CN110763835A - Application of idiotype heterogeneity intensity of allergen-specific IgE antibody in allergen detection kit - Google Patents
Application of idiotype heterogeneity intensity of allergen-specific IgE antibody in allergen detection kit Download PDFInfo
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- G—PHYSICS
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Abstract
The invention belongs to the field of clinical medicine/experimental diagnosis, relates to experimental diagnosis of allergic diseases, and particularly provides a novel experimental diagnosis index for the allergic diseases, namely idiotypic heterogeneity of an allergen specific IgE (sIgE) antibody. The magnitude of the idiotypic heterogeneity of the allergen sIgE antibodies correlates with the clinical manifestations of the patient or the severity of the symptoms of the allergy. In particular to the application of the idiotype heterogeneity intensity of an allergen-specific IgE antibody in a kit for detecting the allergen.
Description
Technical Field
The invention belongs to the field of clinical medicine/experimental diagnosis, relates to experimental diagnosis of allergic diseases, and particularly provides a novel experimental diagnosis index for the allergic diseases, namely idiotypic heterogeneity of an allergen specific IgE (sIgE) antibody. The magnitude of the idiotypic heterogeneity of the allergen sIgE antibodies correlates with the clinical manifestations of the patient or the severity of the symptoms of the allergy. In particular to the application of the idiotype heterogeneity intensity of an allergen-specific IgE antibody in a kit for detecting the allergen.
Background
(1) Allergic diseases
Hypersensitivity refers to pathological or abnormal immune response caused by the sensitized organism when the sensitized organism encounters the stimulation of the same antigen again. Hypersensitivity reactions are classified into 4 types according to clinical manifestations and mechanisms of occurrence, wherein type i hypersensitivity reactions are mediated by immunoglobulin e (ige), mast cells and basophils are involved, and most of the cases occur rapidly, also known as "immediate" hypersensitivity reactions. Clinical medicine refers to type i hypersensitivity as "allergy", which has the meaning of "abnormal", corresponding to a "normal" immune response in the body. Antigens that cause allergies are called "allergens" or "allergens" and are widely distributed, including inhalant allergens (e.g., various pollens), ingestible allergens (e.g., eggs, milk), certain drugs, insect venoms, and the like. Allergic reactions can cause anaphylactic shock, asthma attacks, rhinitis, vomiting, abdominal pain, topical eczema, skin rashes, etc., and diseases caused by allergic reactions are called allergic diseases. Allergic diseases have been a frequently encountered and common disease, such as allergic rhinitis, allergic asthma, allergic rash, allergic gastroenteritis, etc. Allergic diseases have developed into worldwide health problems, affect human health and disturb normal life. Immunoglobulin E is an important mediator of allergic diseases and plays an important role in the development of such diseases. Immunoglobulin E antibodies (IgE) are cytophilic in that their Fc fragment binds to a mast cell or basophil surface receptor (fcer), causing sensitization of the mast cell or basophil. When a cell in a sensitized state encounters a corresponding allergen, IgE antibodies located on the cell surface combine with corresponding epitope through a variable region (VH/VL) to form an antigen bridge, so that a complex cascade reaction occurs on a cell membrane, the sensitized cell releases bioactive media in a degranulation mode, and the active media act on a target tissue or organ to cause clinical symptoms.
(2) Routine experimental diagnosis method
Currently, the diagnosis of allergic diseases relies on interrogation of medical history, skin pricks and laboratory diagnosis. The experimental diagnosis is relatively simple and easy to implement, and is highly valued clinically. The experimental diagnosis includes serum total IgE level detection, allergen specific IgE (sIgE) level detection, peripheral blood Basophil Activation Test (BAT) and the like.
Total IgE levels are often used as an auxiliary diagnostic indicator for allergic diseases. General cases such as adult serum total IgE (tIgE) levels greater than 110IU/ml suggest the possibility of an allergic reaction, but do not suggest the allergen class and do not allow clinical diagnosis. The allergen sIgE antibody refers to a characteristic antibody of a single species, can determine the type of the allergen, and is clinically used as an important basis for etiological diagnosis. However, in the current diagnostic reagent used in clinical laboratories, the detection result of sIgE cannot be directed to a specific protein (antigen), and typically combinatorial proteins with significance are used as capture antigens, and the result represents a mixed antibody. In recent years, foreign researchers have proposed single-component diagnostics (CRD) or Molecular Diagnostics (MD) for inhalant allergens and/or ingestible allergens, in which single antigens are used as capture antigens, respectively, and then bound to IgE antibodies of the corresponding antigens, respectively, and then detected by anti-human IgE antibodies labeled with signal molecules. Despite molecular diagnostic levels, sIgE does not go deep to the most basic level. Because the basic unit of antigen recognition receptors (mIgM and mIgD) distributed on the surface of B cells is an antigenic epitope, it is not a complete antigenic molecule. In other words, an sIgE antibody directed against a single protein is also a mixture of a set of antibodies directed against different epitopes.
Unlike the detection of both tge and sIgE, the Basophil Activation Test (BAT) is based on a cell biology approach that simulates the activation process of truly sensitized cells in vivo, and the detection target is a "binding" antibody that binds to the cell surface, i.e., an sIgE antibody on the surface of sensitized cells. More importantly, the cell biology method can identify the binding capacity (affinity) of the allergen sIgE antibody and an antigen epitope, and the high-affinity antibody has stronger capacity of bridging the allergen and shows higher pathological activity of activating basophils. Clinical application shows that BAT can quantitatively analyze the activation degree and the functional state of basophils, and is suitable for evaluating the specific desensitization treatment effect, the risk of food exposure and the like.
(3) Routine experimentation to diagnose defects
Both the tge and sIgE detection methods rely on the principle of antigen-antibody specific binding assays, immunochemical techniques. the tIgE is determined by taking the IgE to be detected as a protein in a double-antibody sandwich mode, and the result only represents the content of serum IgE and does not represent the real pathological activity in vivo. The sIgE is detected by using a known antigen-antibody to be detected-labeled anti-antibody analysis mode by using the sIgE to be detected as an antibody, and the result only represents the relative content of the sIgE antibody and also cannot represent the real in-vivo 'pathological activity'. More importantly, the currently used clinical products for detection of sIgE do not reflect the situation of ige antibodies directed to epitope levels or detect the degree of idiotypic epitope heterogeneity of sIgE antibodies.
BAT is an ideal assay that reflects the pathological activity of IgE antibodies in vivo. However, in the cytobiological test, fresh peripheral blood and extraction of leukocytes are required, and blood is separately collected at a predetermined time. Meanwhile, the whole detection process is complex, a flow cytometer is needed, the technical requirement on operators is high, and the detection cost is high, so that the detection method is not suitable for being popularized and used as a conventional detection method.
Disclosure of Invention
In view of the above, the present invention provides a new indicator for experimental diagnosis of allergic diseases, specifically, the new indicator is the idiotype heterogeneity intensity of allergen sIgE antibody, and the application of the indicator in a kit.
The heterogeneous intensity can be detected by a liquid phase suspension chip method; the magnitude of heterogeneity is correlated with the ability of the allergen sIgE antibodies to cross-link the allergen, i.e. is positively correlated with the biological activity of the allergen sIgE antibodies. By "idiotypic heterogeneity intensity" is meant the number of types of epitopes in an allergen, single allergen sIgE antibodies in the serum of an allergic patient, that can be recognized.
The detection principle is as follows: the liquid phase suspension chip adopts a plurality of coded magnetic microspheres as a solid phase material to coat streptavidin molecules (MB-SA); the microsphere carries two fluorescein molecules (APC and APC-Cy7), but the fluorescence intensity is different, and the microspheres with different codes can be distinguished by a Flow Cytometer (FCM); because of being coated with streptavidin, the microsphere can be combined with a polypeptide fragment with biotin to prepare a series of microspheres indirectly coated with antigen epitopes. Selecting an allergen (such as lactoglobulin) sIgE antibody as a detection object, synthesizing the defined epitope by an artificial synthesis mode, and labeling an amino terminal or a carboxyl terminal with a biotin molecule (Bio-E) in the artificial synthesis process; different epitopes (epitope, E) (polypeptide fragments) are respectively combined with different coded magnetic microspheres to prepare magnetic microsphere solutions with different epitopes, the solution is used as an R1 reagent, the R1 reagent consists of a group of solutions with different epitopes, and the microspheres are coded differently and expressed as R101, R102, R103, R104 … R148 and R149, and idiotypic epitopes of 49 sIgE antibodies can be detected. The microsphere is specifically composed of magnetic microsphere-streptavidin-biotin-epitope (polypeptide), and is expressed as MB (01-49) -SA-Bio-E (01-49), and microspheres with different codes are mixed at equal concentration. In addition, Phycoerythrin (PE) labeled mouse Anti-human IgE monoclonal antibody (PE-Anti-hIgE) was used as a labeled antibody, and this labeled antibody solution was used as R2 reagent.
A group of MB-SA-Bio-E microsphere solution (R1) is jointly combined with serum to be detected (containing sIgE antibodies in warm bath, sIgE antibodies with different idiotypic epitopes are respectively combined with corresponding peptide segments on the surfaces of the microspheres one by one to form immune complexes, a magnetic separation mode is adopted to remove unconjugated serum protein components, a labeled antibody (PE-Anti-hIgE) solution (R2) is added, the labeled antibody is combined with IgE molecules on the surfaces of the microspheres to finally form a polypeptide antigen-to-be-detected sIgE antibody-labeled antibody complex, a magnetic separation mode is adopted to wash and remove free labeled antibodies, the solution is detected by a flow cytometer, firstly, the coded microspheres are identified and selected through the fluorescence intensity of APC and APC-Cy7, different codes represent different antigen epitope information, secondly, whether each coded microsphere displays the fluorescence intensity of Phycoerythrin (PE) is observed, if the fluorescence intensity is higher than the set threshold value, the sIgE idiotype antibody containing the corresponding antigen epitope in the sample is indicated, and if the fluorescence intensity is not higher than the set threshold value, the sIgE idiotype antibody without the corresponding antigen epitope in the sample is indicated. Finally, the fluorescence of Phycoerythrin (PE) on the surface of the microsphere is calculated, which indicates how many sIgE monoclonal antibodies (idiotypic antibodies) against the allergen are contained in the serum of the patient, and the quantity is the heterogeneity intensity of the sIgE idiotypic antibodies of the patient.
The liquid phase suspension chip detection principle is applied to prepare the allergen detection kit, the kit mainly comprises an R1 solution, an R2 solution and a washing buffer solution, and concretely,
(1) preparation of R1 solution
Comprises the steps that different coded fluorescent Microspheres (MB) coated with SA are respectively combined with different biotin-labeled antigen epitopes (E); the specific method comprises the following steps: 1mL (concentration is 1mg/mL) of fluorescent microspheres and 100 mu L of Bio-E (concentration is 1mg/mL) are mixed uniformly, incubated for 1 hour by shaking, the microspheres are washed, and the volume is restored to the original volume by using fluorescent microsphere preservation solution. Mixing the fluorescent microspheres coated with different antigen epitopes and coded differently in equal proportion, and adjusting the concentration of the microspheres to 0.01mg/ml to obtain an R1 solution.
(2) Preparation of R2 solution
The R2 solution is Phycoerythrin (PE) labeled mouse Anti-human IgE monoclonal antibody (PE-Anti-hIgE), and the concentration of the PE-Anti-hIgE is 5 muL/100 muL of reaction solution.
(3) The washing buffer (PBST) was formulated from the following raw materials:
finally adding 0.05 percent of Tween-20
The following is a detailed description of the heterogeneity of sIgE idiotype antibodies:
(1) idiotypes of sIgE antibodies
The chemical nature of the antibody is immunoglobulin, and that of allergy-associated IgE antibodies is globulin with its heavy chain epsilon. Immunoglobulins have antigenic specificity and are classified into isotype, allotype, and idiotypic epitopes. The invention relates to an idiotypic epitope, referred to as "idiotypic epitope" for short. Idiotypic epitopes (IDs) refer to the specific amino acid sequences and spatial conformation shown of the variable regions (VH/VL) of an immunoglobulin molecule that determine the type of epitope recognized and bound. That is, the idiotypic epitope is located in the VH/VL region of the antibody and represents the strength of binding to the corresponding epitope. An idiotypic epitope of an antibody molecule refers to the unique antigenic specificity exhibited by VH/VL, which results from the production of an immunoglobulin by a single B cell clone. The magnitude of idiotypic epitope heterogeneity refers to the number of allergen sIgE antibodies contained by an allergic patient against a single epitope, or the number of food allergen-activated B cell clones in vivo. Each B cell clone contains a single antigen receptor (BCR), and the secreted immunoglobulins have the same idiotypic antigen specificity. The idiotype of the sIgE antibody in the invention refers to a specific amino acid sequence and a specific spatial conformation of the immunoglobulin E (IgE) CH/VL, and the idiotype of the antibody and an antigen epitope are in a spatial complementary relationship and are specifically combined with each other. The use of specific epitopes (peptide fragments) is known to allow the detection of the idiotype of the corresponding sIgE antibody.
(2) Heterogeneity of sIgE idiotypes
The specific IgE antibodies are important mediators of allergic reactions, however, the antigen against which any specific IgE antibody is directed can be understood as three layers, antibodies directed against a specific species, such as milk sIgE antibodies, or egg sIgE antibodies, antibodies directed against a specific molecule (protein), such as milk β -LG-sIgE antibodies or egg OVA-sIgE antibodies, antibodies directed against a specific epitope, such as β -LG-sIgE antibodies, or a group of antibodies directed against different epitopes, in fact, the basic unit of antibody recognition antigen is not an intact antigen molecule, but rather is a specific amino acid sequence distributed among antigen molecules, which determines the specificity of the antigen, defined as the epitope (antigeen epitope) but not the same, the complexity of antigen is also the basic unit of B cell recognition and T cell recognition, and is distributed among antigen molecules, a number of distinct amino acid sequences distributed among antigen molecules, which the specific epitope is defined as being an epitope of allergen (allergen) only, as such, as the complexity of antigen is that the basic unit of antibody recognition is not an intact antigen, but is distributed among a number of distinct antigen molecules, thus, the number of distinct single allergen antigen molecules, which is different from the antigen, the number of allergen molecules, which is different from the number of allergen molecules, thus, the antigen is derived from a single allergen, the antigen is derived from the antigen, the antigen is derived from the antigen, the number of allergen, the antigen is derived from the antigen, the antigen is derived from the antigen, the antigen is derived from the antigen, the antigen is derived from the.
(3) Analysis of causes of idiotypic heterogeneity in sIgE
The cause of idiotypic heterogeneity of sIgE antibodies is two major factors, internal and external. In terms of external factors, whether inhalation allergy or ingestion allergy, the protein composition of substances causing allergy is very complex, and most of them are not single proteins. On the protein molecule level, the complex protein structure is complex and often contains a plurality of antigen epitopes. Meanwhile, allergic diseases have geographical features, different environmental ecology and some differences in species. Different dietary habits and cooking characteristics can also lead to the change of antigenicity of food proteins. In terms of internal factors, the basic unit of antigen recognition by immune cells is an antigenic epitope, the antigenic epitope can be recognized by T lymphocytes only by combining with HLA molecules, and HLA complexes have abundant polymorphism and have very obvious individual difference. For food allergy, food is digested and degraded in vivo to induce allergic reaction, and the differences of digestive ability and gastrointestinal probiotic flora of different children patients also cause the occurrence and development of allergic reaction. Based on the above analysis, there was significant variability in the idiotype of sIgE antibodies, both at the protein molecule level and at the epitope level, among different patients allergic to the same substance.
(4) Clinical value for detecting idiotypic heterogeneity of sIgE
It is well known that the clinical symptoms of allergic patients are caused by active mediators released from mast cells or basophils. Histamine has the effect of dilating capillaries, causing a reduction in blood pressure leading to anaphylactic shock, local edema, or asphyxia. Leukotrienes have the effect of promoting smooth muscle contraction, even spasm, resulting in bronchospasm, resulting in asthma attacks, or gastrointestinal pain, vomiting, etc. However, the initiating factor that leads to the activation of degranulation by sensitized cells is allergen-bridged mast cells or basophil surface IgE antibody molecules. The surface of mast cells or basophils has receptors for the Fc fragment (FcR) of IgE antibodies, and IgE has cytotropic properties, and the two combine to cause sensitization of effector cells. Meanwhile, IgE antibodies bind to cell surface receptors, independent of IgE antibody specificity. However, the "bridging antigen" process of cell surface IgE antibodies is closely related to the affinity of IgE antibodies to the epitopes, and the number and nature of epitopes to which IgE antibodies may bind. If the affinity factor is not considered, the more different the idiotypes of the sIgE antibody on the surface of the sensitized cell are, or the stronger the heterogeneity of the idiotype epitope is, the more chance the sIgE antibody is combined with the antigen epitope on the allergen molecule is, the higher the probability of forming an "antigen bridge" is, the easier the sensitized cell is to activate, and the stronger the corresponding degree of releasing the medium from the particles is. For this reason, we believe that the heterogeneity of the idiotypic epitopes of the allergen sIgE antibodies is closely related to the pathological activity of the sIgE antibodies in the patient and to the allergic symptoms or clinical manifestations of the patient. Therefore, the application proposes the idiotype heterogeneity of the sIgE antibody as a novel experimental diagnosis index or an experimental diagnosis method which can reflect the pathological activity of the sIgE antibody and is closely related to the allergic symptoms or clinical manifestations of patients. Furthermore, recent studies have shown that the clinical symptoms of allergic patients are closely related to the number and type of epitopes recognized by the sIgE antibodies in the serum of the patients. These reports also confirm our above comments.
Compared with the prior art, the invention has the following advantages:
the problem of the idiotypic epitope heterogeneity of the allergen sIgE antibody is firstly solved, and the heterogeneity intensity is related to clinical symptoms caused by sIgE mediated sensitization cell activation and biological medium release. The heterogeneity intensity of the idiotypic epitope of the allergen sIgE antibody reflects the pathological activity of the sIgE antibody, is expected to be used as an innovative index for reflecting the clinical symptoms of patients, and can be applied to an anaphylaxis detection kit. Different from the clinical routine allergen sIgE antibody detection, the clinical value of the kit can be equal to that of a peripheral blood Basophil Activation Test (BAT), and the kit has good correlation with the detection result of the peripheral blood BAT.
Drawings
FIG. 1 selection of peripheral basophils;
FIG. 2 percent activation of peripheral basophils after stimulation with milk allergen;
in the figure 2, each patient is subjected to negative control and positive control detection to prove the reliability of the result, the result is judged according to the result of an experimental group, the left side is a patient sensitive to milk, the right side is a patient insensitive to milk, the negative control tubes of the two patients do not express CD63, and on the contrary, the cells of the positive control tubes all express CD63 with a certain proportion to indicate that the experiment is established, at the moment, the expression proportion of the tube cell CD63 is counted and determined, namely the activation degree of basophilic granulocyte of the patient to be detected by β -lactoglobulin;
FIG. 3 is a correlation analysis of the results of example 3 and comparative example 1.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The term "idiotype" as used herein refers to the particular amino acid sequence and structure of the variable domains (VH/VL) of an antibody molecule, which exhibits immunoglobulin antigen specificity, as evidenced by the difference in the epitopes recognized by the antibody. Herein, "idiotypic heterogeneity" refers to the significant variability in the number of species of the idiotypic epitopes (sIgE antibodies against the same allergen, different antigenic epitopes) of an allergen sIgE antibody among different individuals and the same allergen.
The present invention will be described in detail with reference to examples.
EXAMPLE 1 preparation of the kit
The kit mainly comprises an R1 solution, an R2 solution and a washing buffer solution, and is specifically prepared by the following steps:
(1) preparation of R1 solution
Comprises the steps of combining different coded fluorescent microspheres (MB, obtained from Merck company) coated with SA with different biotin-labeled antigen epitopes (E) respectively; the specific method comprises the following steps: 1mL (concentration is 1mg/mL) of fluorescent microspheres and 100 mu L of Bio-E (concentration is 1mg/mL) are mixed uniformly, incubated for 1 hour by shaking, the microspheres are washed, and the volume is restored to the original volume by using fluorescent microsphere preservation solution. Mixing the fluorescent microspheres coated with different antigen epitopes and coded differently in equal proportion, and adjusting the concentration of the microspheres to 0.01mg/ml to obtain an R1 solution.
(2) Preparation of R2 solution
The R2 solution was Phycoerythrin (PE) -labeled mouse Anti-human IgE monoclonal antibody (PE-Anti-hIgE, available from biolegend, USA), and the concentration of the PE-Anti-hIgE was 5. mu.L/100. mu.L reaction solution.
(3) The washing buffer (PBST) was formulated from the following raw materials:
finally adding 0.05 percent of Tween-20
Example 2 example 1 method for testing the use of a kit
The detection steps are as follows:
(1) diluting the serum sample with 0.01M phosphate buffer (containing 5% bovine serum albumin) pH 7.2 at a ratio of 1:10, such as 180. mu.L buffer + 20. mu.L serum;
(2) adding 100 mu L of a serum sample diluted by 1:10 and 100 mu L of a mixed reagent 1 indirectly coated with different antigen epitopes and different fluorescence codes into a 96-well plate, and carrying out warm bath on a flat plate oscillator at 37 ℃ for 1h to ensure that sIgE antibodies with different idiotypic epitopes in the serum to be detected are respectively combined with corresponding peptide segments on the surfaces of microspheres one by one to form an immune complex;
(3) placing the 96-well plate on a magnetic field for 3 minutes, and removing the unbound serum protein in the supernatant; after washing three times with the washing buffer, the supernatant was discarded and 100. mu.L of phosphate buffer (same as above) was added to resuspend the immunocomplexes;
(4) adding 5 mu L Phycoerythrin (PE) labeled mouse Anti-human IgE monoclonal antibody (PE-Anti-hIgE), namely reagent 2, continuously bathing for 0.5h at 37 ℃ on a flat plate oscillator, and combining the labeled antibody with sIgE molecules on the surface of the microsphere to form a polypeptide antigen-sIgE antibody to be detected-PE labeled antibody compound;
(5) placing the 96-well plate on a magnetic field for 3 minutes, removing free labeled antibodies in the supernatant, washing with washing buffer solution for three times, and then resuspending the fluorescent microspheres with 200 mu L of phosphate;
(6) flow cytometry detection
The most important maps in the stream are three point maps: one is a forward angle scattered light FSC versus a plot of the lateral angle scattered light SSC; one is a double-fluorescence dot diagram of microsphere fluorescence APC to APC-Cy 7; the other is a single parameter histogram of the intensity of PE surrounding a particular fluorescently encoded microsphere versus the corresponding cell number. The voltage was adjusted in FSC/SSC dot plots through a "zero tube" and the instrument was calibrated with FlowCheck fluorescent microspheres (Beckman Coulter) before each detection. In FSC/SSC dot plotsFinding the positions of the fluorescent microspheres, determining an analysis gate according to the positions, shooting the gate in an APC/APC-Cy7 dot diagram (circle selection), analyzing signals only in the gate, finding the positions of 01-49 different fluorescent microspheres according to the fluorescence intensities of APC and APC-Cy7, simultaneously recording the PE intensities of the different fluorescent microspheres, and setting the PE fluorescence intensity threshold value to be 102And if the threshold value is exceeded, the PE is positive.
And (3) analyzing a detection result:
the PE fluorescence intensity exceeds a set threshold value, which indicates that the sample contains sIgE idiotype antibodies of corresponding antigen epitopes, and otherwise, the sample does not contain sIgE idiotype antibodies of corresponding antigen epitopes. And counting the number and the types of the epitope recognized by each serum sample to obtain the idiotype heterogeneity intensity.
Example 3 detection of the heterogeneity of milk allergen β -lactoglobulin sIgE idiotypic epitope Using the kit of example 1 and the detection method of example 2
The detection process is as follows:
(1) the information for the selected epitopes is as follows:
TABLE β -lactoglobulin epitope amino acid sequence
The amino-terminal was labeled with biotin (Bio-E) and the 8 polypeptides were synthesized by Shanghai Jier biochemistry.
(2) Marking: 8 different encoded fluorescent microspheres (MB 01-08) coated with SA were purchased, 1mL (concentration: 1mg/mL) of each was added to an EP tube, 100. mu.L (concentration: 1mg/mL) of the above biotin-labeled epitope was added thereto, and incubated at 37 ℃ for 1 hour with shaking. Removing unbound polypeptides through a magnetic field, washing PBST for three times, and restoring the original volume by using fluorescent microsphere preservation solution to obtain the magnetic microsphere solution with different antigen epitopes.
(3) Mixing: and mixing the different magnetic microsphere solutions in equal proportion, wherein the working concentration of the solution is 0.01mg/ml, namely diluting the original solution by 100 times, and obtaining the reagent 1.
(4) Reaction: 100 mu L of the reagent 1 is added into a 96-well plate, 25 mu L of a serum sample (1:10) is added, and shaking incubation is carried out at 37 ℃ for 1h, so that an sIgE antibody-epitope complex is formed. Unbound serum proteins were removed by magnetic field and after three PBST washes, 100 μ Ι _ PBS was resuspended.
(5) Antibody staining: adding 5 mu L of Phycoerythrin (PE) labeled mouse Anti-human IgE monoclonal antibody (PE-Anti-hIgE), namely the reagent 2 according to the instruction, shaking and incubating for 0.5h at 37 ℃, and combining the labeled antibody with sIgE molecules on the surface of the microsphere to finally form a polypeptide antigen-to-be-detected sIgE antibody-labeled antibody compound. Unbound labeled antibody was removed by magnetic field, washed three times with PBST, and resuspended in 200. mu.L PBS.
(6) Detecting the percentage of the PE positive magnetic spheres by a flow cytometer: in the FSC/SSC dot plot, the position of the fluorescent microspheres is found and the analysis gate is determined accordingly. Then, the gate is shot in the APC/APC-Cy7 dot diagram, only the signal in the gate is analyzed, and the positions of the 01-08 different encoded fluorescent microspheres are found according to the fluorescence intensity of APC and APC-Cy 7. The instrument was calibrated with FlowCheck fluorescent microspheres (Beckman Coulter) before each measurement. And finally, selecting specific coded fluorescent microspheres, detecting the surfaces of the eight kinds of fluorescent microspheres one by one, marking the fluorescent intensity of the antibody (PE), and displaying the number of the fluorescent microspheres under different PE intensities in a single-parameter mode in distribution. Counting each serum sample to be detected, and detecting how many microspheres in the eight coded fluorescent microspheres show PE fluorescent signals, namely explaining how many sIgE antibodies of lactoglobulin are contained in the sample.
Conclusion analysis:
a total of 30 specimens were tested at this time, and the results are shown in the following table.
Comparative example 1 test procedure for detection of milk allergen β -lactoglobulin in peripheral blood specific allergen Basophil Activation Test (BAT)
Specific allergen Basophil Activation Tests (BATs) use specific allergens to stimulate peripheral blood basophil activation and degranulation, Flow Cytometry (FCM) is adopted, a marker (CD63) for basophil activation is identified by a fluorescence-labeled specific antibody, the number of activated basophils is quantitatively analyzed, the activation degree and degranulation state of basophils can be reflected, and the test has an important value for diagnosing allergic diseases.
[ Experimental principles ]
In the embodiment, peripheral blood of a patient to be detected is mixed and incubated with a β -lactoglobulin (Bos d 5) solution, if the patient is allergic to β -lactoglobulin, the basophil is activated and expresses CD63 molecules, anti-CD 63-FITC/anti-CD 123-PE/anti-HLA-DR PerCP fluorescence labeled antibody is added and incubated, supernatant is centrifuged, analyzed by a flow cytometer, and basophils (CD 123) are selected by setting a gate (CD123 is used for selecting the basophils)+、HLA-DR-) The percentage of basophils activated by milk in the patient to be examined can be determined by whether the CD63 molecule is expressed.
[ Main reagents and Equipment ]
1. Fresh (within 4 hours) peripheral blood of a sample to be detected, and EDTA or heparin anticoagulation.
2. Fluorescent-labeled antibodies Fluorescein Isothiocyanate (FITC) -labeled anti-human CD63 antibodies, Phycoerythrin (PE) -labeled anti-human CD123 antibodies, and dinoflagellate chlorophyll protein (PerCP) -labeled anti-human HLA-DR antibodies.
β -lactoglobulin is used for stimulating basophils, and a positive control (sheep anti-human IgE antibody) and a negative control buffer (PBS) are included.
4. General reagents hemolytic agents, hemolytic agents for lysing erythrocytes, e.g. BD FACSTMDissolving Solution; 20mM EDTA; 0.5% Paraformaldehyde (PFA).
5. Flow cytometry, incubator, microsampler, test tube, flow tube, etc.
[ operating methods ]
1. Assay tubes 20. mu.l of β -lactoglobulin-containing (10ng/ml in PBS) were added to the bottom of 1ml EP tubes, negative control tubes 20. mu.l PBS without allergen, positive control tubes 20. mu.l goat anti-human IgE antibody (5. mu.g/ml) with NFormyl Met Leu Phe (N-formyl-methylinyl-leucyl-phenylanine).
2. Add 100. mu.l fresh whole blood (EDTA anticoagulated) to each tube and vortex in a water bath at 37 ℃ for 10-15 minutes.
3. Immediately after incubation, the cells were transferred to an ice bath to stop degranulation, and 10. mu.l of 20mM EDTA was added and left at room temperature for 5 minutes.
4. The tubes were centrifuged and the supernatant carefully discarded.
5. Mu.l of anti-CD 63-FITC, anti-CD 123 PE and anti-HLA-DR PerCP antibody (final antibody concentration of 5. mu.g/ml) were added to each tube in sequence, and the tubes were vortexed in a dark room for 20 minutes or 15 minutes at room temperature.
6. Add 2ml of 1 XBD FACS to each tubeTMThe sample was dissolved by dissolving Solution and allowed to react for 15 minutes at room temperature.
7. The samples were centrifuged at 300 Xg for 5 minutes, the supernatant discarded and washed 1 time with 1ml Phosphate Buffered Saline (PBS) containing 1% BSA.
8. After centrifugation, the supernatant was discarded, and 0.3ml of 0.5% Paraformaldehyde (PFA) was added to resuspend the sample.
9. By BD FACS at 488nmTMThe flow cytometer sequentially analyzes the samples.
[ result judgment ]
The samples were analyzed by flow cytometry and data collected for the following analyses:
first, a basophil population is selected based on the side scatter light signal (SSC), CD123-PE and HLA-DR-PerCP fluorescence signals, i.e., the side scatter light signal is lower than a threshold value and CD123 is selected+、HLA-DR-. As shown in fig. 1.
Next, the activation of basophils in this specimen was determined under β -lactoglobulin stimulation based on the CD63-FITC fluorescence signal, and the percentage of activation was plotted and calculated using the CD123-PE and CD63-FITC double parameters, as shown in FIG. 2.
Correlation analysis of results of example 3 and comparative example 1
30 cases of β -lactoglobulin-allergic patients were tested in example 3, and β -lactoglobulin was used as a stimulating allergen in the BAT test in comparative example 1. the number of epitopes recognized by the serum of the patients and the percentage of activated cells in the BAT test were subjected to correlation analysis, and the correlation coefficient (r) was 0.82, indicating that they have correlation, as shown in FIG. 3.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (6)
1. Use of the idiotypic heterogeneity intensity of an allergen-specific IgE antibody in a kit for the detection of an allergen.
2. The use according to claim 1, characterized in that the magnitude of the idiotypic heterogeneity is positively correlated with the biological activity of the allergen sIgE antibodies.
3. The use according to claim 1, wherein the kit comprises a solution of R1, a solution of R2, and a buffer solution;
the preparation method of the R1 solution comprises the steps of combining different coding fluorescent microspheres coated with SA with different biotin-labeled antigen epitopes (E) respectively;
the R2 solution is Phycoerythrin (PE) marked mouse anti-human IgE monoclonal antibody.
4. The use according to claim 3, wherein the solution of R1 is prepared by the following steps: 1mL (concentration is 1mg/mL) of fluorescent microspheres and 100 mu L of Bio-E (concentration is 1mg/mL), uniformly mixing, vibrating, incubating, washing the microspheres, and restoring the original volume by using fluorescent microsphere preservation solution; mixing the fluorescent microspheres coated with different antigen epitopes and coded differently in equal proportion, and adjusting the concentration of the microspheres to 0.01mg/ml to obtain an R1 solution.
5. The use according to claim 3, wherein the R2 solution is used at a concentration of 5. mu.L per 100. mu.L of reaction solution.
6. The use according to claims 1-5, wherein the method of the kit for detecting allergens is the liquid suspension chip method.
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Application publication date: 20200207 |