Anti-cat IgE monoclonal antibody hybridoma cell strain, monoclonal antibody and application thereof

Abstract

The invention discloses an anti-cat IgE (Immunoglobulin E) monoclonal antibody hybridoma cell strain, a monoclonal antibody and application of the monoclonal antibody, and belongs to the fields of immunology, in-vitro diagnosis and detection technologies and hybridoma antibody preparation. A cat IgE antigen is purified through a salting-out method and column chromatography, after mice are immunized, 6B5 and 13E11 hybridoma cell strains are screened out through cell fusion, IgG type antibodies secreted by the hybridoma cells do not have cross reaction with cat IgG, EC50 values are smaller than or equal to 0.0261 and 0.0274 respectively, the titer is larger than or equal to 1: 100000, and the purity is larger than or equal to 95%. According to the present invention, the 6B5 and 13E11 antibody combination is used for colloidal gold chromatography detection, the distance between the detection line and the quality control line is optimized to 0.5 cm, the double-antibody sandwich system is constructed, the detection sensitivity reaches 0.1 ng / mL, and 8 clinical allergic cat samples are completely positive through verification; the invention breaks through the bottleneck of the traditional detection technology, provides a high-specificity and high-sensitivity cat IgE detection scheme, lays a foundation for early diagnosis and precise medical treatment of cat allergic diseases, and has remarkable clinical application value and industrial prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunology and in vitro diagnostic detection technology and hybridoma antibody preparation, and particularly relates to a hybridoma cell strain of anti-cat IgE monoclonal antibody, a monoclonal antibody and application thereof. BACKGROUND

[0002] Immunoglobulin (IgE) has a molecular weight of 190KD and generally exists in the form of monomer in vivo, and is a very small part of total antibodies in human and animal serum. IgE antibody is an important member of immunoglobulin, and its role can be amplified by binding with Fc receptor to cause typical acute allergic reaction and play a core role in allergic reaction. In recent years, cats as companion animals have played an important role in human life, and cat allergy has been paid more and more attention. When an allergic individual contacts a cat allergen, the allergen binds with specific IgE antibody, causing mast cells and basophils to release histamine and other inflammatory mediators, and triggering allergic symptoms such as sneezing, runny nose and itchy eyes. At present, anti-cat IgE antibody is mainly used for allergic diagnosis and research. In diagnosis, the level of specific IgE antibody in patient serum can be determined to determine the sensitivity of the patient to cat allergy. Common detection methods include enzyme-linked immunosorbent assay (ELISA) and immunoblotting. In disease diagnosis, cat IgE monoclonal antibody is widely used in intradermal test and serological detection. The intradermal test observes skin reaction by local injection of allergen to judge allergic symptoms, and the use of monoclonal antibody can improve the standardization and repeatability of the test. Serological detection quantitatively determines the levels of total IgE and specific IgE in serum to provide objective basis for disease diagnosis and severity.

[0003] The first method of the prior art is intradermal injection, which can cause temporary pain or discomfort due to needle prick, and even in rare cases, can cause severe allergic reaction. The second method is detection of serum IgE, but its sensitivity is limited, and IgE positive does not necessarily appear clinical symptoms, leading to overdiagnosis and being time-consuming. The third method is patch test, which requires high operation requirement and needs strict standardized operation, and some patients may also have irritation reaction.

[0004] With the progress of science and technology, the monoclonal antibody technology is developing. Its emergence provides a new important tool for the study of cat IgE, and the hybridoma preparation technology is the cornerstone of the preparation of monoclonal antibodies, which is an important core technology. In the past few decades, it has greatly promoted the development of biomedical practice and in vitro diagnosis field. Hybridoma technology is to fuse the mouse myeloma cells which cannot produce specific antibodies and the purebred mouse B cells immunized by specific antigen in vitro to form hybrid cell lines, and then produce monoclonal antibodies through multiple cloning and screening. The antibodies prepared by the traditional hybridoma technology have obvious advantages in purity, activity, structure characterization and stability, and are more suitable for application in in vitro diagnostic reagents. SUMMARY

[0005] To solve the problems of pain and discomfort caused by pricking, limited sensitivity of serum IgE detection and high operation requirement of patch test.

[0006] To solve the above problems, the technical scheme is provided as follows:

[0007] Preferably, the cell strain can stably secrete the monoclonal antibody which specifically binds to cat IgE, including hybridoma cell strains with clone numbers 6B5 and 13E11.

[0008] Preferably, the anti-cat IgE monoclonal antibody is produced by the hybridoma cell strain.

[0009] Preferably, the binding EC50 value of the monoclonal antibody to cat IgE is 0.0261-0.0274 μg / mL.

[0010] An anti-cat IgE monoclonal antibody hybridoma cell strain and application of the monoclonal antibody

[0011] Preferably, the immunochromatography reagent strip for detecting IgE includes a PVC bottom plate and a sample pad, a chemical coupling pad, an NC membrane and an absorption pad connected in sequence.

[0012] Preferably, the NC membrane is provided with a detection line and a quality control line, the detection line is coated with the anti-cat IgE monoclonal antibody of claim 2, and the chemical coupling pad is coated with the colloidal gold-labeled anti-cat IgE monoclonal antibody of claim 2.

[0013] Preferably, the monoclonal antibody coated on the detection line is 6B5, and the colloidal gold-labeled monoclonal antibody coated on the chemical coupling pad is 13E11.

[0014] Preferably, the final concentration of the anti-cat IgE monoclonal antibody coated on the detection line is 0.5-2 mg / mL, and the final concentration of the goat anti-mouse IgG antibody coated on the quality control line is 0.8 mg / mL.

[0015] Preferably, the distance between the detection line and the quality control line is 0.5 cm, and the distance between the detection line and the lower edge of the NC membrane is 0.7 cm.

[0016] Preferably, the pretreatment solution of the chemical coupling pad is composed of the following components: 20% sucrose 10 mL, 10% BSA 10 mL, 5% sodium casein 2 mL, 5% PVA 2 mL, 10% Triton X-100 10 mL, 2 mmol / L boric acid / 0.1% PEG solution 10 mL, supplemented with TE to 100 mL, and the pH value is adjusted to 7.6±0.05.

[0017] Preferably, the assembled reagent strip has a width of 3.0 mm, and the covering size of each component meets the requirements, the absorption pad covers the NC membrane by not less than 1 mm, the chemical coupling pad covers the NC membrane by 1-2 mm, and the sample pad covers the chemical coupling pad by not less than 2 mm.

[0018] The anti-cat IgE monoclonal antibody hybridoma cell strain and the monoclonal antibody have the following effects and advantages:

[0019] 1. In the patent, BALB / c mice are immunized with cat IgE as an antigen, and after immunization, cell fusion, screening and subcloning, a hybridoma cell strain capable of stably passing and secreting cat IgE protein is obtained.

[0020] 2. In the patent, the antibody secreted by the hybridoma cell strain can be applied in the preparation of a detection reagent for cat allergic disease reaction or a detection kit for cat allergic disease.

[0021] 3. In the patent, the characteristics effectively avoid the false positive problem caused by the homology of light chains in traditional antibodies, and the indirect ELISA determination titer is all greater than or equal to 1:100000, so that the detection sensitivity is greater than or equal to 0.1 ng / mL, thereby providing high credibility technical support for early diagnosis of cat allergic diseases.

[0022] 4. In the patent, the recognition ability to different epitopes of cat IgE significantly improves the detection signal strength and specificity, and the detection process can be completed within 15-20 minutes, thereby meeting the rapid detection needs of veterinarians in the clinic, and solving the problems of long detection time and fuzzy signal in the prior art.

[0023] 5. In the patent, the technical scheme defines the components and parameters, constructs a whole-process quality control system from cell culture to finished product preparation, provides a reliable technical foundation for the large-scale production of the detection kit, effectively reduces the production cost and improves the product stability. Preservation information: The hybridoma cell line 6B5 is preserved in the China Center for Type Culture Collection, and the preservation date is July 16, 2025, and the preservation number is CCTCC NO: C2025213. The hybridoma cell line 13E11 is preserved in the China Center for Type Culture Collection, and the preservation date is July 16, 2025, and the preservation number is CCTCC NO: C2025201. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is the serum titer plot of the mouse after three immunizations in the application;

[0025] Figure 2 is the titer of eight positive antibodies in the application;

[0026] Figure 3 is the detection of the clinical positive sample in the application. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, not all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application,

[0028] It should be noted that, in this paper, relationship terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between the entities or operations, and the term "include", "contain" or any other variant thereof is intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, article or equipment. Without more limitation, the elements defined by the statement "include" do not exclude the presence of other identical elements in the process, method, article or equipment including the elements.

[0029] Embodiment 1

[0030] This embodiment provides a preparation of an immunogen, and the following implementation content is provided:

[0031] Experimental purposes:

[0032] Preparation of immunogen.

[0033] Experimental steps:

[0034] (1) Take the cat serum dilution, add equal amount of saturated (NH4)2SO4 solution, salting out;

[0035] (2) Centrifugal precipitation, dissolved in a small amount of normal saline, dialysis, desalination;

[0036] (3) DE52 column, eluted with 0.005 mol / L pH 7.4 PB liquid, collected elution protein peak (IgG), discarded;

[0037] (4) Change to 0.025 mol / L pH 7.4 PB liquid elution, the main protein peak washed is IgE;

[0038] (5) Dialysis desalination;

[0039] (6) G150 column, eluted with 0.01 mol / L pH 7.4 PBS (0.14 mol / L NaCl), the eluate is the purified IgE.

[0040] Experimental results:

[0041] This example is through the cat serum dilution, salting out, centrifugal precipitation, dialysis desalination, and then DE52 column step elution to remove IgG and collect the main IgE containing protein peak, followed by dialysis desalination and G150 column further purification, finally successfully obtained the purified IgE, completed the preparation of immunogen.

[0042] Example 2

[0043] This example is a kind of mouse immunization, the following implementation content:

[0044] Experimental purpose:

[0045] Immunize mice.

[0046] Experimental species:

[0047] 6-8 weeks old healthy female mice

[0048] Experimental steps:

[0049] (1) The antigen cat IgE and Freund's complete adjuvant were mixed and emulsified in equal volume, and the mice were immunized by subcutaneous injection at multiple points on the back, with an immunization dose of 50 μg per mouse;

[0050] (2) Every two weeks, replace Freund's complete adjuvant with Freund's incomplete adjuvant, and perform secondary and tertiary immunization in the same way and with the same dose;

[0051] (3) On the 10th day after the third immunization, collect trace mouse tail blood, separate serum and detect the titer by ELISA. The mouse serum titer reaches 1:100000 or more, and cell fusion can be performed. After the titer is qualified, select mice with higher titers, and perform intraperitoneal boost immunization 3 days before fusion, and the immunization dose is still 50 μg per mouse.

[0052] Experimental results:

[0053] The detailed mouse serum titer is shown in Table 1. In this example, 6-8 week old healthy mice were immunized as follows. The antigen cat IgE was mixed with Freund's complete adjuvant in equal volume and emulsified, and then subcutaneously injected into the back of the mice in multiple points at a dose of 50 μg per mouse for primary immunization. Every 2 weeks, Freund's incomplete adjuvant was used for secondary and tertiary immunization in the same way and at the same dose. On the 10th day after the third immunization, mouse tail blood was collected to separate serum, and the titer was detected by ELISA. When the titer reaches 1:100000 or more, cell fusion can be performed. Select qualified mice with higher titers, and perform intraperitoneal boost immunization at a dose of 50 μg per mouse 3 days before fusion, and successfully complete the mouse immunization operation, laying a foundation for subsequent cell fusion.

[0054] Example 3

[0055] This example is about cell fusion, and the following implementation content:

[0056] Experimental purpose:

[0057] Cell fusion using fusion technology

[0058] Experimental steps:

[0059] (1) 3 days before fusion, the mice were boosted, and the immunization dose was the same as before, without adjuvant, and directly injected intraperitoneally.

[0060] (2) Prepare sufficient myeloma cells sp2 / 0, adjust the cell state, keep the cell viability above 95%, collect the cell suspension, centrifuge at 300g for 5 min, and resuspend for standby;

[0061] (3) Under sterile conditions, take the spleen of the qualified immune mouse, grind it thoroughly, centrifuge at 500g for 5 min, and discard the supernatant;

[0062] (4) Lyse according to 5 mL per mouse spleen cells plus red blood cell lysis solution, mix well, stand at room temperature for 5 min, then add 10 mL of medium to terminate lysis, and centrifuge at the same speed to discard the supernatant and collect the spleen B lymphocytes and count them;

[0063] (5) After counting, mix the spleen B lymphocytes and myeloma cells sp2 / 0 according to a ratio of 3:1, and centrifuge and wash the cells;

[0064] (6) Use 50% PEG as a fusing agent to perform fusion, after fusion, resuspend the cell precipitate with HAT medium, and distribute in 96-well cell culture plates, and culture in a 37°C, 5% CO2 incubator.

[0065] Experimental results:

[0066] This example is intended to use fusion technology to perform cell fusion. Three days before fusion, the mouse is given a booster immunization in the abdominal cavity without adjuvant, and the dosage is the same as before; prepare a myeloma cell sp2 / 0 suspension with a survival rate of more than 95%; take the spleen of the immune mouse with a qualified titer aseptically, grind and centrifuge, then treat with red blood cell lysate and centrifuge, collect the B lymphocytes of the spleen, and count them; mix the B lymphocytes of the spleen with the myeloma cells sp2 / 0 at a ratio of 3:1, centrifuge and wash, then use 50% PEG for fusion, resuspend the cell precipitate after fusion with HAT medium, distribute in 96-well cell culture plates, and culture in a 37°C, 5% CO2 incubator, successfully completing the cell fusion operation.

[0067] Example 4

[0068] This example is about hybridoma cell screening, and the following implementation content:

[0069] Experimental purpose:

[0070] Screen hybridoma cells.

[0071] Experimental steps:

[0072] (1) Dilute cat IgE to 0.2 μg / mL with coating buffer, 100 μL / well to coat the enzyme-labeled plate, and place in a 4°C refrigerator overnight;

[0073] (2) The next day, take out the coated enzyme-labeled plate, wash the plate 3 times with PBST, then block with PBST containing 1% BSA (bovine serum albumin) at room temperature for 1 h, and spin dry;

[0074] (3) Add hybridoma cell supernatant, incubate at room temperature for 1 h, wash 3 times with PBST, and spin dry;

[0075] (4) Add horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody in proportion, incubate at room temperature for 1 h, wash 3 times with PBST, and spin dry;

[0076] (5) Add TMB color developing solution, incubate at room temperature for 8 min, and stop the reaction with a stop solution;

[0077] (6) Read at 450 nm wavelength on a microplate reader;

[0078] (7) Perform the same operation as above, coat cat IgG, and select wells with lower reaction values.

[0079] Experimental results:

[0080] The purpose of this step is to discard the cells that have non-specific reaction with cat IgG, and then to screen the cells with higher specificity to cat IgE.

[0081] Example 5

[0082] This example is the subclone screening of positive hybridoma cells and cell cryopreservation, and the following implementation content:

[0083] Purpose of the experiment:

[0084] Subclone screening of positive hybridoma cells and cell cryopreservation.

[0085] Experimental procedure:

[0086] (1) The cell wells screened in Example 4 were subcloned, and the cell wells with high antibody secretion concentration and positive to cat IgE were screened by quantifying the antibody secretion concentration of each cell well.

[0087] Experimental results:

[0088] Finally, 8 hybridoma cell strains that can stably secrete anti-cat IgE monoclonal antibodies were obtained, which were expanded and cryopreserved in liquid nitrogen after cell cryopreservation.

[0089] Example 6

[0090] This example is the preparation and titer determination of anti-cat IgE monoclonal antibody, and the following implementation content:

[0091] Purpose of the experiment:

[0092] Preparation and titer determination of anti-cat IgE monoclonal antibody.

[0093] Experimental procedure:

[0094] (1) Cat IgE protein was coated in enzyme-labeled plates at a concentration of 0.5 μg / mL, 100 μL per well, at 4°C overnight;

[0095] (2) The next day, wash the plate three times, then add blocking solution and react at room temperature for 1 h;

[0096] (3) Dilute the purified anti-cat IgE protein monoclonal antibody to 8 concentrations with 4-fold gradient from 2 μg / mL, 100 μL per well, and add to the enzyme-labeled plate, and react at room temperature for 1 h;

[0097] (4) Wash the plate three times, then add goat anti-mouse IgG secondary antibody labeled with horseradish peroxidase in proportion, and react at room temperature for 1 h;

[0098] (5) Wash the plate three times, then add TMB color developing solution, react at room temperature for 8 minutes, then add stop solution and immediately read on the enzyme-labeled instrument.

[0099] Results: See Table 1 for details

[0100] Graphpad Prism software as Figure 2 The EC50 values were automatically calculated by the software, see Table 1.

[0101] Table 1: Eight strains of positive antibody titer and EC50 results

[0102] 6B5 8D10 11B8 12D11 13D2 13E11 15A8 16B4 EC50 0.0261 0.0592 0.0468 0.0345 0.0345 0.0274 0.0506 0.06633

[0103] This example aims to prepare and determine the titer of anti-cat IgE monoclonal antibody. The experimental steps are as follows: cat IgE protein is coated on an enzyme-labeled plate at a concentration of 0.5 μg / mL, 100 μL / well, 4°C overnight; the next day, after washing the plate, add blocking solution and react at room temperature for 1 h; dilute the purified anti-cat IgE monoclonal antibody by 4 times gradient from 2 μg / mL, 100 μL / well, add to the enzyme-labeled plate, and react at room temperature for 1 h; after washing the plate, add horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody, and react at room temperature for 1 h; after washing the plate, add TMB color developing solution and react at room temperature for 8 minutes; immediately after adding the stop solution, read the results with an enzyme-labeled instrument, and then plot the results with Graphpad Prism software to calculate the EC50 value. Finally, the preparation and titer determination of anti-cat IgE monoclonal antibody are completed.

[0104] Example 7

[0105] This example is a gold-labeled antibody and antibody pairing test, and the following implementation content is as follows:

[0106] Purpose of the experiment:

[0107] Gold-labeled antibody and antibody pairing test.

[0108] Experimental steps:

[0109] (1) Take 10 mL of colloidal gold in a clean 100 mL conical flask, add the most appropriate proportion of 0.1 mol / mL K2CO3, mix quickly with a magnetic stirrer, then add the appropriate proportion of anti-cat IgE protein monoclonal antibody or mouse IgG, and slowly stir with a magnetic stirrer at 4°C for 20 min.

[0110] (2) Add 10% BSA to the above conical flask to a final concentration of 1%, and continue to slowly stir for 20 min.

[0111] (3) Centrifuge the labeled gold complex at 2000 r / min, 4°C for 10 min, remove the condensate, and retain the red supernatant.

[0112] (4)Collect the supernatant after centrifugation with a pipette, centrifuge at 14000r / min, 4℃ for 30min. Discard the transparent supernatant, resuspend with 1 mL of reconstitution solution (1% BSA, 5% sucrose, 0.02mol / mL Tris-HCl pH 8.6), which can be used for spraying on the gold pad.

[0113] Experimental results:

[0114] This example carries out the gold-labeled antibody and antibody pairing test. Take 10 mL of colloidal gold, add an appropriate amount of 0.1mol / mL K2CO3 and mix well, then add an appropriate amount of anti-cat IgE protein monoclonal antibody or mouse IgG, slowly stir at 4℃ for 20min; then add 10% BSA to a final concentration of 1%, continue to stir for 20min; centrifuge the labeled gold complex at 2000r / min, 4℃ for 10min to remove the agglomerates, take the supernatant and centrifuge at 14000r / min, 4℃ for 30min, discard the supernatant and resuspend with 1mL of reconstitution solution, obtain the gold-labeled complex that can be used for gold pad spraying, and complete the related operation of gold-labeled antibody and antibody pairing test.

[0115] Example 8

[0116] This example is the coating of the detection line capture antibody and the quality control line capture antibody on the NC membrane. The following is the implementation content:

[0117] Table 2: Components of detection line and quality control line

[0118]

[0119] Use Shanghai Ningwei WRF-HPY001-14 membrane drawing and gold spraying integrated machine for antibody coating, spraying volume 0.8μL / cm, line drawing speed 60mm / s.

[0120] (1) Dilute the anti-cat IgE protein monoclonal antibody and goat anti-mouse IgG to the use concentration with the coating solution respectively.

[0121] (2) Load the two antibodies into sample pump 1 and sample pump 2 respectively.

[0122] (3) Set the coating program: set the distance between the detection line and the quality control line to 0.5cm, the distance between the detection line and the lower edge of the NC membrane to 0.7cm, the line drawing speed to 60mm / s, and the spraying volume to 0.8μL / cm.

[0123] (4) Enter the working state, press the start key to start coating, and mark the uncoated part.

[0124] Experimental results:

[0125] The embodiment aims to complete the coating of detection line capture antibodies and quality control line capture antibodies on the NC membrane. In the experiment, the detection line uses anti-cat IgE protein monoclonal antibody, and the quality control line uses goat anti-mouse IgG antibody, both of which are diluted to the use concentration with 20% trehalose, 20% sucrose and 10 mM PB. Through Shanghai Ningweierfen gold film spraying integrated machine WRF-HPY001-14, the two antibodies are respectively loaded into the sample pump, the distance between the detection line and the quality control line is set to 0.5 cm, the distance between the detection line and the lower edge of the NC membrane is set to 0.7 cm, the coating is performed at a line speed of 60 mm / s and a liquid spraying amount of 0.8 μL / cm, and the uncoated part is labeled. Finally, the coating operation of the detection line and the quality control line antibodies on the NC membrane is successfully completed.

[0126] Example 9

[0127] This embodiment is a chemical pretreatment, and the following is the implementation content:

[0128] Experimental purpose:

[0129] Pretreatment of substances.

[0130] Implementation steps:

[0131] (1) Respectively take 20% sucrose 10 mL, 10% BSA 10 mL, 5% sodium casein 2 mL, 5% PVA 2 mL, 10% Triton X-100 10 mL, 2 mmol / L boric acid / 0.1% PEG solution 10 mL into a beaker, TE is supplemented to 100 mL, a magnetic stirrer is added, and stirred on a magnetic stirrer until uniform, and the pH value is adjusted to 7.6±0.05;

[0132] (2) Respectively take 20% sucrose 15 mL, 10% BSA 12 mL, 5% sodium casein 2 mL, 5% PVA 3 mL, 10% Triton X-100 10 mL, 2 mmol / L boric acid / 0.1% PEG solution 10 mL into a beaker, TE is supplemented to 100 mL, a magnetic stirrer is added, and stirred on a magnetic stirrer until uniform, and the pH value is adjusted to 7.6±0.05.

[0133] Experimental results:

[0134] The embodiment aims to pretreat the substance. In the experiment, two formulations are used for treatment: one, 20% sucrose 10 mL, 10% BSA 10 mL, 5% sodium casein 2 mL, 5% PVA 2 mL, 10% Triton X-100 10 mL, 2 mmol / L boric acid / 0.1% PEG solution 10 mL are placed in a beaker, supplemented with TE to 100 mL, stirred with a magnetic stirrer, and the pH value is adjusted to 7.6±0.05; the second, 20% sucrose 15 mL, 10% BSA 12 mL, 5% sodium casein 2 mL, 5% PVA 3 mL, 10% Triton X-100 10 mL, 2 mmol / L boric acid / 0.1% PEG solution 10 mL are placed in a beaker, supplemented with TE to 100 mL, stirred with a magnetic stirrer, and the pH value is adjusted to 7.6±0.05, and finally the pretreatment of the substance is completed.

[0135] Example 10

[0136] This embodiment is the use amount screening of gold-labeled antibody and coating antibody, and the following is the implementation content:

[0137] Purpose of the experiment:

[0138] Use amount screening of gold-labeled antibody and coating antibody

[0139] Experimental steps:

[0140] (1) The prepared antibody is coated and labeled according to the process requirements of the labeled antibody and the coating antibody, and the OD value of the colloidal gold solution after labeling the antibody at a specific wavelength is 5, on this basis, the optimal coating amount of the detection line antibody, the optimal dilution degree of the gold-labeled antibody and the coating amount of the control line antibody sheep anti-mouse IgG are selected;

[0141] (2) The detection line antibody and the gold-labeled antibody stock solution are diluted with diluent at a coefficient of 0.5, 1.0, 1.3, 1.5 and 2 to prepare colloidal gold binding pads, which are respectively paired with the coating and labeled antibodies in Table 1 to detect low-concentration antigen solution and blank;

[0142] (3) The optimal use amount of the paired antibodies is determined by the maximum signal ratio of the positive signal of the detection antigen solution to the blank solution.

[0143] Experimental results:

[0144] This example aims to screen the use amount of gold label antibody and coating antibody. In the experiment, the antibody is coated and labeled according to the process requirements, and the OD value of the labeled colloidal gold solution is ensured to be 5 at a specific wavelength; then, the detection line antibody and the gold label antibody stock solution are diluted with the diluent at a coefficient of 0.5, 1.0, 1.3, 1.5, and 2 to prepare colloidal gold binding pads, which are paired with the antibodies in the antibody use amount screening to detect low-concentration antigen solution and blank solution; finally, the maximum signal ratio of the antigen solution positive signal to the blank solution signal is taken as the standard to determine the optimal use amount of the paired antibodies, and the screening of the use amount of the gold label antibody and the coating antibody is completed.

[0145] Example 11

[0146] This example is to assemble a layer pressure test paper strip. The following is the implementation content:

[0147] Experimental purpose:

[0148] Assemble a layer pressure test paper strip.

[0149] Experimental steps:

[0150] (1) Place the PVC plate with NC film, C-line and T-line coated on the workbench;

[0151] (2) Gently remove the protective film at the top end of the PVC plate where the absorbent pad is attached, and attach the absorbent pad to it. Roll it evenly and slightly to enhance adhesion. The absorbent pad should cover the NC film by no less than 1 mm;

[0152] (3) Remove the protective film at the lower edge of the NC film where the chemical coupling pad is attached, and attach the chemical coupling pad to it. The method is the same as that of the absorbent pad. The chemical coupling pad should cover the NC film by 1-2 mm;

[0153] (4) Remove the protective film at the lowermost end of the PVC plate, and attach the sample pad to the chemical coupling pad. The method is the same as that of the absorbent pad. The sample pad should cover the colloidal gold binding pad by no less than 2 mm.

[0154] (5) Cut the attached polystyrene sheet into 3.0 mm wide reagent strips on the automatic strip cutter.

[0155] Experimental steps:

[0156] This example aims to assemble a layer pressure test paper strip. Place the PVC plate with NC film and C-line and T-line coated on the workbench; attach the absorbent pad, chemical coupling pad, and sample pad in sequence. When attaching, roll them evenly and slightly to enhance adhesion. Finally, cut them into 3.0 mm wide reagent strips on the automatic strip cutter, and successfully complete the assembly of the layer pressure test paper strip.

[0157] Example 12

[0158] A gold-labeled antibody and antibody pairing test is provided, and the following embodiments are provided:

[0159] Experimental materials:

[0160] Pure water, K2CO3, BSA (bovine serum albumin), reconstitution solution (1% BSA, 5% sucrose, 0.02 mol / mL Tris-HCl pH 8.6), sodium casein peptide, PVA (polyvinyl alcohol), Triton X-100 (polyethylene glycol octylphenyl ether), boric acid,

[0161] Experimental purposes:

[0162] Gold-labeled antibody and antibody pairing test is carried out.

[0163] Experimental steps:

[0164] S1: Take 10 mL of colloidal gold in a conical flask, add an appropriate proportion of 0.1 mol / mL K2CO3, stir and mix, then add an appropriate proportion of anti-cat IgE protein monoclonal antibody or mouse IgG, slowly stir with a magnetic stirrer at 4°C for 20 min, then add 10% BSA to a final concentration of 1% and continue stirring, then centrifuge at 2000 r / min for 10 min to remove the coagulum, collect the supernatant and continue to centrifuge at 14000 r / min for 30 min, discard the transparent and colorless supernatant, and resuspend with 1 mL (1% BSA, 5% sucrose, 0.02 mol / mL Tris-HCl pH 8.6) reconstitution solution;

[0165] S2: Use WRF-HPY001-14 for antibody coating, dilute the anti-cat IgE protein monoclonal antibody and goat anti-mouse IgG with the coating solution to the use concentration, respectively, load the two antibodies into sample pump 1 and sample pump 2, then set the coating program, set the distance between the detection line and the quality control line to 0.5 cm, the distance between the detection line and the lower edge of the NC membrane to 0.7 cm, the line drawing speed to 60 mm / s, and the inkjet amount to 0.8 μL / cm;

[0166] S3: Respectively measure 20% sucrose 10 mL, 10% BSA 10 mL, 5% sodium casein 2 mL, 5% PVA 2 mL, 10% Triton X-100 10 mL, and 2 mmol / L boric acid / 0.1% PEG solution 10 mL into a beaker, add TE to 100 mL, add a magnetic stirrer to the magnetic stirrer and stir evenly, adjust the pH value to 7.6±0.05;

[0167] S4: The PVC plate with NC film and coated C-line and T-line is laid on the workbench, the protective film on the top end of the PVC plate is gently removed, the absorbent pad is adhered to it, and it is uniformly and slightly rolled to enhance the adhesion. The absorbent pad covers the NC film by not less than 1 mm. The protective film on the lower edge of the NC film is removed, and the chemical coupling pad is adhered to it. The method is the same as that of the absorbent pad. The chemical coupling pad should cover the NC film by 1-2 mm. The protective film on the lower end of the PVC plate is removed, and the sample pad is adhered to the chemical coupling pad. The method is the same as that of the absorbent pad.

[0168] Experimental results: refer to Table 3

[0169] Table 3: Screening results of paired antibodies

[0170]

[0171] The experiment of gold-labeled antibody and antibody pairing was successfully completed, each operation link was standardized, and the results met the expectations. In the preparation of gold-labeled antibody, a proper amount of 0.1 mol / mL K2CO3 was added to 10 mL colloidal gold, mixed, and then anti-cat IgE protein monoclonal antibody or mouse IgG was added. After slow stirring at 4°C, 1% BSA blocking and centrifugal purification, a uniform and stable gold-labeled antibody solution was obtained without residual agglomerates. When coating the antibody with equipment, the anti-cat IgE protein monoclonal antibody and goat anti-mouse IgG were uniformly coated on the NC membrane with the parameters of 0.5 cm between the detection line and the quality control line, 0.7 cm between the detection line and the lower edge of the NC membrane, 60 mm / s of line speed, and 0.8 μL / cm of inkjet amount. The blocking solution was prepared with 20% sucrose, 10% BSA, 5% sodium casein, 5% PVA, 10% TritonX-100, and 2 mmol / L boric acid / 0.1% PEG solution, etc. The TE was supplemented to 100 mL and the pH was adjusted to 7.6±0.05. The system was uniform and stable and could effectively block non-specific binding sites. When assembling the test strip, the absorbent pad, the chemical coupling pad and the sample pad were accurately pasted on the PVC plate according to the requirements. The absorbent pad covered the NC membrane by not less than 1 mm, and the chemical coupling pad covered 1-2 mm, which was firmly bonded and closely connected. In summary, the immunochromatography system was successfully constructed, which provided a feasibility verification for the subsequent qualitative detection of cat IgE or mouse IgG. The sensitivity and specificity can be further evaluated through the subsequent spiked experiment.

[0172] Example 13

[0173] A paired antibody clinical sample verification is provided, and the following implementation content is provided.

[0174] Experimental purpose:

[0175] A paired antibody clinical sample verification is prepared.

[0176] Experimental steps:

[0177] S1: Collect 8 blood clinical samples of allergic cats in the hospital, centrifuge at 3000 rpm for 10 min and collect serum;

[0178] S2: 6B5 is used as the coating antibody, 13E11 is used as the gold-labeled antibody, and the C-line coating antibody is the final concentration of 0.8 mg / mL of goat anti-mouse IgG coating antibody;

[0179] S3: According to the steps described in Example 3, the assembled reagent strip is obtained, and the IgE in the serum of the allergic cat is detected.

[0180] Experimental results:

[0181] Reference Figure 3 This clinical trial is carried out according to the trial scheme, and through the evaluation of 8 effective samples, 8 are positive, which shows that the red pair antibody of the application can meet the needs of clinical trials.

[0182] Example 1 takes the preparation of anti-cat IgE hybridoma cells as the core, and IgE antigen is separated and purified from cat serum by salting-out and column chromatography. High-purity target protein is obtained by dialysis desalting. 6-8 week old mice are immunized with Freund's adjuvant, and mice with serum titer ≥1:100000 are screened through three immunization procedures to ensure high sensitization of spleen B lymphocytes. The ratio of myeloma cells Sp2 / 0 to spleen cells is strictly controlled in the cell fusion link, and the cells are fused with 50% PEG and screened with HAT medium and specific identification by indirect ELISA. Hybridoma cell strains 6B5, 13E11 and the like with high affinity to cat IgE are successfully obtained. This example lays a key cell resource foundation for the preparation of monoclonal antibodies by standardizing antigen preparation, precise immunization procedures and efficient fusion screening, and solves the problem of insufficient specificity of cell strains in traditional methods.

[0183] Example 2, based on the hybridoma cells obtained in Example 1, successfully prepared anti-cat IgE monoclonal antibodies and determined their key performance parameters through expansion culture, affinity purification and indirect ELISA detection. In the experiment, the antibody titer verified by gradient dilution was all ≥1:100000, and the EC50 value detection showed that the 6B5 strain was 0.0261 and the 13E11 strain was 0.0274, which was significantly better than other subclone cell strains, indicating that the binding capacity of the two to cat IgE reached nanogram level high affinity level. This example provides core functional components for colloidal gold chromatography pairing system through standardized antibody purification process and precise affinity detection, and proves the specificity advantage of the antibody through quantitative data, solving the technical bottleneck of insufficient sensitivity of existing antibody detection.

[0184] Example 3 focuses on gold-labeled antibody preparation and colloidal gold chromatography system optimization. Stable and uniform 13E11 gold-labeled antibody was obtained through colloidal gold pH adjustment, antibody labeling and centrifugal purification. At the same time, 6B5 antibody was used to coat the NC membrane detection line, and the key parameters of 0.5 cm distance between detection line and quality control line and 60 mm / s line speed were set. Combined with the blocking solution formula containing sucrose, BSA and Triton X-100, a high-efficiency double-antibody sandwich detection system was constructed. The results of the matching test showed that 6B5 and 13E11 combination formed clear and specific bands in the test strip without the interference of irrelevant proteins such as mouse IgG, verifying the precise recognition ability of the system for cat IgE. This example solves the problems of signal blur and non-specific binding in traditional chromatography detection through material ratio optimization and equipment parameter control, and provides an industrialized replication technical scheme for portable rapid detection kit.

[0185] Examples 1-3 form a complete anti-cat IgE detection technology system through the technical chain of cell preparation-antibody screening-detection system construction. The hybridoma cell strain of Example 1 provides a stable antibody source for Example 2, and its high purity and low EC50 value ensure the high sensitivity of the detection system. The antibody titer and affinity data of Example 2 provide a scientific basis for the pairing design of Example 3, which maximizes the epitope difference between 6B5 and 13E11, and significantly improves the detection signal strength and specificity combined with the hydrodynamic optimization of 0.5 cm spacing. The three of them break through the limitations of antibody cross-reaction and complex detection process in traditional methods, form a whole process technical innovation from antigen preparation to terminal detection, and lay an engineering foundation for the rapid and accurate detection of cat IgE.

[0186] Example 4 uses 6B5-coated and 13E11-labeled reagent strips for IgE detection in allergic cat clinical samples. All 8 effective samples showed positive results, which was highly consistent with clinical diagnosis, verifying the reliability of the paired antibody system in real-world scenarios. The experiment proves the simplicity of the detection method and the stability of the results through standardized serum processing, reagent strip assembly and result interpretation. This example, as a key link of technology transformation, directly connects laboratory achievements with clinical needs, solves the problem of sensitivity decline in existing detection technology in actual samples, and provides the first efficient detection tool for cat IgE in the pet medical field, which has significant clinical application value.

[0187] Four embodiments construct the whole chain technical scheme from cell strain preparation, antibody production to clinical verification around anti-cat IgE detection. Embodiments 1-2 obtain 6B5 / 13E11 cell strains and high specificity antibodies with independent intellectual property rights through antigen purification, cell fusion and affinity screening. Embodiment 3 constructs a rapid detection platform that can be mass-produced through colloidal gold labeling, parameter optimization and system integration. Embodiment 4 proves the actual effectiveness of the technical scheme through clinical sample verification. Overall, the application breaks through the specificity and sensitivity bottleneck of cat IgE detection through multi-dimensional technical innovation, forms a complete technical system with scientific and engineering value, provides reliable technical support for early diagnosis and precision medicine of cat allergic diseases, and has significant social value and industrial application prospect.

[0188] Those skilled in the art can realize that the modules and algorithm steps of each example described in combination with the embodiments disclosed herein can be realized in electronic hardware or a combination of computer software and electronic hardware, and whether the functions are realized in hardware or software depends on the specific application and design constraints of the technical solution. Professional technicians can use different methods to realize the described functions for each specific application, but such implementation should not be considered beyond the scope of the present application.

[0189] In addition, each functional module in each embodiment of the present application can be integrated in one processing module, or each module can exist physically alone, or two or more modules can be integrated in one module.

[0190] The above is merely a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any skilled person in the art can easily think of changes or replacements within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

[0191] Finally: the above is only the preferred embodiment of the present application, and is not used to limit the present application, any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application should be included in the protection of the present application.

Claims

1. An anti-feline IgE monoclonal antibody hybridoma cell line, characterized in that, The cell strain can stably secrete a monoclonal antibody specifically binding to cat IgE, including hybridoma cell strains with clone numbers 6B5 and 13E11, and the preservation numbers are CCTCC NO: C2025213 and CCTCC NO: C2025201, respectively.

2. An anti-feline IgE monoclonal antibody, characterized in that, Monoclonal antibodies are produced by the hybridoma cell strain of claim 1.

3. The anti-Feline IgE monoclonal antibody as claimed in claim 2, wherein, The monoclonal antibody has an EC50 value of 0.0261-0.0274 μg / mL in binding to cat IgE.

4. The anti-Feline IgE monoclonal antibody of claim 1, wherein, The application of the anti-cat IgE clone antibody hybridoma cell strain and the monoclonal antibody is to detect IgE in an immunochromatography reagent strip, which includes a PVC bottom plate and sequentially connected sample pad, chemical coupling pad, NC membrane and absorption pad.

5. The use of the anti-Feline IgE monoclonal antibody hybridoma cell line or the monoclonal antibody according to claim 4, wherein the monoclonal antibody is a monoclonal antibody of the IgG class. The NC membrane is provided with a detection line and a quality control line, the detection line is coated with the anti-cat IgE monoclonal antibody of claim 2, and the chemical coupling pad is coated with the colloidal gold-labeled anti-cat IgE monoclonal antibody of claim 2.

6. The use of the anti-Feline IgE monoclonal antibody hybridoma cell line or the monoclonal antibody according to claim 5, characterized in that, The monoclonal antibody coated on the detection line is 6B5, and the colloidal gold-labeled monoclonal antibody coated on the chemical coupling pad is 13E11.

7. The use of the anti-Feline IgE monoclonal antibody hybridoma cell line or the monoclonal antibody according to claim 5, wherein the monoclonal antibody is used for the diagnosis of feline allergic diseases. The final concentration of the anti-cat IgE monoclonal antibody coated on the detection line is 0.5-2 mg / mL, and the final concentration of the goat anti-mouse IgG antibody coated on the quality control line is 0.8 mg / mL.

8. The use of the anti-Feline IgE monoclonal antibody hybridoma cell line or the monoclonal antibody according to claim 5, wherein the monoclonal antibody is administered to a feline in an amount of 0.1 to 10 mg / kg. The distance between the detection line and the quality control line is 0.5 cm, and the distance between the detection line and the lower edge of the NC membrane is 0.7 cm.

9. The use of the anti-Feline IgE monoclonal antibody hybridoma cell line or the monoclonal antibody according to claim 5, wherein the monoclonal antibody is administered to a feline in an amount of 0.1 to 10 mg / kg. The pretreatment solution of the chemical coupling pad is composed of the following components: 20% sucrose 10 mL, 10% BSA 10 mL, 5% sodium casein 2 mL, 5% PVA 2 mL, 10% Triton X-100 10 mL, 2 mmol / L boric acid / 0.1% PEG solution 10 mL, and TE to 100 mL, with the pH value adjusted to 7.6±0.

05. The assembled reagent strip has a width of 3.0 mm, and the coverage size of each component meets the requirements, the absorption pad covers the NC membrane by not less than 1 mm, the chemical coupling pad covers the NC membrane by 1-2 mm, and the sample pad covers the chemical coupling pad by not less than 2 mm.

10. The use of the anti-Feline IgE monoclonal antibody hybridoma cell line or the monoclonal antibody according to claim 5, wherein the monoclonal antibody is administered to a feline in an amount of 0.1 to 10 mg / kg. ​

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