WO2022110257A1

WO2022110257A1 - Antiallergic nanobody composition, antibody assay method, and spray

Info

Publication number: WO2022110257A1
Application number: PCT/CN2020/133203
Authority: WO
Inventors: 刁波琼; 周翔; 李胜华
Original assignee: 华科同济干细胞基因工程有限公司
Priority date: 2020-11-24
Filing date: 2020-12-02
Publication date: 2022-06-02
Also published as: CN112480244A

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

A kind of anti-allergic nanobody composition, antibody determination method and spray

technical field

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.

Background technique

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.

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.

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.

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.

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.

SUMMARY OF THE INVENTION

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.

In order to solve the above-mentioned technical problems, a technical scheme adopted in the present invention is:

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.

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 .

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.

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.

besides,

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.

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.

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.

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,

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.

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.

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.

In a preferred embodiment of the present invention, the preparation process of the polypeptide-coated nano-magnetic suspension is as follows:

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;

The cleaned magnetic nanoparticles were dispersed in 0.01M phosphate buffer with pH 7.8.

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.

Another technical scheme adopted by the present invention is:

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) 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) 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) adding a luminescent substrate to the second solution and reacting, and measuring the luminescence intensity;

(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.

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.

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.

In a preferred embodiment of the present invention, the method includes the steps of:

(1), put the nanobody composition into the reagent chamber of the automatic chemiluminescence instrument and identify;

(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) Place the quality control substance or the sample to be tested in the sample compartment of the instrument, and edit the testing information;

(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.

Another technical solution adopted in the present invention is to provide a rhinitis anti-allergic nanobody spray:

An anti-allergic rhinitis spray, the 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.

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.

Description of drawings

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:

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.

Detailed ways

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.

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.

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;

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;

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,

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.

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.

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.

Example 1:

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 anti-allergic Nanobody composition includes the following reagents:

(1) Allergen solution with biomarker protein molecules at a concentration of 0.1-1.0ug/mL;

(2) Polypeptide-coated nano-magnetic levitation liquid, the concentration is 0.1-1.0mg/mL;

(3) IgG3 antibody solution with fluorescein label, the concentration is 0.1-2.0ug/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) Two different concentration levels of the second chemical reagent calibration reagent: 0.7IU/ml, 17.5IU/ml.

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.

Embodiment 2:

Another technical scheme adopted by the present invention is:

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) Preparation buffer:

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.

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.

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.

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) 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) 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) 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) 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) 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) 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.

As shown in Figure 2, in a preferred embodiment of the present invention, 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.

Embodiment three:

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) 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) 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) 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.

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) 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) Add 100ul of fluorescein-labeled IgG3 antibody solution, mix well, and incubate at 45°C for 5 minutes.

(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) 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) 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.

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;

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:

Performance evaluation of the Nanobody composition:

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. 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,

The concentration of IgG antibodies to a single allergen in the serum of normal non-allergic adults is less than 0.35 IU/mL.

If the measured value is between 0.35 and 0.7IU/mL, it is the first-grade allergy to the allergen;

If the measured value is between 0.7 and 3.5IU/mL, it is a grade 2 allergy to the allergen;

If the measured value is between 3.5 and 17.5IU/mL, it is a grade 3 allergy to the allergen;

If the measured value is between 17.5 and 50IU/mL, it is a grade 4 allergy to the allergen;

If 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.

The beneficial effects of the present invention are:

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;

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;

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;

4. The advantage of using AMPPD luminescent substrate is high sensitivity and long platform stability period.

As shown in accompanying drawing 1, another technical scheme adopted for the present invention is to provide a rhinitis anti-allergic nanobody spray:

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.

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

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;

The nanobody includes one or more of active nanobody, nanobody fragment, multi-targeted nanobody polymer linker, nanobody-protein conjugate or nanobody-drug conjugate.
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;

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.
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.
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.
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.
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:

Precipitate the streptavidin-labeled magnetic nanoparticles with a magnetic separator;

Resuspend with 0.01M phosphate buffer with pH 7.8, mix evenly and magnetically separate the precipitate, and repeat the washing;

The cleaned magnetic nanoparticles were dispersed in 0.01M phosphate buffer with pH 7.8.
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) 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) 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) adding a luminescent substrate to the second solution and reacting, and measuring the luminescence intensity;

(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.
A method for measuring serum antibody performance based on nano-magnetic chemiluminescence method according to claim 7, characterized in that,

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;

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;

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;

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;

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.
A method for measuring serum antibody based on nano-magnetic chemiluminescence method according to claim 7, characterized in that, comprising the steps of:

(1) Put the nanobody composition into the reagent chamber of the automatic chemiluminescence instrument and identify it;

(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) Place the quality control substance or the sample to be tested in the sample compartment of the instrument, and edit the testing information;

(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.
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.