阅读说明：本技术 一种血清亲细胞性免疫球蛋白e的定量检测试剂盒 (Quantitative detection kit for serum cytotropic immunoglobulin E ) 是由 李会强 佘钿田 于洋 李军普 崔亚琼 黄伦辉 李柳栩 张蓓 于 2019-09-11 设计创作，主要内容包括：本发明提供了一种血清亲细胞性免疫球蛋白E的定量检测试剂盒,包括如下试剂,亲合素预包被酶标反应板、生物素标记重组人FcεRⅠ-α亚基、酶标记抗体；本发明基于受体-待检IgE分子-抗IgE抗体分析模式,利用受体作为捕获分子,能够捕获具有亲细胞活性IgE分子,从而致使检测结果具有生物活性的意义,更好指导临床的诊断和治疗。(The invention provides a quantitative detection kit for serum cytotropic immunoglobulin E, which comprises a reagent, an avidin precoated enzyme-labeled reaction plate, a biotin-labeled recombinant human Fc epsilon RI-alpha subunit and an enzyme-labeled antibody; the invention is based on the receptor-IgE molecule to be detected-anti-IgE antibody analysis mode, and the receptor is used as a capture molecule, so that the IgE molecule with the cytotropic activity can be captured, the detection result has the significance of biological activity, and the clinical diagnosis and treatment can be better guided.)

1. The recombinant human Fc epsilon RI-alpha subunit is applied to a quantitative detection kit of serum cytotropic immunoglobulin E.

2. A quantitative detection kit for serum cytotropic immunoglobulin E is characterized in that: the kit comprises a reagent, an avidin pre-coated enzyme-labeled reaction plate, a biotin-labeled recombinant human Fc epsilon RI-alpha subunit and an enzyme-labeled antibody.

3. The kit for quantitatively detecting serum cytophilic immunoglobulin E according to claim 2, characterized in that: the biotin-labeled recombinant human Fc epsilon RI-alpha subunit is an immunoglobulin E which can be combined with serum cytotropic.

4. The kit for quantitatively detecting serum cytophilic immunoglobulin E according to claim 2, characterized in that: the enzyme-labeled antibody is a horse radish peroxidase-labeled goat anti-human IgE polyclonal antibody.

5. The kit for quantitatively detecting serum cytophilic immunoglobulin E according to claim 2, characterized in that: also includes enzyme substrate, stop solution and washing solution.

6. The kit for the quantitative determination of serum cytophilic immunoglobulin E according to claim 5, characterized in that: the enzyme substrate is TMB-H₂O₂A solution; the stop solution is a dilute sulfuric acid solution; the washing solution is PBS solution and Tween-20 solution.

7. The use of the kit for quantitative determination of serum cytotropic immunoglobulin E according to any one of claims 2 to 6 in the preparation of a reagent for detecting the level of cytotropic IgE.

8. Use according to claim 7, characterized in that it comprises the following steps:

1) adding biotin-labeled recombinant human Fc epsilon RI-alpha subunit (final concentration is 0.8-1.2 micrograms/ml, preferably, final concentration is 1 microgram/ml) into a micro-porous plate, wherein the concentration is 50 microliters/hole, and simultaneously adding a calibrator solution or a sample to be detected, and the concentration is 50 microliters/hole;

2) uniformly mixing, performing water bath at 37 ℃ for 0.8-1.2 hours, preferably performing water bath for 1 hour;

3) washing for 4-6 times by using a washing liquid and a plate washing machine, and spin-drying liquid in holes, preferably washing for 5 times;

4) adding 100 microliter/well of horse radish peroxidase labeled goat anti-human IgE polyclonal antibody (HRP-anti-hIgE) (dilution ratio, 1:5000) into the well;

5) uniformly mixing, performing water bath at 37 ℃ for 0.8-1.2 hours, preferably performing water bath for 1 hour;

6) repeat "3";

7) adding a bi-component TMB color developing solution: adding 50 microliters/hole of each of the substrate color development liquid A and the substrate color development liquid B (or mixing the A, B liquid and then adding the mixture into the hole, 100 microliters/hole), gently mixing, and incubating for 10-15 minutes at room temperature (22-25 ℃) or 37 ℃ in the dark until the color development is carried out to the expected depth;

8) adding a stop solution into the holes to stop the reaction, wherein the color of blue changes into yellow at 50 microliter/hole;

9) the absorbance was measured at 450nm using a microplate reader over 30 minutes.

Technical Field

The invention belongs to the technical field of serum immunoglobulin E (IgE) analysis, and particularly relates to a quantitative detection kit and a method for serum cytophilic immunoglobulin E, in particular to quantitative analysis of the protein level of IgE (Cyto-IgE) which can be combined with mast cells or basophils and has cytophilic activity.

Background

Under normal conditions, the human immune system generates an immune response to pathogens, inducing the body to develop resistance to the pathogens, thereby protecting the body from infectious diseases. However, in abnormal situations, some particular individuals develop an immune response against inhaled substances (e.g. pollen) or ingested foods (e.g. milk) that is detrimental to their health, resulting in life-threatening conditions such as skin rash, asthma, gastroenteritis, and even anaphylactic shock, clinically known as "allergic disease". The mechanism of development of most allergic diseases in clinic is mediated by antibodies of the immunoglobulin E (IgE) class, and mast cells or basophils are involved. 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 substance, IgE antibodies located on the cell surface bind to the corresponding epitope via the variable region (VH/VL) to form an "antigen bridge" to generate a cascade reaction, so that the cell degranules release bioactive mediators, and the active mediators act on target tissues or organs to cause clinical symptoms.

Clinical diagnosis of allergic diseases relies mainly on medical history and allergen identification, while quantitative analysis of serum IgE is also an important indicator of clinical diagnosis. The quantitative analysis of serum IgE can be used as a primary screening test of allergic diseases and also can be used as an important index for differential diagnosis of allergic diseases.

Studies have shown that immunoglobulin E antibodies (IgE) are the major immunoglobulin mediating allergic diseases. In vivo, IgE is partially distributed in serum in a Free state (Free-IgE), partially bound to mast cells or basophils, and in a bound state (Binding-IgE). The sensitized organism again encounters an allergen, bound state IgE antibodies, Binding to the allergen causes the development of an allergic reaction leading to clinical symptoms, in other words, bound state IgE (Binding-IgE) is the state of antibodies directly associated with allergic diseases.

At present, the quantitative detection method of serum immunoglobulin E mainly comprises a scattering immune turbidimetry method and a luminescence immunoassay method. The immunoturbidimetry adopts latex microspheres coated by anti-human IgE antibodies as a main reagent, the latex microspheres are combined with IgE to be detected in serum, the latex microspheres are crosslinked to change scattered light, and the level of the IgE of a serum sample to be detected is obtained by detecting the intensity of the scattered light. In the luminescence immunoassay, no matter chemiluminescence or electrochemiluminescence, a double-antibody sandwich mode is generally adopted, namely, a pair of anti-human IgE antibodies are respectively used as a capture antibody (connected with a solid phase carrier) and a labeled antibody (coupled with a luminescent agent), an IgE molecule to be detected and two reagents form a double-antibody sandwich compound, and the level of the IgE of a serum sample to be detected is obtained by detecting a signal of the labeled reagent. Both the immunoturbidimetry and the double-antibody sandwich method adopt a specific antibody (anti-human IgE antibody) as a recognition receptor, and are combined with an IgE molecule to be detected, namely, the IgE molecule to be detected is detected as protein, and cannot reflect that the IgE to be detected is detected as a functional molecule (combined cell). Therefore, the existing detection method has certain defects that the biological activity of the IgE antibody to be detected in the body cannot be reflected, so that the detection result and clinical symptoms sometimes have contradiction.

Disclosure of Invention

Based on the current serum IgE antibody detection method adopting immunochemical analysis principle, the invention has the defects of cell-philic activity which can not be reflected in human body and real biological activity which can not be reflected in human body, the invention is based on the receptor-IgE molecule to be detected-anti-IgE antibody analysis mode, uses the recombinant human Fc epsilon RI-alpha subunit as the capture molecule, and can capture the IgE molecule with cell-philic activity, thereby leading the detection result to have the significance of biological activity and better guiding the clinical diagnosis and treatment.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

the invention provides an application of a recombinant human Fc epsilon RI-alpha subunit in a quantitative detection kit of serum cytotropic immunoglobulin E.

The invention also provides a quantitative detection kit for serum cytotropic immunoglobulin E, which comprises the following reagents, an avidin pre-coated enzyme-labeled reaction plate, biotin labeled recombinant human Fc epsilon RI-alpha subunit and an enzyme labeled antibody.

Preferably, the biotin-labeled recombinant human fcsri-alpha subunit is an immunoglobulin E capable of binding to serum cytotropic cells.

Preferably, the enzyme-labeled antibody is a horseradish peroxidase-labeled goat anti-human IgE polyclonal antibody.

Preferably, the enzyme substrate, the stop solution and the washing solution are also included.

Preferably, the enzyme substrate is TMB-H₂O₂A solution; the stop solution is a dilute sulfuric acid solution; the washing solution is PBS solution and Tween-20 solution.

The invention also provides application of the quantitative detection kit for serum cytotropic immunoglobulin E in preparing a cytotropic IgE level detection reagent.

Preferably, the method comprises the following steps:

1) adding biotin-labeled recombinant human Fc epsilon RI-alpha subunit (final concentration is 0.8-1.2 micrograms/ml, preferably, final concentration is 1 microgram/ml) into a micro-porous plate, wherein the concentration is 50 microliters/hole, and simultaneously adding a calibrator solution or a sample to be detected, and the concentration is 50 microliters/hole;

2) mixing, water bathing at 37 deg.C for 1 hr;

3) washing with washing liquid for 5 times, and spin-drying the liquid in the pores;

4) adding 100 microliter/well of horse radish peroxidase labeled goat anti-human IgE polyclonal antibody (HRP-anti-hIgE) (dilution ratio, 1:5000) into the well;

5) mixing, water bathing at 37 deg.C for 1 hr;

6) repeat "3";

7) adding a bi-component TMB color developing solution: adding 50 microliters/hole of each of the substrate color development liquid A and the substrate color development liquid B (or mixing the A, B liquid and then adding the mixture into the hole, 100 microliters/hole), gently mixing, and incubating for 10-30 minutes at room temperature (22-25 ℃) or 37 ℃ in the dark until the color development is carried out to the expected depth;

8) adding a stop solution into the holes to stop the reaction, wherein the color of blue changes into yellow at 50 microliter/hole;

9) the absorbance was measured at 450nm using a microplate reader over 30 minutes.

The IgE bound in the compound is cytotropic IgE, and the quantity of the IgE is in direct proportion to the color development depth and the absorbance value.

The innovation point of the invention is that recombinant human Fc epsilon RI-alpha subunit (IgE FC receptor) is adopted as a receptor for capturing IgE molecules to be detected to replace an anti-human IgE antibody in the traditional detection method. In this alternative, the recombinant human fcsri- α subunit captures IgE molecules that bind to cells, whereas those IgE molecules that do not bind strongly to cells are not. For this reason, this approach only detects biologically active IgE molecules, i.e. cytotropic IgE molecules (Cyto-IgE).

Compared with the prior art, the quantitative detection kit for the serum cytotropic immunoglobulin E has the following advantages:

the IgE FC receptor is used as a capture antibody to directly capture the cytotropic IgE molecules in the serum to be detected, the cytotropic IgE molecules (Cyto-IgE) are immunoglobulin directly causing the sensitization of mast cells or basophils, and the link is an important link causing clinical symptoms. Therefore, the detection method of the technical invention can reflect the cell-philic activity of the IgE molecule to be detected and can partially reflect the biological activity of the IgE molecule, and the biological activity is directly related to the pathogenic activity of the IgE molecule. In other words, the invention adopts the principle of receptor-IgE molecule combination to achieve the clinical significance of cell biological detection.

Drawings

FIG. 1 is a schematic diagram of the detection of the present invention;

1 HRP-labeled goat anti-human-IgE antibody; 2, biotin labeling recombinant human Fc epsilon RI-alpha subunit (receptor); 3 pre-coating a streptavidin micropore plate; 4 IgE molecules to be detected (Cyto-IgE)

FIG. 2 is a graph of the calibration curve of parent cell tlgE obtained in the first embodiment of the present invention;

FIG. 3 is a graph showing the correlation between the results obtained by the conventional method (immunoscattering turbidimetry) and in example one, wherein R2 is 0.77;

FIG. 4 is a graph showing the correlation between the results obtained by the conventional method (immunoscattering turbidimetry) and the results obtained in example I, in which R2 is 0.98, after removing specimens having large differences.

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 present invention will be described in detail with reference to examples.