WO2022110257A1 - Antiallergic nanobody composition, antibody assay method, and spray - Google Patents
Antiallergic nanobody composition, antibody assay method, and spray Download PDFInfo
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- WO2022110257A1 WO2022110257A1 PCT/CN2020/133203 CN2020133203W WO2022110257A1 WO 2022110257 A1 WO2022110257 A1 WO 2022110257A1 CN 2020133203 W CN2020133203 W CN 2020133203W WO 2022110257 A1 WO2022110257 A1 WO 2022110257A1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K9/10—Dispersions; Emulsions
- A61K9/12—Aerosols; Foams
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/02—Nasal agents, e.g. decongestants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
Definitions
- the present application relates to the technical field of medical anti-allergic antibody research and development, in particular, to an anti-allergic nanobody composition, an antibody assay method and a spray.
- Nanobodies are the smallest units known to bind target antigens.
- the crystal size of VHH is 2.5nm ⁇ 4nm, the molecular weight is only 12KD-15KD, its molecular structure is relatively stable, it can withstand high temperature and maintain activity in extremely harsh environments.
- VHH kept 80% of the biological activity at 45°C for 1 week, indicating that the nanobody is quite stable at room temperature, which makes it easier to store and transport than conventional antibodies.
- Nanobodies have strong and fast tissue penetration ability, which is beneficial for them to enter dense tissues such as solid tumors to play their role, and can effectively penetrate the blood-brain barrier, providing a new method for brain drug delivery.
- nanobodies have reversible refolding ability, that is, easy renaturation.
- Tests show that nanobodies still maintain high activity after high temperature treatment at 90 °C, and can regain antigen-binding ability.
- all conventional antibodies lost their activity after being treated at 90°C and underwent irreversible polymerization. Under harsh conditions, such as chaotropic agents, the presence of proteases and extreme pH denaturation, normal antibodies fail or break down, while Nanobodies remain highly stable.
- Measures should be taken to prolong its half-life in the antibody nanobody composition and the human body. Through transformation measures such as nanobody fusion with albumin, Fc fusion, and PEGylation, the half-life of the nanobody in the antibody nanobody composition and in the human body will be greatly improved. extend.
- nanobody drugs With the continuous development of bioengineering technology, a large number of nanobody drugs will continue to emerge. At present, the main dosage forms of its clinical trials are injections and oral preparations. Due to the small molecular weight of nanobody drugs, poor in vivo stability, and easy to be affected by the first-pass effect of gastrointestinal enzyme degradation and liver enzyme system when taken orally, to achieve effective drug therapeutic concentration, patients need long-term repeated injections or oral administration. .
- Type I allergic disease mediated by IgG antibodies is quite common. Anti-allergic disease is when patients inhale, ingest or inject substances containing sensitizing ingredients to trigger the body's B cells to produce specific immunoglobulin G, thereby triggering allergic reactions. disease and related symptoms.
- the main technical problem solved by the present invention is to provide an anti-allergic nanobody composition, an antibody assay method and a spray.
- the nanobody composition is labeled and tracked by biological macromolecular protease, etc., and the allergic antigen is tracked and reacted to report,
- the chemical test results were recorded to carry out the preparation and derivation of anti-allergic verification drugs based on Nanobodies.
- An anti-allergic nanobody composition comprising a nanobody and a fluorescein-labeled IgG3 antibody solution, an allergen solution with a biomarker protein molecule, a polypeptide-coated nanometer magnetic suspension, and a specific nanobody with biological activity, As well as maintaining antibody nanobody compositions, immunosuppressants, non-strain allergic antigens that increase nanobody stability and tissue penetration;
- the nanobody includes one or more of active nanobody, nanobody fragment, multi-targeted nanobody polymer linker, nanobody-protein conjugate or nanobody-drug conjugate.
- Nanobodies contain only one heavy chain variable region VHH and two conventional CH2 and CH3 regions, which are the smallest known units that can bind to target antigens.
- the conjugate of nanobody and protein can make the target protein antibody bind clearly, which can be widely used in the development of therapeutic antibody drugs, diagnostic reagent antibody binding .
- Polypeptide-coated nano-magnetic suspension can be used for antibody drug target tracking.
- Polypeptide-coated nanomagnets are commonly used in modern biology, and immunoassay is an important method in modern bioanalytical technology, which can be used to quantitatively analyze proteins, antigens, antibodies and cells.
- a method of transfecting magnetic nanoparticles bound with carrier DNA into cells under the influence of an external magnetic field is a method of transfecting magnetic nanoparticles bound with carrier DNA into cells under the influence of an external magnetic field.
- the magnetic separation method basically only includes two steps: 1. Labeling target molecules or cells with magnetic nanoparticles; 2. Separating target molecules or cells by a magnetic separation device.
- One example of separation using magnetic nanoparticles is the binding of specific antibodies to magnetic nanoparticles, which can be attached to specific cells, and the magnetic nanoparticles can be rapidly isolated or immunoassayed by applying a magnetic field. Such a method has high specificity, rapid separation, and good reproducibility.
- the nanobody composition needs to be calibrated for the performance of the antibody-antigen binding after a preset time, and the chemical result needs to be subjected to a buffer test.
- a chemical reagent that is resistant to allergen-reactive protein is used for calibration. test.
- Two reagents with different standard contents are prepared here: the first chemical reagent calibration reagent and the second chemical reagent calibration reagent.
- the first chemical reagent calibration reagent and the second chemical reagent calibration reagent are used to perform calibration data testing on the hypersensitive response protein reaction solution.
- the concentration of the allergen solution with biomarker protein molecules is 0.35-1.188ug/ml; the concentration of the streptavidin-labeled polypeptide-coated nano-magnetic suspension solution is 0.35-1.188mg/ml; the concentration of the IgG3 antibody solution with fluorescein label is 0.25-3.75ug/ml.
- the labeled IgG3 antibody with a fluorescein label is an antibody that can specifically bind to IgG protein in the human body
- the preparation process of the fluorescein-labeled IgG3 antibody solution is as follows: after mixing the alkaline phosphatase solution with a concentration of 1.75-6.25 mg/mL and the fluorescein-labeled IgG3 antibody in a molar ratio of 1-4:1 And purifying to obtain IgG3 antibody, the purification is equilibrated with bicarbonate buffer with pH 8-9 and eluted, and then UV detection and recording of purification spectrum, and then use 0.05M, pH value of 3.75 MES buffer to equilibrate and elute. IgG antibody dilution.
- the immunosuppressive agents include cyclophosphamide, mycophenolate mofetil, cyclosporine a, tacrolimus, methotrexate, azathioprine, leflunomide, One or more of hydroxychloroquine corticosteroid, adenosine receptor agonist, phosphodiesterase 4 inhibitor, HDAC inhibitor or proteasome inhibitor.
- the non-strained allergic antigen comprises a medical biological protease
- the medical biological protease is a therapeutic protein or a therapeutic polynucleotide, including temorelin, ocrelizumab One or more of anti-, belimumab, pegylated recombinant uricase, talisidase alfa, agalsidase alfa, or glucocerebrosidase alfa.
- the preparation process of the polypeptide-coated nano-magnetic suspension is as follows:
- streptavidin-labeled magnetic nanoparticles were precipitated with a magnetic separator, resuspended with 0.01M phosphate buffer with a pH value of 7.8, mixed uniformly and the precipitate was magnetically separated, and washed repeatedly;
- the cleaned magnetic nanoparticles were dispersed in 0.01M phosphate buffer with pH 7.8.
- a method for determining the performance of serum antibodies based on nano-magnetic chemiluminescence method, the anti-allergic nano-antibody composition is ingested in the serum comprising the steps of:
- the reaction conditions in step (1) are 5 minutes at 45° C., and the standing time is 3 minutes; step (1) also includes: The solution was cleaned with a cleaning solution, placed in a magnetic field and left to stand and the supernatant was removed, and the cleaning was repeated twice; the reaction conditions in step (2) were 5 minutes at 45° C., and the standing time was 3 minutes ; In step (2), it is also included that the second solution is cleaned with a cleaning solution, placed in a magnetic field and left to stand and remove the supernatant, and the cleaning is repeated 2 times; the reaction condition described in the step (3) is at 45 °C. The reaction was carried out for 2 minutes, and the luminescence intensity was measured in a chemiluminescence analyzer/meter.
- Active substances such as biologically active adsorbents or other ligands attached to the surface of magnetic nanoparticles can specifically bind to specific biomolecules or cells and separate under the action of an external magnetic field.
- the method includes the steps of:
- An anti-allergic rhinitis spray comprises an ethylene glycol solution containing the anti-allergic nanobody composition.
- the beneficial effects of the present invention are: the anti-allergic nanobody composition and method of the present invention, the nanobody composition is labeled and tracked by biological macromolecular protease, etc., the allergic antigen is tracked and reported, and the chemical test results are recorded and The preparation and derivation of anti-allergic verification drugs based on nanobodies were carried out.
- Each reagent component in the nanobody composition has good stability, high detection sensitivity and good specificity, has a perfect and unified process, and is produced in strict accordance with standard production operating procedures and quality control procedures.
- Fig. 1 is the schematic diagram of the anti-allergic nano-body rhinitis spray prepared by the anti-allergic nano-body composition of the present invention
- FIG. 2 is a flow chart of the present invention for measuring IgG3 antibody solution by the method for measuring serum antibody performance based on nano-magnetic chemiluminescence method.
- the nanobody composition needs to be calibrated for the performance of antibody-antigen binding after a preset time, and the chemical result needs to be subjected to a buffer test.
- a chemical reagent that is resistant to allergen-reactive protein is used for calibration. test.
- Two reagents with different standard contents are prepared here: the first chemical reagent calibration reagent and the second chemical reagent calibration reagent.
- the first chemical reagent calibration reagent and the second chemical reagent calibration reagent are used to perform calibration data testing on the hypersensitive response protein reaction solution.
- the concentration of the first chemical reagent calibration reagent is: 0IU/ml, 0.35IU/ml, 0.7IU/ml, 3.5IU/ml, 17.5IU/ml, 50IU/ml, 100IU/ml
- the first chemical reagent calibration reagent is prepared by IgG protein and 0.1M Tris-HCl buffer with a pH value of 7.8;
- the concentration of the second chemical reagent calibration reagent is: 0.7IU/ml, 17.5IU/ml, and the second chemical reagent calibration reagent is prepared by IgG protein and 0.1M Tris-HCl buffer with a pH value of 7.8 .
- Antibody selections are as follows: Fluorescein-labeled IgG3 antibody solution;
- the antigens were selected as follows: inflammatory allergens;
- the inflammatory allergen is an antigen that can bind to inflammation-specific IgG proteins in the human body
- the preparation process of the allergen solution with the biomarker protein molecules is as follows: adding the inflammatory allergen into biotin dissolved in dimethylformamide at a molar ratio of 1:20, and then using 0.1M, pH 7.8 Dialyzed against phosphate buffer.
- the immunosuppressive agents include cyclophosphamide, mycophenolate mofetil, cyclosporine a, tacrolimus, methotrexate, azathioprine, leflunomide, hydroxychloroquine corticosteroids , one or more of an adenosine receptor agonist, a phosphodiesterase 4 inhibitor, an HDAC inhibitor, or a proteasome inhibitor.
- the non-strain allergic antigen comprises a medical biological protease
- the medical biological protease is a therapeutic protein or a therapeutic polynucleotide, including temorelin, ocrelizumab, Bailey One or more of mulimumab, pegylated recombinant uricase, talisidase alfa, agalsidase alfa, or glucocerebrosidase alfa.
- the method provides an anti-allergic nanobody composition, comprising a fluorescein-labeled IgG3 antibody solution, and the nanobody composition includes: an allergen solution with biomarker protein molecules, a polypeptide-coated nanomagnetic suspension, a Biologically active specific Nanobodies, as well as maintaining antibody Nanobody compositions that increase Nanobody stability and tissue penetration, immunosuppressants, non-strain allergic antigens;
- the nanobody includes one or more of active nanobody, nanobody fragment, multi-targeted nanobody polymer linker, nanobody-protein conjugate or nanobody-drug conjugate.
- Polypeptide-coated nano-magnetic suspension can be used for antibody drug target tracking.
- Polypeptide-coated nanomagnets are commonly used in modern biology, and immunoassay is an important method in modern bioanalytical technology, which can be used to quantitatively analyze proteins, antigens, antibodies and cells.
- a method of transfecting magnetic nanoparticles bound with carrier DNA into cells under the influence of an external magnetic field is a method of transfecting magnetic nanoparticles bound with carrier DNA into cells under the influence of an external magnetic field.
- the magnetic separation method basically only includes two steps: 1. Labeling target molecules or cells with magnetic nanoparticles; 2. Separating target molecules or cells by a magnetic separation device.
- One example of separation using magnetic nanoparticles is the binding of specific antibodies to magnetic nanoparticles, which can be attached to specific cells, and the magnetic nanoparticles can be rapidly isolated or immunoassayed by applying a magnetic field. Such a method has high specificity, rapid separation, and good reproducibility.
- the anti-allergic Nanobody composition includes the following reagents:
- IgG3 antibody solution with fluorescein label the concentration is 0.1-2.0ug/mL
- the nanobody includes one or more of active nanobody, nanobody fragment, multi-targeted nanobody polymer linker, nanobody-protein conjugate or nanobody-drug conjugate.
- Polypeptide-coated nano-magnetic suspension can be used for antibody drug target tracking.
- Polypeptide-coated nanomagnets are commonly used in modern biology, and immunoassay is an important method in modern bioanalytical technology, which can be used to quantitatively analyze proteins, antigens, antibodies and cells.
- a method of transfecting magnetic nanoparticles bound with carrier DNA into cells under the influence of an external magnetic field is a method of transfecting magnetic nanoparticles bound with carrier DNA into cells under the influence of an external magnetic field.
- immunosuppressive agents such as adenosine receptor agonists and non-strain allergic antigen packages such as pegylated recombinant uricase are added to allow the reagents to stand.
- Embodiment 2 is a diagrammatic representation of Embodiment 1:
- a method for measuring serum antibody performance based on nano-magnetic chemiluminescence method, the anti-allergic nano-antibody composition is ingested in serum comprising the following steps:
- Preparation of 0.01M, pH 7.8 phosphate buffer take a beaker with a capacity of 1L, add 800ml of deionized water, weigh 2.56g Na 2 HPO 4 ⁇ 12H 2 O, 0.44g NaH 2 PO 4 ⁇ 2H 2 O and 9g NaCl, add it to the beaker, stir to dissolve it fully, adjust the pH value to 7.8 ⁇ 0.05 with HCl or NaOH, add 2% mannitol and 1% glycerol, stir for 0.5h to complete Dissolve, add deionized water to make up to 1L.
- Tris-HCl buffer with pH 7.8 Take a beaker with a capacity of 1L, add 800ml of deionized water, weigh 12.11g of Tris (tris (tris(hydroxymethylaminomethane)) into the beaker, and stir to make it fully Dissolve, adjust the pH to 7.8 ⁇ 0.05 with HCl or NaOH, add 2% BSA, 0.3% Proclin300, stir for 0.5h until completely dissolved, add deionized water to make up to 1L.
- Tris tris (tris (tris(hydroxymethylaminomethane)
- Preparation of 0.05M MES buffer with pH value of 3.75 Take a beaker with a capacity of 1L, add 800ml deionized water, weigh 9.76g MES (2-(N-morpholino)ethanesulfonic acid), 9g NaCl and add it to the solution. In the beaker, stir to make it fully dissolved, adjust the pH value to 3.75 ⁇ 0.05 with HCl or NaOH, add 1% BSA, 0.1% Tween-20, stir for 0.5h until completely dissolved, add deionized Make up to 1L with water.
- Second Chemical Reagent Calibration Reagent Take IgG protein, and prepare 0.7, 17.5IU/ml second chemical reagent calibration reagent with 0.1M Tris-HCl buffer with pH value of 7.8.
- Nanobody composition assembly The allergen solution with biomarker protein molecules, the streptavidin-labeled polypeptide-coated nano-magnetic suspension, the fluorescein-labeled IgG3 antibody solution, the first chemical reagent calibration reagent and The second chemical reagent calibration reagent is assembled into the nanobody composition.
- the method includes the steps of:
- the reagent components (such as cleaning solution, some necessary buffers, etc.) that are not mentioned in detail in the Nanobody composition of the present invention, the outer packaging of the Nanobody composition, and the individual packaging containers for each reagent component can be all listed according to their respective Routine operations in the field can be carried out in accordance with relevant industry regulations.
- This example provides a method for determining the performance of serum antibodies based on nano-magnetic chemiluminescence method.
- the anti-allergic nano-antibody composition is ingested in the serum, and the detection is carried out on an automatic chemiluminescence instrument MAGICL6800 series.
- the operation steps are as follows:
- nanobody composition information is input into the instrument system through the barcode scanner, and can also be set through the instrument supporting software.
- the present invention provides a method for measuring serum antibody performance based on nano-magnetic chemiluminescence method.
- the anti-allergic nano-antibody composition is ingested in serum, which can also be realized on a semi-automatic chemiluminescence instrument.
- the specific sample detection steps As follows: (1) Add 40ul of serum to be tested or IgG first chemical reagent calibration reagent, 50ul of allergens with biomarker protein molecules, and 50ul of streptavidin-labeled magnetic nanoparticles into the detection tube in turn, and after mixing, After co-incubating at 45°C for 5 minutes, the inflammation-specific IgG antibodies in the samples were captured and immobilized on the surface of the magnetic nanoparticles.
- the serum sample to be tested is co-incubated with allergens and streptavidin-labeled nano-magnetic particles with biomarker protein molecules
- the inflammation-specific IgG antibodies in the serum sample to be tested are incubated with the allergens with biomarker protein molecules. Allergen-specific binding, and with the aid of the biotin-streptavidin amplification system, the inflammatory allergen-specific IgG in serum samples is bound to the solid-phase carrier magnetic nanoparticles; under the action of an external magnetic field, magnetic separation and washing remove excess biological hormone-labeled inflammatory allergens;
- IgG3 antibody was added to form an immune complex structure of allergen-inflammatory specific IgG-anti-IgG3 antibody with biomarker protein molecules; under the action of an external magnetic field, the unbound enzyme-labeled antibody and other impurities were removed by repeated magnetic separation and washing; Enzyme-catalyzed chemiluminescence substrate to measure the luminescence intensity; use the first chemical reagent of known concentration to calibrate the standard curve of the reagent, and calculate the content of inflammation-specific IgG antibody in the sample to be tested according to the luminescence intensity and the standard curve;
- Embodiment 4 is a diagrammatic representation of Embodiment 4:
- the concentration of IgG antibodies to a single allergen in the serum of normal non-allergic adults is less than 0.35 IU/mL.
- the measured value is between 0.35 and 0.7IU/mL, it is the first-grade allergy to the allergen
- the measured value is between 0.7 and 3.5IU/mL, it is a grade 2 allergy to the allergen
- the measured value is between 3.5 and 17.5IU/mL, it is a grade 3 allergy to the allergen
- the measured value is between 17.5 and 50IU/mL, it is a grade 4 allergy to the allergen
- the measured value is between 50 and 100 IU/mL, it is a grade 5 allergy to the allergen
- the measured value is greater than 100IU/mL for the allergen grade 6 allergy.
- alkaline phosphatase is used as the labeling enzyme, the antibody is labeled by chemical reaction, and the unreacted enzyme, antibody or antigen is separated by gel chromatography to improve the sensitivity of the reaction;
- immunomagnetic particles as the solid phase and streptavidin-coupled magnetic microspheres as a general separation reagent, it not only makes the immune reaction easier to mix and separate, but also greatly improves the reaction speed;
- the new chemiluminescence substrate AMPPD is used as the substrate.
- the substrate is a glow-type substrate, and it reaches the plateau phase quickly, which is beneficial to the detection of the signal and improves the sensitivity and specificity of the final nanobody composition; and
- the chemiluminescence enhancement system is further optimized to ensure that the final product has high signal sensitivity, good stability and small variation;
- the advantage of using AMPPD luminescent substrate is high sensitivity and long platform stability period.
- An anti-allergic rhinitis spray comprises an ethylene glycol solution containing the anti-allergic nanobody composition.
- Adding the anti-allergic nanobody composition proposed by the present invention to the anti-allergic rhinitis spray can perform anti-inflammatory treatment for patients with low cost.
- the allergic antibody test reaction verification of patients with inflammation can be carried out, and the actual verification antibody reaction structure can be directly traced to demonstrate the clinical medical effect.
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Abstract
Provided are an antiallergic nanobody composition, an assay method for measuring the performance of a serum antibody based on a nanomagnetic chemiluminescence method, and an antiallergic rhinitis spray. The composition comprises a nanobody, an IgG3 antibody solution having a fluorescein label, an allergen solution having a biomarker protein molecule, a polypeptide-coated nanomagnetic levitation solution, a specific nanobody having bioactivity, and, for maintaining and increasing the stability and tissue penetration of the nanobody, an antibody-nanobody composition, an immunosuppressant, and a non-allergic antigen.
Description
本申请涉及医疗抗过敏抗体研发技术领域,具体而言,涉及一种抗过敏性纳米抗体组合物、抗体测定方法及喷雾剂。The present application relates to the technical field of medical anti-allergic antibody research and development, in particular, to an anti-allergic nanobody composition, an antibody assay method and a spray.
纳米抗体是目前已知的可结合目标抗原的最小单位。VHH晶体大小2.5nm×4nm,分子量只有12KD-15KD,其分子结构比较稳定,能够耐高温并在极端苛刻的环境中保持活性。Nanobodies are the smallest units known to bind target antigens. The crystal size of VHH is 2.5nm×4nm, the molecular weight is only 12KD-15KD, its molecular structure is relatively stable, it can withstand high temperature and maintain activity in extremely harsh environments.
VHH在45℃放置1周仍能保持80%的生物活性,表明纳米抗体在室温下保存相当稳定,这使其比常规抗体更易于储藏和运输。纳米抗体具有强而快的组织穿透能力,利于它们进入致密组织如实体瘤中发挥作用,而且能够有效的穿透血脑屏障,为脑部给药提供了新方法。VHH kept 80% of the biological activity at 45°C for 1 week, indicating that the nanobody is quite stable at room temperature, which makes it easier to store and transport than conventional antibodies. Nanobodies have strong and fast tissue penetration ability, which is beneficial for them to enter dense tissues such as solid tumors to play their role, and can effectively penetrate the blood-brain barrier, providing a new method for brain drug delivery.
同时,纳米抗体具有可逆的重折叠能力即易复性,试验表明,纳米抗体在90℃高温处理后,仍然保持了较高的活性,能重新获得抗原结合能力。而所有常规抗体在90℃处理后都丧失了活性,发生了不可逆的聚合。在恶劣条件,如在离液剂、存在蛋白酶和极度pH值变性的条件下,正常抗体会失效或分解,而纳米抗体仍具有高度的稳定性。At the same time, nanobodies have reversible refolding ability, that is, easy renaturation. Tests show that nanobodies still maintain high activity after high temperature treatment at 90 °C, and can regain antigen-binding ability. However, all conventional antibodies lost their activity after being treated at 90°C and underwent irreversible polymerization. Under harsh conditions, such as chaotropic agents, the presence of proteases and extreme pH denaturation, normal antibodies fail or break down, while Nanobodies remain highly stable.
要采取措施延长其在抗体纳米抗体组合物及人体里的半衰期,通过纳米抗体与白蛋白融合、Fc融合、PEG化等改造措施,纳米抗体存在于抗体纳米抗体组合物及人体内的半衰期将大大延长。Measures should be taken to prolong its half-life in the antibody nanobody composition and the human body. Through transformation measures such as nanobody fusion with albumin, Fc fusion, and PEGylation, the half-life of the nanobody in the antibody nanobody composition and in the human body will be greatly improved. extend.
随着生物工程技术的不断发展,大量纳米抗体药物将不断涌现。目前其临床试验的主要剂型为注射剂和口服剂。由于纳米抗体药物分子量小,体内稳定性差,且口服时易受胃肠道酶降解和肝脏酶***的首过效应影响,要达到有效的药物治疗浓度,病人需要长期多次重复注射或口服给药。With the continuous development of bioengineering technology, a large number of nanobody drugs will continue to emerge. At present, the main dosage forms of its clinical trials are injections and oral preparations. Due to the small molecular weight of nanobody drugs, poor in vivo stability, and easy to be affected by the first-pass effect of gastrointestinal enzyme degradation and liver enzyme system when taken orally, to achieve effective drug therapeutic concentration, patients need long-term repeated injections or oral administration. .
IgG抗体介导的I型过敏反应性疾病相当普遍,抗过敏性疾病是患者吸入、 摄食入或者注入含有致敏成分的物质后触发机体的B细胞产生特异性免疫球蛋白G,从而引发过敏反应的疾病及相关症状。Type I allergic disease mediated by IgG antibodies is quite common. Anti-allergic disease is when patients inhale, ingest or inject substances containing sensitizing ingredients to trigger the body's B cells to produce specific immunoglobulin G, thereby triggering allergic reactions. disease and related symptoms.
而目前,对于炎症比如鼻炎抗过敏处理上,并未有成效的纳米抗体抗过敏反应试剂进行临床试验论证。At present, for the anti-allergic treatment of inflammation such as rhinitis, there is no clinical trial demonstration of effective nanobody anti-allergic reaction reagents.
发明内容SUMMARY OF THE INVENTION
本发明主要解决的技术问题是提供一种抗过敏性纳米抗体组合物、抗体测定方法及喷雾剂,该纳米抗体组合物通过生物大分子蛋白酶等进行标记跟踪,对过敏性抗原进行追踪反应报告,记载化学试验结果而进行基于纳米抗体的抗过敏性验证药物制备推导。The main technical problem solved by the present invention is to provide an anti-allergic nanobody composition, an antibody assay method and a spray. The nanobody composition is labeled and tracked by biological macromolecular protease, etc., and the allergic antigen is tracked and reacted to report, The chemical test results were recorded to carry out the preparation and derivation of anti-allergic verification drugs based on Nanobodies.
为解决上述技术问题,本发明采用的一个技术方案是:In order to solve the above-mentioned technical problems, a technical scheme adopted in the present invention is:
一种抗过敏性纳米抗体组合物,包括纳米抗体以及带荧光素标签的IgG3抗体溶液、具备生物标记蛋白分子的过敏原溶液、多肽包覆性纳米磁浮液、具有生物活性的特异性纳米抗体,以及维护增加纳米抗体稳定性和组织穿透性的抗体纳米抗体组合物、免疫抑制剂、非应变性过敏抗原;An anti-allergic nanobody composition, comprising a nanobody and a fluorescein-labeled IgG3 antibody solution, an allergen solution with a biomarker protein molecule, a polypeptide-coated nanometer magnetic suspension, and a specific nanobody with biological activity, As well as maintaining antibody nanobody compositions, immunosuppressants, non-strain allergic antigens that increase nanobody stability and tissue penetration;
所述纳米抗体包括:活性纳米抗体、纳米抗体片段、多靶向纳米抗体聚合链接体、纳米抗体与蛋白的偶联体或纳米抗体与药物的偶联体中的一种或多种。The nanobody includes one or more of active nanobody, nanobody fragment, multi-targeted nanobody polymer linker, nanobody-protein conjugate or nanobody-drug conjugate.
纳米抗体只包含一个重链可变区VHH和两个常规的CH2与CH3区,是已知的可结合目标抗原的最小单位。Nanobodies contain only one heavy chain variable region VHH and two conventional CH2 and CH3 regions, which are the smallest known units that can bind to target antigens.
作为本发明的一个较佳实施例,作为本次炎症试剂用抗体,采用纳米抗体与蛋白的偶联体可以使得靶向蛋白抗体明确结合,可以广泛用于开发治疗性抗体药物、诊断试剂抗体结合。As a preferred embodiment of the present invention, as the antibody used for this inflammatory reagent, the conjugate of nanobody and protein can make the target protein antibody bind clearly, which can be widely used in the development of therapeutic antibody drugs, diagnostic reagent antibody binding .
多肽包覆性纳米磁浮液可以用于抗体药物靶向追踪。Polypeptide-coated nano-magnetic suspension can be used for antibody drug target tracking.
多肽包覆性纳米磁常用语现代生物,免疫分析是现代生物分析技术中重要 的一种方法,利用它可对蛋白质、抗原、抗体及细胞进行定量分析。Polypeptide-coated nanomagnets are commonly used in modern biology, and immunoassay is an important method in modern bioanalytical technology, which can be used to quantitatively analyze proteins, antigens, antibodies and cells.
将结合有载体DNA的磁性纳米粒子在外界磁场影响下转染到细胞内的方法。A method of transfecting magnetic nanoparticles bound with carrier DNA into cells under the influence of an external magnetic field.
磁性纳米粒子表面连接的具有生物活性的吸附剂或其他配体等活性物质可与特定生物分子或细胞特异性结合,在外加磁场作用下分离。磁性分离方法基本只包括2个步骤:1.用磁性纳米粒子标记目标分子或细胞;2.通过磁分离装置分离出目标分子或细胞。利用磁性纳米粒子分离的例子之一是把特异性抗体与磁性纳米粒子结合,可将磁性纳米粒子连接在特定细胞上,外加磁场即可快速将结合磁性纳米粒子的细胞分离或进行免疫分析。这样的方法特异性高、分离迅速、重现性好。Active substances such as biologically active adsorbents or other ligands attached to the surface of magnetic nanoparticles can specifically bind to specific biomolecules or cells and separate under the action of an external magnetic field. The magnetic separation method basically only includes two steps: 1. Labeling target molecules or cells with magnetic nanoparticles; 2. Separating target molecules or cells by a magnetic separation device. One example of separation using magnetic nanoparticles is the binding of specific antibodies to magnetic nanoparticles, which can be attached to specific cells, and the magnetic nanoparticles can be rapidly isolated or immunoassayed by applying a magnetic field. Such a method has high specificity, rapid separation, and good reproducibility.
除此之外,besides,
本纳米抗体组合物在是需要经过预设时间后进行抗体抗原结合产生的性能进行标定测试,化学结果需要进行缓冲测试,在本实施例,采用对过敏原反应蛋白具有抗性的化学试剂进行标定测试。The nanobody composition needs to be calibrated for the performance of the antibody-antigen binding after a preset time, and the chemical result needs to be subjected to a buffer test. In this embodiment, a chemical reagent that is resistant to allergen-reactive protein is used for calibration. test.
本处配置两者不同标准含量的试剂:第一化学试剂标定试剂和第二化学试剂标定试剂。Two reagents with different standard contents are prepared here: the first chemical reagent calibration reagent and the second chemical reagent calibration reagent.
第一化学试剂标定试剂和第二化学试剂标定试剂来对过敏反应蛋白反应溶液进行标定数据测试。The first chemical reagent calibration reagent and the second chemical reagent calibration reagent are used to perform calibration data testing on the hypersensitive response protein reaction solution.
在本发明一个较佳实施例中,所述具备生物标记蛋白分子的过敏原溶液的浓度为0.35~1.188ug/ml;所述链霉亲和素标记的多肽包覆性纳米磁浮液的浓度为0.35~1.188mg/ml;所述带荧光素标签的IgG3抗体溶液的浓度为0.25~3.75ug/ml。In a preferred embodiment of the present invention, the concentration of the allergen solution with biomarker protein molecules is 0.35-1.188ug/ml; the concentration of the streptavidin-labeled polypeptide-coated nano-magnetic suspension solution is 0.35-1.188mg/ml; the concentration of the IgG3 antibody solution with fluorescein label is 0.25-3.75ug/ml.
在本发明一个较佳实施例中,所述标记的带荧光素标签的IgG3抗体为能与人体内IgG蛋白特异结合的抗体,In a preferred embodiment of the present invention, the labeled IgG3 antibody with a fluorescein label is an antibody that can specifically bind to IgG protein in the human body,
所述带荧光素标签的IgG3抗体溶液的制备过程为:将浓度为1.75-6.25mg/mL的碱性磷酸酶溶液与带荧光素标签的IgG3抗体按摩尔比为1-4:1进行混合后并纯化,得到IgG3抗体,所述纯化是用pH为8-9的碳酸氢盐缓冲液平衡并洗脱,再紫外检测和记录纯化图谱,再用0.05M、pH值为3.75的MES缓冲液对IgG抗体稀释。The preparation process of the fluorescein-labeled IgG3 antibody solution is as follows: after mixing the alkaline phosphatase solution with a concentration of 1.75-6.25 mg/mL and the fluorescein-labeled IgG3 antibody in a molar ratio of 1-4:1 And purifying to obtain IgG3 antibody, the purification is equilibrated with bicarbonate buffer with pH 8-9 and eluted, and then UV detection and recording of purification spectrum, and then use 0.05M, pH value of 3.75 MES buffer to equilibrate and elute. IgG antibody dilution.
在本发明一个较佳实施例中,所述免疫抑制剂包括环磷酰氨、霉酚酸酯、环孢素a、他克莫司、甲氨蝶呤、硫唑嘌呤、来氟米特、羟氯喹皮质类固醇、腺苷受体激动剂、磷酸二酯酶4抑制剂、HDAC抑制剂或蛋白酶体抑制剂的一种或多种。In a preferred embodiment of the present invention, the immunosuppressive agents include cyclophosphamide, mycophenolate mofetil, cyclosporine a, tacrolimus, methotrexate, azathioprine, leflunomide, One or more of hydroxychloroquine corticosteroid, adenosine receptor agonist, phosphodiesterase 4 inhibitor, HDAC inhibitor or proteasome inhibitor.
在本发明一个较佳实施例中,所述非应变性过敏抗原包含医疗用生物蛋白酶,所述医疗用生物蛋白酶是治疗性蛋白质或治疗性多核苷酸,包括替莫瑞林、奥瑞珠单抗、贝利木单抗、聚乙二醇化重组尿酸酶、他利苷酶α、阿加糖酶α或葡糖脑苷脂酶α的一种或多种。In a preferred embodiment of the present invention, the non-strained allergic antigen comprises a medical biological protease, and the medical biological protease is a therapeutic protein or a therapeutic polynucleotide, including temorelin, ocrelizumab One or more of anti-, belimumab, pegylated recombinant uricase, talisidase alfa, agalsidase alfa, or glucocerebrosidase alfa.
在本发明一个较佳实施例中,所述多肽包覆性纳米磁浮液的制备过程为:In a preferred embodiment of the present invention, the preparation process of the polypeptide-coated nano-magnetic suspension is as follows:
将链霉亲和素标记的纳米磁微粒用磁分离器沉淀,再用0.01M、pH值为7.8的磷酸盐缓冲液重悬,混匀磁分离沉淀,并重复清洗;The streptavidin-labeled magnetic nanoparticles were precipitated with a magnetic separator, resuspended with 0.01M phosphate buffer with a pH value of 7.8, mixed uniformly and the precipitate was magnetically separated, and washed repeatedly;
清洗后的纳米磁微粒分散于0.01M、pH值为7.8的磷酸盐缓冲液。The cleaned magnetic nanoparticles were dispersed in 0.01M phosphate buffer with pH 7.8.
为了检测人体在吸入本技术提出的一种抗过敏性纳米抗体组合物后,血清中的抗体和抗原性能参数,便于后续药物跟踪分析。In order to detect the performance parameters of antibodies and antigens in serum after inhalation of an anti-allergic nanobody composition proposed by the present technology, it is convenient for subsequent drug tracking analysis.
本发明采用的另一个技术方案是:Another technical scheme adopted by the present invention is:
一种基于纳米磁化学发光法测定血清抗体性能的方法,血清中摄入有所述的抗过敏性纳米抗体组合物,包括步骤为:A method for determining the performance of serum antibodies based on nano-magnetic chemiluminescence method, the anti-allergic nano-antibody composition is ingested in the serum, comprising the steps of:
(1)将多肽包覆性纳米磁浮液、具备生物标记蛋白分子的过敏原和待检样本混匀并反应,反应后置于磁场中静置并去上清,得到第一溶液;(1) Mixing and reacting the polypeptide-coated nano-magnetic levitation liquid, the allergen with biomarker protein molecules and the sample to be tested, and after the reaction, place it in a magnetic field to stand and remove the supernatant to obtain the first solution;
(2)向所述第一溶液中加入IgG3抗体混匀并反应,反应后置于磁场中静置并去上清,得到第二溶液;(2) adding IgG3 antibody to the first solution, mixing and reacting, placing it in a magnetic field to stand and removing the supernatant after the reaction to obtain a second solution;
(3)向所述第二溶液中加入发光底物并反应,测发光强度;(3) adding a luminescent substrate to the second solution and reacting, and measuring the luminescence intensity;
(4)利用已知浓度的第一化学试剂标定试剂绘制发光强度标准曲线,根据步骤(3)得到的发光强度对照所述标准曲线,计算得出待测血清样本中炎症特异性IgG的含量。(4) Draw a standard curve of luminescence intensity using the first chemical reagent calibration reagent of known concentration, and compare the standard curve with the luminescence intensity obtained in step (3) to calculate the content of inflammation-specific IgG in the serum sample to be tested.
在本发明一个较佳实施例中,步骤(1)中所述反应的条件为45℃下反应5分钟,所述静置的时间为3分钟;步骤(1)中还包括对所述第一溶液用清洗液进行清洗,置于磁场中静置并去上清,清洗重复2次;步骤(2)中所述反应的条件为45℃下反应5分钟,所述静置的时间为3分钟;步骤(2)中还包括对所述第二溶液用清洗液进行清洗,置于磁场中静置并去上清,清洗重复2次;步骤(3)中所述反应的条件为45℃下反应2分钟,在化学发光分析/测定仪中测定发光强度。In a preferred embodiment of the present invention, the reaction conditions in step (1) are 5 minutes at 45° C., and the standing time is 3 minutes; step (1) also includes: The solution was cleaned with a cleaning solution, placed in a magnetic field and left to stand and the supernatant was removed, and the cleaning was repeated twice; the reaction conditions in step (2) were 5 minutes at 45° C., and the standing time was 3 minutes ; In step (2), it is also included that the second solution is cleaned with a cleaning solution, placed in a magnetic field and left to stand and remove the supernatant, and the cleaning is repeated 2 times; the reaction condition described in the step (3) is at 45 ℃. The reaction was carried out for 2 minutes, and the luminescence intensity was measured in a chemiluminescence analyzer/meter.
磁性纳米粒子表面连接的具有生物活性的吸附剂或其他配体等活性物质可与特定生物分子或细胞特异性结合,在外加磁场作用下分离。Active substances such as biologically active adsorbents or other ligands attached to the surface of magnetic nanoparticles can specifically bind to specific biomolecules or cells and separate under the action of an external magnetic field.
在本发明一个较佳实施例中,所述方法包括步骤为:In a preferred embodiment of the present invention, the method includes the steps of:
(1)、将所述纳米抗体组合物放入全自动化学发光仪的试剂仓里并进行识别;(1), put the nanobody composition into the reagent chamber of the automatic chemiluminescence instrument and identify;
(2)、将第一化学试剂标定试剂置于所述全自动化学发光仪的仪器样本仓,识别第一化学试剂标定试剂信息,分配第一化学试剂标定试剂位置;(2), placing the first chemical reagent calibration reagent in the instrument sample compartment of the automatic chemiluminescence instrument, identifying the first chemical reagent calibration reagent information, and assigning the first chemical reagent calibration reagent position;
(3)、将质控物或待测样本置于仪器样本仓,编辑检测信息;(3) Place the quality control substance or the sample to be tested in the sample compartment of the instrument, and edit the testing information;
(4)、启动运行程序,所有第一化学试剂标定试剂/质控物/待测样本自动进行处理得到结果。(4), start the running program, all the first chemical reagent calibration reagents/quality control substances/samples to be tested are automatically processed to obtain the results.
本发明采用的另一个技术方案是提供一种鼻炎抗过敏性纳米抗体喷雾剂:Another technical solution adopted in the present invention is to provide a rhinitis anti-allergic nanobody spray:
一种抗过敏性鼻炎喷雾剂,所述抗过敏性鼻炎喷雾剂包含乙二醇溶液,所述乙二醇溶液中包含有所述的抗过敏性纳米抗体组合物。An anti-allergic rhinitis spray, the anti-allergic rhinitis spray comprises an ethylene glycol solution containing the anti-allergic nanobody composition.
本发明的有益效果是:本发明的检测抗过敏性纳米抗体组合物及方法,该纳米抗体组合物通过生物大分子蛋白酶等进行标记跟踪,对过敏性抗原进行追踪反应报告,记载化学试验结果而进行基于纳米抗体的抗过敏性验证药物制备推导。The beneficial effects of the present invention are: the anti-allergic nanobody composition and method of the present invention, the nanobody composition is labeled and tracked by biological macromolecular protease, etc., the allergic antigen is tracked and reported, and the chemical test results are recorded and The preparation and derivation of anti-allergic verification drugs based on nanobodies were carried out.
本纳米抗体组合物中各试剂组分稳定性良好,检测灵敏度高、特异性能好,得到完善统一工艺,并严格按照标准生产操作规程和质量控制规程进行生产。Each reagent component in the nanobody composition has good stability, high detection sensitivity and good specificity, has a perfect and unified process, and is produced in strict accordance with standard production operating procedures and quality control procedures.
可以用于较大范围的炎症试用,比如鼻炎等炎症。费用成本低二适用于国内广大患者。It can be used for a wider range of inflammation trials, such as rhinitis and other inflammations. The low cost and low cost are suitable for the majority of domestic patients.
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其它的附图,其中:In order to illustrate the technical solutions in the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the description of the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, under the premise of no creative work, other drawings can also be obtained from these drawings, wherein:
图1为本发明采用抗过敏性纳米抗体组合物制得的抗过敏性纳米抗体鼻炎喷雾剂示意图;Fig. 1 is the schematic diagram of the anti-allergic nano-body rhinitis spray prepared by the anti-allergic nano-body composition of the present invention;
图2为本发明采用基于纳米磁化学发光法测定血清抗体性能的方法测定IgG3抗体溶液的流程图。FIG. 2 is a flow chart of the present invention for measuring IgG3 antibody solution by the method for measuring serum antibody performance based on nano-magnetic chemiluminescence method.
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例,都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
本纳米抗体组合物在是需要经过预设时间后进行抗体抗原结合产生的性能进行标定测试,化学结果需要进行缓冲测试,在本实施例,采用对过敏原反应蛋白具有抗性的化学试剂进行标定测试。The nanobody composition needs to be calibrated for the performance of antibody-antigen binding after a preset time, and the chemical result needs to be subjected to a buffer test. In this embodiment, a chemical reagent that is resistant to allergen-reactive protein is used for calibration. test.
本处配置两者不同标准含量的试剂:第一化学试剂标定试剂和第二化学试剂标定试剂。Two reagents with different standard contents are prepared here: the first chemical reagent calibration reagent and the second chemical reagent calibration reagent.
第一化学试剂标定试剂和第二化学试剂标定试剂来对过敏反应蛋白反应溶液进行标定数据测试。The first chemical reagent calibration reagent and the second chemical reagent calibration reagent are used to perform calibration data testing on the hypersensitive response protein reaction solution.
在本发明一个较佳实施例中,所述第一化学试剂标定试剂的浓度为:0IU/ml、0.35IU/ml、0.7IU/ml、3.5IU/ml、17.5IU/ml、50IU/ml、100IU/ml,所述第一化学试剂标定试剂是IgG蛋白与0.1M、pH值为7.8的Tris-HCl缓冲液配制得到的;In a preferred embodiment of the present invention, the concentration of the first chemical reagent calibration reagent is: 0IU/ml, 0.35IU/ml, 0.7IU/ml, 3.5IU/ml, 17.5IU/ml, 50IU/ml, 100IU/ml, the first chemical reagent calibration reagent is prepared by IgG protein and 0.1M Tris-HCl buffer with a pH value of 7.8;
所述第二化学试剂标定试剂的浓度为:0.7IU/ml、17.5IU/ml,所述第二化学试剂标定试剂是IgG蛋白与0.1M、pH值为7.8的Tris-HCl缓冲液配制得到的。The concentration of the second chemical reagent calibration reagent is: 0.7IU/ml, 17.5IU/ml, and the second chemical reagent calibration reagent is prepared by IgG protein and 0.1M Tris-HCl buffer with a pH value of 7.8 .
抗体选择如下:荧光素标签的IgG3抗体溶液;Antibody selections are as follows: Fluorescein-labeled IgG3 antibody solution;
抗原选择如下:炎症过敏原;The antigens were selected as follows: inflammatory allergens;
在本发明一个较佳实施例中,炎症过敏原为能与人体内炎症特异性IgG蛋白结合的抗原,In a preferred embodiment of the present invention, the inflammatory allergen is an antigen that can bind to inflammation-specific IgG proteins in the human body,
所述具备生物标记蛋白分子的过敏原溶液的制备过程为:将炎症过敏原按1:20摩尔比加入到溶于二甲基甲酰胺的生物素中,再用0.1M、pH值为7.8的磷酸盐缓冲液透析得到。The preparation process of the allergen solution with the biomarker protein molecules is as follows: adding the inflammatory allergen into biotin dissolved in dimethylformamide at a molar ratio of 1:20, and then using 0.1M, pH 7.8 Dialyzed against phosphate buffer.
在本实施例中,所述免疫抑制剂包括环磷酰氨、霉酚酸酯、环孢素a、他克莫司、甲氨蝶呤、硫唑嘌呤、来氟米特、羟氯喹皮质类固醇、腺苷受体激动剂、磷酸二酯酶4抑制剂、HDAC抑制剂或蛋白酶体抑制剂的一种或多种。In this embodiment, the immunosuppressive agents include cyclophosphamide, mycophenolate mofetil, cyclosporine a, tacrolimus, methotrexate, azathioprine, leflunomide, hydroxychloroquine corticosteroids , one or more of an adenosine receptor agonist, a phosphodiesterase 4 inhibitor, an HDAC inhibitor, or a proteasome inhibitor.
在本实施例中,所述非应变性过敏抗原包含医疗用生物蛋白酶,所述医疗用生物蛋白酶是治疗性蛋白质或治疗性多核苷酸,包括替莫瑞林、奥瑞珠单抗、贝利木单抗、聚乙二醇化重组尿酸酶、他利苷酶α、阿加糖酶α或葡糖脑苷脂酶α的一种或多种。In this embodiment, the non-strain allergic antigen comprises a medical biological protease, and the medical biological protease is a therapeutic protein or a therapeutic polynucleotide, including temorelin, ocrelizumab, Bailey One or more of mulimumab, pegylated recombinant uricase, talisidase alfa, agalsidase alfa, or glucocerebrosidase alfa.
实施例一:Example 1:
本方法提供一种抗过敏性纳米抗体组合物,包括带荧光素标签的IgG3抗体溶液,所述纳米抗体组合物包括:具备生物标记蛋白分子的过敏原溶液、多肽包覆性纳米磁浮液、具有生物活性的特异性纳米抗体,以及维护增加纳米抗体稳定性和组织穿透性的抗体纳米抗体组合物、免疫抑制剂、非应变性过敏抗原;The method provides an anti-allergic nanobody composition, comprising a fluorescein-labeled IgG3 antibody solution, and the nanobody composition includes: an allergen solution with biomarker protein molecules, a polypeptide-coated nanomagnetic suspension, a Biologically active specific Nanobodies, as well as maintaining antibody Nanobody compositions that increase Nanobody stability and tissue penetration, immunosuppressants, non-strain allergic antigens;
所述纳米抗体包括:活性纳米抗体、纳米抗体片段、多靶向纳米抗体聚合链接体、纳米抗体与蛋白的偶联体或纳米抗体与药物的偶联体中的一种或多种。The nanobody includes one or more of active nanobody, nanobody fragment, multi-targeted nanobody polymer linker, nanobody-protein conjugate or nanobody-drug conjugate.
多肽包覆性纳米磁浮液可以用于抗体药物靶向追踪。Polypeptide-coated nano-magnetic suspension can be used for antibody drug target tracking.
多肽包覆性纳米磁常用语现代生物,免疫分析是现代生物分析技术中重要的一种方法,利用它可对蛋白质、抗原、抗体及细胞进行定量分析。Polypeptide-coated nanomagnets are commonly used in modern biology, and immunoassay is an important method in modern bioanalytical technology, which can be used to quantitatively analyze proteins, antigens, antibodies and cells.
将结合有载体DNA的磁性纳米粒子在外界磁场影响下转染到细胞内的方法。A method of transfecting magnetic nanoparticles bound with carrier DNA into cells under the influence of an external magnetic field.
磁性纳米粒子表面连接的具有生物活性的吸附剂或其他配体等活性物质可与特定生物分子或细胞特异性结合,在外加磁场作用下分离。磁性分离方法基本只包括2个步骤:1.用磁性纳米粒子标记目标分子或细胞;2.通过磁分离装置分离出目标分子或细胞。利用磁性纳米粒子分离的例子之一是把特异性抗体与磁性纳米粒子结合,可将磁性纳米粒子连接在特定细胞上,外加磁场即可快速将结合磁性纳米粒子的细胞分离或进行免疫分析。这样的方法特异性高、分离迅速、重现性好。Active substances such as biologically active adsorbents or other ligands attached to the surface of magnetic nanoparticles can specifically bind to specific biomolecules or cells and separate under the action of an external magnetic field. The magnetic separation method basically only includes two steps: 1. Labeling target molecules or cells with magnetic nanoparticles; 2. Separating target molecules or cells by a magnetic separation device. One example of separation using magnetic nanoparticles is the binding of specific antibodies to magnetic nanoparticles, which can be attached to specific cells, and the magnetic nanoparticles can be rapidly isolated or immunoassayed by applying a magnetic field. Such a method has high specificity, rapid separation, and good reproducibility.
抗过敏性纳米抗体组合物包括如下试剂:The anti-allergic Nanobody composition includes the following reagents:
(1)具备生物标记蛋白分子的过敏原溶液,浓度为0.1-1.0ug/mL;(1) Allergen solution with biomarker protein molecules at a concentration of 0.1-1.0ug/mL;
(2)多肽包覆性纳米磁浮液,浓度为0.1-1.0mg/mL;(2) Polypeptide-coated nano-magnetic levitation liquid, the concentration is 0.1-1.0mg/mL;
(3)带荧光素标签的IgG3抗体溶液,浓度为0.1-2.0ug/mL;(3) IgG3 antibody solution with fluorescein label, the concentration is 0.1-2.0ug/mL;
(4)6个不同浓度水平的第一化学试剂标定试剂:0IU/ml,0.35IU/ml,0.7IU/ml,3.5IU/ml,17.5IU/ml,50IU/ml,100IU/ml;(4) Six different concentration levels of the first chemical reagent calibration reagent: 0IU/ml, 0.35IU/ml, 0.7IU/ml, 3.5IU/ml, 17.5IU/ml, 50IU/ml, 100IU/ml;
(5)2个不同浓度水平的第二化学试剂标定试剂:0.7IU/ml,17.5IU/ml。(5) Two different concentration levels of the second chemical reagent calibration reagent: 0.7IU/ml, 17.5IU/ml.
所述纳米抗体包括:活性纳米抗体、纳米抗体片段、多靶向纳米抗体聚合链接体、纳米抗体与蛋白的偶联体或纳米抗体与药物的偶联体中的一种或多种。The nanobody includes one or more of active nanobody, nanobody fragment, multi-targeted nanobody polymer linker, nanobody-protein conjugate or nanobody-drug conjugate.
多肽包覆性纳米磁浮液可以用于抗体药物靶向追踪。Polypeptide-coated nano-magnetic suspension can be used for antibody drug target tracking.
多肽包覆性纳米磁常用语现代生物,免疫分析是现代生物分析技术中重要的一种方法,利用它可对蛋白质、抗原、抗体及细胞进行定量分析。Polypeptide-coated nanomagnets are commonly used in modern biology, and immunoassay is an important method in modern bioanalytical technology, which can be used to quantitatively analyze proteins, antigens, antibodies and cells.
将结合有载体DNA的磁性纳米粒子在外界磁场影响下转染到细胞内的方法。A method of transfecting magnetic nanoparticles bound with carrier DNA into cells under the influence of an external magnetic field.
配置完毕,加入免疫抑制剂如腺苷受体激动剂和非应变性过敏抗原包如聚乙二醇化重组尿酸酶进行试剂静置。After the configuration is completed, immunosuppressive agents such as adenosine receptor agonists and non-strain allergic antigen packages such as pegylated recombinant uricase are added to allow the reagents to stand.
实施例二:Embodiment 2:
为了检测人体在吸入本技术提出的一种抗过敏性纳米抗体组合物后,血清中的抗体和抗原性能参数,便于后续药物跟踪分析。In order to detect the performance parameters of antibodies and antigens in serum after inhalation of an anti-allergic nanobody composition proposed by the present technology, it is convenient for subsequent drug tracking analysis.
本发明采用的另一个技术方案是:Another technical scheme adopted by the present invention is:
一种基于纳米磁化学发光法测定血清抗体性能的方法,血清中摄入有所述的抗过敏性纳米抗体组合物,包括如下步骤:A method for measuring serum antibody performance based on nano-magnetic chemiluminescence method, the anti-allergic nano-antibody composition is ingested in serum, comprising the following steps:
(1)配制缓冲液:(1) Preparation buffer:
配制0.01M、pH值为7.8的磷酸盐缓冲液,0.1M、pH值为7.8的Tris-HCl缓冲液,0.05M、pH值为3.75的MES缓冲液。Prepare 0.01M phosphate buffer at pH 7.8, 0.1M Tris-HCl buffer at pH 7.8, and 0.05M MES buffer at pH 3.75.
0.01M、pH值为7.8的磷酸盐缓冲液的配制:取容量1L的烧杯,加入800ml去离子水,称取2.56g Na
2HPO
4·12H
2O、0.44g NaH
2PO
4·2H
2O和9g NaCl,加入到烧杯中,搅拌使其充分溶解,用HCl或NaOH调节pH值至7.8±0.05,加入质量分数为2%的甘露醇、质量分数为1%的甘油,搅拌0.5h至完全溶解,加去离子水定容至1L。
Preparation of 0.01M, pH 7.8 phosphate buffer: take a beaker with a capacity of 1L, add 800ml of deionized water, weigh 2.56g Na 2 HPO 4 ·12H 2 O, 0.44g NaH 2 PO 4 · 2H 2 O and 9g NaCl, add it to the beaker, stir to dissolve it fully, adjust the pH value to 7.8±0.05 with HCl or NaOH, add 2% mannitol and 1% glycerol, stir for 0.5h to complete Dissolve, add deionized water to make up to 1L.
0.1M、pH值为7.8的Tris-HCl缓冲液的配制:取容量1L的烧杯,加入800ml去离子水,称取12.11g Tris(三羟甲基氨基甲烷)加入到烧杯中,搅拌使其充分溶解,用HCl或NaOH调节pH值至7.8±0.05,加入质量分数为2%的BSA、质量分数为0.3%的Proclin300,搅拌0.5h至完全溶解,加去离子水定容至1L。Preparation of 0.1M Tris-HCl buffer with pH 7.8: Take a beaker with a capacity of 1L, add 800ml of deionized water, weigh 12.11g of Tris (tris (tris(hydroxymethylaminomethane)) into the beaker, and stir to make it fully Dissolve, adjust the pH to 7.8±0.05 with HCl or NaOH, add 2% BSA, 0.3% Proclin300, stir for 0.5h until completely dissolved, add deionized water to make up to 1L.
0.05M、pH值为3.75的MES缓冲液的配制:取容量1L的烧杯,加入800ml去离子水,称取9.76g MES(2-(N-吗啉代)乙磺酸)、9g NaCl加入到烧杯中,搅拌使其充分溶液,用HCl或NaOH调节pH值至3.75±0.05,加入质量分数为1%的BSA、质量分数为0.1%的Tween-20,搅拌0.5h至完全溶解,加去离子水定容至1L。Preparation of 0.05M MES buffer with pH value of 3.75: Take a beaker with a capacity of 1L, add 800ml deionized water, weigh 9.76g MES (2-(N-morpholino)ethanesulfonic acid), 9g NaCl and add it to the solution. In the beaker, stir to make it fully dissolved, adjust the pH value to 3.75±0.05 with HCl or NaOH, add 1% BSA, 0.1% Tween-20, stir for 0.5h until completely dissolved, add deionized Make up to 1L with water.
(2)制备具备生物标记蛋白分子的过敏原溶液,将炎症过敏原以1:20的比例加入到溶于二甲基甲酰胺的生物素中,室温条件下充分混合反应30分钟,反应后的溶液用0.1M、pH值为7.8的磷酸盐缓冲液透析。BCA法测定具备生物标记蛋白分子的过敏原溶液的浓度,并调整其浓度到0.1-1.0ug/ml。(2) Prepare an allergen solution with biomarker protein molecules, add inflammatory allergens to biotin dissolved in dimethylformamide at a ratio of 1:20, and thoroughly mix and react at room temperature for 30 minutes. The solution was dialyzed against 0.1 M phosphate buffer pH 7.8. The concentration of the allergen solution with biomarker protein molecules was determined by BCA method, and the concentration was adjusted to 0.1-1.0ug/ml.
(3)制备多肽包覆性纳米磁浮液,将链霉亲和素标记的纳米磁微粒用磁分离器沉淀出来,用0.01M、pH值为7.8的磷酸盐缓冲液重悬,充分混匀5分钟后磁分离沉出,弃去上清,再用0.01M、pH值为7.8的磷酸盐缓冲液重悬,如此重复清洗5次,清洗后的纳米磁微粒分散于0.01M、pH值为7.8的磷酸盐 缓冲液,浓度为0.1-1.0mg/ml。(3) Prepare the polypeptide-coated nano-magnetic suspension, precipitate the streptavidin-labeled nano-magnetic particles with a magnetic separator, resuspend with 0.01M phosphate buffer with a pH value of 7.8, and mix thoroughly for 5 After 10 minutes, the magnetic separation and sedimentation were carried out, the supernatant was discarded, and then resuspended with 0.01M phosphate buffer with a pH value of 7.8. Repeat the cleaning for 5 times. The cleaned magnetic nanoparticles were dispersed in 0.01M, a pH value of 7.8. of phosphate buffered saline at a concentration of 0.1-1.0 mg/ml.
(4)制备带荧光素标签的IgG3抗体溶液;用0.05M、pH值为3.75的MES缓冲液将IgG3抗体稀释到0.1-2.0ug/ml。(4) Prepare a fluorescein-labeled IgG3 antibody solution; dilute the IgG3 antibody to 0.1-2.0ug/ml with 0.05M MES buffer at pH 3.75.
(5)制备第一化学试剂标定试剂取IgG蛋白,用0.1M、pH值为7.8的Tris-HCl缓冲液配制成0、0.35、0.7、3.5、17.5、50、100IU/ml的校准点。(5) Preparation of the first chemical reagent Calibration reagent Take IgG protein, and prepare the calibration point of 0, 0.35, 0.7, 3.5, 17.5, 50, 100 IU/ml with 0.1 M Tris-HCl buffer with pH value of 7.8.
(6)制备第二化学试剂标定试剂取IgG蛋白,用0.1M、pH值为7.8的Tris-HCl缓冲液配制成0.7、17.5IU/ml的第二化学试剂标定试剂。(6) Preparation of Second Chemical Reagent Calibration Reagent Take IgG protein, and prepare 0.7, 17.5IU/ml second chemical reagent calibration reagent with 0.1M Tris-HCl buffer with pH value of 7.8.
(7)纳米抗体组合物组装将具备生物标记蛋白分子的过敏原溶液、链霉亲和素标记的多肽包覆性纳米磁浮液、带荧光素标签的IgG3抗体溶液、第一化学试剂标定试剂和第二化学试剂标定试剂组装成纳米抗体组合物。(7) Nanobody composition assembly The allergen solution with biomarker protein molecules, the streptavidin-labeled polypeptide-coated nano-magnetic suspension, the fluorescein-labeled IgG3 antibody solution, the first chemical reagent calibration reagent and The second chemical reagent calibration reagent is assembled into the nanobody composition.
如附图2所示,在本发明一个较佳实施例中,所述方法包括步骤为:As shown in Figure 2, in a preferred embodiment of the present invention, the method includes the steps of:
(1)、将所述纳米抗体组合物放入全自动化学发光仪的试剂仓里并进行识别;(1), put the nanobody composition into the reagent chamber of the automatic chemiluminescence instrument and identify;
(2)、将第一化学试剂标定试剂置于所述全自动化学发光仪的仪器样本仓,识别第一化学试剂标定试剂信息,分配第一化学试剂标定试剂位置;(2), placing the first chemical reagent calibration reagent in the instrument sample compartment of the automatic chemiluminescence instrument, identifying the first chemical reagent calibration reagent information, and assigning the first chemical reagent calibration reagent position;
(3)、将质控物或待测样本置于仪器样本仓,编辑检测信息;(3) Place the quality control substance or the sample to be tested in the sample compartment of the instrument, and edit the testing information;
(4)、启动运行程序,所有第一化学试剂标定试剂/质控物/待测样本自动进行处理得到结果。(4), start the running program, all the first chemical reagent calibration reagents/quality control substances/samples to be tested are automatically processed to obtain the results.
本发明的纳米抗体组合物中未详细提及的试剂组分(例如清洗液、一些必要的缓冲液等)、纳米抗体组合物的外包装以及各试剂组分的独立包装容器等均可以按照所属领域的常规操作进行,符合相关行业规定即可。The reagent components (such as cleaning solution, some necessary buffers, etc.) that are not mentioned in detail in the Nanobody composition of the present invention, the outer packaging of the Nanobody composition, and the individual packaging containers for each reagent component can be all listed according to their respective Routine operations in the field can be carried out in accordance with relevant industry regulations.
实施例三:Embodiment three:
本实施例提供了一种基于纳米磁化学发光法测定血清抗体性能的方法,血清中摄入有所述的抗过敏性纳米抗体组合物,检测在全自动化学发光仪MAGICL6800系列上进行,具体仪器操作步骤如下:This example provides a method for determining the performance of serum antibodies based on nano-magnetic chemiluminescence method. The anti-allergic nano-antibody composition is ingested in the serum, and the detection is carried out on an automatic chemiluminescence instrument MAGICL6800 series. The operation steps are as follows:
(1)将纳米抗体组合物放入全自动化学发光分析/测定仪试剂仓相应位置,纳米抗体组合物信息通过条形码扫描仪输入仪器***,也可通过仪器配套软件设定。(1) Put the nanobody composition into the corresponding position of the reagent chamber of the automatic chemiluminescence analyzer/determiner. The nanobody composition information is input into the instrument system through the barcode scanner, and can also be set through the instrument supporting software.
(2)将第一化学试剂标定试剂置于仪器样本仓。通过条形码扫描仪识别第一化学试剂标定试剂信息,并在仪器***中分配第一化学试剂标定试剂位置。(3)将质控物/待测样本置于仪器样本仓,通过仪器配套软件编辑相应检测信息。(2) Place the first chemical reagent calibration reagent in the instrument sample compartment. The information of the first chemical reagent calibration reagent is identified by the barcode scanner, and the position of the first chemical reagent calibration reagent is allocated in the instrument system. (3) Place the quality control substance/sample to be tested in the sample compartment of the instrument, and edit the corresponding test information through the instrument supporting software.
(4)启动运行程序,所有第一化学试剂标定试剂/质控物/待检样本处理步骤将自动执行。(4) Start the running program, and all the processing steps of the first chemical reagent calibration reagent/quality control substance/sample to be tested will be automatically executed.
本发明提供的一种基于纳米磁化学发光法测定血清抗体性能的方法,血清中摄入有所述的抗过敏性纳米抗体组合物,还可以在半自动化学发光仪上实现,具体的样本检测步骤如下:(1)在检测管中依次加入40ul待测血清或IgG第一化学试剂标定试剂、50ul具备生物标记蛋白分子的过敏原、50ul链霉亲和素标记的纳米磁微粒,混匀后,在45℃下共孵育5分钟,样本中炎症特异性IgG抗体被捕获,并固着在纳米磁微粒表面。The present invention provides a method for measuring serum antibody performance based on nano-magnetic chemiluminescence method. The anti-allergic nano-antibody composition is ingested in serum, which can also be realized on a semi-automatic chemiluminescence instrument. The specific sample detection steps As follows: (1) Add 40ul of serum to be tested or IgG first chemical reagent calibration reagent, 50ul of allergens with biomarker protein molecules, and 50ul of streptavidin-labeled magnetic nanoparticles into the detection tube in turn, and after mixing, After co-incubating at 45°C for 5 minutes, the inflammation-specific IgG antibodies in the samples were captured and immobilized on the surface of the magnetic nanoparticles.
(2)外加磁场作用下,磁微粒沉降下来,去除上清,加入500ul清洗液,去除磁场后,震荡使磁微粒重悬;重复此清洗步骤3次以除去未结合的生物素标记的鼠抗人IgG抗体。(2) Under the action of an external magnetic field, the magnetic particles settle down, remove the supernatant, add 500 ul of cleaning solution, and after removing the magnetic field, shake to resuspend the magnetic particles; repeat this cleaning step 3 times to remove unbound biotin-labeled mouse antibody Human IgG antibodies.
(3)加入100ul带荧光素标签的IgG3抗体溶液,混匀,45℃下孵育5分钟。(3) Add 100ul of fluorescein-labeled IgG3 antibody solution, mix well, and incubate at 45°C for 5 minutes.
(4)外加磁场作用下,磁微粒沉降下来,去除上清,加入500ul清洗液,去除磁场后,震荡使磁微粒重悬;重复此清洗步骤3次以除去未结合的酶标抗 体及其它杂质。(4) Under the action of an external magnetic field, the magnetic particles settle down, remove the supernatant, add 500 ul of cleaning solution, and after removing the magnetic field, shake to resuspend the magnetic particles; repeat this cleaning step 3 times to remove unbound enzyme-labeled antibodies and other impurities .
(5)加入150ul酶促化学发光底物,去除磁场,充分混匀后,在45℃下孵育2分钟测定发光值。(5) Add 150ul of enzymatic chemiluminescence substrate, remove the magnetic field, mix well, incubate at 45°C for 2 minutes to measure the luminescence value.
(6)数据的处理:通过第一化学试剂标定试剂的浓度值和检测到的发光值,通过四参数非线性拟合获得标准曲线,将样本的发光值代入图1所示的标准曲线获得相应的浓度值。(6) Data processing: the concentration value of the reagent and the detected luminescence value are calibrated by the first chemical reagent, the standard curve is obtained by four-parameter nonlinear fitting, and the luminescence value of the sample is substituted into the standard curve shown in Figure 1 to obtain the corresponding concentration value.
本发明的工作原理:待测血清样本与具备生物标记蛋白分子的过敏原、链霉亲和素标记的纳米磁微粒共孵育,待测血清样本中炎症特异性IgG抗体与具备生物标记蛋白分子的过敏原特异性结合,同时借助生物素-链霉亲和素放大体系,血清样本中炎症过敏原特异性IgG结合到固相载体纳米磁微粒上;外加磁场作用下,磁分离洗涤去除多余的生物素标记的炎症过敏源;The working principle of the present invention: the serum sample to be tested is co-incubated with allergens and streptavidin-labeled nano-magnetic particles with biomarker protein molecules, and the inflammation-specific IgG antibodies in the serum sample to be tested are incubated with the allergens with biomarker protein molecules. Allergen-specific binding, and with the aid of the biotin-streptavidin amplification system, the inflammatory allergen-specific IgG in serum samples is bound to the solid-phase carrier magnetic nanoparticles; under the action of an external magnetic field, magnetic separation and washing remove excess biological hormone-labeled inflammatory allergens;
加入IgG3抗体,形成具备生物标记蛋白分子的过敏原-炎症特异性IgG-抗IgG3抗体的免疫复合物结构;外加磁场作用下,多次磁分离洗涤去除未结合的酶标抗体及其它杂质;加入酶促化学发光底物,测定发光强度;使用已知浓度的第一化学试剂标定试剂标准曲线,根据发光强度对照标准曲线,计算得出待测样本中炎症特异性IgG抗体的含量;IgG3 antibody was added to form an immune complex structure of allergen-inflammatory specific IgG-anti-IgG3 antibody with biomarker protein molecules; under the action of an external magnetic field, the unbound enzyme-labeled antibody and other impurities were removed by repeated magnetic separation and washing; Enzyme-catalyzed chemiluminescence substrate to measure the luminescence intensity; use the first chemical reagent of known concentration to calibrate the standard curve of the reagent, and calculate the content of inflammation-specific IgG antibody in the sample to be tested according to the luminescence intensity and the standard curve;
实施例四:Embodiment 4:
所述纳米抗体组合物的性能评估:Performance evaluation of the Nanobody composition:
1、具备抗过敏性纳米抗体(D2)检测纳米抗体组合物(纳米磁化学发光法)分析性能和稳定性,具备抗过敏性纳米抗体(D2)检测纳米抗体组合物(纳米磁化学发光法)与进口试剂的性能对比;1. It has the analytical performance and stability of anti-allergic nanobody (D2) detection nanobody composition (nanomagnetic chemiluminescence method), and has anti-allergic nanobody (D2) detection nanobody composition (nanomagnetic chemiluminescence method) Performance comparison with imported reagents;
3、具备抗过敏性纳米抗体(D2)检测纳米抗体组合物(纳米磁化学发光法)临床参考值范围,以正常人样本检测值的双侧95百分位数作为参考值。95%计算基本范围(mIU/mL),具体见下,3. It has the clinical reference value range of anti-allergic nanobody (D2) detection nanobody composition (nanomagnetic chemiluminescence method), and the double-sided 95th percentile of the detection value of normal human samples is used as the reference value. 95% Calculated basic range (mIU/mL), see below for details,
正常非过敏反应成人的血清中单个过敏原IgG抗体浓度小于0.35IU/mL。The concentration of IgG antibodies to a single allergen in the serum of normal non-allergic adults is less than 0.35 IU/mL.
测值介于0.35~0.7IU/mL为该过敏原1级过敏;If the measured value is between 0.35 and 0.7IU/mL, it is the first-grade allergy to the allergen;
测值介于0.7~3.5IU/mL为该过敏原2级过敏;If the measured value is between 0.7 and 3.5IU/mL, it is a grade 2 allergy to the allergen;
测值介于3.5~17.5IU/mL为该过敏原3级过敏;If the measured value is between 3.5 and 17.5IU/mL, it is a grade 3 allergy to the allergen;
测值介于17.5~50IU/mL为该过敏原4级过敏;If the measured value is between 17.5 and 50IU/mL, it is a grade 4 allergy to the allergen;
测值介于50~100IU/mL为该过敏原5级过敏;If the measured value is between 50 and 100 IU/mL, it is a grade 5 allergy to the allergen;
测值大于100IU/mL为该过敏原6级过敏。The measured value is greater than 100IU/mL for the allergen grade 6 allergy.
本发明的有益效果是:The beneficial effects of the present invention are:
一、本发明以碱性磷酸酶(AP)为标记酶,通过化学反应标记抗体,并使用凝胶层析分离未反应的酶、抗体或抗原,提高反应的灵敏度;1. In the present invention, alkaline phosphatase (AP) is used as the labeling enzyme, the antibody is labeled by chemical reaction, and the unreacted enzyme, antibody or antigen is separated by gel chromatography to improve the sensitivity of the reaction;
二、以免疫磁微粒为固相,以链霉亲和素偶联磁性微球,作为通用的分离试剂,不仅使免疫反应更容易混匀和分离,而且大大提高了反应速度;2. Using immunomagnetic particles as the solid phase and streptavidin-coupled magnetic microspheres as a general separation reagent, it not only makes the immune reaction easier to mix and separate, but also greatly improves the reaction speed;
三、以新型化学发光底物AMPPD为底物,该底物是辉光型底物,而且快速达到平台期,有利于信号的检测,提高了最终纳米抗体组合物的灵敏度和特异性性能;并进一步优化了化学发光增强体系,保证了终产品的信号灵敏度高和稳定性好、变异小;3. The new chemiluminescence substrate AMPPD is used as the substrate. The substrate is a glow-type substrate, and it reaches the plateau phase quickly, which is beneficial to the detection of the signal and improves the sensitivity and specificity of the final nanobody composition; and The chemiluminescence enhancement system is further optimized to ensure that the final product has high signal sensitivity, good stability and small variation;
四、采用AMPPD发光底物的优点在于灵敏度高和平台稳定期长。4. The advantage of using AMPPD luminescent substrate is high sensitivity and long platform stability period.
如附图1所示,为本发明采用的另一个技术方案是提供一种鼻炎抗过敏性纳米抗体喷雾剂:As shown in accompanying drawing 1, another technical scheme adopted for the present invention is to provide a rhinitis anti-allergic nanobody spray:
一种抗过敏性鼻炎喷雾剂,所述抗过敏性鼻炎喷雾剂包含乙二醇溶液,所述乙二醇溶液中包含有所述的抗过敏性纳米抗体组合物。An anti-allergic rhinitis spray, the anti-allergic rhinitis spray comprises an ethylene glycol solution containing the anti-allergic nanobody composition.
在抗过敏性鼻炎喷雾剂中加入本发明提出的抗过敏性纳米抗体组合物,可以为患者进行消炎处理,成本低。Adding the anti-allergic nanobody composition proposed by the present invention to the anti-allergic rhinitis spray can perform anti-inflammatory treatment for patients with low cost.
可以进行炎症患者的过敏性抗体试验反应验证,直接追踪实际的验证抗体 反应结构,论证临床医疗效果。The allergic antibody test reaction verification of patients with inflammation can be carried out, and the actual verification antibody reaction structure can be directly traced to demonstrate the clinical medical effect.
以上所述仅为本申请的优选实施例而已,并不用于限制本申请,对于本领域的技术人员来说,本申请可以有各种更改和变化。凡在本申请的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本申请的保护范围之内。The above descriptions are only preferred embodiments of the present application, and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of this application shall be included within the protection scope of this application.
Claims (10)
- 一种抗过敏性纳米抗体组合物,其特征在于,包括纳米抗体以及带荧光素标签的IgG3抗体溶液、具备生物标记蛋白分子的过敏原溶液、多肽包覆性纳米磁浮液、具有生物活性的特异性纳米抗体,以及维护增加纳米抗体稳定性和组织穿透性的抗体纳米抗体组合物、免疫抑制剂、非应变性过敏抗原;An anti-allergic nanobody composition is characterized in that, it comprises nanobody and IgG3 antibody solution with fluorescein label, allergen solution with biomarker protein molecule, polypeptide-coated nanometer magnetic suspension, specific biological activity Antibody Nanobodies, and Antibody Nanobody Compositions, Immunosuppressants, Non-Strain Allergic Antigens that Maintain Increased Nanobody Stability and Tissue Penetration;所述纳米抗体包括:活性纳米抗体、纳米抗体片段、多靶向纳米抗体聚合链接体、纳米抗体与蛋白的偶联体或纳米抗体与药物的偶联体中的一种或多种。The nanobody includes one or more of active nanobody, nanobody fragment, multi-targeted nanobody polymer linker, nanobody-protein conjugate or nanobody-drug conjugate.
- 根据权利要求1所述的一种抗过敏性纳米抗体组合物,其特征在于,所述具备生物标记蛋白分子的过敏原溶液的浓度为0.35~1.188ug/ml;The anti-allergic nanobody composition according to claim 1, wherein the concentration of the allergen solution with biomarker protein molecules is 0.35-1.188ug/ml;所述多肽包覆性纳米磁浮液的浓度为0.35~1.188mg/ml;所述带荧光素标签的IgG3抗体溶液的浓度为0.25~3.75ug/ml。The concentration of the polypeptide-coated nano-magnetic suspension liquid is 0.35-1.188 mg/ml; the concentration of the IgG3 antibody solution with fluorescein label is 0.25-3.75 ug/ml.
- 根据权利要求1所述的一种抗过敏性纳米抗体组合物,其特征在于,所述免疫抑制剂包括环磷酰氨、霉酚酸酯、环孢素a、他克莫司、甲氨蝶呤、硫唑嘌呤、来氟米特、羟氯喹皮质类固醇、腺苷受体激动剂、磷酸二酯酶4抑制剂、HDAC抑制剂或蛋白酶体抑制剂的一种或多种。The anti-allergic nanobody composition according to claim 1, wherein the immunosuppressant comprises cyclophosphamide, mycophenolate mofetil, cyclosporine a, tacrolimus, methotrexate One or more of oxazine, azathioprine, leflunomide, hydroxychloroquine corticosteroid, adenosine receptor agonist, phosphodiesterase 4 inhibitor, HDAC inhibitor or proteasome inhibitor.
- 根据权利要求1所述的一种抗过敏性纳米抗体组合物,其特征在于,所述非应变性过敏抗原包含医疗用生物蛋白酶,所述医疗用生物蛋白酶是治疗性蛋白质或治疗性多核苷酸,包括替莫瑞林、奥瑞珠单抗、贝利木单抗、聚乙二醇化重组尿酸酶、他利苷酶α、阿加糖酶α或葡糖脑苷脂酶α的一种或多种。The anti-allergic nanobody composition according to claim 1, wherein the non-strain allergic antigen comprises a medical biological protease, and the medical biological protease is a therapeutic protein or a therapeutic polynucleotide , including one or more of temorelin, ocrelizumab, belimumab, pegylated recombinant uricase, talisidase alfa, agalsidase alfa, or glucocerebrosidase alfa kind.
- 根据权利要求1所述的一种抗过敏性纳米抗体组合物,其特征在于,所述标记的带荧光素标签的IgG3抗体为能与人体内IgG蛋白特异结合的抗体,所述带荧光素标签的IgG3抗体溶液的制备过程为:将浓度为1.75-6.25mg/mL的碱性磷酸酶溶液与带荧光素标签的IgG3抗体按摩尔比为1-4:1进行混合后并纯化,得到IgG3抗体,所述纯化是用pH为8-9的碳酸氢盐缓冲液平衡并洗脱,再紫外检测和记录纯化图谱,再用0.05M、pH值为3.75的MES缓冲液 对IgG抗体稀释。The anti-allergic nanobody composition according to claim 1, wherein the labeled IgG3 antibody with a fluorescein label is an antibody that can specifically bind to IgG protein in the human body, and the labeled IgG3 antibody with a fluorescein label The preparation process of the IgG3 antibody solution is as follows: the alkaline phosphatase solution with a concentration of 1.75-6.25 mg/mL is mixed with the fluorescein-labeled IgG3 antibody in a molar ratio of 1-4:1 and purified to obtain the IgG3 antibody , the purification is equilibrated and eluted with bicarbonate buffer with pH 8-9, and then the purification spectrum is detected and recorded by UV, and then the IgG antibody is diluted with 0.05M MES buffer with pH value of 3.75.
- 根据权利要求1所述的一种抗过敏性纳米抗体组合物,其特征在于,所述多肽包覆性纳米磁浮液的制备过程为:A kind of anti-allergic nanobody composition according to claim 1, is characterized in that, the preparation process of described polypeptide-coated nanometer magnetic suspension is:将链霉亲和素标记的纳米磁微粒用磁分离器沉淀;Precipitate the streptavidin-labeled magnetic nanoparticles with a magnetic separator;再用0.01M、pH值为7.8的磷酸盐缓冲液重悬,混匀磁分离沉淀,并重复清洗;Resuspend with 0.01M phosphate buffer with pH 7.8, mix evenly and magnetically separate the precipitate, and repeat the washing;清洗后的纳米磁微粒分散于0.01M、pH值为7.8的磷酸盐缓冲液。The cleaned magnetic nanoparticles were dispersed in 0.01M phosphate buffer with pH 7.8.
- 一种基于纳米磁化学发光法测定血清抗体性能的方法,其特征在于,血清中摄入有权利要求1-6中任一项所述的抗过敏性纳米抗体组合物,包括步骤为:A method for measuring serum antibody performance based on nano-magnetic chemiluminescence method, characterized in that the anti-allergic nano-antibody composition according to any one of claims 1-6 is ingested in the serum, comprising the steps of:(1)将多肽包覆性纳米磁浮液、具备生物标记蛋白分子的过敏原和待检样本混匀并反应,反应后置于磁场中静置并去上清,得到第一溶液;(1) Mixing and reacting the polypeptide-coated nano-magnetic levitation liquid, the allergen with biomarker protein molecules and the sample to be tested, and after the reaction, place it in a magnetic field to stand and remove the supernatant to obtain the first solution;(2)向所述第一溶液中加入IgG3抗体混匀并反应,反应后置于磁场中静置并去上清,得到第二溶液;(2) adding IgG3 antibody to the first solution, mixing and reacting, placing it in a magnetic field to stand and removing the supernatant after the reaction to obtain a second solution;(3)向所述第二溶液中加入发光底物并反应,测发光强度;(3) adding a luminescent substrate to the second solution and reacting, and measuring the luminescence intensity;(4)利用已知浓度的第一化学试剂标定试剂绘制发光强度标准曲线,根据步骤(3)得到的发光强度对照所述标准曲线,计算得出待测血清样本中炎症特异性IgG的含量。(4) Draw a luminescence intensity standard curve using the first chemical reagent calibration reagent of known concentration, and compare the standard curve with the luminescence intensity obtained in step (3) to calculate the content of inflammation-specific IgG in the serum sample to be tested.
- 根据权利要求7所述的一种基于纳米磁化学发光法测定血清抗体性能的方法,其特征在于,A method for measuring serum antibody performance based on nano-magnetic chemiluminescence method according to claim 7, characterized in that,步骤(1)中所述反应的条件为45℃下反应5分钟,所述静置的时间为3分钟;The condition of the reaction described in the step (1) is 5 minutes at 45 ° C, and the time of the standing is 3 minutes;步骤(1)中还包括对所述第一溶液用清洗液进行清洗,置于磁场中静置 并去上清,清洗重复2次;In step (1), also comprise that described first solution is cleaned with cleaning solution, place in magnetic field and leave standstill and remove supernatant, and cleaning is repeated 2 times;步骤(2)中所述反应的条件为45℃下反应5分钟,所述静置的时间为3分钟;The condition of the reaction described in the step (2) is 5 minutes at 45 ° C, and the time of the standing is 3 minutes;步骤(2)中还包括对所述第二溶液用清洗液进行清洗,置于磁场中静置并去上清,清洗重复2次;Step (2) also includes cleaning the second solution with a cleaning solution, placing it in a magnetic field for standing and removing the supernatant, and repeating the cleaning twice;步骤(3)中所述反应的条件为45℃下反应2分钟,在化学发光分析/测定仪中测定发光强度。The conditions of the reaction in step (3) are to react at 45° C. for 2 minutes, and measure the luminescence intensity in a chemiluminescence analyzer/determiner.
- 根据权利要求7所述的一种基于纳米磁化学发光法测定血清抗体性的方法,其特征在于,包括步骤为:A method for measuring serum antibody based on nano-magnetic chemiluminescence method according to claim 7, characterized in that, comprising the steps of:(1)将所述纳米抗体组合物放入全自动化学发光仪的试剂仓里并进行识别;(1) Put the nanobody composition into the reagent chamber of the automatic chemiluminescence instrument and identify it;(2)将第一化学试剂标定试剂置于所述全自动化学发光仪的仪器样本仓,识别第一化学试剂标定试剂信息,分配第一化学试剂标定试剂位置;(2) placing the first chemical reagent calibration reagent in the instrument sample compartment of the automatic chemiluminescence instrument, identifying the first chemical reagent calibration reagent information, and assigning the first chemical reagent calibration reagent position;(3)将质控物或待测样本置于仪器样本仓,编辑检测信息;(3) Place the quality control substance or the sample to be tested in the sample compartment of the instrument, and edit the testing information;(4)启动运行程序,所有第一化学试剂标定试剂/质控物/待测样本自动进行处理得到结果。(4) Start the running program, and all the first chemical reagent calibration reagents/quality control substances/samples to be tested are automatically processed to obtain the results.
- 一种抗过敏性鼻炎喷雾剂,其特征在于,所述抗过敏性鼻炎喷雾剂包含乙二醇溶液,所述乙二醇溶液中包含有权利要求1-6中任一项所述的抗过敏性纳米抗体组合物。An anti-allergic rhinitis spray, characterized in that the anti-allergic rhinitis spray comprises an ethylene glycol solution, and the ethylene glycol solution contains the anti-allergy described in any one of claims 1-6 Sexual Nanobody Composition.
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