CN111808188A

CN111808188A - ELISA kit for H7N9 influenza virus hemagglutinin protein

Info

Publication number: CN111808188A
Application number: CN202010853522.1A
Authority: CN
Inventors: 谢良志; 张�杰; 李雁; 王旭曼; 张娜娜; 高然; 王娜; 任为; 杨茜; 李学仁
Original assignee: Beijing Yiqiao Shenzhou Polytron Technologies Inc
Current assignee: Beijing Yiqiao Shenzhou Polytron Technologies Inc; Sino Biological Inc
Priority date: 2020-08-24
Filing date: 2020-08-24
Publication date: 2020-10-23

Abstract

The invention discloses an H7N9 influenza virus hemagglutinin protein double-antibody sandwich ELISA kit. The kit comprises a solid phase carrier coated with monoclonal antibody, a rabbit polyclonal antibody marked by horseradish peroxidase, an H7N9 hemagglutinin protein standard, a sample diluent, a washing solution, a substrate color developing solution and a reaction termination solution, has good sensitivity, can carry out quantitative detection on H7N9 influenza hemagglutinin protein, and specifically identifies H7N9 and H7N7 influenza viruses, and has no cross reaction with other main subtypes of influenza A viruses, such as H1N1, H2N2, H3N2, H4N6, H5N1, H6N1, H8N4, H9N2, H10N8, H11N2, H12N5, H13N8, H15N8, H16N3, H18N11 and hemagglutinin protein of influenza B viruses. The kit is simple to operate, can be used for simultaneously and rapidly detecting a large number of samples, can be used for supporting the basic research of the H7N9 influenza virus, and has important significance for developing the epidemiological research of the influenza virus.

Description

ELISA kit for H7N9 influenza virus hemagglutinin protein

Technical Field

The invention belongs to the field of immunology, and particularly relates to preparation of a specific monoclonal antibody of H7N9 influenza virus hemagglutinin protein, and a double-antibody sandwich ELISA (enzyme-Linked immuno sorbent assay) detection kit for quantitative H7N9 influenza virus hemagglutinin protein.

Background

The influenza virion coat is covered by two surface glycoproteins, one hemagglutinin (i.e., H) and one neuraminidase (i.e., N), H being subdivided into 18 subtypes and N into 11 subtypes. All human influenza viruses can cause avian influenza, but not all avian influenza viruses can cause human influenza, and among the avian influenza viruses, H5, H7 and H9 can be transmitted to human beings.

H7N9 is a subtype of avian influenza virus, and has not been found to infect humans, even though it was only found between birds. China first found H7N9 infected people at the end of 3 months in 2013, and the cases were located in Shanghai and Anhui. Through investigation, the gene of the avian influenza virus H7N9 is generated by gene recombination, and the virus sequence is from wild birds in east Asia region and chickens in Shanghai, Zhejiang and Jiangsu in China. The discovered cases are distributed in Beijing, Shanghai, Jiangsu, Zhejiang, Anhui, Henan, Fujian, etc. China reports that people are infected with the H7N9 avian influenza virus for the first time in 3 months in 2013, and China has already experienced the prevalence of 5-wave epidemic situations: 19 cases of avian influenza infection and 1 case of death caused by H7N9 infection in 2013; 330 cases of H7N9 avian influenza infection in 2014, 135 cases of deaths and 41% of deaths; 196 cases of avian influenza infected with H7N9 in 2015, 92 deaths and 47% of deaths; 264 cases of avian influenza infection with H7N9 in 2016, 73 deaths and 28% mortality; the incidence of H7N9 avian influenza infection in 2017 is increased sharply to 589 cases, 259 cases of death, and the mortality rate is 44%. The incidence of the avian influenza virus is greatly reduced in 2018, and the data show that the number of Chinese infected with H7N9 avian influenza is 3 and the number of dead people is 2 from 2018 to 2019 in 8 months.

Since 2013, avian influenza virus subtype H7N9 has caused over 1000 human infections and has a high mortality rate, and the world health organization states that "since 2013 only one possible human case has been detected, limited interpersonal transmission is still likely to occur". Influenza viruses often have strong variability, and although the incidence of H7N9 is reduced in recent two years, once pathogenicity is obviously increased due to certain mutations, immeasurable influence is generated on the life safety and social stability of human beings, so that the research on the H7N9 influenza virus is of great significance. At present, the detection of H7N9 is mainly a quantitative PCR method based on nucleic acid detection, and the method has higher requirements on environment, samples, instruments and equipment and operators, is not easy to popularize and realize high-throughput detection; therefore, a quantitative detection reagent with simple operation, good sensitivity and high specificity is urgently needed.

Disclosure of Invention

The invention aims to provide a H7N9 influenza virus hemagglutinin protein double-antibody sandwich method detection kit which is high in detection sensitivity, strong in accuracy and low in cost.

The detection kit provided by the invention comprises a solid phase carrier coated with a monoclonal antibody, an enzyme-labeled detection polyclonal antibody, a protein standard, a sample diluent, a washing solution, a substrate developing solution and a reaction stopping solution.

Wherein the monoclonal antibody is a rabbit monoclonal antibody, and the heavy chain and light chain amino acid sequences of the monoclonal antibody are SEQ ID NO. 9 and SEQ ID NO. 10 respectively; the enzyme-labeled detection polyclonal antibody is a rabbit polyclonal antibody marked by Horse Radish Peroxidase (HRP); the protein standard is recombinant H7N9 avian influenza virus hemagglutinin protein.

The rabbit monoclonal antibody is prepared by immunizing an animal by adopting recombinant H7 hemagglutinin protein and then selecting a rabbit with high serum titer through a phage display technology; the enzyme-labeled rabbit polyclonal antibody adopts recombinant H7N9 avian influenza virus hemagglutinin protein to immunize animals. The purified polyclonal antibody is prepared by protein A and antigen affinity purification technology, and then is obtained by labeling the antibody with HRP according to a conventional method.

The technical scheme of the invention is that a monoclonal antibody with high sensitivity and high specificity and an enzyme-labeled polyclonal antibody are preferably matched and combined, the monoclonal antibody is taken as a coated antibody and is adsorbed on a solid phase carrier, the coated antibody can specifically capture H7N9 hemagglutinin protein and a protein standard product in a sample, an enzyme-labeled detection antibody is added to form a coated antibody, antigen and detection antibody compound, a corresponding substrate is stopped after color development, the light absorption value of the sample is read, and the content of the H7N9 avian influenza hemagglutinin protein in the sample can be obtained by comparing with a standard curve.

The hemagglutinin protein is a key functional protein of host cell infected by influenza virus, and is also an important target spot for developing influenza therapeutic drugs and vaccines. The coating and detection antibodies which are key components of the kit are independently developed, so that the kit is low in cost, can be produced in batches, is in place in one step, is high in plasticity, and has wide market prospect and great economic and social benefits.

Drawings

FIG. 1, H7N9 avian influenza virus hemagglutinin protein ELISA kit standard curve chart

Detailed Description

The invention will be further described and illustrated with reference to specific examples

Example 1 component preparation of H7N9 avian influenza Virus hemagglutinin protein ELISA kit

1. Preparation of protein standards

The standard substance in the kit is recombinant H7N9 avian influenza virus hemagglutinin protein from Beijing Yiqian Shenzhou science and technology corporation (Cat No. 40104-V08H), the protein is high-purity protein obtained by in vitro expression by utilizing a human source cell expression system and then purification, and specific purity data can be referred to the description of the website of the Yi qian Shenzhou corporation on the product.

2. Preparation of rabbit monoclonal antibodies:

1) animal immunization

New Zealand big ear white rabbits are selected as immune animals, recombinant H7 hemagglutinin protein (11082-V08B) produced by Yi Qiao Shen science and technology GmbH is taken as immunogen, and the immune dose is 500ug of protein per rabbit each time. Preparing an immunogen and an equal amount of complete Freund's adjuvant into an emulsifier during first immunization, performing subcutaneous multi-point injection on the abdomen, taking the same dose of immunogen and an equal amount of incomplete Freund's adjuvant to prepare the emulsifier after 2-3 weeks, performing enhanced immunization for 3-4 times, measuring the serum titer by using an indirect ELISA method after the last immunization, selecting a rabbit with the highest titer after the serum titer reaches 1:25000, and taking a spleen for phage library construction.

2) Establishment of phage antibody library

Taking rabbit spleen, grinding the tissue in liquid nitrogen, extracting total RNA of animal tissue by Trizol method, detecting quality by electrophoresis, and measuring concentration by UV. Reverse transcription of mRNA into cDNA was performed using RT reverse transcriptase from Hibiscus syriacus technologies, Inc., and frozen at-20 ℃ for later use. The reverse transcription reaction system is as follows: RNA (25. mu.g), oligdT₂₀5.0μL，dNTP mix(10mmol/L)5.0μL，10×reaction buffer 10μL，RNase inhibitor(20U/μL)0.6μL，MgCl₂20.0 μ L (0.1M), 6.0 μ L RT (Yinqiao), 10.0 μ L DTT (0.1M), and 100.0 μ L total reaction volume. Respectively obtaining the variable region sequences of the light chain and the heavy chain of the antibody by PCR by taking cDNA obtained by reverse transcription as a template, wherein the reaction system is as follows: 4.0. mu.L of 5 XPrimeSTAR Buffer, 1.6. mu.L of 2.5mM dNTP, 1.6. mu.L of 10. mu.M primer pair, 1.0. mu.L cDNA, 0.2. mu.L PrimeSTAR, 20.0. mu.L reaction system. Amplification conditions: 5min at 94 ℃; 12s at 98 ℃, 15s at 62 ℃, 30s at 72 ℃ and 30 cycles; and (3) adjusting the cycle number to make each light chain and each heavy chain belt uniform at 72 ℃ for 5min, and obtaining the scFv fragment by splicing PCR. The scFv fragment obtained after recovery and purification of the gel is subjected to single enzyme digestion with sfi I and then purified again, and the purification operation is carried outThe purified scFv fragment was inserted into a phage expression vector using T4 ligase from Promega's Wizard SV Gel and PCR clean-up System purification kit, Hippocampus Proteus technologies Co., Ltd., and the expression vector was used to transform XL1-Blue allelochemicals, and the obtained transformed bacteria were amplified to OD in 2YT medium₆₀₀After 0.5, 20 times of the total bacterial amount of helper phage was added for infection to obtain a single-chain phage antibody library.

3) Elutriation identification

The phage containing anti-H7N 9 protein scFv were screened using a microplate screening method, which is well known to those skilled in the art. Using 100. mu.L of 0.05M Na containing 10. mu.g/mL H7N9 recombinant protein₂CO₃-NaHCO₃Nunc culture plates are coated with coating solution at 4 ℃, TBS/Tween20(20mM Tris-HCl pH7.5, 250mM NaCl, 0.1% Tween20) is washed once, after 3% skimmed milk powder is blocked at 37 ℃ for 1h, TBS/Tween20(20mM Tris-HCl, pH7.5, 250mM NaCl, 0.1% Tween20) is washed twice, concentrated phage is added, the mixture is incubated at 37 ℃ for 2h, TBS/Tween20 washing solution is washed for several times to remove unbound phage, 100 mu L of solution buffer (0.13mol/L Glycine, pH2.2) is added to each well, the phage adhered to the microplate is eluted at room temperature for 7min, and then 10 mu L (2mol/L Tris, pH7.4) of the neutralized washed phage solution is added to each well. And infecting escherichia coli in logarithmic growth phase by using the eluted phage solution, culturing and amplifying to enrich the specific scFv phagemid expressing the anti-H7N 9 recombinant protein, and adding the auxiliary phage to infect to obtain the phage containing the anti-H7N 9 scFv by the method. The process of adsorption, elution and amplification was repeated 4 more times. The panning of each round of the pool was tested by ELISA. Finally adding auxiliary phage for infection, carrying out specificity identification on the screened phage single clone by an ELISA method, sequencing a plurality of phage antibodies with higher antigen binding activity, and obtaining a heavy-light chain variable region sequence. The heavy chain variable region sequence and the light chain variable region sequence were digested with ScaI + KpnI and ScaI + BamHI, respectively, and ligated to vectors having respective constant regions to construct a heavy and light chain full length antibody sequence, and a pair of plasmid-transfected cells containing the heavy and light chain full length antibody after sequencing were used for antibody production. See example 3.1. Using orthogonal assaysThe optimal antibody combination and working concentration of the enzyme-linked immunoassay kit are groped, a rabbit monoclonal antibody is preferably selected, and sequencing results and sequence analysis show that the heavy chain amino acid sequence of the antibody is SEQID NO. 9; the light chain amino acid sequence is SEQ ID NO 10.

3) Production and purification of monoclonal antibodies

Transfecting HEK293 cells by the plasmids, culturing the cells by using a culture bottle or a bioreactor, collecting cell culture supernatant, purifying by using a conventional protein A affinity chromatography column, identifying the purity and specificity of the antibody by SDS-PAGE electrophoresis and an indirect ELISA method, subpackaging, and storing at a low temperature of-20 ℃ for later use.

3. Preparation of Rabbit polyclonal antibody

New Zealand white rabbits with big ears are selected as immune animals, recombinant H7N9 avian influenza hemagglutinin protein produced by Yi Qiao Shen science and technology GmbH is taken as immunogen, and the immune dose is 500ug of protein per rabbit each time. Preparing an immunogen and an equal amount of complete Freund's adjuvant into an emulsifier during first immunization, performing abdominal subcutaneous multi-point injection, taking the same dose of immunogen and an equal amount of incomplete Freund's adjuvant at intervals of 2-3 weeks to prepare the emulsifier, enhancing the immunization, performing the immunization for 3-4 times, determining the serum titer by using an indirect ELISA method after the last immunization, taking blood from the heart after the serum titer reaches 1:25000, performing two-step purification by using a conventional protein A affinity chromatography column and an H7N9 hemagglutinin protein antigen affinity column to obtain a purified polyclonal antibody, subpackaging, and storing at the low temperature of-20 ℃ for preparation of the enzyme-labeled antibody. The hemagglutinin protein antigen affinity purification comprises the following specific steps:

a) 0.7g of cyanogen bromide-activated agarose gel (GE Co.) was weighed, swollen with 1mM HCl, and then washed three times with 1mM HCl for use;

b) replacing the protein buffer solution with 0.1M NaHCO by ultrafiltration method from 2mg of hemagglutinin protein₃pH8.3, and controlling the volume to be 1-2 mL;

c) adding the protein solution into the activated agarose gel washed in the step a), and shaking for 4 hours at room temperature;

d) blocking unreacted groups with Tris buffer, pH8.0, 0.1M;

e) loading a gravity column with the bed volume of about 2mL, washing with PBS, and balancing;

f) the protein A purified polyclonal antibody column is taken, unbound antibodies are washed by PBS, specifically bound antibodies are eluted by citric acid buffer solution with pH3.0, and the eluate is neutralized by Tris buffer solution to pH7.0-7.5.

4. Preparing an enzyme-labeled antibody;

a) 5mg of HRP is weighed and dissolved in 0.5mL of distilled water;

b) 0.5mL of freshly prepared 0.1M NaIO was added₄The solution was stirred at room temperature for 20 minutes in the absence of light.

c) Putting the solution into a dialysis bag, dialyzing with 1mM sodium acetate buffer solution (pH4.4), and standing at 4 deg.C overnight;

d) adding 5mg of affinity purified polyclonal antibody into 1mL of 0.01M carbonate buffer solution for later use;

e) adding 0.2M carbonate buffer solution (pH9.5) into the solution c) to increase the pH to 9.0-9.5, immediately adding into the solution d), and lightly stirring at room temperature in a dark place for 2 hours;

f) 0.2mL of freshly prepared 4mg/mL NaBH was added₄Mixing the solutions, and standing at 4 deg.C for 2 hr.

g) The above solution was filled into a dialysis bag and dialyzed against 0.15M PBS at pH7.4 at 4 ℃ overnight.

Example 2 construction of H7N9 avian influenza hemagglutinin protein ELISA kit

The constructed ELISA kit contains the following reagents:

a) rabbit monoclonal antibodies;

b) HRP-labeled rabbit polyclonal antibody;

c) H7N9 avian influenza hemagglutinin protein standard;

d) coating buffer solution: 0.05mol/L carbonate buffer solution with the pH value of 9.6;

e) sealing liquid: tris buffer containing 2% bovine serum albumin;

f) sample diluent: phosphate buffer containing 0.1% bovine serum albumin

g) Washing liquid: phosphate buffer containing 0.1% tween;

h) substrate color developing solution: the color developing solution A is hydrogen peroxide or carbamide peroxide, and the color developing solution B is tetramethyl biphenyl;

i) stopping liquid: 2mol/L sulfuric acid.

Example 3 preparation of H7N9 avian influenza hemagglutinin protein ELISA kit

1. Optimum antibody combination and working concentration of enzyme-linked immunosorbent assay kit are groped by using orthogonal test

The concentrations of the antibody and antigen were measured according to the ultraviolet spectrophotometer method. Different anti-H7N 9 avian influenza hemagglutinin protein monoclonal antibodies are diluted to the concentrations of 4 mug/mL, 2 mug/mL, 1 mug/mL and 0.5 mug/mL, the recombinant hemagglutinin protein concentration is diluted to 2ng/mL, 1ng/mL and 0.5ng/mL, and the HRP-labeled rabbit polyclonal antibody is diluted to the concentrations of 2 mug/mL, 1 mug/mL, 0.5 mug/mL and 0.25 mug/mL by adopting an orthogonal test method and searching for the optimal antibody combination and the optimal antibody using concentration. Comprehensively considering blank hole background and light absorption value of a positive experimental hole, preferably selecting a heavy chain amino acid sequence as SEQ ID NO. 9; the rabbit monoclonal antibody with the light chain amino acid sequence of SEQ ID NO. 10 is used as a coating antibody, and the optimal working concentration is confirmed to be 0.5 mu g/mL, and the working concentration of the HRP-labeled polyclonal antibody is confirmed to be 0.5 mu g/mL.

2. Batch preparation of kits

1) Coating an enzyme label plate: coating buffer with carbonate (3.18 g Na was weighed)₂CO₃，5.86g NaHCO₃，1g Na₂N₃And diluting the avian influenza hemagglutinin protein monoclonal antibody against H7N9 to the concentration of 0.5 mu g/mL and 100 mu L/hole by using deionized water to fix the volume to 2L, adjusting the pH value to 9.6), coating an enzyme label plate, incubating overnight at 4 ℃, and washing the plate for 1 time and 200 mu L/hole by using a washing solution.

2) And (3) sealing: adding 300 mu L of blocking solution into each hole to block the non-specific binding sites, and incubating for 1 hour at room temperature; then washing the plate 2 times with a washing solution at 200. mu.L/well; and (4) after being dried, packaging by a pinhole packaging machine, and storing at 4 ℃ for later use.

3) Preparation of protein standard: and (3) diluting the H7N9 avian influenza hemagglutinin protein by using a freeze-drying buffer solution, subpackaging, freeze-drying and storing at 4 ℃ for later use.

4) Preparation of enzyme-labeled antibody: and (3) labeling the rabbit polyclonal antibody subjected to affinity purification with HRP, adding 50% glycerol, subpackaging, and storing at-20 ℃ for later use.

Example 4 determination of major parameters of H7N9 avian influenza hemagglutinin protein ELISA kit

1. Precision determination of the kit

Selecting 3 samples containing different H7N9 hemagglutinin protein concentrations, detecting in the same ELISA plate, and repeating each sample for 20 times; and simultaneously, detection is carried out among different enzyme-labeled plates, each sample is repeated for 5 times, each enzyme-labeled plate is respectively provided with a standard substance control curve, the variation coefficient C.V of each detection result is respectively calculated, the C.V is (standard deviation SD/Mean value Mean) × 100%, the precision measurement in the plate is shown in table 1, the precision measurement among the plates is shown in table 2, the Mean variation coefficient is 2.1% (when C.V is more than 15%, the difference among different groups is larger), and the prepared kit has good repeatability.

TABLE 1 in-plate precision determination of H7N9 hemagglutinin protein ELISA kits

TABLE 2 measurement of precision between plates of H7N9 hemagglutinin protein ELISA kit

2. Determination of accuracy of kit

The accuracy of the kit is detected by adding a recovery experiment, recombinant H7N9 hemagglutinin proteins with different concentrations are added into human serum, the concentrations of the added H7N9 hemagglutinin proteins are 0.15ng/mL, 0.3ng/mL and 0.6ng/mL, then the added samples are detected by using the prepared kit, the H7N9 hemagglutinin protein content in each sample is obtained by calculation according to a standard curve, the H7N9 hemagglutinin protein content in blank serum is subtracted to obtain the measured H7N9 hemagglutinin protein content, and the recovery rate is obtained by dividing the theoretical amount of the added protein. The results are shown in Table 3, and show that the addition recovery rate is 97-100%, which indicates that the prepared kit has good accuracy and the serum substrate does not have obvious interference on detection.

TABLE 3 determination of accuracy of H7N9 hemagglutinin protein ELISA kit

3. Minimum detection Limit determination of the kit

And (3) taking the sample diluent as a blank sample, repeating the sample for 20 times, detecting the blank sample by using the prepared kit, recording the absorbance value of each blank sample, and calculating the lowest detection limit of the kit by dividing 3 times of the blank standard deviation by the slope of the standard curve. The calculation shows that the lowest detection limit of the kit is 0.003ng/mL, which indicates that the kit has higher detection sensitivity.

4. Specific assay of the kit

The recombinant hemagglutinin proteins of different influenza virus subtypes and different strains of the H7N9 subtype are diluted to 50ng/mL and are detected by using an ELISA kit for H7N9 avian influenza hemagglutinin protein, and the result shows that the kit specifically recognizes the hemagglutinin proteins of the H7N9 and the H7N7 influenza viruses, and has no cross reaction with other main subtypes of the influenza A viruses including H1N1, H2N2, H3N2, H4N6, H5N1, H6N1, H8N4, H9N2, H10N8, H11N2, H12N5, H13N8, H15N8, H16N3, H18N11 and the hemagglutinin proteins of the influenza B viruses (Table 4), thereby proving that the kit has good specificity, can be used for supporting epidemiological research of the H7N9 influenza and has important practical application value for basic research of the influenza viruses.

TABLE 4 specificity assay of influenza hemagglutinin protein ELISA kit of H7N9

Influenza virus proteins of different subtypes	OD value
		H7N9(A/Anhui/1/2013)HAProtein	1.419
H7N9(A/Shanghai/1/2013) HA protein	1.6615
		H7N9(A/Hangzhou/1/2013) HA protein	1.4195
H7N9(A/Pigeon/Shanghai/S1069/2013) HA protein	1.5285
		H7N9(A/ZHejiang/1/2013) HA protein	1.314
H7N9(A/Shanghai/4664T/2013) HA protein	0.951
		H7N9(A/Shanghai/2/2013) HA protein	1.0475
H7N9(A/ZHejiang/DTID-ZJU10/2013) HA protein	1.467
		H7N7(A/Netherlands/219/03) HA protein	2.6365
Influenza B virus (B/Florida/4/2006) HA protein	0.002
		Influenza B (B/Brisbane/60/2008) HA protein	0.026
H1N1(A/Brisbane/59/2007) HA protein	-0.003
		H1N1(A/Puerto Rico/8/34) HA protein	0
H1N1(A/California/04/2009) HA protein	0.0025
		H2N2(A/Canada/720/2005) HA protein	-0.0005
H3N2(A/Brisbane/10/2007) HA protein	-0.001
		H3N2(A/Aichi/2/1968) HA protein	0.0485
H4N6(A/Swine/Ontario/01911-1/99) HA protein	0.002
		H5N1(A/Anhui/1/2005) HA protein	-0.0015
H5N1(A/chicken/VietNam/NCVD-016/2008) HA protein	0.055
		H6N1(A/northern shever/California/HKWF 115/2007) HA protein	0.001
H8N4(A/pintail duck/Alberta/114/1979) HA protein	-0.0025
		H9N2(A/Hong Kong/1073/99) HA protein	0.001
H10N8(A/Jiangxi-Donghu/346/2013) HA protein	0.008
		H11N2(A/duck/Yangzhou/906/2002) HA protein	-0.0025
H12N5 (A/green-bound protein/ALB/199/1991) HA protein	-0.0005
		H13N8(A/black-headed gun/Netherlands/1/00) HA protein	0.0015
H15N8(A/duck/AUS/341/1983) HA protein	0.0215
		H16N3(A/black-headed gun/Sweden/5/99) HA protein	0.003
H18N11(A/flat-faced bat/Peru/033/2010) HA protein	0.0235

Example 5 detection procedure of H7N9 influenza Virus hemagglutinin protein ELISA kit

1. Sample application

1) And taking out the coated ELISA plate and the freeze-dried standard substance, and adding 1mL of sample diluent to dissolve the standard substance. Left at room temperature for 20 minutes. Diluting the standard substance by 2 times from 1ng/mL for 7 points, and adding 100 mu L of each diluted standard substance into a 96-hole enzyme label plate;

2) taking a sample to be detected, adding 100 mu L/hole into the reaction hole, and incubating for 2 hours at room temperature;

3) the plate is washed 3 times by washing liquid, 200 mu L/hole, and the ELISA plate is patted dry.

2. Adding detection antibody

Diluting the rabbit polyclonal antibody marked by HRP to 0.5 mu g/mL by using a sample diluent, adding the diluted antibody into a reaction hole, incubating at room temperature for 1 hour, washing the plate for 3 times by using a washing solution, washing the plate for 200 mu L/hole, and patting the plate dry.

3. Color development

1) Adding 200 mu L of freshly prepared substrate color developing solution, reacting for 20 minutes at room temperature, and then adding 50 mu L of stop solution to stop the reaction;

2) and reading the light absorption value by a microplate reader at the wavelength of 450 nm.

4. Establishment of a Standard Curve

And establishing a standard curve (figure 1) by taking the concentration of the hemagglutinin protein standard as a horizontal coordinate and the OD value as a vertical coordinate, and calculating the content of the H7N9 hemagglutinin protein in the sample according to the measured OD value of the sample.

While the present invention has been described in connection with the present embodiments, it is not to be considered as limited in scope by the appended claims. In addition, various changes or modifications of the method, steps or conditions of the present invention, which are within the scope of the present invention as defined by the appended claims, will be made by those skilled in the art and these changes or modifications also fall within the scope of the present invention.

Sequence listing

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130 135 140

Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn

145 150 155 160

Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser

165 170 175

Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys

180 185 190

Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val Ala

195 200 205

Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro Pro Glu Leu Leu Gly

210 215 220

Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

225 230 235 240

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

245 250 255

Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val

260 265 270

Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile

275 280 285

Arg Val Val Ser Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly

290 295 300

Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile

305 310 315 320

Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val

325 330 335

Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser

340 345 350

Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu

355 360 365

Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Ala

370 375 380

Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val

385 390 395 400

Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met

405 410 415

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser

420 425 430

Pro Gly Lys

435

<210>10

<211>214

<212>PRT

<213> New Zealand big ear white rabbit (Oryctolagus cuniculus)

<400>10

Glu Leu Asp Leu Thr Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly

1 5 10 15

Gly Thr Val Thr Ile Ser Cys Gln Ser Ser Glu Ser Val Tyr Lys Asn

20 25 30

Asn Asn Leu Ala Trp Phe Gln Gln Lys Leu Gly Gln Pro Pro Thr Leu

35 40 45

Leu Ile Trp Asp Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu

65 70 75 80

Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Asp Tyr Ser Gly

85 90 95

Val Asn Tyr Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys Gly Asp

100 105 110

Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp Gln Val

115 120 125

Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr Phe Pro

130 135 140

Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr Thr Gly

145 150 155 160

Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr Tyr Asn

165 170 175

Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser His Lys

180 185 190

Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val Gln Ser

195 200 205

Phe Asn Arg Gly Asp Cys

210

Claims

1. An isolated antibody or antigen-binding fragment thereof against the hemagglutinin protein of an H7N9 influenza virus comprising a) a heavy chain variable region having a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively; and/or b) the light chain variable region having light chain CDR1, light chain CDR2, and light chain CDR3 of SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, comprising: a) a heavy chain variable region having the sequence of SEQ ID NO. 7 or at least 90%, 95%, 98% or 99% sequence identity thereto; and/or b) a light chain variable region having the sequence of SEQ ID NO 8 or at least 90%, 95%, 98% or 99% sequence identity thereto.

3. The antibody or antigen binding fragment thereof of claim 1 or 2, having a heavy chain amino acid sequence of SEQ ID No. 9 or at least 90%, 95%, 98% or 99% sequence identity thereto; the light chain amino acid sequence is SEQ ID NO 10 or at least 90%, 95%, 98% or 99% sequence identity thereto.

4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, which is a rabbit derived antibody.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, which is a monoclonal antibody.

6. An ELISA kit for detecting H7N9 influenza virus hemagglutinin protein, comprising:

1) an elisa plate coated with the H7N9 influenza virus hemagglutinin protein monoclonal antibody or antigen-binding fragment as described in any one of claims 1 to 5;

2) an enzyme-labeled H7N9 polyclonal antibody of influenza virus hemagglutinin protein, preferably a horseradish peroxidase-labeled rabbit polyclonal antibody.

7. The kit according to claim 6, wherein the concentration of the monoclonal antibody recognizing the hemagglutinin protein of the H7N9 influenza virus is 0.5-4 μ g/mL, preferably 0.5 μ g/mL; the concentration of the enzyme-labeled polyclonal antibody is 0.25-2. mu.g/mL, preferably 0.5. mu.g/mL.

8. The kit according to any one of claims 6 to 7, further comprising a H7N9 influenza virus hemagglutinin protein standard, a sample diluent, a washing solution, a substrate developing solution and a reaction stop solution;

preferably, the H7N9 influenza virus hemagglutinin protein standard is a recombinant expressed hemagglutinin protein; the sample diluent is a phosphate buffer solution containing 0.1 percent of bovine serum albumin; the washing solution is phosphate buffer solution containing 0.1% Tween; the substrate color developing solution consists of a color developing solution A and a color developing solution B, wherein the color developing solution A is hydrogen peroxide or carbamide peroxide, and the color developing solution B is tetramethyl benzidine; the reaction termination liquid is 2mol/L sulfuric acid.

9. The kit of any one of claims 6 to 8, wherein the enzyme-labeled rabbit polyclonal antibody against hemagglutinin protein of influenza virus H7N9 is prepared as follows: the recombinant H7N9 influenza virus hemagglutinin protein is used for immunizing a New Zealand big ear white rabbit by 0.5 mg/rabbit, subcutaneous multipoint immunization is carried out at intervals of 2-3 weeks, four times of immunization are carried out totally, blood is taken from heart, purified polyclonal antibody is obtained after affinity purification of protein A and H7N9 influenza virus hemagglutinin protein antigen, and the purified polyclonal antibody is marked by horseradish peroxidase.

10. The kit of any one of claims 6-9, comprising recombinant H7N9 influenza virus hemagglutinin protein as a standard for quantitative detection of H7N9 influenza virus hemagglutinin protein.

11. The kit of any one of claims 6 to 10, which specifically recognizes influenza a H7N9 and H7N7 hemagglutinin proteins, without cross-reacting with other major subtypes of influenza a virus, including H1N1, H2N2, H3N2, H4N6, H5N1, H6N1, H8N4, H9N2, H10N8, H11N2, H12N5, H13N8, H15N8, H16N3, H18N11, and hemagglutinin proteins of influenza b virus.