WO2021128948A1 - Anti-mhin2 protein antibody, and use thereof and kit containing same - Google Patents

Abstract

Provided in the present invention are an anti-mHI N2 protein antibody, and the use thereof and a kit containing same. The antibody contains a heavy chain and a light chain, wherein the heavy chain and the light chain contain three CDR regions, respectively, and wherein the amino acid sequences of the CDR regions of the heavy chain are respectively as shown in SEQ ID NOs: 1-3 or 7-9, and the amino acid sequences of the CDR regions of the light chain are respectively as shown in SEQ ID NOs: 4-6 or 10-12.

Description

Anti-mHI N2 protein antibody, application thereof and kit containing the same Technical field

The invention belongs to the field of biotechnology, and relates to a monoclonal antibody and its application.

Background technique

Staphylococcus aureus α-hemolysin (Hla) is an exotoxin, which is the most important pathogenic factor of Staphylococcus aureus infection. It is often caused by pathogenic Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus ( MRSA) is generated. The iron surface determinant B subunit (IsdB) is an important outer membrane anchoring protein of Staphylococcus aureus, which plays an important role in the process of assisting bacteria to absorb and metabolize heme iron, as well as bacterial adhesion and colonization. mHI _N2 and a molecular fusion E. coli expression of genetic engineering technology, the S. aureus -hemolysin nontoxic mutant α- (mHla _H35L) active fragment of factor B N2 (IsdB-N2) and iron surface subunit decision Molecular fusion and soluble expression of recombinant fusion protein.

Using recombinant mHI _{N2 protein as the immunogen, monoclonal antibodies against multiple active epitopes of mHI N2} obtained after immunization, screening and identification can be used to prepare specific therapeutic antibody drugs for immune prevention and treatment of Staphylococcus aureus infections and detection A kit for detecting _{mHI N2} antigen content in recombinant Staphylococcus aureus vaccine. No corresponding antibody currently exists.

Summary of the invention

Based on the above-mentioned reasons, the present invention provides an anti-mHI _N2 protein antibody, which can effectively detect the mHI _N2 protein-containing reagent, such as _{the content of mHI N2} protein in a vaccine, to solve the above-mentioned problems in the prior art.

In the first aspect, the present invention provides an _{antibody against mHI N2} protein, the antibody comprising a heavy chain and a light chain, the heavy chain and the light chain each comprising 3 CDR regions, wherein:

The amino acid sequence of the heavy chain CDR1 region is GYTFTSYW (SEQ ID NO:1);

The amino acid sequence of the heavy chain CDR2 region is INPTNGHT (SEQ ID NO: 2);

The amino acid sequence of the heavy chain CDR3 region is ARDGYDDGSWLAY (SEQ ID NO: 3); or

The amino acid sequence of the heavy chain CDR1 region is GFTFSSFG (SEQ ID NO: 7);

The amino acid sequence of the heavy chain CDR2 region is INSDSSNI (SEQ ID NO: 8);

The amino acid sequence of the heavy chain CDR3 region is ARSGTTVVQDH (SEQ ID NO: 9); and

The amino acid sequence of the light chain CDR1 region is QRIGTN (SEQ ID NO: 4);

The amino acid sequence of the light chain CDR2 region is YAS (SEQ ID NO: 5);

The amino acid sequence of the light chain CDR3 region is QQSNSWPYS (SEQ ID NO: 6); or

The amino acid sequence of the light chain CDR1 region is QDISNY (SEQ ID NO: 10);

The amino acid sequence of the light chain CDR2 region is YTS (SEQ ID NO: 11);

The amino acid sequence of the light chain CDR3 region is QQGNTLPWT (SEQ ID NO: 12).

Preferably, the antibody comprises a heavy chain and a light chain, wherein,

The amino acid sequence of the heavy chain CDR1 region is GYTFTSYW (SEQ ID NO:1);

The amino acid sequence of the heavy chain CDR2 region is INPTNGHT (SEQ ID NO: 2);

The amino acid sequence of the heavy chain CDR3 region is ARDGYDDGSWLAY (SEQ ID NO: 3);

The amino acid sequence of the light chain CDR1 region is QRIGTN (SEQ ID NO: 4);

The amino acid sequence of the light chain CDR2 region is YAS (SEQ ID NO: 5);

The amino acid sequence of the light chain CDR3 region is QQSNSWPYS (SEQ ID NO: 6); or

The amino acid sequence of the heavy chain CDR1 region is GFTFSSFG (SEQ ID NO: 7);

The amino acid sequence of the heavy chain CDR2 region is INSDSSNI (SEQ ID NO: 8);

The amino acid sequence of the heavy chain CDR3 region is ARSGTTVVQDH (SEQ ID NO: 9);

The amino acid sequence of the light chain CDR1 region is QDISNY (SEQ ID NO: 10);

The amino acid sequence of the light chain CDR2 region is YTS (SEQ ID NO: 11);

The amino acid sequence of the light chain CDR3 region is QQGNTLPWT (SEQ ID NO: 12).

Further preferably, the antibody is antibody 5A8, and its heavy chain and light chain sequences are respectively:

Heavy chain:

Light chain:

DILLTQSPAILSVSPGERVSFSCRASQRIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPYSFGGGTKLEI (SEQ ID NO: 14); or

The antibody is antibody 7G4, and its heavy chain and light chain sequences are:

Heavy chain:

Light chain:

Preferably, the antibody is an IgG antibody.

In the second aspect, the present invention provides a kit for detecting the _{content of mHI N2} protein, said kit comprising the antibody of the present invention. Preferably, the antibodies are antibodies 5A8 and 7G4.

Preferably, the kit also includes human IgG, lotion, substrate color development solution A (EDTA-Na, citric acid, glycerin, TMB), substrate color development solution B (sodium acetate, citric acid, 30% H ₂ O ₂ ), stop solution (2M H ₂ SO ₄ ) and BSA.

Preferably, the kit further includes an enzyme-linked plate and a sealing film.

Preferably, the kit also includes mHI _N2 protein standards.

More preferably, the antibody 5A8 is a detection antibody, and the antibody 7G4 is a coating antibody.

In the third aspect, the present invention provides a method for detecting the _{content of mHI N2} protein, including the use of antibody 5A8 and antibody 7G4, and double antibody sandwich method for ELISA detection.

Preferably, the method includes:

1) Use antibody 7G4 to coat the ELISA plate;

2) React the test sample with antibody 7G4;

3) Use antibody 5A8 for detection and color reaction, and measure the absorbance at 450nm;

4) Use the mHI _N2 protein standard to prepare a standard curve, and calculate the mHI _N2 protein content in the tested sample according to the standard curve.

Preferably, the mHI _N2 protein content detected by the method ranges from 0.046875 to 3 μg/ml.

The fourth aspect provides the application of the anti-mHI _N2 protein antibody of the present invention in detecting the content of _{mHI N2 protein.}

In the present invention, the _{sequence of the mHI N2} protein is:

GPLGSMADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKILVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDSGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQ LFMKTRNGSMKAAENFLDPNKASSLLSSGFSPDFATVITMDRKATKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTNGGGGSKMTDLQDTKYVVYESVENNESMMDAFVKHPIKTGMLNGKKYMVMETTNDDYWKDFMVEGQRVRTISKDAKNNTRTIIFPYVEGKTLYDAIVKVHVKTIDYDGQYHVRIVDKEAFTKANLE (SEQ ID NO: 17). For _{specific information of mHI N2} protein, including preparation and other content, refer to CN103695508A. _{The amino acid sequence of mHI N2} protein is shown in SEQ ID NO: 1 of the patent document. The entire content of CN103695508A is incorporated herein by reference.

The anti-mHI _N2 protein antibody of the _{present invention can specifically bind to mHI N2} protein, effectively detecting _{the content of mHI N2} protein, with high sensitivity, reaching 0.046875μg/mL, wide detection range, 0.046875-3μg/mL, which can meet vaccine quantification Testing requirements.

Description of the drawings

Figure 1: 1# mouse monoclonal hybridoma cell culture and screening results;

Figure 2: 2# mouse monoclonal hybridoma cell culture screening results;

Figure 3: Linear fitting results of standard products.

Detailed ways

The content of the present invention will be described below in conjunction with specific embodiments, but the scope of the present invention is not limited thereto.

If there is no special instructions, the reagents and instruments used in the following examples are all conventional reagents and instruments in the art, and can be obtained commercially; the methods used are all conventional experimental methods, and those skilled in the art can do nothing according to the content of the examples. Undoubtedly implement the program and obtain the corresponding results.

The present invention uses mHI _N2 recombinant protein as the immunogen to immunize mice. Through multiple cell fusions and screenings, multiple strains _{of mouse monoclonal antibodies that specifically recognize mHI N2} protein are obtained, and high-affinity mouse monoclonal antibodies are selected for ascites preparation. After protein purification, HRP labeling was performed separately.

After paired detection and optimization of conditions, the paired antibodies (coating antibody 7G4, detection antibody 5A8) of the kit were obtained, and the detection conditions and standard curve were determined. The detection sensitivity reached 0.046875 μg/mL, and the linear range was 0.046875-3 μg/mL. Meet the requirements for quantitative testing of vaccines.

Main reagent

Main instrument

name	factory	model
Pipette	Eppendorf	To
Pipette	Thermo Fisher Scientific	Twelve Ways
Constant temperature incubator	Beijing Yongguangming Medical Instrument Factory	DHP-420 type
Microplate Oscillator	Zhongyi Guoke Co., Ltd.	SC60-4
Plate washer	Beijing Tuopu Analytical Instrument Co., Ltd.	DEM-3 type
Microplate reader	Thermo Scientific	Multiskan Mk3

Experimental animal

Balb/C mice: SPF (Specific Pathogen Free) Balb/C mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

Buffer preparation

The substrate color developing solution A (A solution) was prepared as follows: EDTA-Na 0.2g, citric acid 0.95g, glycerol 50ml, TMB (dissolved with DMSO to 10mg/ml and then added) 0.2g, dH ₂ O 500ml.

The substrate color developing solution B (B solution) was prepared as follows: sodium acetate 13.6 g, citric acid 1.6 g, H ₂ O ₂ (30%) 0.3 ml, and dH ₂ O 500 ml.

The formula of 20× lotion is: NaCl 818g, Na ₂ HPO ₄ .12H ₂ O 358g, KCl 205g, H ₂ O is fixed to 5L, and the pH value is 7.4-7.6.

The formula of the glycine eluent with pH 2.7 is: 1.9 g of glycine, _{constant volume of H 2} O to 500 mL, and pH value of 2.68～2.72.

The formula of the pH 1.9 glycine eluent is: 1.9 g of glycine, _{constant volume of H 2} O to 500 mL, and pH value of 1.88 to 1.92.

Example 1: Preparation and screening of mouse monoclonal antibodies that _{specifically recognize mHI N2 protein}

The mHI _N2 recombinant protein (1.4mg/mL) was mixed with the adjuvants CFA and AD11.15 to prepare the immunogen, that is, the recombinant protein was first mixed with CFA in a volume ratio of 5:6, as immunogen A, and the recombinant protein was then mixed with AD11.15 was mixed in a volume ratio of 1:1 as immunogen B. Immunogen A is the primary immunization, and immunogen B is the booster immunization. Three mice were immunized intramuscularly, and the tail blood of the mice was drawn on the 14th day after immunization, and the antibody titer of the tail blood was evaluated by indirect ELISA method.

Use mHI _N2 recombinant protein to coat the ELISA plate (1μg/mL), add 100μL to each well, react overnight at 4°C; wash the plate 3 times with PBS solution, block with 5% milk-PBS at room temperature for 1hr; then wash with PBS solution After plate 1 time, add mouse tail blood diluted with 5% milk-PBS solution, react at room temperature for 1hr; then wash the plate 3 times with PBS solution and pat dry, add 1:2000 diluted HRP-labeled sheep Anti-mouse IgG (Fc) secondary antibody, react at room temperature for 1 hr; wash the plate with PBS solution 5 times, pat dry, add equal volumes of solution A and solution B, and react for 20 minutes at room temperature in the dark; then add 50 μL stop solution ( 2M H ₂ SO _{4 ), read the OD 450} value on the microplate reader after mixing. Table 1 shows the results of indirect ELISA evaluation of mouse tail blood on the 14th day after immunization.

Table 1: Evaluation of antibody titer in mouse tail blood on day 14 after immunization

It can be seen from the results that the antibody titer of 3# mouse tail blood recognizing HI recombinant protein is the highest, reaching 1:50000, followed by the antibody titer of 1# mouse. Therefore, 1# and 3# mice were selected for cell fusion with myeloma cells SP2/0. After fusion, 564 and 752 monoclonal hybridoma cells were picked and cultured, and the positive clones were screened by conventional ELISA. The screening criterion was that the OD value was 2.1 times the NC value. The screening results are shown in Figure 1 and Figure 2, respectively.

From the screening results, 35 positive clones were selected for the screening confirmation experiment. The experimental process is as follows: _{100ng/well of mHI N2} protein was coated, and the supernatant of each clone was diluted 1:1 as the primary antibody, and then goat anti-mouse was added. The secondary antibody retains the clones that are still positive, and the results are shown in Table 2.

Table 2: Confirmation results of positive clones

Clone number	1D3	1E2	2A6	2A9	2B5	2B6	2G7	2H1	2H2	3C11	NC	NC
OD 450	1.485	0.708	0.411	0.282	0.387	0.679	0.814	0.277	1.777	0.706	0.12	0.179
Clone number	3F8	4G10	4A12	4C3	4D11	4D12	4H1	4H2	5A7	5A8	NC	NC
OD 450	1.013	0.722	0.187	0.28	3.38	0.545	0.129	999	0.714	3.238	0.104	0.11
Clone number	5A9	6A7	6E1	6E4	7F10	7F9	7G4	7G8	8C4	8D11	NC	NC
OD 450	0.458	1.567	0.306	1.657	0.383	0.278	3.143	0.441	0.812	1.667	0.098	0.099
Clone number	8E3	8E4	8E5	8E7	8E10	NC	NC	NC	NC	NC	NC	PC
OD 450	0.69	0.401	0.596	1.577	0.79	0.107	0.106	0.1	0.096	0.09	0.101	2.312

Note: NC is a negative control, 5% milk-PBS; PC is a positive control, 1# serum; an OD ₄₅₀ value of 999 means that the OD ₄₅₀ value is greater than 3.5.

From the re-screening results of positive clones listed in Table 2, it can be seen that the 4 positive clones with the highest affinity are 4D11, 4H2, 5A8, and 7G4.

Example 2: Preparation of purified antibody

1. Ascites preparation

The 4 positive clones (4D11, 4H2, 5A8, 7G4) obtained above were subjected to ascites preparation. Approximately 10 were injected into ¹⁰⁷ cells injected intraperitoneally two previously Balb IFA adjuvant / C mice, and then about 10 days later, ascites were generated for each positive clone, then 4 ℃, 12000rpm centrifuged 15min, charged The supernatant is used for the next step of protein G purification.

2. Purification of mouse monoclonal antibodies

Take 1mL of the column material coupled with protein G and add it to an empty column. After washing with PBS solution, dilute 2mL of ascites with 8mL of PBS and apply it to the column, and then reload the flow-through solution onto the column again; then use pH 2.7 The glycine eluate is eluted, and each 1mL eluate is collected in one tube (100μL neutralization solution is added in advance, and the composition of the neutralization solution is 1M Tris-HCl, 10mM EDTA, 1.5M NaCl, pH8.0-8.38). Collect 5 tubes; then use pH 1.9 glycine eluent for elution, collect one tube per 1 mL of eluate (pre-add 300 μL neutralization solution), collect 3 tubes; then perform elution on each tube separately OD ₂₈₀ reading, _{mix the eluates with OD 280} greater than 0.5, and then re-measure the OD _{280 of the} mixture after mixing, and calculate the antibody concentration according to the coefficient of 1.4; antibody concentration = OD ₂₈₀ /1.4.

The G protein purified mouse monoclonal antibody was evaluated using the indirect ELSIA method. The results are shown in Table 3.

Table 3: Evaluation of purified mouse monoclonal antibodies

Note: An OD ₄₅₀ value of 999 means that the OD ₄₅₀ value is greater than 3.5; NC is a negative control; PC is the respective cell supernatant.

It can be seen from the results in Table 3 that the detection sensitivity of the four purified antibodies is between 0.0005 and 0.005 μg/mL.

Example 3: Antibody pair screening

The four purified antibodies were labeled with HRP using the sodium periodate oxidation method, and _{the binding ability of the labeled antibodies with the mHI N2} recombinant protein was evaluated by ELISA. The results are shown in Table 4.

Table 4: Evaluation results of HRP-labeled antibodies

It can be seen from Table 4 that _{the binding ability of HRP-labeled antibody to mHI N2} protein is not significantly reduced compared with the antibody before labeling. The next pairing experiment can be performed (see Table 5), and the selection can be used The matching antibody for mHI _{N2 protein detection.}

Table 5: Pairwise experiment of antibodies

According to the matching results in Table 5 and the comprehensive evaluation of the yields of the four purified mouse monoclonal antibodies, the best matching results were selected to select mouse monoclonal antibody 7G4 as the coating antibody and mouse monoclonal antibody 5A8 as the detection antibody.

After sequencing analysis, the heavy chain sequence of antibody 5A8 is shown in SEQ ID NO: 13, the light chain sequence is shown in SEQ ID NO: 14; the heavy chain sequence of antibody 7G4 is shown in SEQ ID NO: 15, and the light chain sequence is shown in SEQ ID NO: 16. Further analysis, the sequence of the 3 CDR regions of the heavy chain of antibody 5A8 are shown in SEQ ID NO: 1 to 3 respectively, and the sequences of the 3 CDR regions of the light chain are shown in SEQ ID NO: 4 to 6 respectively; the sequence of the heavy chain of antibody 7G4 The sequences of the three CDR regions are shown in SEQ ID NOs: 7 to 9 respectively, and the sequences of the three CDR regions of the light chain are shown in SEQ ID NOs: 10 to 12, respectively.

Example 4: Preparation and use of _{mHI N2 protein content detection kit}

Based on the above results, 5A8 and 7G4 are selected as antibody preparation kits. The reagents and items in the kit can include:

1. Antibody 5A8 coated enzyme-linked plate, 8 wells × 12 strips

2. Enzyme conjugate (antibody 7G4) 120μl×1 tube (for 1:100 dilution)

3.BSA 3g/bag×1bag

4. Human IgG 10μg/ml, 120μl×1 tube (for 1:100 dilution)

5. 20×Lotion 50ml×1 bottle

6. Substrate color solution A (EDTA-Na, citric acid, glycerin, TMB) 7ml×1 bottle

7. Substrate color developing solution B (sodium acetate, citric acid, 30% H ₂ O ₂ ) 7ml×1 bottle

8. Stop solution (2M H ₂ SO ₄ ) 7ml×1 bottle

9. Sealing film 2 sheets

10. Instruction manual 1 copy

In addition, a single-component mHI _N2 protein was used as a standard product.

Kit operation steps

1. Equilibrium: Move the required reagents to room temperature (18～25℃) and equilibrate for 30 minutes.

2. Liquid preparation:

①.1× Washing Solution: Take 1 bottle of 20× Washing Solution, dilute to 1000ml with deionized water, mix well and set aside.

②. Enzyme conjugate diluent (3% BSA): completely dissolve BSA (3g/bag) into 100ml ① 1× washing solution, and mix thoroughly for later use.

③. Enzyme conjugate working solution: take the required enzyme conjugate and dilute it with the enzyme conjugate diluent prepared in ②, and mix thoroughly for later use.

④. Standard product and test product diluent: take an appropriate amount of human IgG, dilute the human IgG 100 times with the diluent ② and use it for later use.

3. Adding samples: Take out the enzyme-linked plate coated with antibody 5A8 from the sealed bag, dilute the standard and the sample to be tested, add 100μl to each well, and set a negative control at the same time. After sealing the plate with a sealing film, incubate at 37°C for 60 minutes.

4. Washing: Discard the liquid in each well, fill the micropores with washing liquid, and discard the liquid in the well after standing for 30 seconds; repeat 3 times, and pat dry on facial tissue after the last wash.

5. Add enzyme: add 100μl of enzyme conjugate working solution to each well, seal the plate with a sealing film and incubate at 37°C for 60 minutes.

6. Washing: Discard the liquid in each well, fill the micropores with washing liquid, and discard the liquid in the well after standing for 30 seconds; repeat 3 times, pat dry on facial tissue after the last wash.

7. Color development: add 50μl of Substrate Chromogenic Solution A and 50μl of Substrate Chromogenic Solution B to each well, shake and mix gently, and place in the dark at room temperature for 10 minutes.

8. Determination: add 50μl of stop solution to each well and mix slightly. Choose the microplate reader wavelength 450nm, and measure the absorbance value (OD value) of each well.

The logX-LogY fitting method is used to linearly fit the results of the standard products to obtain a standard curve. The results are shown in Table 6 and Figure 3.

Table 6: Standard curve

It can be seen from the above results that the detection sensitivity of the kit reaches 0.046875μg/mL, and the linear range is 0.046875-3μg/mL.

Example 5: Kit detection experiment

_{Use this kit to detect the protein content of mHI N2} antigen in the 20180903 batch of Staphylococcus aureus vaccine. The detection steps are as follows:

1. Sample pretreatment: Take 20 finished vaccines, mix 600μL of each vaccine in a 1.5mL EP tube, centrifuge at 5000rpm for 10min, remove 300μL of supernatant, add 300μL of 2mol/L Na ₂ CO ₃ solution and mix well , After the solution is clear, centrifuge to take appropriate amount of supernatant for later use;

2. Detect the sample according to the operation steps of Example 4;

3. The results of standard products are linearly fitted using logX-LogY fitting method to obtain a standard curve. Substitute the absorbance value (OD value) of the test sample into the standard curve to calculate the sample concentration. The test results are as follows:

Table 7: Results of detection of mHIN2 protein content by the kit

From the above results, it can be seen that the mHI _N2 protein content in the 20 samples tested by this kit is greater than 42.75μg/mL, and the RSD is 5.66% and less than 10%, indicating that the kit can be used for mHI _N2 protein detection. Quantitative detection and good applicability.

Claims (10)

1. An antibody against mHI _N2 protein, said antibody comprising a heavy chain and a light chain, the heavy chain and light chain each comprising 3 CDR regions, wherein:

   The amino acid sequence of the heavy chain CDR1 region is GYTFTSYW (SEQ ID NO:1);

   The amino acid sequence of the heavy chain CDR2 region is INPTNGHT (SEQ ID NO: 2);

   The amino acid sequence of the heavy chain CDR3 region is ARDGYDDGSWLAY (SEQ ID NO: 3); or

   The amino acid sequence of the heavy chain CDR1 region is GFTFSSFG (SEQ ID NO: 7);

   The amino acid sequence of the heavy chain CDR2 region is INSDSSNI (SEQ ID NO: 8);

   The amino acid sequence of the heavy chain CDR3 region is ARSGTTVVQDH (SEQ ID NO: 9); and

   The amino acid sequence of the light chain CDR1 region is QRIGTN (SEQ ID NO: 4);

   The amino acid sequence of the light chain CDR2 region is YAS (SEQ ID NO: 5);

   The amino acid sequence of the light chain CDR3 region is QQSNSWPYS (SEQ ID NO: 6); or

   The amino acid sequence of the light chain CDR1 region is QDISNY (SEQ ID NO: 10);

   The amino acid sequence of the light chain CDR2 region is YTS (SEQ ID NO: 11);

   The amino acid sequence of the light chain CDR3 region is QQGNTLPWT (SEQ ID NO: 12).
2. The antibody of claim 1, which comprises a heavy chain and a light chain, wherein:

   The amino acid sequence of the heavy chain CDR1 region is GYTFTSYW (SEQ ID NO:1);

   The amino acid sequence of the heavy chain CDR2 region is INPTNGHT (SEQ ID NO: 2);

   The amino acid sequence of the heavy chain CDR3 region is ARDGYDDGSWLAY (SEQ ID NO: 3);

   The amino acid sequence of the light chain CDR1 region is QRIGTN (SEQ ID NO: 4);

   The amino acid sequence of the light chain CDR2 region is YAS (SEQ ID NO: 5);

   The amino acid sequence of the light chain CDR3 region is QQSNSWPYS (SEQ ID NO: 6); or

   The amino acid sequence of the heavy chain CDR1 region is GFTFSSFG (SEQ ID NO: 7);

   The amino acid sequence of the heavy chain CDR2 region is INSDSSNI (SEQ ID NO: 8);

   The amino acid sequence of the heavy chain CDR3 region is ARSGTTVVQDH (SEQ ID NO: 9);

   The amino acid sequence of the light chain CDR1 region is QDISNY (SEQ ID NO: 10);

   The amino acid sequence of the light chain CDR2 region is YTS (SEQ ID NO: 11);

   The amino acid sequence of the light chain CDR3 region is QQGNTLPWT (SEQ ID NO: 12).
3. The antibody of claim 2, wherein:

   The antibody is antibody 5A8, and its heavy chain and light chain sequences are:

   Heavy chain:

   VQLQESGAELVKPGASVKLSCKASGYTFTSYWIHWVKQRPGQGLEWIGEINPTNGHTNFNEKFKNRATLTVDKSSNTAYMQLSILTSEDSAVYYCARDGYDDGSWLAYWGQGTTVTVSS(SEQ ID NO: 13);

   Light chain:

   DILLTQSPAILSVSPGERVSFSCRASQRIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPYSFGGGTKLEI (SEQ ID NO: 14); or

   The antibody is antibody 7G4, and its heavy chain and light chain sequences are:

   Heavy chain:

   VKLQQSGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYINSDSSNIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARSGTTVVQDHWGQGTTVTVSS(SEQ ID NO: 15);

   Light chain:

   DIVLTQTPSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSKLHSGVPSRFSGSGSGTDYSLTISSLEQEDIATYFCQQGNTLPWTFGGGTKLEIK (SEQ ID NO: 16).
4. The antibody according to any one of claims 1 to 3, wherein the antibody is an IgG antibody.
5. A kit for detecting the _{content of mHI N2} protein, the kit comprising the antibody according to any one of claims 1 to 4.
6. The kit according to claim 5, further comprising human IgG, washing solution, substrate color developing solution A, substrate color developing solution B, stop solution and BSA.
7. The kit according to claim 6, wherein the kit further comprises an enzyme-linked plate and a sealing film and/or mHI _N2 protein standard.
8. A method for detecting the _{content of mHI N2} protein includes the use of antibody 5A8 and antibody 7G4, and the double antibody sandwich method for ELISA detection.
9. The method according to claim 8, comprising:

   1) Use antibody 7G4 to coat the ELISA plate;

   2) React the test sample with antibody 7G4;

   3) Use antibody 5A8 for detection and color reaction, and measure the absorbance at 450nm;

   4) Use mHI _N2 protein standard to prepare a standard curve, and calculate the mHI _N2 protein content in the tested sample according to the standard curve;

   Preferably, the mHI _N2 protein content detected by the method ranges from 0.046875 to 3 μg/ml.
10. The use of the anti-mHI _N2 protein antibody of any one of claims 1 to 4 in detecting the content of _{mHI N2 protein.}

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