Monoclonal antibody aiming at cat Fel d7 and application thereof

Abstract

The invention belongs to the technical field of biological immunity, and particularly relates to a monoclonal antibody for cat Fel d7 and application of the monoclonal antibody. By screening the high-affinity monoclonal antibody aiming at the Fel d7, the detection of the Fel d7 as low as nanogram level (ng / mL) is realized, meanwhile, the cross reaction with other proteins in a cat body is avoided by utilizing the high uniformity of the monoclonal antibody and only recognizing a certain specific epitope, so that the detection specificity is improved, and compared with a polyclonal antibody, the Fel d7 monoclonal antibody is more stable and stronger in specificity; according to the present invention, the monoclonal antibody is adopted to detect the cat Fel d7, the interference of the non-specific reaction is avoided, the double-antibody sandwich ELISA kit formed based on the monoclonal antibody and used for detecting the cat Fel d7 is adopted as the coating antibody and the detection antibody so as to achieve the synergistic effect to amplify the detection signal, and the biotin-streptavidin-HRP three-stage signal amplification system is adopted so as to achieve the high-precision detection of the Fel d7;

Description

Technical Field

[0001] This invention belongs to the technical field of bioimmunology, specifically relating to a monoclonal antibody against feline Fel d7 and its applications. Background Technology

[0002] Cat allergy is one of the fastest-growing allergic diseases worldwide, affecting approximately 10%-30% of the population. Among patients with rhinoconjunctivitis and / or asthma due to exposure to cats, approximately 31-54% of cat allergy sufferers are allergic to Fel d7.

[0003] Fel d7 is a member of the lipid transporter family, possessing typical lipid transporter domains. It has a molecular weight of approximately 17.5 kDa and is primarily secreted by the salivary glands, submandibular glands, and sebaceous glands of cats, distributed in saliva, dander, and fur. Fel d7 differs significantly in structure from other cat allergens (such as Fel d1), thus the likelihood of cross-reactivity is low. This means that some people allergic to Fel d1 may be insensitive to Fel d7, and vice versa.

[0004] Fel d7 protein is extremely small and easily adheres to clothing, furniture, and airborne particles, triggering symptoms through inhalation or contact. Therefore, rapid detection of Fel d7 levels in the environment or in cats is crucial for allergy monitoring, pet health management, and drug development.

[0005] Currently, ELISA (Enzyme-Linked Immunosorbent Assay) has become the mainstream technique for detecting Fel d7 due to its high sensitivity, ease of operation, and high throughput. ELISA is mainly divided into direct methods, indirect methods, double-antibody sandwich methods, and competitive methods. Direct ELISA has fewer steps and is faster, but has lower sensitivity and higher background interference; indirect ELISA has high sensitivity and low cost, but carries the risk of cross-reactivity and has a longer experimental cycle; competitive ELISA is suitable for impure samples and has high data stability, but is complex to operate and has lower sensitivity. Double-antibody sandwich ELISA, due to its high specificity and sensitivity, has become the ideal method for detecting Fel d7.

[0006] No relevant information was found for existing Fel d7 detection kits in China or abroad. With the number of pet cats in China reaching 69.8 million (2023 data) and the adoption rate continuing to rise, the development of highly sensitive and specific double-sandwich ELISA kits is of great significance for filling the domestic market gap, allergy diagnosis and desensitization treatment, development of low-allergenic products (such as low-allergenic cat food), and environmental and public health monitoring. Summary of the Invention

[0007] To address the above-mentioned problems, the present invention aims to provide a monoclonal antibody against feline Fel d7 and its application.

[0008] The technical content of this invention is as follows: The present invention also provides a monoclonal antibody or antigen-binding fragment thereof targeting feline Fel d7, wherein the monoclonal antibody or antigen-binding fragment thereof comprises: A monoclonal antibody: The heavy chain variable region has three heavy chain complementarity-determining regions as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, or sequences having at least 95%, 96%, 97%, 98%, or 99% homology with them, respectively; And light chain variable regions, which have three light chain complementarity-determining regions as shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or sequences having at least 95%, 96%, 97%, 98% or 99% homology with them, respectively; And / or, B monoclonal antibodies: The heavy chain variable region has three heavy chain complementarity-determining regions as shown in SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 or sequences having at least 95%, 96%, 97%, 98% or 99% homology with them, respectively; And a light chain variable region, which has three light chain complementarity-determining regions as shown in SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22 or sequences having at least 95%, 96%, 97%, 98% or 99% homology with them.

[0009] The monoclonal antibody or its antigen-binding fragment includes: A monoclonal antibody: The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.7 or a sequence having at least 95%, 96%, 97%, 98% or 99% homology thereto; and the light chain variable region has an amino acid sequence as shown in SEQ ID NO.8 or a sequence having at least 95%, 96%, 97%, 98% or 99% homology thereto; and / or B monoclonal antibodies: The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.23 or a sequence having at least 95%, 96%, 97%, 98% or 99% homology thereto; and the light chain variable region has an amino acid sequence as shown in SEQ ID NO.24 or a sequence having at least 95%, 96%, 97%, 98% or 99% homology thereto.

[0010] The present invention also provides a nucleotide sequence encoding the above-described monoclonal antibody or its antigen-binding fragment; The nucleotide sequence includes: Nucleotides encoding the heavy chain complementarity-determining region (CMR) of the amino acid sequence as shown in SEQ ID NO. 1-3, DNA molecules or their corresponding mRNA molecules containing the nucleotide sequences shown in SEQ ID NO. 9-11; nucleotides encoding the light chain CMR of the amino acid sequence as shown in SEQ ID NO. 4-6, DNA molecules or their corresponding mRNA molecules containing the nucleotide sequences shown in SEQ ID NO. 12-14; and / or, Nucleotides encoding the heavy chain complementarity-determining region as shown in SEQ ID NO. 17~19, DNA molecules or their corresponding mRNA molecules containing nucleotide sequences as shown in SEQ ID NO. 25~27; nucleotides encoding the light chain complementarity-determining region as shown in SEQ ID NO. 20~22, DNA molecules or their corresponding mRNA molecules containing nucleotide sequences as shown in SEQ ID NO. 28~30. Also includes: Nucleotides encoding the heavy chain variable region with the amino acid sequence shown in SEQ ID NO. 7, DNA molecules containing the nucleotide sequence shown in SEQ ID NO. 15 or their corresponding mRNA molecules; and nucleotides encoding the light chain variable region with the amino acid sequence shown in SEQ ID NO. 8, DNA molecules containing the nucleotide sequence shown in SEQ ID NO. 16 or their corresponding mRNA molecules; and / or, Nucleotides encoding the heavy chain variable region as shown in SEQ ID NO. 23, DNA molecules containing the nucleotide sequence shown in SEQ ID NO. 31 or their corresponding mRNA molecules; and nucleotides encoding the light chain variable region as shown in SEQ ID NO. 8, DNA molecules containing the nucleotide sequence shown in SEQ ID NO. 32 or their corresponding mRNA molecules.

[0011] Furthermore, the present invention also provides a polynucleotide encoding a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect above. This polynucleotide is not limited to any particular method of its production and can be obtained using genetic engineering recombination techniques or chemical synthesis methods.

[0012] Furthermore, the present invention also provides a nucleic acid construct comprising a polynucleotide as described in the second aspect above, and optionally, at least one expression regulatory element operatively linked to the polynucleotide.

[0013] Furthermore, the present invention also provides a recombinant vector comprising the polynucleotide as described in the second aspect above, or the nucleic acid construct as described in the third aspect above.

[0014] Furthermore, the vector of the present invention can be a cloning vector or an expression vector, for example, it can be a plasmid, a granule, a bacteriophage, etc.

[0015] In some preferred embodiments, the recombinant vector is a recombinant expression vector, preferably a eukaryotic expression vector.

[0016] Furthermore, the present invention also provides a transformed host cell, wherein the transformation is performed with polynucleotides, nucleic acid constructs, or recombinant vectors as described above. The host cells include, but are not limited to, one of BL21(DE3), SHuffle T7 E.coli, or ArcticExpress(DE3)pRARE2.

[0017] Furthermore, the present invention also provides the use of monoclonal antibodies or antigen-binding fragments thereof as described above, polynucleotides as described above, nucleic acid constructs as described above, recombinant vectors as described above, and / or host cells transformed as described above in the preparation of detection reagents or kits for detecting feline Fel d7.

[0018] The present invention also provides the use of monoclonal antibodies or antigen-binding fragments thereof, polynucleotides, nucleic acid constructs, recombinant vectors and / or host cells as described above in the preparation of detection products for detecting feline Fel d7; The detection products for detecting feline Fel d7 include one or more of the following: Western blot assay reagents, immunofluorescence assay reagents, immunoprecipitation assay reagents, ELISA assay reagents, colloidal gold assay reagents, chemiluminescent immunoassay reagents, and flow cytometry assay reagents. The ELISA detection method includes double antibody sandwich detection.

[0019] The beneficial effects of the present invention are as follows: This invention relates to monoclonal antibodies or antigen-binding fragments targeting feline Fel d7. By screening for high-affinity monoclonal antibodies against Fel d7, Fel d7 detection at nanogram levels (ng / mL) is achieved. Simultaneously, the high homogeneity of monoclonal antibodies and their recognition of only a specific antigenic epitope avoids cross-reactions with other proteins in the feline body, thus improving detection specificity. Multiclonal antibodies are more stable and have stronger specificity, avoiding interference from non-specific reactions. A dual-antibody sandwich ELISA kit for detecting feline Fel d7, based on monoclonal antibodies, serves as both the coating and detection antibodies, synergistically amplifying the detection signal. The biotin-streptavidin-HRP three-stage signal amplification system enables high-precision detection of Fel d7. The pre-coated plates and ready-to-use reagents simplify the operation process, allowing for testing without professional training, resulting in high efficiency and large-scale detection. This addresses the challenges of existing feline Fel d7 detection methods. The limitations of existing d7 detection kits, such as insufficient specificity and sensitivity, and reliance on imported products, have led to this invention. This invention utilizes a domestically produced monoclonal antibody preparation process, reducing kit production costs and promoting technology adoption. It fills the market gap for double-antibody sandwich ELISA kits in the field of Fel d7 detection, providing a technological foundation for extending Fel d7 detection from professional laboratories to homes and veterinary clinics, as well as for precise allergen control. It also provides a reliable tool for home self-testing of feline allergies, clinical triage and diagnosis, and environmental trace Fel d7 monitoring. Attached Figure Description

[0020] Figure 1 This is a Western blot (WB) image of the recombinant protein in Example 1; Figure 2 This is a sensitivity analysis chart for a double-antibody sandwich ELISA. Detailed Implementation

[0021] The present invention will be further described in detail below through specific implementation examples and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.

[0022] Unless otherwise specified, all raw materials and reagents used in this invention are from the conventional market.

[0023] Example 1 Preparation of feline allergy protein antigen The amino acid sequence of the feline allergy protein Fel d7 was retrieved from the NCBI database (GenBank: NP_001191706), and then reverse-translated to synthesize the corresponding gene sequence. To achieve expression of feline Fel d7, the pET30a prokaryotic expression system was constructed, and BL21(DE3) cells were used as the host cell. The recombinant protein Fel d7 was successfully expressed, as shown below. Figure 1 As shown, the molecular weight of the Fel d7 protein is predicted to be approximately 17.57 kDa.

[0024] Next, mice were immunized with recombinant feline Fel d7 protein to produce specific antibodies.

[0025] Example 2 mouse immunization Balb / c strain mice (purchased from the Experimental Animal Management Center of Southern Medical University) were used as the immunization subjects; all were healthy mice aged 6 weeks. Feline Fel d7, prepared in Example 1, was used as the immunogen, and a total of 20 mice were immunized. The entire immunization process was carried out in three stages: First immunization: The immunogen cat Fel d7 was injected into mice at a dose of 50 μg / mouse; Second immunization: Three weeks after the first immunization, the immunogen dose was halved to 25 μg / mouse; Three-stage immunization: Two weeks after the second immunization, mice were immunized with 25 μg of immunogen per mouse.

[0026] One week after the third immunization, blood samples were collected from the mice via eyelid sampling. Subsequently, the antibody titer was measured using an indirect ELISA method to assess the strength of the immune response.

[0027] Based on the test results shown in Table 1, mice with high antibody titers (high serum dilution factor and large OD value) were selected as experimental subjects for subsequent spleen cell fusion experiments.

[0028] Table 1. Detection results using the chessboard method

[0029] Example 3 Screening of monoclonal antibody cell lines 1. Fusion of mouse spleen cells 1) Spleen cell sampling: Under aseptic conditions, the spleen is removed, the membranous tissue is peeled off, and the spleen is placed in an incomplete culture medium. The incomplete culture medium is drawn up with a syringe and injected into the spleen to release the free spleen cells. The remaining spleen tissue is ground with a cell sieve, and the cells are collected and counted. 2) SP2 / 0 collection: SP2 / 0 cells are round and clear. Collect the cells in 50mL centrifuge tubes and count them. 3) Adjust the ratio of SP2 / 0 cells to spleen cells to 1:10-1:3; 4) Take out the PEG1450 preheated at 37℃, rotate the centrifuge tube, add 1 mL of PEG solution within 60 s, and let stand for 1 min; slowly add 3 mL of DMEM medium over 3 min, and then gradually increase the adding speed, adding DMEM medium to a total volume of 30 mL over 5 min; let stand in a 37℃ water bath for 5 min. 5) Centrifuge at 1000 rpm for 5 min, remove the supernatant, add an appropriate amount of HAT medium to resuspend the cells, and seed 200 μL per well into a 96-well cell culture plate (the 96-well plate was pre-added with peritoneal macrophages the day before, 10,000 cells / well), and culture in a 5% CO2 incubator at 37°C.

[0030] 2. Indirect ELISA screening of positive wells 1) Coating: Dilute cat Fel d7 protein with 1× coating buffer to prepare a coating solution of 2 μg / mL. Add 50 μL of the coating solution to each well of the detection plate and incubate at 2-8℃ for at least 12 hours. 2) Blocking: Wash the coated plate 4 times with ELISA plate washing buffer to remove the residual liquid, add 100 μL of blocking buffer to each well, seal, and incubate at 37°C for 2 hours; wash the plate 4 times with washing buffer and set aside. 3) Sample incubation: Take the supernatant of the fused cell culture plate for detection, use the serum of immunized mice as a positive control, the cell culture medium as a negative control, and PBS as a blank control. Load 100 μL of sample into each well and incubate at 37℃ for 1 h. 4) Secondary antibody incubation: Wash the detection plate 4 times with ELISA plate washing buffer, dilute goat anti-mouse secondary antibody (Celvi, HRP*Goat Anti-Mouse IgG (H+L)) 1:10000 with blocking buffer, load 100 μL per well, and incubate at 37℃ for 1 h; 5) Color development and termination: Wash the test plate 4 times with ELISA plate washing buffer to remove residual liquid, add 100 μL of color development solution to each well, and incubate at room temperature for 10 minutes; add 50 μL of stop solution to all wells of the ELISA reaction plate. 6) Reading: The absorbance value is read at 450nm using the microplate reader; 7) Select wells with high test values ​​as suspected positive wells. If the positive value is still high after repeated testing the next day, the wells are considered positive wells.

[0031] Based on the above tests, five positive wells (2C4, 2D9, 3A6, 8D3, and 14H3) were found in the cat Fel d7 screening.

[0032] 3. Monocloning 1) First round of monoclonal cell culture: Collect cells from the above positive wells, dilute the cells to a single cell / 200 μL using monoclonal cell culture medium, add the cell suspension to a 96-well cell culture plate, culture for 8 days, and then detect the cells using the indirect ELISA method described above. 2) Second round of monocloning: Analyze the results of the first round of subcloning ELISA. If the positive well rate is 100%, the cell purity can be determined to be sufficient. Otherwise, select the positive wells for this round of monocloning. The operation of the second round of monocloning is the same as that of the first round of monocloning. 3) Once the purity of the hybridoma cells reaches 100% after multiple rounds of monoclonalization of the above-mentioned positive well cells, the monoclonalization process can be terminated.

[0033] The cell lines obtained through the above screening were named according to the plate number and well number, respectively: Fel d7: 2C4, 2D9, 3A6, 8D3, 14H3 cell lines, and their corresponding antibodies are named 2C4, 2D9, 3A6, 8D3, 14H3 antibodies.

[0034] 4. Screening of paired antibodies The above cell lines were injected into the peritoneal cavity of mice, with 5 mice injected with each cell line. Ascites fluid was collected and the antibody was purified using a protein A affinity chromatography column. A portion of the purified antibody was labeled with biotin and used as a detection antibody. The unlabeled portion of the purified antibody was used as a coating antibody. Follow the sandwich ELISA experimental procedure below and refer to the specific operational details such as intermediate ELISA plate washing mentioned above to screen sandwich antibody pairing combinations: 1) Coat the plates with 100 ng / well of antibody and incubate at 2-8°C for at least 12 hours; 2) Incubate cat Fel d7 at 5 μg / mL × 50 μL at 37℃ for 0.5 h; 3) Load 100 μL of biotin-labeled detection antibody into each well (the detection titers of the detection antibodies need to be adjusted to be consistent before loading), and incubate for 0.5 h; 4) HRP-labeled streptavidin (Selva, C050109-HRP-SA) was diluted 1:4000, 100 μL was loaded into each well, and incubated at 37℃ for 0.5 h; 5) Color development and termination; 6) Reading: The absorbance value is read at 450nm using the microplate reader.

[0035] Table 2. Detection results using the chessboard method

[0036] As shown in Table 2, 14H3 as the coating antibody and 2C4 as the detection antibody have the best detection effect. 14H3 and 2C4 antibodies can be used as an antibody combination for the detection of feline Fel d7.

[0037] The selected antibody combination was then tested as follows: 1. Specific detection To test the specificity of the 14H3 and 2C4 antibody combination in feline Fel d7, two samples of feline Fel d1 protein, two samples of feline Fel d4 protein, and two samples of feline serum albumin (FSA) were used as test samples, all at a concentration of 1 μg / mL. PBS was used as a negative control, and 1 μg / mL feline Fel d7 solution was used as a positive control. The samples were tested according to the sandwich ELISA procedure in Example 3, and the results are shown in the table below: Table 3 Specific detection results

[0038] Cross-reactivity rate = (OD value of interfering protein - OD value of negative control) × 100% / (OD value of feline Fel d7 protein - OD value of negative control); A well is considered a positive well if its ratio S / N is ≥ 2.1 (where S is the OD value of the test sample and N is the OD value of the negative sample); otherwise, it is considered a negative well. As shown in Table 3, the positive control (feline Fel d7 protein) had an S / N > 2.1, while the feline Fel d1 protein, feline Fel d4 protein, and feline serum albumin (FSA) had an S / N < 2.1 and a cross-reactivity rate ≤ 1.40%. This indicates that the combination of 14H3 and 2C4 antibodies in feline Fel d7 has high specificity for the detection of feline Fel d7 and has no significant cross-reactivity with feline Fel d1 protein, feline Fel d4 protein, or other feline proteins.

[0039] Based on the results of the pairing experiments and specificity tests described above, the combination of antibodies 14H3 and 2C4 in feline Fel d7 can be developed into a detection reagent for feline Fel d7. Therefore, subsequent work included the determination of the variable region sequence of the antibodies and optimization of the detection conditions for the antibody combination.

[0040] 2. Antibody sequencing Hybridoma cells containing the 14H3 and 2C4 antibody combinations from feline Fel d7 were collected separately, with each hybridoma cell containing approximately 1 × 10⁻⁶ cells. 5 ~1×10 6Cells were lysed, mRNA was extracted and reverse transcribed into cDNA, antibody genes were amplified and cloned into a vector, and the Sanger sequencing method was used to obtain antibody nucleic acid sequences. Biological information analysis of the sequences yielded capture antibody-related sequences (Table 4 below) and detection antibody-related sequences (Table 5 below). Table 4 Information on capture antibodies

[0041] Table 5 Information on antibody detection

[0042] Note: The relevant amino acids and nucleic acids in the capture antibodies and detection antibodies in Tables 4 and 5 are named in the same way, but their sequences are different.

[0043] Among them, the amino acid sequence of SEQ ID NO.5 is DTS, the nucleotide sequence of SEQ ID NO.13 is GACACATCC, the amino acid sequence of SEQ ID NO.21 is GAT, and the nucleotide sequence of SEQ ID NO.29 is GGTGCAACC, which are shown as skipped sequences in the sequence listing file.

[0044] The sequences for capturing and detecting antibodies described above also include sequences that are at least 95%, 96%, 97%, 98%, or 99% homologous to them.

[0045] 3. Optimization of detection conditions for the double-antibody sandwich ELISA kit 3.1 Determination of the optimal working concentration of the antibody 1) The purified 14H3 monoclonal antibody was diluted to different concentrations (0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL) using coating buffer with a pH of 9.2. The biotin-conjugated 2C4 monoclonal antibody was diluted to different concentrations (0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL) using antibody dilution buffer (containing 5% goat serum and 2.5% sucrose phosphate buffer, pH 7.6). 2) 14H3 monoclonal antibody was coated at different concentrations, 100 μL / well, and incubated overnight at 4°C.

[0046] 3) Discard the liquid in the wells, add 200 μL / well of ELISA plate washing buffer (pH 7.6) and wash the plate 3 times, then pat dry; 4) Antigen incubation: 0.5 μg / mL cat Fel d7, 100 μL / well, incubate at 37℃ for 1 h; 5) Wash the board as in step 3). 6) Add 100 μL of the detection antibody 2C4-biotin at four different dilutions to each well and incubate at 37°C for 1 hour. 7) Wash the plate, same as step 3). 8) HRP-labeled streptavidin (Selvey, C050109-HRP-SA) was diluted 1:4000 with 0.01M PBS (pH 7.4) containing 1% BSA, 100 μL / well, and incubated at 37°C for 1 h; 9) Wash the plate, same as step 3). 10) Add TMB to the microplate, 100 μL / well, incubate at 37°C in the dark for 5 minutes, and immediately place it in a microplate reader to measure the absorbance at 450 nm.

[0047] Follow the steps above: The results are shown in Table 6. The optimal coating concentration was determined to be 4 μg / mL for 14H3 monoclonal antibody and 1 μg / mL for 2C4 monoclonal antibody. Under these conditions, the antigen detection value was the highest.

[0048] Table 6. Determination of Optimal Antibody Concentration by Checkerboard Titration

[0049] 3.2 Determination of antibody incubation time Based on the optimal feline Fel d7 monoclonal antibody coating concentration and detection concentration mentioned above, different antigen incubation times (15 min, 30 min, 45 min, 1 h, 1.5 h) were set, with other conditions kept constant. The results of detecting 0.5 μg / mL feline Fel d7 are shown in the table below: Table 7 Determination of Optimal Antigen Incubation Time

[0050] As shown in Table 7, the optimal incubation time for the feline Fel d7 antigen is 1.5 h based on the S / N value.

[0051] Based on the optimal coating concentration and detection concentration of feline Fel d7 monoclonal antibody, as well as the optimal antigen incubation time, different incubation times (15 min, 30 min, 45 min, 1 h, 1.5 h) were set for detecting 0.5 μg / mL feline Fel d7. The results are shown in the table below: Table 8 Determination of Optimal Antibody Incubation Time

[0052] As shown in Table 8, the optimal antibody incubation time for detecting feline Fel d7 is 1 hour, based on the S / N value.

[0053] Based on the optimal coating concentration and detection concentration of feline Fel d7 monoclonal antibody, as well as the optimal incubation time for antigen and detection antibody, different HRP-labeled streptavidin incubation times were set to detect 0.5 μg / mL feline Fel d7. The results are shown in the table below: Table 9 Determination of Optimal Streptococcus Incubation Time

[0054] As shown in Table 9, based on the S / N value, the optimal incubation time for HRP-labeled streptavidin for feline Fel d7 is 30 min.

[0055] 4. Sensitivity Test Using the aforementioned paired antibodies and optimized conditions, specifically an antibody combination of 4 μg / mL feline Fel d7-14H3-coated antibody and 1 μg / mL feline Fel d7-2C4 detection antibody, was employed to detect feline Fel d7. Feline Fel d7 was prepared at different concentrations, and sensitivity tests were conducted. The test results are shown in the table below: Table 10 Sensitivity Test Results

[0056] As shown in Table 10, a good linear relationship exists between antigen concentrations in the range of 30-400 ng / mL. Within this range, the equation y = 0.0028x + 0.0722 with R² = 0.9948 can be generated. Figure 2 As shown, the horizontal axis represents the antigen concentration value, and the vertical axis represents the absorbance value. Furthermore, to determine the detection limit, when the feline Fel d7 concentration is as low as 40 ng / mL, the OD value is 0.17, and the S / N ratio is >2.1; therefore, the above-mentioned antagonistic assay can detect feline Fel d7 at 40 ng / mL, demonstrating high sensitivity.

[0057] 5. Detection method of a double-sandwich ELISA kit for detecting feline Fel d7 based on monoclonal antibodies. 1) Coating ELISA microplates: Dilute the coating antibody 14H3 to 4 μg / mL with coating buffer at pH 9.2, 100 μL / well, and coat overnight at 4℃; 2) Blocking: Discard the liquid in the wells, add 200 μL / well of ELISA plate washing buffer (pH 7.6), wash 3-5 times, then add 100 μL / well of blocking buffer (containing 10% goat serum and 5% sucrose phosphate buffer, pH 7.6), block at 37℃ for 2 hours, wash 3-5 times, and then dry. 3) Sample incubation: The ELISA plate is configured with sample wells, positive control wells, and negative control wells. Add 100 μL of feline Fel d7 standard protein to the positive control wells; add 100 μL of 1×PBS buffer to the blank wells; add 100 μL of the sample to be tested to the sample wells. Incubate at 37°C for 1.5 h, then add 200 μL of ELISA plate washing buffer (pH 7.6) to each well and wash 3-5 times, then agitate dry. 4) Antibody incubation: Dilute the detection antibody 2C4-biotin to 1 μg / mL with detection antibody dilution buffer (containing 5% goat serum and 2.5% sucrose phosphate buffer, pH 7.6), 100 μL / well, incubate at 37℃ for 1 h, then add 200 μL / well of ELISA plate washing buffer (pH 7.6) and wash 3-5 times, then spin dry; 5) Signal amplification: Add HRP-labeled streptavidin: Add HRP-labeled streptavidin to the microplate, dilute it 4000 times with 0.01 M PBS (pH 7.4) containing 1% BSA, 100 μL / well, incubate at 37℃ for 30 min, then add 200 μL / well of ELISA microplate washing buffer (pH 7.6) and wash 5-6 times, then spin dry; 6) Color development and reading: Add TMB to the microplate, 100 μL / well, develop at 37°C in the dark for 5 minutes, and immediately place it in a microplate reader to detect the absorbance at 450 nm.

[0058] 6. Application of the feline Fel d7 double antibody sandwich ELISA detection kit Five different breeds of domestic cats (Persian, Siamese, Ragdoll, British Shorthair, and Chinese domestic cat) were fed a diet containing egg yolk antibody IgY for 20 consecutive days. Saliva samples were collected from the cats on an empty stomach on day 0 of the intervention and day 28 after the intervention ended. The samples were diluted 1:500 and the feline Fel d7 double-sandwich ELISA kit was used to evaluate its practicality according to the method described in section 5. The results are shown in Table 11. The feline Fel d7 antigen was detected before and after feeding (S / N > 2.1), and the Fel d7 content decreased significantly after feeding. It is preliminarily judged that the detection method has clinical applicability.

[0059] Table 11 Results of Feline Oral Allergen Fel d7 Content Test

Claims

1. A monoclonal antibody or its antigen-binding fragment against feline Fel d7, characterized in that, The monoclonal antibody or its antigen-binding fragment includes: A monoclonal antibody: The heavy chain variable region has three heavy chain complementarity-determining regions as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, or sequences having at least 95%, 96%, 97%, 98%, or 99% homology with them, respectively; And light chain variable regions, which have three light chain complementarity-determining regions as shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or sequences having at least 95%, 96%, 97%, 98% or 99% homology with them, respectively; and / or B monoclonal antibodies: The heavy chain variable region has three heavy chain complementarity-determining regions as shown in SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 or sequences having at least 95%, 96%, 97%, 98% or 99% homology with them, respectively; And a light chain variable region, which has three light chain complementarity-determining regions as shown in SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22 or sequences having at least 95%, 96%, 97%, 98% or 99% homology with them.

2. The monoclonal antibody against feline Fel d7 or its antigen-binding fragment according to claim 1, characterized in that, The monoclonal antibody or its antigen-binding fragment includes: A monoclonal antibody: The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.7 or a sequence having at least 95%, 96%, 97%, 98% or 99% homology thereto; and the light chain variable region has an amino acid sequence as shown in SEQ ID NO.8 or a sequence having at least 95%, 96%, 97%, 98% or 99% homology thereto; and / or B monoclonal antibodies: The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.23 or a sequence having at least 95%, 96%, 97%, 98% or 99% homology thereto; and the light chain variable region has an amino acid sequence as shown in SEQ ID NO.24 or a sequence having at least 95%, 96%, 97%, 98% or 99% homology thereto.

3. A polynucleotide, characterized in that, It encodes the monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2.

4. A nucleic acid construct, characterized in that, It comprises the polynucleotide as described in claim 3, and at least one expression regulatory element operatively linked to the polynucleotide.

5. The use of the polynucleotide of claim 3 in constructing expression vectors, preparing recombinant antibodies, preparing gene therapy products and vaccines.

6. A recombinant vector, characterized in that, It comprises the polynucleotide as described in claim 3, or the nucleic acid construct as described in claim 4.

7. A transformed host cell, characterized in that, It comprises the polynucleotide as described in claim 3, the nucleic acid construct as described in claim 4, or the recombinant vector as described in claim 6.

8. The use of the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 or 2, the polynucleotide as described in claim 3, the nucleic acid construct as described in claim 4, the recombinant vector as described in claim 6, and / or the transformed host cell as described in claim 7 in the preparation of a detection product for detecting feline Fel d7.

9. The application according to claim 8, characterized in that, The detection products for detecting feline Fel d7 include one or more of the following: Western blot assay reagents, immunofluorescence assay reagents, immunoprecipitation assay reagents, ELISA assay reagents, colloidal gold assay reagents, chemiluminescent immunoassay reagents, and flow cytometry assay reagents.

10. The application according to claim 9, characterized in that, The ELISA assay includes the use of a double-antibody sandwich assay kit.

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