CN112480244A - Anti-allergic nano antibody composition, antibody determination method and spray - Google Patents
Anti-allergic nano antibody composition, antibody determination method and spray Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- A61P37/08—Antiallergic agents
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- 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
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Abstract
The invention discloses an anti-allergic nano antibody composition, a serum determination method based on a nano-magnetization chemiluminescence method for determining serum antibody performance and an anti-allergic rhinitis spray. The nanometer antibody composition has the advantages of good stability of each reagent component, high detection sensitivity and good specificity, is a perfect and unified process, and is produced strictly according to standard production operation regulations and quality control regulations. Can be used for the trial of inflammation in a larger range, such as rhinitis and the like, and is suitable for the majority of patients in China with low cost. The anti-allergic rhinitis spray provided by the invention can be effectively used for rhinitis.
Description
Technical Field
The application relates to the technical field of research and development of medical anti-allergic antibodies, in particular to an anti-allergic nano antibody composition, an antibody determination method and a spray.
Background
Nanobodies are the smallest unit currently known to bind to the antigen of interest. The VHH crystal has the size of 2.5nm multiplied by 4nm, the molecular weight is only 12KD-15KD, the molecular structure is relatively stable, and the VHH crystal can resist high temperature and keep activity in extremely harsh environment.
VHH retained 80% of biological activity after 1 week at 45 ℃, indicating that nanobodies were fairly stable when stored at room temperature, making them easier to store and transport than conventional antibodies. The nano-antibody has strong and fast tissue penetration capability, is beneficial to entering compact tissues such as solid tumors to play a role, can effectively penetrate blood brain barriers, and provides a new method for brain administration.
Meanwhile, the nano antibody has reversible refolding capacity, namely easy renaturation, and tests show that the nano antibody still keeps higher activity after being treated at the high temperature of 90 ℃ and can regain the antigen binding capacity. Whereas all conventional antibodies lost activity after treatment at 90 ℃ and irreversibly polymerized. Under harsh conditions, such as in chaotropic agents, the presence of proteases and extreme pH denaturation, normal antibodies will fail or break down, while nanobodies still have a high degree of stability.
The half-life period of the nano antibody in the antibody nano antibody composition and the human body is prolonged by taking measures, and the half-life period of the nano antibody existing in the antibody nano antibody composition and the human body is greatly prolonged by the modification measures of fusion of the nano antibody and albumin, Fc fusion, PEG and the like.
With the continuous development of bioengineering technology, a large number of nano antibody drugs will emerge continuously. At present, the main dosage forms of the clinical trials are injection and oral preparation. Because the nano antibody drug has small molecular weight and poor in-vivo stability, and is easily influenced by gastrointestinal enzyme degradation and the first-pass effect of a liver enzyme system when being orally taken, patients need to repeatedly inject or orally take drugs for a long time to achieve effective drug treatment concentration.
IgG antibody-mediated type I allergic diseases are quite common, and the anti-allergic diseases are diseases and related symptoms which are caused by that patients inhale, ingest or inject substances containing sensitizing components to trigger B cells of organisms to generate specific immunoglobulin G, so that allergic reactions are caused.
At present, the nano antibody anti-allergic reaction reagent which has no effect on the anti-allergic treatment of inflammation such as rhinitis is proved by clinical tests.
Disclosure of Invention
The invention mainly solves the technical problem of providing an anti-allergic nano antibody composition, an antibody measuring method and a spraying agent, wherein the nano antibody composition carries out label tracking through biomacromolecule protease and the like, carries out tracking reaction report on allergic antigen, records chemical test results and carries out preparation derivation of an anti-allergic verification medicament based on a nano antibody.
In order to solve the technical problems, the invention adopts a technical scheme that:
an anti-allergic nano antibody composition comprises a nano antibody, an IgG3 antibody solution with a fluorescein label, an allergen solution with a biomarker protein molecule, a polypeptide-coated nano magnetic suspension, a specific nano antibody with bioactivity, an antibody nano antibody composition for maintaining and increasing the stability and tissue penetrability of the nano antibody, an immunosuppressant and an unstrained allergic antigen;
the nanobody includes: one or more of active nano-antibody, nano-antibody fragment, multi-targeting nano-antibody polymer linker, conjugate of nano-antibody and protein, or conjugate of nano-antibody and drug.
Nanobodies comprise only one heavy chain variable region VHH and two conventional CH2 and CH3 regions, which are the smallest units known to bind to the antigen of interest.
As a preferred embodiment of the present invention, as the antibody for the present inflammation reagent, a conjugate of a nanobody and a protein is used to specifically bind a target protein antibody, and the antibody can be widely used for antibody binding in the development of therapeutic antibody drugs and diagnostic reagents.
The polypeptide-coated nano magnetic suspension can be used for targeting tracking of antibody drugs.
Polypeptide-coated nanomagnets are commonly used in modern biology, and immunoassay is an important method in modern biological analysis technology, and can be used for quantitative analysis of proteins, antigens, antibodies and cells.
A method for transfecting magnetic nanoparticles bound with carrier DNA into cells under the influence of an external magnetic field.
Active substances such as adsorbents or other ligands with biological activity connected to the surfaces of the magnetic nanoparticles can be specifically combined with specific biomolecules or cells and separated under the action of an external magnetic field. The magnetic separation method essentially comprises only 2 steps: 1. labeling a target molecule or cell with a magnetic nanoparticle; 2. separating out target molecules or cells by a magnetic separation device. One example of the separation using magnetic nanoparticles is to combine specific antibodies with magnetic nanoparticles, attach the magnetic nanoparticles to specific cells, and apply a magnetic field to rapidly separate or perform an immunoassay on the cells combined with the magnetic nanoparticles. The method has high specificity, rapid separation and good reproducibility.
In addition to this, the present invention is,
the nano antibody composition needs to perform a calibration test on the performance generated by antibody-antigen combination after a preset time, a chemical result needs to perform a buffer test, and in the embodiment, a chemical reagent with resistance to the allergen-reactive protein is used for the calibration test.
The reagent with two different standard contents is prepared: a first chemical reagent calibration reagent and a second chemical reagent calibration reagent.
The first chemical reagent calibration reagent and the second chemical reagent calibration reagent are used for performing calibration data test on the anaphylactic reaction protein reaction solution.
In a preferred embodiment of the invention, the concentration of the allergen solution with biomarker protein molecules is 0.35-1.188 ug/ml; the concentration of the streptavidin-labeled polypeptide-coated nano magnetic suspension is 0.35-1.188 mg/ml; the concentration of the IgG3 antibody solution with the fluorescein label is 0.25-3.75 ug/ml.
In a preferred embodiment of the invention, the labeled fluorescein-labeled IgG3 antibody is an antibody that specifically binds to human IgG protein,
the preparation process of the fluorescein-labeled IgG3 antibody solution comprises the following steps: mixing alkaline phosphatase solution with the concentration of 1.75-6.25mg/mL and fluorescein-labeled IgG3 antibody according to the molar ratio of 1-4: 1, equilibrated and eluted with bicarbonate buffer at pH 8-9, followed by uv detection and recording of the purification pattern, and dilution of the IgG antibody with MES buffer 0.05M, pH value 3.75, gave IgG3 antibody.
In a preferred embodiment of the invention, the immunosuppressant comprises one or more of cyclophosphamide, mycophenolate mofetil, cyclosporine a, tacrolimus, methotrexate, azathioprine, leflunomide, hydroxychloroquine corticosteroids, adenosine receptor agonists, phosphodiesterase 4 inhibitors, HDAC inhibitors or proteasome inhibitors.
In a preferred embodiment of the invention, the unstrained allergic antigen comprises a therapeutic biological protease that is a therapeutic protein or therapeutic polynucleotide comprising one or more of temorelin, ocrelizumab, belimumab, pegylated recombinant uricase, talisidase alpha, acarbosidase alpha, or glucocerebrosidase alpha.
In a preferred embodiment of the invention, the preparation process of the polypeptide-coated nano magnetic suspension comprises the following steps:
precipitating the streptavidin-labeled nano magnetic particles by using a magnetic separator, then re-suspending by using a phosphate buffer solution with the value of 0.01M, pH being 7.8, uniformly mixing, magnetically separating and precipitating, and repeatedly cleaning;
the washed nano magnetic particles are dispersed in a phosphate buffer solution with a value of 0.01M, pH being 7.8.
In order to detect the antibody and antigen performance parameters in the serum of a human body after the human body inhales the anti-allergic nano antibody composition, the follow-up drug tracking analysis is facilitated.
The invention adopts another technical scheme that:
a method for measuring the antibody performance of serum based on a nano-magnetic chemiluminescence method, wherein the anti-allergic nano-antibody composition is taken into the serum, and the method comprises the following steps:
(1) uniformly mixing and reacting the polypeptide-coated nano magnetic suspension, the allergen with the biomarker protein molecules and a sample to be detected, standing in a magnetic field after reaction, and removing supernatant to obtain a first solution;
(2) adding an IgG3 antibody into the first solution, mixing uniformly, reacting, placing in a magnetic field, standing, and removing supernatant to obtain a second solution;
(3) adding a luminescent substrate into the second solution, reacting, and measuring the luminous intensity;
(4) and (4) drawing a standard curve of the luminous intensity by using a first chemical reagent calibration reagent with a known concentration, and calculating the content of the inflammation specificity IgG in the serum sample to be detected according to the standard curve of the luminous intensity obtained in the step (3).
In a preferred embodiment of the present invention, the reaction in step (1) is carried out under the conditions of 45 ℃ for 5 minutes, and the standing time is 3 minutes; the step (1) also comprises the steps of cleaning the first solution by using a cleaning solution, placing the first solution in a magnetic field for standing, removing supernatant, and repeating the cleaning for 2 times; the reaction condition in the step (2) is 5 minutes of reaction at 45 ℃, and the standing time is 3 minutes; cleaning the second solution with cleaning solution, placing the second solution in a magnetic field for standing and removing supernatant, and repeating the cleaning for 2 times; the reaction in the step (3) was carried out under the conditions of 45 ℃ for 2 minutes, and the luminescence intensity was measured in a chemiluminescence analyzer/measuring instrument.
Active substances such as adsorbents or other ligands with biological activity connected to the surfaces of the magnetic nanoparticles can be specifically combined with specific biomolecules or cells and separated under the action of an external magnetic field.
In a preferred embodiment of the present invention, the method comprises the steps of:
(1) placing the nano antibody composition into a reagent bin of a full-automatic chemiluminescence apparatus and identifying;
(2) placing a first chemical reagent calibration reagent in an instrument sample bin of the full-automatic chemiluminescence instrument, identifying first chemical reagent calibration reagent information, and distributing a first chemical reagent calibration reagent position;
(3) placing a quality control object or a sample to be detected in an instrument sample bin, and editing detection information;
(4) and starting an operation program, and automatically processing all the first chemical reagent calibration reagents, quality control substances and samples to be detected to obtain results.
The invention adopts another technical scheme that a rhinitis anti-allergic nano antibody spray is provided:
an anti-allergic rhinitis spray comprises a glycol solution, wherein the anti-allergic nano antibody composition is contained in the glycol solution.
The invention has the beneficial effects that: the invention relates to a composition and a method for detecting an anti-allergic nano antibody, wherein the nano antibody composition is marked and tracked by biomacromolecule protease and the like, a tracking reaction report is carried out on an allergic antigen, and a chemical test result is recorded to deduce the preparation of an anti-allergic verification medicament based on a nano antibody.
The nanometer antibody composition has the advantages of good stability of each reagent component, high detection sensitivity and good specificity, is a perfect and unified process, and is produced strictly according to standard production operation regulations and quality control regulations.
Can be used for large-scale inflammation tests, such as rhinitis and other inflammations. The cost is low, and the medicine is suitable for the majority of patients in China.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without inventive efforts, wherein:
FIG. 1 is a schematic view of an anti-allergic nano-antibody rhinitis spray prepared by using the anti-allergic nano-antibody composition according to the present invention;
FIG. 2 is a flow chart of IgG3 antibody solution measured by the method for measuring serum antibody performance based on nano-magnetic chemiluminescence method.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The nano antibody composition needs to perform a calibration test on the performance generated by antibody-antigen combination after a preset time, a chemical result needs to perform a buffer test, and in the embodiment, a chemical reagent with resistance to the allergen-reactive protein is used for the calibration test.
The reagent with two different standard contents is prepared: a first chemical reagent calibration reagent and a second chemical reagent calibration reagent.
The first chemical reagent calibration reagent and the second chemical reagent calibration reagent are used for performing calibration data test on the anaphylactic reaction protein reaction solution.
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 and 100IU/ml, wherein the first chemical reagent calibration reagent is prepared by IgG protein and Tris-HCl buffer solution with the value of 0.1M, pH being 7.8;
the concentration of the second chemical reagent calibration reagent is as follows: 0.7IU/ml and 17.5IU/ml, and the second chemical reagent calibration reagent is prepared by mixing IgG protein with 0.1M, pH value of 7.8 Tris-HCl buffer solution.
The antibodies were selected as follows: fluorescein-labeled IgG3 antibody solution;
the antigens were selected as follows: an inflammatory allergen;
in a preferred embodiment of the invention, the inflammatory allergen is an antigen capable of binding to an inflammation-specific IgG protein in humans,
the preparation process of the allergen solution with the biomarker protein molecules comprises the following steps: adding inflammatory allergen into biotin dissolved in dimethylformamide at a molar ratio of 1:20, and dialyzing with 0.1M, pH value of 7.8 phosphate buffer.
In this embodiment, the immunosuppressive agent comprises one or more of cyclophosphamide, mycophenolate mofetil, cyclosporine a, tacrolimus, methotrexate, azathioprine, leflunomide, hydroxychloroquine corticosteroids, adenosine receptor agonists, phosphodiesterase 4 inhibitors, HDAC inhibitors, or proteasome inhibitors.
In this embodiment, the unstrained allergic antigen comprises a therapeutic biological protease that is a therapeutic protein or a therapeutic polynucleotide comprising one or more of temorelin, ocrelizumab, belimumab, pegylated recombinant uricase, taliosidase a, acarbosidase a, or glucocerebrosidase a.
The first embodiment is as follows:
the method provides an anti-allergic nano antibody composition, which comprises an IgG3 antibody solution with a fluorescein label, and the nano antibody composition comprises: the biological protein allergen antigen-antibody hybrid antibody is characterized by comprising an allergen solution with biomarker protein molecules, a polypeptide-coated nano magnetic suspension liquid, a specific nano antibody with biological activity, an antibody nano antibody composition for maintaining and increasing the stability and tissue penetrability of the nano antibody, an immunosuppressant and an unstrained allergic antigen;
the nanobody includes: one or more of active nano-antibody, nano-antibody fragment, multi-targeting nano-antibody polymer linker, conjugate of nano-antibody and protein, or conjugate of nano-antibody and drug.
The polypeptide-coated nano magnetic suspension can be used for targeting tracking of antibody drugs.
Polypeptide-coated nanomagnets are commonly used in modern biology, and immunoassay is an important method in modern biological analysis technology, and can be used for quantitative analysis of proteins, antigens, antibodies and cells.
A method for transfecting magnetic nanoparticles bound with carrier DNA into cells under the influence of an external magnetic field.
Active substances such as adsorbents or other ligands with biological activity connected to the surfaces of the magnetic nanoparticles can be specifically combined with specific biomolecules or cells and separated under the action of an external magnetic field. The magnetic separation method essentially comprises only 2 steps: 1. labeling a target molecule or cell with a magnetic nanoparticle; 2. separating out target molecules or cells by a magnetic separation device. One example of the separation using magnetic nanoparticles is to combine specific antibodies with magnetic nanoparticles, attach the magnetic nanoparticles to specific cells, and apply a magnetic field to rapidly separate or perform an immunoassay on the cells combined with the magnetic nanoparticles. The method has high specificity, rapid separation and good reproducibility.
The anti-allergic nanobody composition comprises the following reagents:
(1) the allergen solution with biomarker protein molecules has the concentration of 0.1-1.0 ug/mL;
(2) the concentration of the polypeptide-coated nano magnetic suspension is 0.1-1.0 mg/mL;
(3) IgG3 antibody solution with fluorescein labels, the concentration is 0.1-2.0 ug/mL;
(4)6 different concentration levels of a first chemical reagent calibration reagent: 0IU/ml, 0.35IU/ml, 0.7IU/ml, 3.5IU/ml, 17.5IU/ml, 50IU/ml and 100 IU/ml;
(5)2 different concentration levels of a second chemical calibration reagent: 0.7IU/ml and 17.5 IU/ml.
The nanobody includes: one or more of active nano-antibody, nano-antibody fragment, multi-targeting nano-antibody polymer linker, conjugate of nano-antibody and protein, or conjugate of nano-antibody and drug.
The polypeptide-coated nano magnetic suspension can be used for targeting tracking of antibody drugs.
Polypeptide-coated nanomagnets are commonly used in modern biology, and immunoassay is an important method in modern biological analysis technology, and can be used for quantitative analysis of proteins, antigens, antibodies and cells.
A method for transfecting magnetic nanoparticles bound with carrier DNA into cells under the influence of an external magnetic field.
After preparation, the reagents are allowed to stand with the addition of an immunosuppressive agent such as an adenosine receptor agonist and a non-allergenic allergen package such as pegylated recombinant uricase.
Example two:
in order to detect the antibody and antigen performance parameters in the serum of a human body after the human body inhales the anti-allergic nano antibody composition, the follow-up drug tracking analysis is facilitated.
The invention adopts another technical scheme that:
a method for measuring the antibody performance of serum based on a nano-magnetic chemiluminescence method, wherein the anti-allergic nano-antibody composition is taken into the serum, and the method comprises the following steps:
(1) preparing a buffer solution:
phosphate buffer with a value of 0.01M, pH of 7.8, Tris-HCl buffer with a value of 0.1M, pH of 7.8 and MES buffer with a value of 0.05M, pH of 3.75 are prepared.
Preparation of 0.01M, pH value of 7.8 phosphate buffer: taking a beaker with the capacity of 1L, adding 800ml of deionized water, and weighing 2.56g of Na2HPO4·12H2O、0.44g NaH2PO4·2H2Adding O and 9g of NaCl into a beaker, stirring to fully dissolve the O and the 9g of NaCl, adjusting the pH value to 7.8 +/-0.05 by using HCl or NaOH, adding mannitol with the mass fraction of 2% and glycerol with the mass fraction of 1%, stirring for 0.5h until the mannitol and the glycerol are completely dissolved, and adding deionized water to the volume of 1L.
Preparation of Tris-HCl buffer with 0.1M, pH value of 7.8: taking a beaker with the capacity of 1L, adding 800ml of deionized water, weighing 12.11g of Tris (Tris-hydroxymethyl aminomethane) and adding the Tris into the beaker, stirring to fully dissolve the Tris, adjusting the pH value to 7.8 +/-0.05 by using HCl or NaOH, adding 2 mass percent of BSA and 0.3 mass percent of Proclin300, stirring for 0.5h to fully dissolve the BSA, and adding deionized water to fix the volume to 1L.
Preparation of 0.05M, pH MES buffer with value 3.75: adding 800ml of deionized water into a beaker with the capacity of 1L, weighing 9.76g of MES (2- (N-morpholino) ethanesulfonic acid) and 9g of NaCl, adding the MES and the NaCl into the beaker, stirring to obtain a sufficient solution, adjusting the pH value to 3.75 +/-0.05 by using HCl or NaOH, adding 1 mass percent of BSA and 0.1 mass percent of Tween-20, stirring for 0.5h until the BSA and the Tween-20 are completely dissolved, and adding the deionized water to the volume of 1L.
(2) Preparing an allergen solution with biomarker protein molecules, adding inflammatory allergen into biotin dissolved in dimethylformamide according to the ratio of 1:20, fully mixing and reacting for 30 minutes at room temperature, and dialyzing the reacted solution by using a phosphate buffer solution with the value of 0.1M, pH being 7.8. The BCA method measures the concentration of the allergen solution containing the biomarker protein molecules and adjusts the concentration to 0.1-1.0 ug/ml.
(3) Preparing polypeptide-coated nano magnetic suspension, precipitating streptavidin-labeled nano magnetic particles by using a magnetic separator, resuspending the streptavidin-labeled nano magnetic particles by using a phosphate buffer solution with the value of 0.01M, pH of 7.8, carrying out magnetic separation and precipitation after fully and uniformly mixing for 5 minutes, discarding supernatant, then resuspending the streptavidin-labeled nano magnetic particles by using a phosphate buffer solution with the value of 0.01M, pH of 7.8, repeating the washing for 5 times, and dispersing the washed nano magnetic particles in a phosphate buffer solution with the value of 0.01M, pH of 7.8 and the concentration of 0.1-1.0 mg/ml.
(4) Preparing an IgG3 antibody solution with a fluorescein label; IgG3 antibody was diluted to 0.1-2.0ug/ml with 0.05M, pH MES buffer with a value of 3.75.
(5) Preparation of first chemical reagent the calibration reagent was used to prepare IgG proteins using 0.1M, pH value 7.8 Tris-HCl buffer to prepare calibration dots of 0, 0.35, 0.7, 3.5, 17.5, 50, 100 IU/ml.
(6) Preparing a second chemical reagent calibration reagent, namely preparing IgG protein by using a Tris-HCl buffer solution with the value of 0.1M, pH being 7.8 into the second chemical reagent calibration reagent with the concentration of 0.7IU/ml and the concentration of 17.5 IU/ml.
(7) Assembling a nano antibody composition, namely assembling an allergen solution with a biomarker protein molecule, a streptavidin-labeled polypeptide-coated nano magnetic suspension, a fluorescein-labeled IgG3 antibody solution, a first chemical reagent calibration reagent and a second chemical reagent calibration reagent into the nano antibody composition.
In a preferred embodiment of the present invention, as shown in fig. 2, the method comprises the steps of:
(1) placing the nano antibody composition into a reagent bin of a full-automatic chemiluminescence apparatus and identifying;
(2) placing a first chemical reagent calibration reagent in an instrument sample bin of the full-automatic chemiluminescence instrument, identifying first chemical reagent calibration reagent information, and distributing a first chemical reagent calibration reagent position;
(3) placing a quality control object or a sample to be detected in an instrument sample bin, and editing detection information;
(4) and starting an operation program, and automatically processing all the first chemical reagent calibration reagents, quality control substances and samples to be detected to obtain results.
Reagent components (such as cleaning solution, some necessary buffer solution and the like) which are not mentioned in detail in the nano antibody composition of the invention, an outer package of the nano antibody composition, an independent packaging container of each reagent component and the like can be carried out according to the conventional operation in the field, and the nano antibody composition meets the relevant industry regulations.
Example three:
the embodiment provides a method for measuring serum antibody performance based on a nano-magnetic chemiluminescence method, wherein the anti-allergic nano-antibody composition is taken in serum, and the detection is carried out on a full-automatic chemiluminescence apparatus MAGICL6800 series, and the specific apparatus operation steps are as follows:
(1) the nanometer antibody composition is put into a reagent bin corresponding to a full-automatic chemiluminescence analyzer/determinator, and the information of the nanometer antibody composition is input into an instrument system through a bar code scanner or set through instrument matching software.
(2) A first chemical reagent calibration reagent is placed in the instrument sample compartment. The first chemical reagent calibration reagent information is identified by the barcode scanner and a first chemical reagent calibration reagent location is assigned in the instrument system. (3) And (3) placing the quality control material/sample to be detected in an instrument sample bin, and editing corresponding detection information through instrument matching software.
(4) The running program is started, and all the first chemical reagent calibration reagent/quality control substance/sample to be tested processing steps are automatically executed.
The invention provides a method for measuring serum antibody performance based on a nano-magnetic chemiluminescence method, wherein the antiallergic nano-antibody composition is taken in serum, and the method can be realized on a semi-automatic chemiluminescence apparatus, and the specific sample detection steps are as follows: (1) 40ul of a first chemical reagent calibration reagent of serum or IgG to be detected, 50ul of allergen with a biomarker protein molecule and 50ul of streptavidin-labeled nano magnetic particles are sequentially added into a detection tube, mixed uniformly and incubated for 5 minutes at 45 ℃, and the inflammation specific IgG antibody in the sample is captured and fixed on the surface of the nano magnetic particles.
(2) Under the action of an external magnetic field, settling magnetic particles, removing supernatant, adding 500ul of cleaning solution, and after removing the magnetic field, oscillating to resuspend the magnetic particles; this washing step was repeated 3 times to remove unbound biotin-labeled mouse anti-human IgG antibody.
(3) 100ul of fluorescein-labeled IgG3 antibody solution was added, mixed well and incubated at 45 ℃ for 5 minutes.
(4) Under the action of an external magnetic field, settling magnetic particles, removing supernatant, adding 500ul of cleaning solution, and after removing the magnetic field, oscillating to resuspend the magnetic particles; this washing step was repeated 3 times to remove unbound enzyme-labeled antibody and other impurities.
(5) 150ul of an enzymatic chemiluminescent substrate was added, the magnetic field was removed, the mixture was thoroughly mixed and incubated at 45 ℃ for 2 minutes to determine the luminescence value.
(6) And (3) processing data: and (3) obtaining a standard curve through four-parameter nonlinear fitting according to the concentration value of the first chemical reagent calibration reagent and the detected luminous value, and substituting the luminous value of the sample into the standard curve shown in the figure 1 to obtain a corresponding concentration value.
The working principle of the invention is as follows: co-incubating a serum sample to be detected with the allergen with the biomarker protein molecules and the streptavidin-labeled nano-magnetic particles, specifically binding an inflammation-specific IgG antibody in the serum sample to be detected with the allergen with the biomarker protein molecules, and simultaneously binding the inflammation-allergen-specific IgG in the serum sample to the solid-phase carrier nano-magnetic particles by virtue of a biotin-streptavidin amplification system; under the action of an external magnetic field, magnetically separating and washing to remove the redundant biotin-labeled inflammation allergen;
adding IgG3 antibody to form an immune complex structure of allergen-inflammation specificity IgG-anti-IgG 3 antibody with biomarker protein molecules; under the action of an external magnetic field, carrying out magnetic separation and washing for multiple times to remove unbound enzyme-labeled antibody and other impurities; adding an enzymatic chemiluminescence substrate, and measuring the luminous intensity; calibrating a reagent standard curve by using a first chemical reagent with known concentration, and calculating the content of the inflammation specificity IgG antibody in the sample to be detected according to the luminous intensity contrast standard curve;
example four:
performance evaluation of the nanobody composition:
1. the method has the analysis performance and stability of the anti-allergic nano antibody (D2) detection nano antibody composition (nano magnetic chemiluminescence method), and has the performance comparison of the anti-allergic nano antibody (D2) detection nano antibody composition (nano magnetic chemiluminescence method) and an imported reagent;
3. the method has a clinical reference value range of an anti-allergic nano antibody (D2) detection nano antibody composition (nano magnetic chemiluminescence method), and takes two-side 95 percentiles of a detection value of a normal human sample as reference values. The basic range (mIU/mL) was calculated at 95%, see in particular below,
the serum concentration of individual allergen IgG antibodies in normal non-allergic adult humans is less than 0.35 IU/mL.
The measured value is 0.35-0.7 IU/mL, which is the grade 1 allergy of the allergen;
the measured value is 0.7-3.5 IU/mL, which is the grade 2 allergy of the allergen;
the measured value is 3.5-17.5 IU/mL, which is the grade 3 allergy of the allergen;
the measured value is 17.5-50 IU/mL, which is the grade 4 allergy of the allergen;
the measured value is 50-100 IU/mL, which is the grade 5 allergy of the allergen;
measured value of more than 100IU/mL is the grade 6 allergy of the allergen.
The invention has the beneficial effects that:
the invention takes Alkaline Phosphatase (AP) as a labeled enzyme, labels an antibody through a chemical reaction, and separates unreacted enzyme, antibody or antigen by using gel chromatography, thereby improving the sensitivity of the reaction;
secondly, the immune magnetic particles are used as a solid phase, and streptavidin coupled magnetic microspheres are used as a general separation reagent, so that immune reaction is easier to mix and separate, and the reaction speed is greatly improved;
thirdly, a novel chemiluminescence substrate AMPPD is used as a substrate, the substrate is a glow substrate, and the plateau period is quickly reached, so that the detection of signals is facilitated, and the sensitivity and the specificity performance of the final nano antibody composition are improved; furthermore, a chemiluminescence enhancement system is further optimized, and high signal sensitivity, good stability and small variation of a final product are ensured;
and fourthly, the AMPPD luminescent substrate has the advantages of high sensitivity and long platform stability.
As shown in the attached figure 1, another technical scheme adopted by the invention is to provide a rhinitis anti-allergic nano antibody spray:
an anti-allergic rhinitis spray comprises a glycol solution, wherein the anti-allergic nano antibody composition is contained in the glycol solution.
The anti-allergic nano antibody composition provided by the invention is added into the anti-allergic rhinitis spray, so that the anti-inflammatory treatment can be performed on a patient, and the cost is low.
The allergic antibody test reaction of an inflammation patient can be verified, the actual antibody reaction structure can be directly tracked, and the clinical medical effect can be demonstrated.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (10)
1. An anti-allergic nano antibody composition is characterized by comprising a nano antibody, an IgG3 antibody solution with a fluorescein label, an allergen solution with a biomarker protein molecule, a polypeptide-coated nano magnetic suspension, a specific nano antibody with bioactivity, an antibody nano antibody composition for maintaining and increasing the stability and tissue penetrability of the nano antibody, an immunosuppressant and an unstrained allergic antigen;
the nanobody includes: one or more of active nano-antibody, nano-antibody fragment, multi-targeting nano-antibody polymer linker, conjugate of nano-antibody and protein, or conjugate of nano-antibody and drug.
2. The anti-allergic nanobody composition according to claim 1, wherein the concentration of the allergen solution having the biomarker protein molecule is 0.35-1.188 ug/ml;
the concentration of the polypeptide-coated nano magnetic suspension is 0.35-1.188 mg/ml; the concentration of the IgG3 antibody solution with the fluorescein label is 0.25-3.75 ug/ml.
3. The anti-allergic nanobody composition according to claim 1, wherein the immunosuppressive agent comprises one or more of cyclophosphamide, mycophenolate mofetil, cyclosporine a, tacrolimus, methotrexate, azathioprine, leflunomide, hydroxychloroquine corticosteroids, adenosine receptor agonists, phosphodiesterase 4 inhibitors, HDAC inhibitors or proteasome inhibitors.
4. An anti-allergic nanobody composition according to claim 1, wherein the unstrained allergic antigen comprises a therapeutic biological protease, which is a therapeutic protein or a therapeutic polynucleotide comprising one or more of temerelin, ocrelizumab, belimumab, pegylated recombinant uricase, talisidase a, acangase a or glucocerebrosidase a.
5. The anti-allergic nanobody composition according to claim 1, wherein the labeled fluorescein-labeled IgG3 antibody is an antibody capable of specifically binding to IgG protein in human body, and the fluorescein-labeled IgG3 antibody solution is prepared by the following steps: mixing alkaline phosphatase solution with the concentration of 1.75-6.25mg/mL and fluorescein-labeled IgG3 antibody according to the molar ratio of 1-4: 1, equilibrated and eluted with bicarbonate buffer at pH 8-9, followed by uv detection and recording of the purification pattern, and dilution of the IgG antibody with MES buffer 0.05M, pH value 3.75, gave IgG3 antibody.
6. The anti-allergic nanobody composition according to claim 1, wherein the polypeptide-coated nanobody suspension is prepared by the following steps:
precipitating the streptavidin-labeled nano magnetic particles by using a magnetic separator;
then re-suspending with phosphate buffer solution with the value of 0.01M, pH being 7.8, mixing uniformly, magnetically separating and precipitating, and repeatedly cleaning;
the washed nano magnetic particles are dispersed in a phosphate buffer solution with a value of 0.01M, pH being 7.8.
7. A method for measuring serum antibody performance based on a nanomagnetic chemiluminescence method, wherein the anti-allergic nanobody composition of any one of claims 1 to 6 is ingested into serum, comprising the steps of:
(1) uniformly mixing and reacting the polypeptide-coated nano magnetic suspension, the allergen with the biomarker protein molecules and a sample to be detected, standing in a magnetic field after reaction, and removing supernatant to obtain a first solution;
(2) adding an IgG3 antibody into the first solution, mixing uniformly, reacting, placing in a magnetic field, standing, and removing supernatant to obtain a second solution;
(3) adding a luminescent substrate into the second solution, reacting, and measuring the luminous intensity;
(4) and (4) drawing a standard curve of the luminous intensity by using a first chemical reagent calibration reagent with a known concentration, and calculating the content of the inflammation specificity IgG in the serum sample to be detected according to the standard curve of the luminous intensity obtained in the step (3).
8. The method for measuring serum antibody performance based on nano-magnetic chemiluminescence method of claim 7,
the reaction condition in the step (1) is 5 minutes of reaction at 45 ℃, and the standing time is 3 minutes;
the step (1) also comprises the steps of cleaning the first solution by using a cleaning solution, placing the first solution in a magnetic field for standing, removing supernatant, and repeating the cleaning for 2 times;
the reaction condition in the step (2) is 5 minutes of reaction at 45 ℃, and the standing time is 3 minutes;
cleaning the second solution with cleaning solution, placing the second solution in a magnetic field for standing and removing supernatant, and repeating the cleaning for 2 times;
the reaction in the step (3) was carried out under the conditions of 45 ℃ for 2 minutes, and the luminescence intensity was measured in a chemiluminescence analyzer/measuring instrument.
9. The method for measuring serum antibody based on nanomagnetic chemiluminescence according to claim 7, comprising the steps of:
(1) placing the nano antibody composition into a reagent bin of a full-automatic chemiluminescence apparatus and identifying;
(2) placing a first chemical reagent calibration reagent in an instrument sample bin of the full-automatic chemiluminescence instrument, identifying first chemical reagent calibration reagent information, and allocating a first chemical reagent calibration reagent position;
(3) placing a quality control object or a sample to be detected in an instrument sample bin, and editing detection information;
(4) and starting an operation program, and automatically processing all the first chemical reagent calibration reagents, quality control substances and samples to be detected to obtain results.
10. An anti-allergic rhinitis spray, which comprises a glycol solution containing the anti-allergic nanobody composition of any one of claims 1 to 6.
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| CN202011327231.5A CN112480244A (en) | 2020-11-24 | 2020-11-24 | Anti-allergic nano antibody composition, antibody determination method and spray |
| PCT/CN2020/133203 WO2022110257A1 (en) | 2020-11-24 | 2020-12-02 | Antiallergic nanobody composition, antibody assay method, and spray |
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Inventor after: Zhou Xiang Inventor after: Li Shenghua Inventor before: Diao Boqiong Inventor before: Zhou Xiang Inventor before: Li Shenghua |
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Application publication date: 20210312 |