Cat allergen Fel d 1 protein monoclonal antibody and its application

Abstract

The present invention provides monoclonal antibodies against the cat allergen FeI d 1 protein and their applications. First, the present invention obtained two hybridoma cell lines, 2A7 and 6B1, that stably secrete FeI d 1 monoclonal antibodies. Furthermore, a double-antibody sandwich ELISA method was established using 2A7 as the capture antibody and biotin-labeled 6B1 as the detection antibody. This method can be used to monitor FeI d 1 protein antigen levels, replacing existing indirect ELISA detection methods. It exhibits high specificity and sensitivity, is simple to operate, and produces clear results.

Description

Technical Field

[0001] The present invention relates to the technical field of biological antibodies, and in particular to a specific monoclonal antibody against cat allergen FeI d 1 protein and an application thereof. Background Art

[0002] Cat allergen (Felis domesticus allergen, Fel d) is responsible for 30% of allergic diseases, with symptoms ranging from mild rhinitis and conjunctivitis to life-threatening asthmatic reactions, making it a global health problem. Fel d 1 is the primary allergen. Fel d 1 is a heat-stable protein found in cats' saliva, anal glands, sebaceous glands, skin, and fur. Existing research suggests that salivary and sebaceous glands are the primary sites of its production. When cats groom, Fel d 1 in their saliva remains on their fur and is subsequently shed with the hair and dander. Fel d 1 is also found in perianal and lacrimal glands. Due to its structure, Fel d 1 can remain airborne for extended periods and adhere to clothing, carpets, and some furniture, making it difficult to remove. Therefore, when dried saliva and dander are dispersed into tiny particles in the environment and contacted or inhaled by specific individuals, they can trigger sensitization and allergic reactions in patients.

[0003] Fel d 1 belongs to the uteroglobin family, is approximately 35 to 39 kDa in size, and is composed of two heterodimers. Each dimer is composed of two chains—chain 1 and chain 2, encoded by genes CH1 and CH2, respectively. Each dimer has a molecular weight of 18 to 19 kDa and is non-covalently linked to form a tetramer. Chain 1 consists of 70 amino acids with a molecular weight of 8 kDa. Chain 2 is a 10 kDa glycoprotein with N-oligosaccharides. It is composed of approximately 90 amino acids. Native Fel d 1 is thought to be a mixture of intact chain 1 and truncated or intact chain 2. Studies have shown that the three-dimensional structure of Fel d 1 is more complex than that of other allergens, with an internal cavity that can accommodate endogenous ligands and two external calcium-binding sites. Therefore, recombinant Feld 1 protein is more difficult to obtain than other allergens. Each chain is first produced in a prokaryotic expression system and then modified with glycosylation using a baculovirus expression system. The final Feld 1 protein molecule has a three-dimensional structure similar to the natural cat allergen Feld 1.

[0004] Over the past few decades, a small number of methods for detecting Fel d 1 protein have been reported. Because the allergic symptoms caused by Fel d 1 are similar to those caused by other allergens, such as rhinitis and asthma, molecular biology and immunological methods are needed to diagnose the disease. The Fel d 1 protein detection method on the market is mainly the indirect ELISA method. Although it has high sensitivity and low cost, it has a high probability of cross-reaction and is easily affected by environmental factors. Compared with the indirect ELISA method, the double antibody sandwich ELISA method has high specificity. The capture antibody and detection antibody used are both detection-specific. It is suitable for complex samples and the antigen does not need to be purified for detection. It has greater flexibility and higher sensitivity. However, it also has the disadvantages of poor antibody accuracy and stability. Summary of the Invention

[0005] To address the above technical issues, the present invention first obtained two anti-cat allergen Fel d 1 protein monoclonal antibodies, which were secreted by hybridoma cell lines 2A7 and 6B1 respectively;

[0006] Among them, the amino acid sequence of the light chain variable region of antibody 2A7 is: QAVVTQESALTTSPGETVTLTCRSSTGAVITSNYANWVQEKPDHLFTGLIGGTNNRGLGVPA RFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVL;

[0007] The amino acid sequence of the heavy chain variable region is: QVQLKESGPGLVAPSQSLSITCTVSGSSLTNYAVHWVRQPPGKGLEWLGVIWPGGTTNYNSPLMSRLSISKDISKSQVFLKMNSLQTDDTAMYYCARDPLYGNSWFAYWGQGTQVTVTA

[0008] The amino acid sequence of the light chain variable region of antibody 6B1 is: DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTHVGWFQQKTGQSPKALIYSASYRYSGVPD RFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNNYPYTFGGGTKVEKK;

[0009] The amino acid sequence of the heavy chain variable region is: QVQLQQPGAELVRPGASVRLSCKASGYTFTNYWINWVKQRPGQGLEWIGNIFPSDSYTNY NHNFKDKASLTVDKSSTTAYMHLSSPTSEDSAVYYCSRRGIGTVLFDYWGQGTTLTVSS.

[0010] Secondly, this application established a double-sandwich ELISA method for detecting the cat allergen Fel d 1 using 2A7 monoclonal antibody as the capture antibody and HRP-labeled 6B1 monoclonal antibody as the detection antibody; the method can be used to monitor Fel d 1 antigen levels, has high specificity and high sensitivity, is simple to operate, does not require laboratory support, and can be used for large-scale antigen detection.

[0011] Furthermore, the present invention provides the use of the monoclonal antibody in the detection or diagnosis of diseases caused by cat allergen FeI d 1.

[0012] Furthermore, the present invention provides a double-antibody sandwich ELISA detection kit comprising monoclonal antibody 2A7, which binds to the cat allergen FeI d 1 protein, secreted by hybridoma cell line 2A7, and biotin-labeled monoclonal antibody 6B1, which binds to the cat allergen FeI d 1 protein, secreted by hybridoma cell line 6B1. Specifically, a double-antibody sandwich ELISA method for detecting the cat allergen FeI d 1 protein was established using 2A7 as the capture antibody and biotin-labeled 6B1 as the detection antibody. This method can be used to monitor FeI d 1 protein antigen levels, exhibiting high specificity, high sensitivity, and low cost, and is suitable for large-scale antigen detection.

[0013] Preferably, in the above double antibody sandwich ELISA detection method, the capture antibody is coated on the enzyme labeling plate, the optimal coating amount of the capture antibody is 4 μg / well, and the working concentration of the detection antibody is 1:2000.

[0014] Furthermore, the present invention provides the use of the above monoclonal antibody in preparing a reagent, a test strip, or a kit for detecting cat allergen FeI d 1 protein.

[0015] Furthermore, the present invention provides a double-antibody sandwich ELISA kit for detecting cat allergen FeI d 1 protein, the kit comprising 2A7 and 6B1 monoclonal antibodies; the 2A7 is a capture antibody, and the 6B1 is a detection antibody.

[0016] Preferably, the kit further comprises an ELISA plate, a blocking solution, a diluent, a washing solution, a color developing solution, and a stop solution.

[0017] Preferably, the blocking solution is 5% skim milk;

[0018] Preferably, the diluent is a PBS solution;

[0019] Preferably, the washing solution is PBST buffer.

[0020] Preferably, the color developing agent is TMB.

[0021] Preferably, the stop solution is 2M sulfuric acid.

[0022] Furthermore, the present invention provides the use of the above ELISA kit for in vitro detection of cat allergen Feld 1 protein antigen for non-disease diagnosis purposes.

[0023] Furthermore, the present invention provides a double-antibody sandwich ELISA method for detecting cat allergen FeI d 1 protein antigen for non-diagnostic purposes, the method comprising:

[0024] The ELISA plate was coated with 2A7, which is anti-cat allergen Fel d 1 protein, and blocked. The sample to be tested was added and incubated. The plate was washed and the 6B1 monoclonal antibody was added for reaction. After washing, a color developer was added to develop the color. The assay was terminated by adding a stop solution. The absorbance at 450 nm was read and the test result was determined based on the P / N ratio.

[0025] Preferably, the method comprises the following steps:

[0026] (1) Coating: Dilute 2A7 to 4 μg / ml with PBS, coat 100 μl per well of the ELISA plate horizontally, and incubate at 37°C for 2 h; (2) Blocking: Wash the ELISA plate 4 times with PBST and block with 5% skim milk at 4°C for 12 h;

[0027] (3) Adding the test sample: After washing, add 100 μl of the test sample to each well, using cat allergen Fel d 1 protein and PBS as positive and negative controls, and incubate at 37°C for 2 h.

[0028] (4) Washing: Remove the ELISA plate, spin dry it, wash it four times with PBST, and spin dry on absorbent paper;

[0029] (5) Add enzyme-labeled monoclonal antibody: dilute horseradish peroxidase-labeled monoclonal antibody 6B1 at 1:2000, add 100 μl to each well, and react at 37°C for 1.5 h;

[0030] (6) Color development: After washing, add 100 μl of TMB to each well and develop color at 37°C for 20 min;

[0031] (7) Termination: Add 50 μl of 2 M sulfuric acid to terminate;

[0032] (8) Result calculation and judgment: Read the absorbance at 450 nm. When the OD value of the test sample is ≥0.062 and P / N ≥2, it is judged as positive, otherwise it is judged as negative.

[0033] The beneficial effects of the present invention are as follows: ① The present invention obtains two hybridoma cell lines, 2A7 and 6B1, which can stably secrete cat allergen Fel d 1 monoclonal antibodies; ② The present invention uses 2A7 as a capture antibody and biotin-labeled 6B1 as a detection antibody to establish a double-antibody sandwich ELISA method; ③ The method can be used to monitor the level of cat allergen Fel d 1 protein antigen, replacing the existing indirect ELISA detection method, with high specificity and high sensitivity, simple operation and clear results. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0035] Figure 1 Western blotting analysis of the reactivity of 2A7 and 6B1 monoclonal antibodies with FeI d 1 protein. M. Protein markers, A. 2A7, B. 6B1.

[0036] Figure 2 Indirect immunofluorescence characterization of 2A7 and 6B1 monoclonal antibodies.

[0037] Figure 3 Epitope identification of 2A7 and 6B1 monoclonal antibodies, M. Protein marker, A. 2A7, B. 6B1.

[0038] Figure 4 Standard curve and fitting equation for sandwich ELISA detection of FeI d 1 protein standard samples. DETAILED DESCRIPTION

[0039] Below describe in detail embodiments of the present invention, the example of described embodiment is shown in the accompanying drawings, wherein identical or similar reference numerals represent identical or similar elements or elements with identical or similar functions throughout.The embodiment described below by reference to the accompanying drawings is exemplary, is intended to be used for explaining the present invention, and is not to be construed as limiting the present invention.Unindicated specific technology or condition person in the embodiment, according to the technology or condition described in the document in this area or according to product specification sheet, carry out.Reagents therefor or instrument do not indicate manufacturer person, all are conventional products that can be obtained by commercial purchase.

[0040] The experimental animals, antigens and cells used in the following examples are: 6-week-old SPF-grade BALB / c female mice; SP2 / 0 cells and cat allergen FeI d 1 protein were stored in this laboratory.

[0041] Example 1 Preparation of Monoclonal Antibodies Against Cat Allergen Fel d 1 Protein

[0042] Fel d 1 protein expression and purification

[0043] The FeI d 1 gene was codon-optimized for E. coli, and restriction enzyme sites BamHI and Xhol I, as well as a His tag, were introduced at both ends of the optimized sequence. Specific primers targeting the FeI d 1 ORF sequence were designed: the upstream primer for pET28a-Fel d 1 was 5'-AATCTCGAGACACAGCGGGCTGGTGTA-3' (SEQ ID NO. 1), and the downstream primer for pET28a-Fel d 1 was 5'-CGTGGATCCATGGTAAAAATGGCTGAA-3' (SEQ ID NO. 2). Expression was achieved in E. coli Rosetta strains, and soluble expression was achieved by optimizing expression conditions. The desired antigen was then purified.

[0044] The pET-28a-Fel d 1 recombinant plasmid was transformed into Rosetta competent cells and cultured in a constant temperature shaker at 37°C. When the OD600nm value of the bacterial solution was 0.6, 0.6 mmol / L IPTG was added to the bacterial solution, and the expression was induced at 25°C, 180 r / min for 15 h. The induced bacteria were collected.

[0045] The protein purification steps after induction are briefly described as follows:

[0046] 1. After the target protein is induced to express in the bacterial solution, centrifuge at 5000 rpm for 10 minutes at 4°C to pellet the bacteria.

[0047] 2. Add 5 ml of non-denaturing lysis buffer (add 10 mM imidazole to reduce the amount of foreign proteins hanging on the column) per gram of wet weight of bacterial pellet to fully resuspend the bacteria.

[0048] 3. Add 0.2 mg / ml lysozyme, mix gently using a vortex shaker, and shake at room temperature for 30 minutes.

[0049] 4. Ultrasonicate the bacteria (200W, sonicate for 3 seconds on, 5 seconds off, place on ice for 30 minutes), centrifuge at 12,000 rpm (maximum possible speed) for 30 minutes at 4°C, and place the supernatant on ice. (Complete ultrasonic disruption and clear supernatant after centrifugation must be free of insoluble matter before proceeding to the next step.)

[0050] 5. BeyoGold™ His-tag Purification Resin (reduction-resistant chelating type) was loaded onto the column and the column was equilibrated with 1 column volume of non-denaturing lysis buffer three times.

[0051] 6. Mix the supernatant from step 4 and the gel from step 5 together, and shake slowly on a side-by-side shaker at 4°C for 60 minutes to allow the protein and gel to fully combine.

[0052] 7. Load the supernatant and gel-bound product from step 6 onto the column.

[0053] 8. Wash the column six times with one column volume of non-denaturing wash buffer (add 10 mM imidazole).

[0054] 9. Wash the column three times with one column volume of gradient non-denaturing eluent (add 100 mM imidazole).

[0055] The solution formula required in the above steps is as follows:

[0056] Non-denaturing lysis buffer: 50 mM NaH2PO4, 300 mM NaCl, pH adjusted to 8.0 with sodium hydroxide

[0057] Non-denaturing wash buffer: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH adjusted to 8.0 with sodium hydroxide

[0058] Non-denaturing eluent: 50 mM NaH2PO4, 300 mM NaCl, 100 mM imidazole, pH adjusted to 8.0 with sodium hydroxide.

[0059] 10. Determine the protein content and purity of the eluate collected in step 9 by SDS-PAGE. Place the purified protein in a dialysis bag and dialyze against PEG 20000 at 4°C to a concentration of approximately 1 mg / ml.

[0060] Animal Immunization

[0061] Balb / c mice were immunized three times using conventional immunization methods. Purified recombinant cat allergen Fel d1 protein was mixed with Freund's complete adjuvant by mass and fully emulsified. The mixture was then injected subcutaneously at multiple points on the back of the mouse at a rate of 0.2 ml per mouse. A total of five mice were immunized. After the initial immunization, the mice were immunized again every two weeks. The recombinant Fel d 1 protein was mixed with Freund's incomplete adjuvant by mass and injected subcutaneously at multiple points on the back of the mouse. A booster immunization was performed intraperitoneally three days before cell fusion.

[0062] Establishment of screening method

[0063] Screening was performed using an indirect ELISA assay, and the optimal antigen coating concentration was determined using a square array titration test. Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was used as the secondary antibody, and the optimal secondary antibody concentration was 1:5000, a standard working concentration in our laboratory. The specific steps were as follows:

[0064] (1) Dilute the antigen to 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, and 32.0 μg / ml, respectively, coat with 100 μl / well, incubate at 37°C for 2 h, and then at 4°C overnight.

[0065] (2) The next day, pat dry the coating solution in the ELISA plate, block with 100 μl / well of blocking solution, incubate at 37°C for 2 h, and pat dry before testing.

[0066] (3) The positive serum was serially diluted from 1:400 to the eighth column (1:51200). At the same time, a negative control group (i.e., non-immunized mouse serum) and a blank control group (i.e., control wells with PBS added) were set up. After adding the sample, the wells were incubated at 37°C for 1 hour and washed 3 times.

[0067] (4) Add enzyme-labeled horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody, 100 μl / well, incubate at 37°C for 1 hour, and wash 3 times. (5) Add TMB 50 μl / well, develop color at 37°C in the dark for 15 minutes, and terminate the reaction with stop solution, 50 μl / well. Measure OD using an enzyme-labeled detector. 450 value.

[0068] When screening with indirect ELISA, the ratio of positive serum to negative serum is the largest and the OD 450 The antigen dilution with a value close to 1 is regarded as the optimal working concentration of the indirect ELISA reaction system. When screening hybridoma cells, the cell supernatant is tested using this system, and a P / N value of ≥ 2.1 is considered positive.

[0069] Determination of mouse titer

[0070] After the indirect ELISA method was established, the optimal coating concentration was selected to test the antibody titer of the immunized mice. The two mice with the highest immune titer were selected for a booster intraperitoneal immunization. Three days later, the spleens of these mice were harvested for cell fusion.

[0071] Establishment of hybridoma cell lines

[0072] Preparation of myeloma cells

[0073] Two weeks before fusion, thaw myeloma SP2 / 0 cells. Remove the cryovial from liquid nitrogen and immediately place in 37°C warm water to dissolve. Once completely dissolved, centrifuge at 800 rpm for 5 minutes. Discard the supernatant and carefully resuspend the pellet in complete DMEM medium supplemented with 20% fetal bovine serum. Mix thoroughly and add to a cell culture flask. Subculture in a 5% CO2, 37°C incubator. Transfer to two T75 flasks and allow the myeloma cells to reach the logarithmic growth phase on the day of fusion before use.

[0074] Preparation of feeder cells

[0075] Prepare feeder cells on the day of cell fusion as follows:

[0076] (1) Take an 8-10 week old healthy Balb / c mouse, remove the eyeball and collect blood to prepare negative serum. Sacrifice by cervical dislocation and disinfect with 75% alcohol.

[0077] (2) Place the mouse in a clean bench with the ventral surface facing up. Lift the mouse's abdominal skin with tweezers, make a small cut in the lower abdomen, peel it off longitudinally to fully expose the peritoneum, and disinfect it with alcohol.

[0078] (3) Use a disposable sterile syringe to draw 5 ml of HAT culture medium and inject it into the mouse's abdominal cavity. Keep the syringe in your right hand still, shake the mouse gently, and then draw out the cell suspension.

[0079] (4) Add the cell suspension to 60 ml of HAT medium, mix well, count, and adjust the cell concentration to 1-5×10 5 pcs / ml.

[0080] If the number of cells is too small, feeder cells can be prepared according to the above steps.

[0081] (5) Transfer 100 μl / well of the solution into a 96-well cell culture plate and place it in a 5% CO2, 37°C constant temperature incubator until ready for use.

[0082] Preparation of splenocytes

[0083] (1) Take mice that were boosted with immunization three days ago, remove their eyeballs and collect blood to prepare positive serum. Kill them by dislocating the neck and disinfect them with 75% alcohol.

[0084] (2) In a clean bench, the spleen was aseptically removed, fat and connective tissue were removed, and the spleen was placed on a 200-mesh sterilized copper grid. The spleen was gently ground with a sterilized syringe core, and serum-free DMEM was slowly added dropwise.

[0085] (3) Collect the spleen cell suspension and transfer it to a 15 ml centrifuge tube. Centrifuge at 800 rpm for 5 min, discard the supernatant, and resuspend in 5 ml of serum-free DMEM culture medium. Count the spleen cells and set aside.

[0086] Cell fusion

[0087] (1) Select two T75 flasks of SP2 / 0 cells in good condition and logarithmically growing. Gently pipette the cells off the flask wall and transfer them to a 50 ml centrifuge tube. Centrifuge at 1000 rpm for 10 min. Discard the supernatant, resuspend the cells in 5 ml of serum-free DMEM, and count the cells.

[0088] (2) Mix SP2 / 0 cells and immune spleen cell suspension. The ratio of spleen cell number to myeloma cell number should be maintained between 5:1 and 10:1.

[0089] (3) Centrifuge at 1000 rpm for 5 min and discard the supernatant.

[0090] (4) Place the fusion tube in the palm of your hand and rub it back and forth to loosen the cell blocks. This step is the key step for fusion.

[0091] (5) Use a pipette to slowly add 1 ml of 50% PEG1450 preheated at 37°C into the centrifuge tube within 60 seconds and let it stand for 60 seconds.

[0092] (6) Add serum-free DMEM preheated at 37°C dropwise over 3 minutes, first slowly and then quickly, to a total of 25 ml. Shake gently to mix, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.

[0093] (7) Resuspend the cells in HAT culture medium, then transfer them into 60 ml of HAT culture medium, mix gently, and then transfer 100 μl / well into a 96-well cell culture plate and culture in a 5% CO2, 37°C constant temperature incubator.

[0094] Hybridoma cell screening

[0095] After fusion, carefully observe the growth of hybridoma cells. Generally, small clones will appear after about three days of culture. On the seventh day, use HAT culture medium for the first medium change. Around the 9th to 10th day, when the cells have grown to 20% to 50% of the bottom of the well, aspirate the supernatant and perform indirect ELISA detection. Use P / N ≥ 2 as the positive judgment standard, and use SP2 / 0 cell culture supernatant and normal mouse serum as negative controls. Expand the culture of cells that test positive and perform cloning culture at the same time. Switch to HT culture medium for the first subclone, and switch to ordinary complete culture medium after two weeks.

[0096] Hybridoma cell cloning culture

[0097] Hybridoma cells that are positive in ELISA tests are cloned and cultured promptly using the limiting dilution method. The steps are as follows:

[0098] (1) Use a pipette to gently pipette the hybridoma cells in the ELISA-positive wells to mix thoroughly. Take a sample and count the cells on a hemocytometer. Then adjust the cell count with HT medium. For subcloning, generally adjust the cell count to 5 cells / ml.

[0099] (2) Inoculate the hybridoma cell suspension into a 96-well cell culture plate, 200 μl per well, and culture at 37°C, 5% CO2, and saturated humidity.

[0100] (3) Observe and record every day whether the cells in each well are monoclonal. When the cells grow to 20% to 50% of the bottom of the well, change the medium and perform indirect ELISA test on the cell supernatant.

[0101] (4) Select the positive wells with monoclonal clones and high OD450 values ​​for re-cloning until the positive rate of all cloned cell wells reaches 100%. It can be determined that the hybridoma cell line secreting specific monoclonal antibodies, i.e., the hybridoma cell of anti-cat allergen Fel d 1 protein monoclonal antibodies, is obtained, and the culture is expanded and preserved in time.

[0102] Hybridoma cell recovery

[0103] Take out a hybridoma cell cryopreservation tube containing a specific anti-cat allergen Fel d 1 protein monoclonal antibody from the liquid nitrogen tank, clamp it with tweezers and quickly place it in a 37℃ water bath. Shake continuously to quickly thaw the cells. Open the cryopreservation tube aseptically and transfer the cell fluid into 5ml RPMI-1640 culture medium. Place it in a 37℃ incubator containing 5% CO2 until the hybridoma cells are completely attached to the wall, and then change the cell medium.

[0104] PCR Amplification and Sequencing of Monoclonal Antibody Variable Region Genes

[0105] The selected 2A7 and 6B1 cell lines were cultured and mRNA was extracted for sequence determination and sent to Nanjing Mingyan Biotechnology Co., Ltd. for sequencing. The results showed:

[0106] The amino acid sequence of the light chain variable region of antibody 2A7 is: QAVVTQESALTTSPGETVTLTCRSSTGAVITSNYANWVQEKPDHLFTGLIGGTNNRGLGVPA RFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVL (SEQ ID NO. 3);

[0107] The amino acid sequence of the heavy chain variable region is: QVQLKESGPGLVAPSQSLSITCTVSGSSLTNYAVHWVRQPPGKGLEWLGVIWPGGTTNYNS PLMSRLSISKDISKSQVFLKMNSLQTDDTAMYYCARDPLYGNSWFAYWGQGTQVTVTA (SEQ ID NO. 4);

[0108] The amino acid sequence of the light chain variable region of antibody 6B1 is: DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTHVGWFQQKTGQSPKALIYSASYRYSGVPD RFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNNYPYTFGGGTKVEKK (SEQ ID NO. 5);

[0109] The amino acid sequence of the heavy chain variable region is: QVQLQQPGAELVRPGASVRLSCKASGYTFTNYWINWVKQRPGQGLEWIGNIFPSDSYTNYNHNFKDKASLTVDKSSTTAYMHLSSPTSEDSAVYYCSRRGIGTVLFDYWGQGTTLTVSS (SEQ ID NO. 6).

[0110] Large-scale production of monoclonal antibodies

[0111] Six-week-old female Balb / c mice were selected and, under sterile conditions, each mouse was intraperitoneally injected with 0.5 ml of paraffin oil. One week later, the prepared hybridoma cell culture medium (DMEM) was injected into the mouse peritoneal cavity.

[0112] Preparation of hybridoma cells: Select hybridoma cells with good growth conditions and subculture them in culture flasks. Under sterile conditions, pour out the culture medium in the cell flask, add 5 ml of serum-free DMEM medium, gently blow up the cells, and transfer all into a centrifuge tube. Centrifuge at room temperature and 800 rpm / min for 5 minutes, discard the supernatant, add an appropriate amount of serum-free DMEM medium to suspend the cells, count the cells, and adjust the cell number to 1×10 6 ~6×10 6 / ml, inject 0.5ml of the obtained cells into the abdominal cavity of each mouse, observe the mice every day, and the abdomen of the mice will gradually bulge. When the mice become depressed, immobile, and on the verge of death after about 7 to 10 days, they are killed by cervical dislocation, and the ascites is collected. Centrifuge at room temperature and 10000 rpm / min for 10 minutes, and the clear liquid in the middle is the desired monoclonal antibody.

[0113] Purification and HRP-labeled monoclonal antibodies

[0114] Purify the monoclonal antibodies using the Protein G column and label them using the HRP labeling kit to obtain HRP-labeled specific monoclonal antibodies. Isotype analysis of the Fel d 1 monoclonal antibodies was performed using the Proteintech Mouse Monoclonal Antibody Isotyping Kit according to the manufacturer's instructions. ELISA analysis revealed that both antibodies 2A7 and 6B1 belonged to the IgG1 subtype, as shown in Table 1.

[0115] Table 1 Antibody typing for 2A7 and 6B1

[0116] Antibody Heavy chain subtype Light chain subtype 2A7 IgG1 λ 6B1 IgG1 κ

[0117] Example 2 Verification of the Binding Specificity of 2A7 and 6B1 Antibodies to FeI d 1 Protein

[0118] Western blot analysis was performed to identify the binding specificity of purified 2A7 and 6B1 antibodies to FeI d 1 protein. Purified recombinant FeI d 1 protein was used as the antigen in the experiment as follows:

[0119] Use 12% SDS-PAGE to separate the prokaryotic expressed and purified Fel d 1 protein, and then use a Bio-rad wet transfer apparatus to transfer the protein on the gel to a PVDF membrane. Block with 5% skim milk dissolved in TBST for 2 hours at room temperature, and then incubate with the primary antibody solution (2A7 and 6B1) diluted (1:2000) with the above 5% skim milk on a shaker at 4°C overnight. Then wash 4 times with TBST, and incubate with the secondary antibody (HRP-labeled goat anti-mouse IgG antibody) solution diluted (1:5000) with TBST on a shaker at room temperature for 1 hour. After washing 5 times with TBST, use ECL luminescent liquid from Saner Company for color development. In the experiment, 2A7 and 6B1 were used as primary antibodies, and HRP-labeled goat anti-mouse IgG was used as secondary antibody to detect the specific binding of the prepared monoclonal antibodies to the recombinant Fel d 1 protein. Figure 1 As shown, both antibodies specifically recognized recombinant FeI d 1 protein, wherein pET-28a was used as a negative control of the experiment.

[0120] Specific primers were designed: pCAGGS-Fel d1-F: 5'-cattttggcaaagaattcgcggccgcGCCGCCACCATGGTAAAAATGGCTGAA-3'; pCAGGS-Fel d1-R: 5'-tcgtcgtcatccttgtaatcctcgagACACAGCGGGCTGGTGTAGA-3'. The primers were double-digested with Not I and Xho I and ligated to the pCAGGS plasmid to construct a Fel d 1 eukaryotic expression vector. 2A7 and 6B1 were transfected with pCAGGS-Feld 1 into 293T cells for indirect immunofluorescence detection, while SP2 / 0 cell supernatant was used as a negative control. The results showed that both monoclonal antibodies exhibited strong specific fluorescence against cells transfected with pCAGGS-Feld 1, while the SP2 / 0 cell supernatant control was negative ( Figure 2 ).

[0121] Example 3 Epitope Identification of Monoclonal Antibodies 2A7 and 6B1

[0122] The antigenic epitopes of the two antibodies shown in the present invention were identified using recombinantly expressed cat allergen FeI d 1 protein truncated sequences and immunoblotting. Overlapping sequences were designed to prevent possible epitope loss.

[0123] Use the corresponding pair of primers F1 and R1 (for domain 1), F2 and R2 (for domain 2), and F3 and R3 (for domain 3) to amplify and ligate into the linearized pGEX6p-1 vector, respectively, and name them F1, F2, and F3. The primer list is as follows:

[0124] Primer name F / R Primer sequence 5' to 3' F1 F 5′-ATGGTAAAAATGGCTGAAACAT-3′ R 5′-CACGCGGCTAATCAGGCCG-3′ F2 F 5′-ATGGGCCTGATTAGCCGCGTGCT-3′ R 5′-TGGGGTGCCGGTCAGAAAC-3′ F3 F 5′-ATGTTTCTGACCGGCACCCCAG-3′ R 5′-ACACAGCGGGCTGGTGTAG-3′

[0125] Transform DH5α competent cells and select using solid LB plates containing ampicillin (100 ng / ml). Single colonies obtained from overnight cultures were cultured in LB medium containing antibiotics, and the plasmids were extracted. Successfully constructed truncated plasmids were then transformed into BL21(DE3) competent cells. Single colonies were selected and cultured overnight in 5 ml of LB medium containing ampicillin at 37°C. The overnight culture was inoculated at a ratio of 1:100 into fresh culture medium of the corresponding type and cultured at 37°C until an OD600 of 0.6 was reached. Expression was then induced for 6 hours by adding 1 mM IPTG.

[0126] Immunoblotting was used to identify the epitopes recognized by 2A7 and 6B1. The simple procedure was as follows: 5 ml of bacterial culture was collected by centrifugation, resuspended in 1 ml of PBS, and boiled in a 99°C metal bath for 10 minutes to prepare protein samples. Prokaryotically expressed proteins were separated by 12% SDS-PAGE and then transferred to a PVDF membrane using a Bio-rad wet transfer apparatus. Blocking was performed with 5% skim milk dissolved in TBST for 2 hours at room temperature. The membrane was then incubated with a primary antibody solution (2A7 and 6B1) diluted 1:2000 in the same 5% skim milk solution on a shaker at 4°C overnight. The membrane was then washed four times with TBST and incubated with a secondary antibody (HRP-conjugated goat anti-mouse IgG antibody) diluted 1:5000 in TBST on a shaker at room temperature for 1 hour. After washing five times with TBST, the membrane was developed with a colorimetric solution. In the experiment, 2A7 and 6B1 were used as primary antibodies, and HRP-labeled goat anti-mouse IgG was used as secondary antibody to identify the epitope region recognized by the cat allergen Fel d 1 monoclonal antibody. The results showed that the epitope recognized by the 6B1 monoclonal antibody was F3 (105aa-163aa); the epitope recognized by the 2A7 monoclonal antibody was F1 (1aa-59aa) ( Figure 3 ).

[0127] Example 4 Highly sensitive double-antibody sandwich ELISA method for detecting cat allergen FeI d 1

[0128] The above results demonstrate that the obtained monoclonal antibody against the cat allergen FeI d 1 can detect native FeI d 1 protein. A highly sensitive double-antibody sandwich ELISA method was developed using the anti-Fel d 1 antibodies 2A7 and 6B1 described herein to detect active FeI d 1 in cat saliva. The method primarily uses 2A7 as the capture antigen, HRP-labeled 6B1 as the detection antigen, and prokaryotically expressed and purified FeI d 1 as the standard to detect active FeI d 1 in cat saliva. Experiments have demonstrated that the sandwich ELISA developed herein has high sensitivity and a wide linear range.

[0129] The specific experimental methods are as follows:

[0130] 1. Sample source: saliva from five healthy cats aged 6-8 months.

[0131] 2. Specific steps:

[0132] (1) ELISA plate coating: Dilute 2A7 to 4 μg / ml with PBS, coat 100 μl per well of the ELISA plate horizontally, and incubate at 37°C for 2 h.

[0133] (2) Blocking: Wash the plate 4 times with PBST and block with 5% skim milk at 4°C for 12 h;

[0134] (3) Sample incubation: A known concentration of FeI d 1 protein was serially diluted with PBS to draw a standard curve. The concentrations after dilution were 4000, 2000, 1000, 500, 200, 100, 50, 10, 1, and 0.1 ng / ml, respectively. The saliva sample to be tested was diluted 10-fold, and 100 μl of the sample to be tested was added to each well. PBS was used as a negative control and the cells were incubated at 37°C for 2 h.

[0135] (4) Washing: Remove the ELISA plate, spin dry it, wash it four times with PBST, and spin dry on absorbent paper;

[0136] (5) Detection antibody incubation: horseradish peroxidase-labeled monoclonal antibody 6B1 was diluted 1:2000, 100 μl was added to each well, and the reaction was carried out at 37°C for 1.5 h;

[0137] (6) Color development: After washing, add 100 μl of TMB to each well and develop color at 37°C for 20 min;

[0138] (7) Termination: Add 50 μl of 2 M sulfuric acid to terminate;

[0139] (8) Result calculation and judgment: Read the absorbance at 450 nm, draw a standard curve based on the standard Fe d 1 concentration, and calculate the concentration of the sample to be tested based on the standard curve.

[0140] Data processing:

[0141] A standard curve was created using known concentrations of Feld 1 protein. The concentration of the diluted Feld 1 protein ranged from 0.1 to 4000 ng / ml. The standard curve was plotted as shown in the figure. Calculations indicate that the method is linear within the detection range of 20 to 500 ng / ml. The standard curve was plotted using the natural logarithm of Feld 1 protein concentration (LN) as the horizontal axis and the OD450nm value as the vertical axis. Figure 4 The equation of the standard curve is y=0.4405x-1.0537 and the correlation coefficient is 0.9959.

[0142] The above description of the embodiments is intended to facilitate understanding and use of the present invention by those skilled in the art. Those skilled in the art will readily be able to make various modifications to these embodiments and apply the general principles described herein to other embodiments without resorting to creative effort. Therefore, the present invention is not limited to the above-described embodiments. Any improvements or modifications made by those skilled in the art based on the principles of the present invention that do not depart from the scope of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against cat allergen Fel d 1 protein, characterized in that The monoclonal antibodies are secreted by hybridoma cell lines 2A7 and 6B1, respectively; Among them, the amino acid sequence of the light chain variable region of antibody 2A7 is: QAVVTQESALTTSPGETVTLTCRSSTGAVITSNYANWVQEKPDHLFTGLIGGTNNRGLGVPA RFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVL; The amino acid sequence of the heavy chain variable region is: QVQLKESGPGLVAPSQSLSITCTVSGSSLTNYAVHWVRQPPGKGLEWLGVIWPGGTTNYNSPLMSRLSISKDISKSQVFLKMNSLQTDDTAMYYCARDPLYGNSWFAYWGQGTQVTVTA The amino acid sequence of the light chain variable region of antibody 6B1 is: DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTHVGWFQQKTGQSPKALIYSASYRYSGVPD RFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNNYPYTFGGGTKVEKK; The amino acid sequence of the heavy chain variable region is: QVQLQQPGAELVRPGASVRLSCKASGYTFTNYWINWVKQRPGQGLEWIGNIFPSDSYTNY NHNFKDKASLTVDKSSTTAYMHLSSPTSEDSAVYYCSRRGIGTVLFDYWGQGTTLTVSS.

2. Use of the monoclonal antibody according to claim 1 in preparing an in vitro detection kit for cat allergen FeI d 1 protein antigen.

3. A double antibody sandwich ELISA kit for detecting cat allergen FeI d 1 protein, characterized in that: The kit comprises the monoclonal antibody according to claim 1.

4. The double antibody sandwich ELISA kit for detecting cat allergen FeI d 1 protein according to claim 3, characterized in that: The kit also includes an ELISA plate, a blocking solution, a diluent, a washing solution, a color developer, and a stop solution.

5. A double antibody sandwich ELISA method for detecting cat allergen FeI d 1 protein antigen for non-diagnostic purposes, characterized in that: The method comprises: coating a monoclonal antibody onto an ELISA plate, blocking the plate, adding a sample to be tested and incubating the plate, washing the ELISA plate, adding an enzyme-labeled monoclonal antibody for reaction, adding a color developer after washing for color development, adding a stop solution for termination, reading the absorbance at 450 nm, and determining the test result based on the P / N ratio, wherein the capture antibody is the 2A7 monoclonal antibody in claim 1, and the detection antibody is the 6B1 monoclonal antibody in claim 1 labeled with HRP.

6. The double antibody sandwich ELISA method according to claim 5, wherein The method comprises the following steps: (1) Coating: Dilute the monoclonal antibody to 4 μg / mL with phosphate buffer (pH 9.6), add 100 μl per well to the ELISA plate, coat at 37°C for 2 h, and wash four times with 200 μl / well PBST. (2) Blocking: Add 100 μl of 5% skim milk to each well, block at 4°C for 12 h, and wash four times with 200 μl / well PBST; (3) Add the sample to be tested: add 100 μl / well of the sample to the ELISA plate, use purified FeI d 1 protein and PBS as positive and negative controls, and incubate at 37°C for 2 h; (4) Washing: Shake the plate dry, wash four times with 200 μl / well PBST, and pat dry on absorbent paper; (5) Add enzyme-labeled monoclonal antibody: dilute HRP-labeled monoclonal antibody 1:2000 with PBS, add 100 μl to each well, and react at 37°C for 1.5 h; (6) Color development: After washing, add 100 μl of TMB to each well and develop color at 37°C in the dark for 20 min; (7) Result calculation and judgment: Add 50 μl 1.5M H2SO4 to terminate the reaction, and OD 450nm Read the results. When the OD value of the test sample is ≥0.062 and the P / N value is ≥2.1, it is judged as positive; otherwise, it is judged as negative.

Images