Anti-H9N2 subtype avian influenza virus paired monoclonal antibody and immunochromatography test paper and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a group of paired monoclonal antibodies for resisting H9N2 subtype avian influenza viruses and a surface enhanced Raman spectroscopy rapid detection test strip established based on the antibodies. The paired monoclonal antibody comprises a labeled antibody 1E9 and a capture antibody 3C10 which are prepared by taking hemagglutinin protein of the H9N2 subtype avian influenza virus as antigens and can be specifically combined with the H9N2 subtype avian influenza virus. According to the invention, an SERS (Surface Enhanced Raman Scattering) probe Au-coated Pt-4ATP is used as a marker and is coupled with a labeled antibody 1E9 to prepare a labeled probe, and the labeled probe is paired with a capture antibody 3C10 to establish an SERS immunochromatography test strip. The test strip has a colorimetric and Raman dual interpretation mode, can be used for qualitative and quantitative detection, has the advantages of strong specificity, high sensitivity, good repeatability and stability and the like, is simple and rapid in clinical detection operation, and provides reliable technical support for on-site rapid screening of subtype viruses.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a group of paired monoclonal antibodies against H9N2 subtype avian influenza virus, their immunochromatographic test strips, and their applications. Background Technology

[0002] Avian influenza is an acute infectious disease of poultry caused by avian influenza virus (AIV). The H9N2 subtype of avian influenza virus, in particular, poses a serious threat to the stable development of the global poultry farming industry and public health security due to its wide spread, rapid mutation rate, and ability to act as a gene fragment donor in viral reassortment. Establishing rapid, sensitive, and accurate on-site detection technologies is crucial for achieving early screening and control of the disease and reducing the risk of transmission.

[0003] Currently, the detection of H9N2 subtype avian influenza virus mainly includes the following categories: (1) Virus isolation and culture: as the gold standard, but the operation is complicated and takes 3-7 days, requiring professional laboratory conditions, which cannot meet the needs of rapid on-site detection; (2) Real-time fluorescence quantitative PCR: has high sensitivity, but relies on expensive instruments and professional operators, and is not suitable for on-site detection in farms, etc.; (3) Enzyme-linked immunosorbent assay (ELISA): the operation is relatively simple, but the detection cycle is about 2-4 hours, the accuracy is limited, and there is a risk of cross-contamination; (4) Colloidal gold strips: fast and convenient, but the sensitivity is low (the detection limit is usually ≥0.1 HAU), which can only detect qualitatively and is prone to false negative results.

[0004] Furthermore, in existing detection technologies, some antibodies target conserved proteins such as NP and M2e, leading to cross-reactivity between different subtypes and affecting detection specificity. The existing SERS-LFIA protocol, combining SERS technology with immunochromatography, suffers from insufficient affinity of paired antibodies and low specificity. Screening for highly specific and high-affinity monoclonal antibodies and combining SERS technology with immunochromatographic test strips to form SERS-LFIA promises to improve signal reading capabilities while maintaining the convenience of test strips, and to enable on-site reading and quantitative analysis using portable Raman spectroscopy. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a set of highly specific paired monoclonal antibodies against H9N2 subtype avian influenza virus, and SERS immunochromatographic test strips based on these antibodies.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A group of paired monoclonal antibodies against H9N2 subtype avian influenza virus, including marker antibody 1E9 and capture antibody 3C10;

[0008] The labeled antibody 1E9 includes a heavy chain variable region and a light chain variable region. The nucleotide sequence of the heavy chain variable region of 1E9 is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2. The nucleotide sequence of the light chain variable region of 1E9 is shown in SEQ ID NO.3, and the encoded amino acid sequence is shown in SEQ ID NO.4.

[0009] The capture antibody 3C10 includes a heavy chain variable region and a light chain variable region. The nucleotide sequence of the heavy chain variable region of 3C10 is shown in SEQ ID NO.5, and the encoded amino acid sequence is shown in SEQ ID NO.6. The nucleotide sequence of the light chain variable region of 3C10 is shown in SEQ ID NO.7, and the encoded amino acid sequence is shown in SEQ ID NO.8.

[0010] The heavy chain variable region and light chain variable region of the labeled antibody 1E9

[0011] The 1E9 heavy chain variable region backbone includes FR-H1, FR-H2, FR-H3, and FR-H4, with sequences shown in SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, and SEQ ID NO. 15, respectively; the 1E9 light chain variable region backbone includes FR-L1, FR-L2, FR-L3, and FR-L4, with sequences shown in SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, and SEQ ID NO. 22, respectively.

[0012] The capture antibody 3C10 includes a heavy chain variable region backbone and a light chain variable region backbone.

[0013] The 3C10 heavy chain variable region backbone includes FR-H1, FR-H2, FR-H3, and FR-H4, with sequences shown in SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, and SEQ ID NO.29, respectively; the 3C10 light chain variable region backbone includes FR-L1, FR-L2, FR-L3, and FR-L4, with sequences shown in SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, and SEQ ID NO.36, respectively.

[0014] The labeled antibody 1E9 also includes a heavy chain complementarity-determining region and a light chain complementarity-determining region.

[0015] The 1E9 heavy chain complementarity-determining region includes CDR-H1, CDR-H2, and CDR-H3, with sequences shown in SEQ ID NO.10, SEQ ID NO.12, and SEQ ID NO.14, respectively; the 1E9 light chain complementarity-determining region includes CDR-L1, CDR-L2, and CDR-L3, with sequences shown in SEQ ID NO.17, SEQ ID NO.19, and SEQ ID NO.21, respectively.

[0016] The capture antibody 3C10 includes a heavy chain complementarity-determining region (CDR) and a light chain complementarity-determining region (LCD). The heavy chain LCD of 3C10 includes CDR-H1, CDR-H2, and CDR-H3, with sequences shown in SEQ ID NO.24, SEQ ID NO.26, and SEQ ID NO.28, respectively. The light chain LCD of 3C10 includes CDR-L1, CDR-L2, and CDR-L3, with sequences shown in SEQ ID NO.31, SEQ ID NO.33, and SEQ ID NO.35, respectively.

[0017] The labeled antibody 1E9 and the capture antibody 3C10 were both prepared using the H9N2 subtype avian influenza virus HA protein expressed in a eukaryotic system as an immunogen, and were able to specifically bind to the HA protein.

[0018] An immunochromatographic test strip based on surface-enhanced Raman spectroscopy utilizing the aforementioned monoclonal antibody comprises a support substrate and an adsorption layer fixed on the support substrate; the adsorption layer, from the test end, consists of: a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent pad; the conjugation pad is coated with a labeled monoclonal antibody 1E9 containing the probe Au@Pt-4ATP; the probe Au@Pt-4ATP is a gold-core-platinum-shell core-shell nanoparticle, and the surface-modified 4-ATP serves as a Raman reporter molecule; the nitrocellulose membrane has a detection line T and a control line C, the detection line T is coated with monoclonal antibody 3C10, and the control line C is coated with SPA.

[0019] The preparation method of the probe Au@Pt-4ATP includes:

[0020] (1) Add 10 mL of 1% (w / v) chloroauric acid solution to 100 mL of boiling deionized water, stir for 5 min, then quickly add 10 mL of 1% (w / v) sodium citrate solution, stir at 100 °C for 15-20 min, cool and store away from light.

[0021] (2) Add 5 mL of 1% (w / v) chloroplatinic acid and 5 mL of 1% (w / v) ascorbic acid to the gold nanoparticle system, stir for 30 min to form Au@Pt core-shell nanoparticles;

[0022] (3) The surface of Au@Pt core-shell nanoparticles was modified with 4-ATP to obtain the probe Au@Pt-4ATP.

[0023] The conjugation pad is a nitrocellulose membrane, which is soaked in PBS buffer and then freeze-dried under vacuum. The PBS buffer contains 1.0% BSA and 5% sucrose. The sample pad is soaked in a pretreatment solution and then dried. The pretreatment solution consists of the following components: 20 mM Tris-HCl buffer, 1.0% BSA, 0.5% Tween 20, 0.5-1.0% NaCl, and 0.02% sodium azide.

[0024] The labeled antibody 1E9 was coupled with the probe Au@Pt-4ATP and then resuspended in a special buffer solution, which consisted of: 0.1% PEG4000, 0.5% BSA, 0.5% Tween 20, 0.5% S-17, 20% sucrose, and 20% trehalose.

[0025] The loading of the pre-coated labeled probe Au@Pt-4ATP-1E9 on the binding pad is 1.0 μL / cm.

[0026] The application of the paired monoclonal antibody in the preparation of immunochromatographic test strips or kits for detecting H9N2 subtype avian influenza virus.

[0027] The present invention has the following beneficial effects:

[0028] (1) The paired monoclonal antibodies of the present invention include labeling antibody 1E9 and capture antibody 3C10, both of which are prepared using hemagglutinin (HA) protein of H9N2 subtype avian influenza virus as antigen and can specifically bind to H9N2 subtype avian influenza virus.

[0029] (2) The titers of the paired monoclonal antibodies of the present invention are all greater than 1: 4.096 × 10 6 The affinity constant of the labeled antibody 1E9 is 4.58 × 10⁻⁶. 9 L / mol, the affinity constant of the capture antibody 3C10 is 2.94 × 10⁻⁶. 9 L / mol, with strong binding ability to HA protein.

[0030] (3) The test strip of the present invention uses the SERS probe Au@Pt4-ATP as a marker, which is conjugated with the above-mentioned labeled antibody 1E9 to prepare a labeled probe, which is then paired with the capture antibody 3C10 to establish a SERS immunochromatographic test strip. This test strip uses HA protein as a target to prepare paired monoclonal antibodies, avoiding cross-reactions between different subtypes of avian influenza viruses and other avian disease viruses (such as Newcastle disease virus and infectious bronchitis virus), and has high specificity;

[0031] (4) The test strip of the present invention utilizes the electromagnetic-chemical synergistic enhancement effect of the Au@Pt-4ATP probe, combined with a high-affinity paired monoclonal antibody, with a detection limit as low as 0.02 HAU and high sensitivity;

[0032] (5) The test strip of the present invention has good stability, the Au@Pt core-shell structure probe has excellent chemical stability, and the special treatment solution for the binding pad and the sample pad can improve the stability of the antibody.

[0033] (6) The test strip has both colorimetric and Raman interpretation modes, and can perform both qualitative and quantitative detection. It has advantages such as high specificity, high sensitivity, good repeatability and stability, high potential for clinical sample detection, and simple and fast operation, providing reliable technical support for rapid on-site screening of this subtype of virus. Attached Figure Description

[0034] Figure 1 The results of SDS-PAGE identification of the purified HA protein.

[0035] Where M is a 180kDa protein marker, and lane 1 is the purified HA protein.

[0036] Figure 2 The results are Western blot analysis of the purified HA protein.

[0037] Where M is a 180kDa protein marker, and lane 1 is the purified HA protein.

[0038] Figure 3 This is the result of viral antigen hemagglutination titer determination.

[0039] Figure 4 The results are from the determination of hemagglutination inhibition titer of monoclonal antibodies.

[0040] Figure 5 The results of SDS-PAGE identification of purified monoclonal antibodies 1E9 and 3C10 are shown.

[0041] Figure 6 This is a double reciprocal curve for determining the affinity constant of a monoclonal antibody.

[0042] Figure 7 This is a schematic diagram illustrating the structure and working principle of the SERS immunochromatographic test strip. Detailed Implementation

[0043] The specific implementation methods of this application are further described in detail below with reference to the embodiments.

[0044] Example 1: Expression and purification of HA protein from H9N2 subtype avian influenza virus

[0045] Gene optimization and vector construction: Referring to the HA protein sequence of H9N2 subtype avian influenza virus in GenBank, accession number PP474085.1, the transmembrane region was removed, the gene sequence was optimized according to the codon preference of HEK293F cells, and cloned into the pcDNA3.1 expression vector to construct the recombinant expression vector pcDNA3.1-HA;

[0046] Protein expression: After resuscitating HEK293F cells, they were cultured to a density of (3.0-4.0) × 10⁶ cells / year. 6 With cells / mL and a viability ≥90%, the recombinant vector was transfected into HEK293F cells using a transfection reagent (Lipofectamine 3000) and cultured at 37 ℃ and 5% CO2 with shaking at 120 rpm for 72 hours.

[0047] Protein purification: Cell culture supernatant was collected, filtered through 0.45 μm and 0.22 μm filters, and a protease inhibitor (PMSF, final concentration 1 mM) was added. Purification was performed using a Ni affinity chromatography column (HisTrap™ Excel). The column was equilibrated with Buffer A (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0) for 15 column volumes. Samples were loaded three times at a rate of 1 mL / min, and then equilibrated with Buffer A for another 15 column volumes. Finally, a gradient elution was performed with Buffer C (20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 8.0). The elution peaks were collected and analyzed by SDS-PAGE. Figure 1 ) and Western blot ( Figure 2 ) identification;

[0048] The results showed that the purified product had a single and clear band, proving that high-purity HA protein was obtained.

[0049] Example 2 Preparation of paired monoclonal antibodies

[0050] Animal immunization: The purified HA protein was emulsified with Freund's adjuvant and immunized subcutaneously at multiple sites on the back of 6-8 week old BALB / c mice (HA protein dose of 20 μg / mouse). The first immunization was with Freund's complete adjuvant, and the subsequent three booster immunizations were with Freund's incomplete adjuvant (2 weeks apart). Blood was collected from the tail vein 3 days after the last immunization, and the serum antibody titer was detected by indirect ELISA.

[0051] Cell fusion: Mouse spleen cells with the highest titer were used to fuse with SP2 / 0 myeloma cells at a ratio of 8:1 using 50% PEG 1500. The fused cells were then screened for hybridoma cells using HAT medium.

[0052] Subcloning and screening: Using HA protein as the coating antigen, positive hybridoma cells were screened by indirect ELISA. Subcloning was performed 2-3 times using the limiting dilution method to obtain hybridoma cell lines 1E9 and 3C10 that stably secrete monoclonal antibodies.

[0053] Ascites preparation and purification: BALB / c mice pretreated with paraffin (500 μL of sterile paraffin injected intraperitoneally, 1 week later) were injected intraperitoneally with hybridoma cells (2 × 10⁻⁶). 6 (each individual), ascites fluid was collected after 7 days and purified using the caprylic acid-ammonium sulfate method: the ascites fluid was mixed with an equal volume of PBS buffer, and saturated ammonium sulfate solution was added dropwise to a final concentration of 20%, allowed to stand for 30 min, centrifuged at 8000 rpm for 20 min, and the precipitate was discarded; saturated ammonium sulfate solution was added to the supernatant to a final concentration of 50%, allowed to stand for 30 min, centrifuged at 8000 rpm for 20 min, and the precipitate was collected; the precipitate was dissolved in PBS, saturated ammonium sulfate solution was added to a final concentration of 33%, allowed to stand for 30 min, centrifuged at 8000 rpm for 20 min, and the supernatant was discarded, and this process was repeated twice; the precipitate was dissolved in 5 mL of PBS, placed in a dialysis bag, dialyzed four times with PBS buffer at 4 ℃, centrifuged at 8000 rpm for 20 min, and the precipitate was discarded, with the supernatant yielding the purified product.

[0054] The purified product was identified by SDS-PAGE, and the results are as follows: Figure 5 As shown.

[0055] The left image shows the SDS-PAGE identification results of purified monoclonal antibody 1E9; the right image shows the SDS-PAGE identification results of purified monoclonal antibody 3C10. It can be seen that the bands of both antibodies are clear after purification of the ascites fluid, indicating that the obtained monoclonal antibodies have high purity.

[0056] Where M is a 180kDa protein marker, lane 1 is before ascites purification, and lane 2 is after ascites purification.

[0057] Example 3 Performance Identification of Monoclonal Antibodies

[0058] Titer assay: Indirect ELISA method. Antibodies were serially diluted from 1:1000 and incubated at 37 ℃ for 30 min. HRP-labeled goat anti-mouse IgG (1:5000 dilution) was incubated at 37 ℃ for 30 min. TMB was used for color development, and the OD450 value was read by a microplate reader. The results showed that the titers of both 1E9 and 3C10 antibodies were greater than 1:4.096×10. 6 ;

[0059] Hemagglutination inhibition titer assay: The antigen hemagglutination titer and antibody hemagglutination inhibition titer were determined using a hemagglutination test, with erythrocyte agglutination as the criterion. The purified virus was serially diluted 2-fold to 2... -10Even at this time, it can still induce erythrocyte aggregation, and its hemagglutination titer is 2. -10 ( Figure 3 Monoclonal antibodies 1E9 and 3C10 were serially diluted 2-fold to 2... -10 At that time, it can effectively inhibit H9N2 virus-induced erythrocyte agglutination, and this result is consistent with Figure 4 The hemagglutination inhibition dilution concentrations shown correspond perfectly, indicating that its hemagglutination inhibition titer is 2. -10 ;

[0060] Affinity assay: The affinity of monoclonal antibodies is usually expressed using the affinity constant "Ka" and is determined by ELISA. The procedure is as follows: Antigens at different dilutions are coated onto plates, i.e., one plate each at concentrations of 1 μg / mL and 2 μg / mL. Purified monoclonal antibodies are used as primary antibodies, serially diluted starting at 1:1000. The secondary antibody is HRP-labeled goat anti-mouse antibody (1:5000 dilution). The OD450 values ​​are read for each coating concentration.

[0061] The x-axis represents antibody concentration, and the y-axis represents OD450 value. The OD value at which the reaction curve flattens is taken as the 100% reading. Then, a double reciprocal curve is plotted with the reciprocal of the monoclonal antibody concentration and the reciprocal of the OD450 value as the x-axis and y-axis, respectively. The monoclonal antibody concentration corresponding to the OD450 value is calculated according to the formula, and its unit is converted to (mol / mL). The affinity constant is then calculated according to the formula.

[0062] Kaff=(n-1) / 2(n[Ab']t-[Ab]t)

[0063] n=[Ag]t / [Ag']t

[0064] [Ag]t corresponds to a high coating concentration, and [Ag′]t corresponds to a low coating concentration; [Ab′]t and [Ab]t represent the molar concentration of monoclonal antibody corresponding to 50% OD450 value. A double reciprocal curve is plotted. Figure 6 The affinity constants of monoclonal antibodies 1E9 and 3C10 were calculated to be 4.58 × 10⁻⁶. 9 L / mol, 2.94×10 9 L / mol.

[0065] Specificity identification: Cross-reactivity tests showed that monoclonal antibodies 1E9 and 3C10 did not cross-react with H3, H6, and H10 subtypes of avian influenza virus, as well as Newcastle disease virus, infectious bronchitis virus, and infectious bursal disease virus, indicating good specificity.

[0066] Example 4: Preparation of SERS probe Au@Pt-4ATP and conjugation with labeled antibody 1E9

[0067] Preparation of gold nanoparticles: Add 100 mL of deionized water to a 250 mL three-necked flask, heat to 100 ℃ and keep boiling under magnetic stirring, accurately add 10 mL of 1% (w / v) chloroauric acid solution, stir for 5 min and then quickly add 10 mL of 1% (w / v) sodium citrate solution, stir at 100 ℃ for 20 min, the solution color changes from pale yellow to wine red, cool and store in the dark to obtain wine red gold nanoparticles;

[0068] Preparation of Au@Pt core-shell nanoparticles: Take 50 mL of gold nanoparticle solution, add 5 mL of 1% (w / v) chloroplatinic acid solution and 5 mL of 1% (w / v) ascorbic acid solution, stir at room temperature for 30 min to form Au@Pt core-shell nanoparticles;

[0069] 4-ATP modification: The above Au@Pt core-shell nanoparticles were centrifuged at 8000 rpm for 10 min, washed twice with deionized water, concentrated to 1 mL, and 20 μL of 1 mM 4-ATP ethanol solution was added while stirring. The mixture was stirred and modified at room temperature for 1 h, centrifuged at 8000 rpm for 10 min, and resuspended in deionized water to obtain the Au@Pt-4ATP probe.

[0070] Coupled with labeled antibody 1E9: Adjust the pH of Au@Pt-4ATP probe solution to 8.5 with 0.25 M K2CO3. Mix 10 mL of this solution with 1 mL of 1 mg / mL 1E9 antibody and incubate at room temperature for 1 h. Add 10 mL of 10% PVP solution and continue incubation for 1 h. Centrifuge at 8000 rpm for 10 min, collect the precipitate, and resuspend it in 100 mL of dedicated buffer (0.1% PEG4000, 0.5% BSA, 0.5% Tween 20, 0.5% S-17, 20% sucrose, 20% trehalose) and store at 4℃ for later use.

[0071] Example 5: Preparation and Performance of SERS Immunochromatographic Test Strips

[0072] 1. The structure of the test strip is as follows: Figure 7 It includes a support base plate and an adsorption layer fixed on the support base plate. The adsorption layer consists of sample pad 1, conjugation pad 4, and absorbent pad 9 from the test end. 2 is the sample to be tested, 3 is the labeled antibody, 5 is the capture antibody, 6 is the detection T line, 7 is the quality control C line, and 8 is the SPA.

[0073] The steps for preparing the test strip are as follows:

[0074] (1) Sample pad: The glass fiber membrane was immersed in the pretreatment solution (20mM Tris-HCl, 1.0% BSA, 0.5% Tween 20, 0.8% NaCl, 0.02% sodium azide, pH 7.4) and dried at 37 ℃ for 2h;

[0075] (2) Binding pad: The nitrocellulose membrane was soaked in PBS buffer (pH 7.2) containing 1.0% BSA and 5% sucrose, and after vacuum freeze-drying, the Au@Pt-4ATP-1E9 labeled probe of Example 4 above was sprayed at a rate of 1.0 μL / cm and dried at 37 °C for 10 h;

[0076] (3) Nitrocellulose membrane: T line (3C10 antibody, 1 mg / mL) and C line (SPA, 1 mg / mL) were sprayed onto the nitrocellulose membrane using a spraying device at a spraying speed of 1.0 μL / cm, and dried at 37 ℃ for 10 h;

[0077] (4) Assembly: The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are overlapped (overlap width 2 mm) and fixed on the PVC base plate, covered with PET protective layer, and cut into 4 mm wide test strips with a programmable cutter. The test strips are sealed and stored at 4 ℃ to obtain the immunochromatographic test strips of the present invention.

[0078] 2. Detection principle and result interpretation

[0079] After the sample to be tested is added to the sample pad, the sample is chromatographically separated along the adsorption layer by capillary action. The target antigen in the sample binds to the labeled probe in the conjugate pad to form a complex. The complex is chromatographically separated to the detection line T and captured by the capture antibody 3C10, forming a sandwich complex and enriching the signal. The uncaptured labeled probe continues to chromatographically separated to the control line C and is captured by SPA.

[0080] The test strips are interpreted qualitatively by colorimetry: when the C line is visible, the test strip is valid; if the C line shows no signal, the test strip is invalid. If the T line is visible when the C line is visible, the result is positive; if the T line is not visible, the result is negative. Quantitative detection can also be achieved by reading the Raman signal of the detection line T using a portable Raman spectrometer.

[0081] 3. Performance Testing

[0082] Sensitivity: The immunochromatographic test strip obtained in this example was used for actual testing. The H9N2 virus stock solution was serially diluted 5 times, and the virus samples of the dilution gradient were tested on the test strip. The results showed that the limit of detection of the immunochromatographic test strip for H9N2 subtype avian influenza virus can reach 0.02 HAU.

[0083] Specificity: When the immunochromatographic test strips were used to detect H3, H6, and H10 subtypes of avian influenza virus, as well as Newcastle disease virus, infectious bronchitis virus, and infectious bursal disease virus, no cross-reactivity was observed, indicating that the test strips have high specificity.

[0084] Practical application testing: The test strip of this invention was used to test 100 poultry fecal swabs and pharyngeal swabs (50 positive and 50 negative samples, verified by RT-qPCR). The results were consistent with those of RT-qPCR, indicating that the test strip of this invention provides reliable test results.

[0085] The sequence in the text is as follows:

[0086] 1. Nucleotide sequence of the variable region of the 1E9 antibody heavy chain (SEQ ID NO.1)

[0087] GTGCAGCTGCAGCAGTCAGGGGCAGAACTTGTGAAATCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCCTCAACATTGAAGACTCCTATATGCACTGGGTGAACAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTA AATATGACCCGAATTTCCAGGTCAAGGCCACTATAACATCAGACACATCCTCCATCACAGCCTACCTGCAGCTCAGTAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGATCGGGAGACCCTTGGCCCTATGCTATGGACTTCTGGGGCCAAGGGACCACGGTC

[0088] 2. Amino acid sequence of the variable region of the 1E9 antibody heavy chain (SEQ ID NO.2)

[0089] VQLQQSGAELVKSGASVKLSCTASGLNIEDSYMHWVKQRPEQGLEWIGRIDPANGNTKYDPNFQVKATITSDTSSITAYLQLSSLTSEDTAVYYCARSGDPWPYAMDFWGQGTTV

[0090] 3. Nucleotide sequence of the variable region of the light chain of the 1E9 antibody (SEQ ID NO.3)

[0091] GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAGAGCCAGCGAAAGTGTTGATAATTATGGCATTAGTTTTATGAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAACCGAGGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATACTGCAATGTATTTCTGTCAGCAAAGTAAGGAGGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0092] 4. Amino acid sequence of the light chain variable region of the 1E9 antibody (SEQ ID NO.4)

[0093] DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNRGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPYTFGGGTKLEIK

[0094] 5. Nucleotide sequence of the heavy chain variable region of the 3C10 antibody (SEQ ID NO.5)

[0095] GTGCAGCTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTAACCAGCTATGGTATACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGACGAATATGGCCTGGTGGAAGCACAAATTATAATTCGGCTCTCATGTCCAGACTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAATAGTCTGCAAACTGATGACACAGCCATGTTCTACTGTGCCAGAGGGGAATATGGTAACTACTTCTATGTTATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCC

[0096] 6. Amino acid sequence of the variable region of the 3C10 antibody heavy chain (SEQ ID NO.6)

[0097] VQLQESGPGLVAPSQSLSITCTVSGFSLTSYGIHWVRQPPGKGLEWLGRIWPGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMFYCARGEYGNYFYVMDYWGQGTTVTVS

[0098] 7. Nucleotide sequence of the variable region of the 3C10 antibody light chain (SEQ ID NO.7)

[0099] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGGGAATATTCACAATTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCT TAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAACCCGAAGATTTTGGGAGTTATTTCTGTCAACATTTTTGGAGTATTCCGTACACGTTCGGAGGGGGGACCAACCTGGAAATAAAA

[0100] 8. Amino acid sequence of the variable region of the 3C10 antibody light chain (SEQ ID NO.8)

[0101] DIQMTQSPASSLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYFCQHFWSIPYTFGGGTNLEIK

[0102] 9. Amino acid sequence of the variable backbone region 1 (FR-H1) of the 1E9 antibody heavy chain (SEQ ID NO.9): VQLQQSGAELVKSGASVKLSCTAS

[0103] 10. 1E9 antibody heavy chain variable region complementarity-determining region 1 (CDR-H1) amino acid sequence (SEQ ID NO.10) GLNIEDSY

[0104] 11. Amino acid sequence of the variable backbone region 2 (FR-H2) of the 1E9 antibody heavy chain (SEQ ID NO.11) MHWVKQRPEQGLEWIGR

[0105] 12. 1E9 antibody heavy chain variable region complementarity-determining region 2 (CDR-H2) amino acid sequence (SEQ ID NO.12) IDPANGNT

[0106] 13. Amino acid sequence of the variable backbone region 3 (FR-H3) of the 1E9 antibody heavy chain (SEQ ID NO.13): KYDPNFQVKATITSDTSSITAYLQLSSLTSEDTAVYYC

[0107] 14. 1E9 antibody heavy chain variable region complementarity-determining region 3 (CDR-H3) amino acid sequence (SEQ ID NO.14) ARSGDPWPYAMDF

[0108] 15. Amino acid sequence of the FR-H4 (variable region 4) of the 1E9 antibody heavy chain (SEQ ID NO.15) WGQGTTV

[0109] 16. Amino acid sequence of the variable region backbone 1 (FR-L1) of the 1E9 antibody light chain (SEQ ID NO.16): DIVLTQSPASLAVSLGQRATISCRAS

[0110] 17. 1E9 antibody light chain variable region complementarity-determining region 1 (CDR-L1) amino acid sequence (SEQ ID NO.17) ESVDNYGISF

[0111] 18. Amino acid sequence of the variable region backbone 2 (FR-L2) of the 1E9 antibody light chain (SEQ ID NO.18): MNWFQQKPGQPPKLLIY

[0112] 19. 1E9 antibody light chain variable region complementarity-determining region 2 (CDR-L2) amino acid sequence (SEQ ID NO.19) AAS

[0113] 20. Amino acid sequence of the variable region 3 (FR-L3) of the 1E9 antibody light chain (SEQ ID NO.20)

[0114] NRGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFC

[0115] 21. Amino acid sequence of complementarity-determining region 3 (CDR-L3) of the 1E9 antibody light chain variable region (SEQ ID NO.21) QQSKEVPYT

[0116] 22. Amino acid sequence of the FR-L4 (variable region backbone region 4) of the 1E9 antibody light chain (SEQ ID NO.22): FGGGTKLEIK

[0117] 23. Amino acid sequence of the variable backbone region 1 (FR-H1) of the 3C10 antibody heavy chain (SEQ ID NO.23) VQLQESGPGLVAPSQSLSITCTVS

[0118] 24. Amino acid sequence of the complementarity-determining region 1 (CDR-H1) of the 3C10 antibody heavy chain variable region (SEQ ID NO.24) GFSLTSYG

[0119] 25. Amino acid sequence of the variable backbone region 2 (FR-H2) of the 3C10 antibody heavy chain (SEQ ID NO.25): IHWVRQPPGKGLEWLGR

[0120] 26. Amino acid sequence of complementarity-determining region 2 (CDR-H2) of the 3C10 antibody heavy chain variable region (SEQ ID NO.26) IWPGGST

[0121] 27. Amino acid sequence of the variable backbone region 3 (FR-H3) of the 3C10 antibody heavy chain (SEQ ID NO.27)

[0122] NYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMFYC

[0123] 28. Amino acid sequence of complementarity-determining region 3 (CDR-H3) of the 3C10 antibody heavy chain variable region (SEQ ID NO.28): ARGEYGNYFYVMDY

[0124] 29. Amino acid sequence of the 4th (FR-H4) backbone region of the 3C10 antibody heavy chain (SEQ ID NO.29) WGQGTTVTVS

[0125] 30. Amino acid sequence of the variable region backbone 1 (FR-L1) of the 3C10 antibody light chain (SEQ ID NO.30): DIQMTQSPASLSASVGETVTITCRAS

[0126] 31. Amino acid sequence of the complementarity-determining region 1 (CDR-L1) of the 3C10 antibody light chain variable region (SEQ ID NO.31) GNIHNY

[0127] 32. Amino acid sequence of the variable region backbone 2 (FR-L2) of the 3C10 antibody light chain (SEQ ID NO.32): LAWYQQKQGKSPQLLVY

[0128] 33. Amino acid sequence of the complementarity-determining region 2 (CDR-L2) of the 3C10 antibody light chain variable region (SEQ ID NO.33) NAK

[0129] 34. Amino acid sequence of the FR-L3 (variable region) backbone of the 3C10 antibody light chain (SEQ ID NO.34)

[0130] TLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYFC

[0131] 35. Amino acid sequence of complementarity-determining region 3 (CDR-L3) of the 3C10 antibody light chain variable region (SEQ ID NO.35) QHFWSIPYT

[0132] 36. Amino acid sequence of the FR-L4 (variable region backbone) of the 3C10 antibody light chain (SEQ ID NO.36): FGGGTNLEIK

Claims

1. A group of paired monoclonal antibodies against H9N2 subtype avian influenza virus, characterized in that, The paired monoclonal antibodies include labeled antibody 1E9 and capture antibody 3C10; The labeled antibody 1E9 includes a heavy chain variable region and a light chain variable region. The nucleotide sequence of the heavy chain variable region of 1E9 is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.

2. The nucleotide sequence of the variable region of the 1E9 light chain is shown in SEQ ID NO.3, and the encoded amino acid sequence is shown in SEQ ID NO.4; The capture antibody 3C10 includes a heavy chain variable region and a light chain variable region. The nucleotide sequence of the heavy chain variable region of 3C10 is shown in SEQ ID NO.5, and the encoded amino acid sequence is shown in SEQ ID NO.

6. The nucleotide sequence of the light chain variable region of 3C10 is shown in SEQ ID NO.7, and the encoded amino acid sequence is shown in SEQ ID NO.

8.

2. The paired monoclonal antibody according to claim 1, characterized in that, The heavy chain variable region and light chain variable region of the labeled antibody 1E9 The 1E9 heavy chain variable region backbone includes FR-H1, FR-H2, FR-H3, and FR-H4, with sequences shown in SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, and SEQ ID NO.15, respectively; the 1E9 light chain variable region backbone includes FR-L1, FR-L2, FR-L3, and FR-L4, with sequences shown in SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, and SEQ ID NO.22, respectively. The capture antibody 3C10 includes a heavy chain variable region backbone and a light chain variable region backbone. The 3C10 heavy chain variable region backbone includes FR-H1, FR-H2, FR-H3, and FR-H4, with sequences shown in SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, and SEQ ID NO.29, respectively; the 3C10 light chain variable region backbone includes FR-L1, FR-L2, FR-L3, and FR-L4, with sequences shown in SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, and SEQ ID NO.36, respectively.

3. The paired monoclonal antibody according to claim 1, characterized in that, The labeled antibody 1E9 also includes a heavy chain complementarity-determining region and a light chain complementarity-determining region. The 1E9 heavy chain complementarity-determining region includes CDR-H1, CDR-H2, and CDR-H3, with sequences shown in SEQ ID NO.10, SEQ ID NO.12, and SEQ ID NO.14, respectively; the 1E9 light chain complementarity-determining region includes CDR-L1, CDR-L2, and CDR-L3, with sequences shown in SEQ ID NO.17, SEQ ID NO.19, and SEQ ID NO.21, respectively. The capture antibody 3C10 includes a heavy chain complementarity-determining region (CDR) and a light chain complementarity-determining region (LCD). The heavy chain LCD of 3C10 includes CDR-H1, CDR-H2, and CDR-H3, with sequences shown in SEQ ID NO.24, SEQ ID NO.26, and SEQ ID NO.28, respectively. The light chain LCD of 3C10 includes CDR-L1, CDR-L2, and CDR-L3, with sequences shown in SEQ ID NO.31, SEQ ID NO.33, and SEQ ID NO.35, respectively.

4. The paired monoclonal antibody according to any one of claims 1-3, characterized in that, The labeled antibody 1E9 and the capture antibody 3C10 were both prepared using the H9N2 subtype avian influenza virus HA protein expressed in a eukaryotic system as an immunogen, and were able to specifically bind to the HA protein.

5. An immunochromatographic test strip based on surface-enhanced Raman spectroscopy using the monoclonal antibody of claim 1, characterized in that, The device comprises a support base plate and an adsorption layer fixed to the support base plate. The adsorption layer, from the test end, consists of: a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. The conjugate pad is coated with a labeled monoclonal antibody 1E9 containing the probe Au@Pt-4ATP. The probe Au@Pt-4ATP is a gold-core-platinum-shell core-shell nanoparticle with surface-modified 4-ATP serving as a Raman reporter molecule. The nitrocellulose membrane has a detection line T and a control line C. The detection line T is coated with a monoclonal antibody 3C10, and the control line C is coated with SPA.

6. The immunochromatographic test strip according to claim 5, characterized in that, The preparation method of the probe Au@Pt-4ATP includes: (1) Add 10 mL of 1% (w / v) chloroauric acid solution to 100 mL of boiling deionized water, stir for 5 min, then quickly add 10 mL of 1% (w / v) sodium citrate solution, stir at 100℃ for 15-20 min, cool and store away from light. (2) Add 5 mL of 1% (w / v) chloroplatinic acid and 5 mL of 1% (w / v) ascorbic acid to the gold nanoparticle system, stir for 30 min to form Au@Pt core-shell nanoparticles; (3) The surface of Au@Pt core-shell nanoparticles was modified with 4-ATP to obtain the probe Au@Pt-4ATP.

7. The immunochromatographic test strip according to claim 5, characterized in that, The conjugation pad is a nitrocellulose membrane, which is soaked in PBS buffer and then freeze-dried under vacuum. The PBS buffer contains 1.0% BSA and 5% sucrose. The sample pad is soaked in a pretreatment solution and then dried. The pretreatment solution consists of the following components: 20 mM Tris-HCl buffer, 1.0% BSA, 0.5% Tween 20, 0.5-1.0% NaCl, and 0.02% sodium azide.

8. The immunochromatographic test strip according to claim 5, characterized in that, After the labeled antibody 1E9 was conjugated with the probe Au@Pt-4ATP, it was resuspended in a special buffer solution, which consisted of: 0.1% PEG4000, 0.5% BSA, 0.5% Tween 20, 0.5% S-17, 20% sucrose, and 20% trehalose.

9. The immunochromatographic test strip according to claim 8, characterized in that, The loading of the pre-coated labeled probe Au@Pt-4ATP-1E9 on the binding pad is 1.0 μL / cm.

10. The use of the paired monoclonal antibody according to any one of claims 1-3 in the preparation of immunochromatographic test strips or kits for detecting H9N2 subtype avian influenza virus.

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