFLLSGTAGAHSEIQLQQTGPELVKPGAPVKISCKASGYSLTDYI
ILWVKQSHGKSLEWIGNINPYFGTTSYNLKFKDKATLTVDKSSSTAYMELNSLTSEDSAVYYCARNDNYAMDY
WGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSS
SVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDI
SKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRP
KAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQP
IMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK。
2. a kind of double-antibody sandwich elisa kit for detecting H1N1 swine influenza virus hemagglutinin, which is characterized in that
Include coating claim 1 monoclonal antibody solid phase carrier, the rabbit polyclonal antibody of horseradish peroxidase-labeled,
H1N1 hemagglutinins standard items, sample diluting liquid, cleaning solution, substrate developing solution and reaction terminating liquid；The monoclonal resists
Body light chain and heavy chain amino acid sequence are respectively SEQ ID NO：1 and SEQ ID NO：2.
3. kit as claimed in claim 2, which is characterized in that using restructuring H1N1 swine influenza virus hemagglutinin conducts
Standard items can carry out H1N1 swine influenza virus hemagglutinin quantitative detection.
4. kit as claimed in claim 2, which is characterized in that specific identification H1N1 swine influenza virus mainly flows
Row strain H1N1, A/California/4/2009 and H1N1, A/California/7/2009 are other with H1N1 influenza viruses
Strain includes Brevig Mission/1/1918, Solomon Islands/3/2006, Ohio/UR06-0091/2007, New
Caledonia/20/1999, Puerto Rico/8/1934 and WSN/1933 no cross reactions, with other influenza virus sub-strain bags
Include H1N2, swine/Guangxi/13/2006, H1N3, duck/NZL/160/1976, H2N2, Canada/720/2005,
H6N1, Cal ifornia/HKWF115/2007 no cross reactions.
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