CN110579592A - Heterogeneous chemiluminescent immunoassay kit for detecting 14-3-3eta protein and its application - Google Patents

Abstract

the invention relates to a heterogeneous chemiluminescence immunoassay kit for detecting 14-3-3eta protein. The kit is prepared by adopting a first antibody and a second antibody which can be specifically combined with 14-3-3eta protein; the chemiluminescence immunoassay platform has the advantages of high sensitivity, wide linear range, good precision and the like by using a double-antibody sandwich mode, improves the diagnosis rate for clinically diagnosing the rheumatoid disease, and provides auxiliary detection for preventing the rheumatoid disease and finding the rheumatoid disease in advance.

Description

Heterogeneous chemiluminescent immunoassay kit for detecting 14-3-3eta protein and its application

technical field

The invention belongs to the technical field of immune analysis, and in particular relates to a heterogeneous chemiluminescence immunoassay kit for detecting 14-3-3eta protein, a preparation method and a use method thereof.

Background technique

14-3-3eta(η) protein is a novel serum/plasma protein marker that induces inflammatory factors such as interleukin (IL)-1 and -6 and is linked to joint damage. Among the seven subtypes, only 14-3-3η protein was highly expressed in RA active inflammatory synovium and serum, and was positively correlated with the expression of RA pro-inflammatory factors and synovial inflammation, and the level of 14-3-3η protein in synovial fluid At least 5 times higher than the serum level, suggesting that synovium is the main source of 14-3-3η protein. 14-3-3η isoforms are expressed at high levels in arthritic patients compared to healthy individuals, which is thought to be directly related to the ability of 14-3-3η proteins to induce associated inflammatory factors and joint damage.

The existing heterogeneous immunoassay method for detecting 14-3-3 protein has low sensitivity and low accuracy. Moreover, no commercial 14-3-3eta protein detection kit has been found in the domestic and foreign markets. It has been reported that enzyme-linked immunosorbent assay (ELISA) is used to detect 14-3-3eta protein in serum of patients, but its sensitivity is low and specificity is poor.

Therefore, there is an urgent need to research and develop a 14-3-3eta protein heterogeneous immunoassay kit with high sensitivity and good specificity.

Contents of the invention

In order to solve the above technical problems, the present invention provides a heterogeneous chemiluminescent immunoassay kit for detecting 14-3-3eta protein. The kit is made of primary antibody and secondary antibody that can specifically bind to 14-3-3eta protein, combined with chemiluminescence technology; the chemiluminescence immunoassay platform has high sensitivity, wide linear range and good precision It can improve the diagnosis rate for the clinical diagnosis of rheumatoid, provide auxiliary detection for the prevention and early detection of rheumatoid, and become a new type of marker for rheumatoid.

To this end, the first aspect of the present invention provides a method for detecting the presence of an immune complex formed by the 14-3-3eta protein or a fragment thereof or said 14-3-3eta protein or a fragment thereof and at least one antibody for use in the preparation of Heterogeneous chemiluminescent immunoassay method for use in a reagent for assessing rheumatoid arthritis in a subject by: a) providing a test sample from a subject suspected of having rheumatoid arthritis; b) Detecting the 14-3-3eta protein or its fragments or the immune complex formed by the 14-3-3eta protein or its fragments and at least one antibody in the test sample; wherein the 14-3-3eta protein or its The presence of fragments or immune complexes indicates rheumatoid arthritis in the subject; wherein the 14-3-3eta protein or fragment thereof comprises at least one 14-3-3eta epitope, and the test sample is selected from blood , blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema effusions and tissues.

In some embodiments of the present invention, the step further includes measuring the content of the 14-3-3eta protein or its fragment or the immune complex formed by the 14-3-3eta protein or its fragment and at least one antibody.

In some preferred embodiments of the present invention, the content of 14-3-3eta protein in the sample to be tested is determined based on the standard working curve of 14-3-3eta protein.

In some embodiments of the present invention, the step further comprises forming an immune complex of the measured 14-3-3eta protein or its fragment or the 14-3-3eta protein or its fragment with at least one antibody The content of the 14-3-3eta protein or its fragment or the 14-3-3eta protein or its fragment in the normal control sample, the rheumatoid arthritis control sample or the pre-treatment sample from the same subject The levels of immune complexes formed by at least one antibody were compared.

According to the present invention, the step includes contacting the sample with an antibody capable of specifically binding to at least one specific epitope of the 14-3-3eta protein or a fragment thereof to form an immune complex.

In some embodiments of the present invention, the antibody comprises a first antibody capable of specifically binding to the first epitope of the 14-3-3eta protein and a second antibody capable of specifically binding to the second epitope of the 14-3-3eta protein The second antibody, wherein said second epitope and said first epitope do not overlap.

In some embodiments of the present invention, one of the first antibody and the second antibody is directly or indirectly bound to the label, and the other is directly or indirectly bound to the solid phase carrier; the label can be combined with the substrate The substrate reacts to generate a chemiluminescent signal, or the catalyzed substrate reacts with a chemiluminescent signal.

In some embodiments of the present invention, the marker is a luminescent marker selected from the group consisting of luminol and its derivatives, isoluminol and its derivatives, acridinium esters and their derivatives, Adamantane, rare earth elements and bipyridyl ruthenium complexes.

In other embodiments of the present invention, the label is a chemiluminescent catalyst selected from horseradish peroxidase and/or alkaline phosphatase.

In the present invention, the solid phase carrier is selected from magnetic microspheres, plastic microspheres, plastic particles, microwell plates, glass, capillary tubes and nylon; preferably magnetic microspheres.

In some embodiments of the present invention, the particle size of the magnetic microspheres is 0.05-50 microns; preferably 0.1-40 microns; more preferably 5-20 microns.

In some embodiments of the present invention, the first antibody and the second antibody are independently selected from monoclonal antibodies and/or polyclonal antibodies, preferably monoclonal antibodies.

In some embodiments of the present invention, the amino acid sequence of the 14-3-3eta protein or a fragment thereof is shown in SEQUENCE NO.1.

In some embodiments of the present invention, the epitope is selected from relatively specific fragments of the amino acid fragments of the 14-3-3eta protein sequence: 1-6aa, 27-38aa, 71-83aa, 112-119aa and 141- 154aa.

The second aspect of the present invention provides a heterogeneous chemiluminescent immunoassay kit for detecting 14-3-3eta protein, which includes:

Component a, which comprises a solid phase carrier and a first antibody or a binding fragment thereof directly or indirectly bound thereto, the first antibody or a binding fragment thereof capable of being specific to the first epitope of the 14-3-3eta protein Combine;

Component b, which comprises a second antibody or a binding fragment thereof capable of reacting with a substrate or catalyzing a substrate to generate a detectable signal label and directly or indirectly binding thereto, said second antibody or a binding fragment thereof capable of binding to The second epitope of the 14-3-3eta protein specifically binds, and the second epitope does not overlap with the first epitope.

According to the present invention, the amino acid sequence of the 14-3-3eta protein is shown in SEQUENCE NO.1.

In some embodiments of the present invention, the second epitope and the first epitope are independently selected from relatively specific fragments of the amino acid fragments of the 14-3-3eta protein sequence: 1-6aa, 27- 38aa, 71-83aa, 112-119aa, and 141-154aa.

In the present invention, the first antibody and the second antibody are independently selected from monoclonal antibodies and/or polyclonal antibodies, preferably monoclonal antibodies.

According to some embodiments of the present invention, the reagent set further includes a pure 14-3-3eta protein as a calibrator, and the calibrator is diluted with a diluent of the calibrator into working calibrator solutions of different concentrations according to a proportional gradient.

In the present invention, the first antibody or its binding fragment binds to one member of the specific binding pair, and the solid phase carrier binds to the other member of the specific binding pair

In some preferred embodiments, the first antibody or its binding fragment is bound to biotin, and the solid phase carrier is bound to streptavidin.

In the present invention, the second antibody or binding fragment thereof binds to one member of the specific binding pair, and the label binds to the other member of the specific binding pair.

In some preferred embodiments, the second antibody or binding fragment thereof is bound to biotin, and the label is bound to streptavidin.

In some embodiments of the present invention, the concentration of the solid phase carrier and the first antibody or its binding fragment bound thereto in the component a is 1-100 mg/mL, preferably 10-50 mg/mL.

In other embodiments of the present invention, the concentration of the label and the second antibody or binding fragment thereof in component b is 1-100 mg/mL, preferably 10-50 mg/mL.

In some embodiments of the present invention, the marker is a chemiluminescent marker selected from luminol and its derivatives, isoluminol and its derivatives, acridinium esters and its derivatives, adamantane, rare earth Elements and bipyridyl ruthenium complexes.

In some embodiments of the present invention, the label is a chemiluminescent catalyst selected from horseradish peroxidase and alkaline phosphatase.

In some preferred embodiments of the present invention, the reagent set further includes component c, a substrate solution.

In some specific embodiments of the present invention, the substrate solution includes A solution and B solution, the A solution is a hydrogen peroxide solution, and the B solution is a sodium hydroxide solution.

The third aspect of the present invention provides a heterogeneous chemiluminescent immunoassay kit for detecting 14-3-3eta protein, which comprises the heterogeneous chemiluminescent immunoassay reagent set described in the second aspect of the present invention.

In some embodiments of the invention, the kit includes the following components:

Component a: it comprises magnetic microspheres directly or indirectly connected to the first antibody;

Component b: it comprises a second antibody directly or indirectly linked to a label or a second label.

In some embodiments of the present invention, the kit further includes component c, which includes a substrate solution containing hydrogen peroxide solution and sodium hydroxide solution.

The fourth aspect of the present invention provides a heterogeneous chemiluminescent immunoassay method for detecting 14-3-3eta protein in a sample to be tested, which includes using the heterogeneous chemiluminescent immunoassay kit as described in the second aspect or Use the heterogeneous chemiluminescent immunoassay kit as described in the third aspect to determine whether 14-3-3eta protein exists in the sample to be tested and/or determine the content of 14-3-3eta protein.

According to the invention, the method comprises:

Step R1, mixing the sample to be tested with component a and combination b to obtain a third mixture;

Step R2, mixing the third mixture with component c to obtain a fourth mixture that produces a detectable signal;

Step R3, detecting the presence and/or intensity of the chemiluminescent signal in step R2, so as to determine whether there is 14-3-3eta protein in the sample to be tested and/or determine the content of 14-3-3eta protein.

According to some embodiments of the present invention, the method further includes the step of preparing a standard working curve of 14-3-3eta protein before step R1.

In some further specific embodiments of the present invention, in step R3, the intensity of the chemiluminescence signal described in step R2 is detected, and based on the 14-3-3eta protein standard working curve to determine the 14-3-3-eta protein in the sample to be tested 3eta protein content.

In the present invention, there are steps of separation and washing between steps R1 and R2 and between steps R2 and R3.

In some embodiments of the present invention, in step R2, the fourth mixture produces emission light with a wavelength of 470 nm.

The fifth aspect of the present invention provides a method for detecting 14-3-3eta protein, characterized in that, using the kit described in the third aspect of the present invention comprises the following steps:

1) Adding the sample for the first time: mix the sample to be tested with component a and incubate to form a complex;

2) Cleaning: apply an external magnetic field to precipitate the above reaction product, remove the supernatant, and wash with buffer;

3) The second sample addition: add component b to the above precipitate, mix evenly, and incubate to form a double-antibody sandwich complex;

4) Detection: Precipitate the above double-antibody sandwich complex by applying an external magnetic field, remove the supernatant, add a luminescent substrate after washing, detect the relative light intensity emitted, and calculate the content of 14-3-3eta protein.

The sixth aspect of the present invention provides a heterogeneous chemiluminescent immunoassay kit as described in the second aspect of the present invention or the heterogeneous chemiluminescent immunoassay kit as described in the third aspect of the present invention or the first 4. Application of the method described in the fifth aspect in detecting the presence and/or content of 14-3-3eta protein in the test sample, wherein the test sample is selected from blood, blood derivatives, serum, plasma, Urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysematous fluid, and tissue.

The seventh aspect of the present invention provides an application of the heterogeneous chemiluminescent immunoassay reagent set as described in the second aspect of the present invention in the preparation of a kit for detecting rheumatoid arthritis, which includes:

Step M1, providing a sample to be tested from a subject to be tested;

Step M2, determining whether there is 14-3-3eta protein in the sample to be tested and/or determining the content of 14-3-3eta protein;

Step M3, comparing it with the content of the 14-3-3eta protein in a normal control sample, a rheumatoid arthritis control sample, or a sample from the same subject before treatment;

Wherein, the sample to be tested is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema effusion and tissue.

In some embodiments of the invention, the presence of 14-3-3eta protein in the test sample compared to a normal control sample is a diagnostic indicator of rheumatoid arthritis in the test subject.

In some embodiments of the present invention, compared with the normal control sample, the increase of the content of 14-3-3eta protein in the test sample is a diagnostic indicator of rheumatoid arthritis in the test subject .

In some preferred embodiments of the present invention, compared with the normal control sample, the increase of 0.2ng/mL in the content of 14-3-3eta protein in the test sample is the result of rheumatoid arthritis in the test subject. diagnostic indicators.

In some embodiments of the present invention, compared with the rheumatoid arthritis control sample, the relative content of 14-3-3eta protein in the test sample is the prognosis of rheumatoid arthritis in the test subject. indicator.

In other embodiments of the present invention, the relative content of 14-3-3eta protein in the test sample compared with the pre-treatment sample from the same test subject indicates the efficacy of the treatment regimen.

According to the method of the present invention, in step M2, the method as described in the fourth aspect of the present invention is used to determine whether there is 14-3-3eta protein in the sample to be tested and/or determine the content of 14-3-3eta protein.

The eighth aspect of the present invention provides a heterogeneous chemiluminescent immunoassay kit as described in the second aspect of the present invention or the heterogeneous chemiluminescent immunoassay kit as described in the third aspect of the present invention or as described in the fourth aspect Or the application of the heterogeneous chemiluminescence immunoassay method described in the fifth aspect in a chemiluminescence immunoassay analyzer.

The ninth aspect of the present invention provides the chemiluminescence immunoassay analyzer described in the above application, which includes:

A sample filling module, which is used to fill the preset position of the chemiluminescence analyzer with the sample to be tested of the subject suspected of suffering from rheumatoid arthritis;

Reagent filling module, which is used to fill the pipetting of various reagents to the preset position of the chemiluminescence analyzer;

The incubation module is used to provide a suitable incubation reaction environment for the immune reaction of the sample to be tested and the reagent;

A magnetic separation module, which is used to clean the magnetic particles in the reaction mixture, and discharge the reaction solution after the incubation reaction, leaving the cleaned magnetic particles;

A detection module, which is used to detect a chemiluminescent signal and determine the concentration of the 14-3-3eta protein in the sample to be tested;

The electrical control module is used to coordinate and control the incubation module, the sample filling module and the reagent filling module, and the magnetic separation module and the detection module operate according to the set program.

The tenth aspect of the present invention provides a method for controlling the chemiluminescence immunoassay analyzer as described in the ninth aspect of the present invention, which includes the following steps:

Step R1, mixing the sample to be tested with component a and combination b to obtain a third mixture;

Step R2, mixing the third mixture with component c to obtain a fourth mixture that produces a detectable signal;

Step R3, detecting the presence and/or intensity of the chemiluminescent signal in step R2, so as to determine whether there is 14-3-3eta protein in the sample to be tested and/or determine the content of 14-3-3eta protein.

In some embodiments of the present invention, the method further includes the step of preparing a standard working curve of 14-3-3eta protein before step R1.

In some further embodiments of the present invention, in step R3, the intensity of the chemiluminescence signal described in step R2 is detected, and the 14-3-3eta protein in the test sample is determined based on the 14-3-3eta protein standard working curve. protein content.

The eleventh aspect of the present invention provides a method for detecting 14-3-3eta protein, which uses the kit described in the third aspect of the present invention and the chemiluminescent immunoassay analyzer described in the ninth aspect of the present invention, Include the following steps:

1) Adding the sample for the first time: mix the sample to be tested with component a and incubate to form a complex;

2) Cleaning: apply an external magnetic field to precipitate the above reaction product, remove the supernatant, and wash with buffer;

3) The second sample addition: add component b to the above precipitate, mix evenly, and incubate to form a double-antibody sandwich complex;

4) Detection: Precipitate the above double-antibody sandwich complex by applying an external magnetic field, remove the supernatant, add a luminescent substrate after washing, detect the relative light intensity emitted, and calculate the content of 14-3-3eta protein.

The twelfth aspect of the present invention provides a heterogeneous chemiluminescent immunoassay kit as described in the second aspect of the present invention or the heterogeneous chemiluminescent immunoassay kit as described in the third aspect of the present invention or as described in this invention The method described in the fourth or fifth aspect of the invention or the chemiluminescence immunoassay analyzer described in the ninth aspect of the present invention, or the method described in the tenth or eleventh aspect is effective in detecting the 14-3-3eta protein in the sample to be tested Application in presence and/or content, wherein, the sample to be tested is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema effusion and tissue .

The heterogeneous chemiluminescent immunoassay kit for detecting 14-3-3eta protein provided by the present invention is made of a first antibody and a second antibody capable of specifically binding to 14-3-3eta protein; The antibody sandwich method utilizes the advantages of high sensitivity, wide linear range, and good precision of the heterogeneous chemiluminescent immunoassay platform to improve the diagnosis rate for clinical diagnosis of rheumatoid, and provide auxiliary detection for the prevention and early detection of rheumatoid.

Applying the kit of the present invention to a heterogeneous immune analyzer has the following advantages: 1. The signal value range is wide, reaching the quantitative test standard; 2. The sensitivity is high, and the sensitivity to early RA and confirmed RA patients is high; 3. Stability Good performance and precision; 4. This kit is suitable for heterogeneous chemiluminescence immunoassay analyzers, becoming the first 14-3-3eta protein detection kit in China. Promote the progress of rheumatoid clinical diagnosis and establish new standards for the industry.

Description of drawings

In order to make the present invention easy to understand, the present invention will be described below in conjunction with the accompanying drawings.

Figure 1 shows the detection results of 14-3-3eta(η) protein in different sample groups in Example 1.

Figure 2 shows the ROC curve in Example 1.

Detailed ways

In order to make the present invention easy to understand, the present invention will be described in detail below. Before the invention is described in detail, however, it is to be understood that the invention is not limited to the particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Where a range of values is provided, it is understood that each intervening value between the upper and lower limits of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any expressly excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Ⅰ. Terminology

"Subject to be tested", "subject" and "patient" are used interchangeably and refer to mammals, such as humans and non-human primates, as well as rabbits, rats, Mice, goats, pigs and other mammalian species.

The English definition of the term "heterogeneous" in the present invention is "heterogeneous", which means that the bound antigen-antibody complex and the remaining free antigen or antibody must be separated before detection can be performed.

The term "sample to be tested" in the present invention refers to a mixture that may contain analytes, including but not limited to proteins, hormones, antibodies or antigens. Typical test samples that can be used in the methods disclosed herein include body fluids and tissues such as blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema fluids and tissues etc.

The terms "14-3-3" and "14-3-3 protein" in the present invention are used interchangeably, and refer to at least one member of the 14-3-3 family of conserved intracellular regulatory molecules generally expressed in eukaryotic cells. a member. 14-3-3 proteins have the ability to bind many functionally diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors. In fact, more than 100 signaling proteins have been reported as ligands for 14-3-3. 14-3-3 proteins can be considered as evolved members of the Tetratrico peptide repeat superfamily. They typically have 9 or 10 alpha helices, often forming homodimeric and/or heterodimeric interactions along their amino-terminal helices. These proteins contain multiple known domains including regions for divalent cation interactions, phosphorylation & acetylation and proteolytic cleavage, among others. Seven different genetically encoded 14-3-3 protein isoforms, each comprising 242-255 amino acids, are known to be expressed in mammals. The seven 14-3-3 protein isoforms are named 14-3-3α/β (alpha/beta), 14-3-3δ/ξ (delta/zeta), 14-3-3ε (epsilon), 14- 3-3γ (gamma), 14-3-3η (eta), 14-3-3τ/θ (tau/theta), and 14-3-3σ (sigma/stratifin). 14-3-3 proteins share a high degree of sequence similarity and are known to undergo post-translational processing such as phosphorylation, citrullination, and the like. See, eg, Megidish et al. (1998) J. Biol. Chem. 273:21834-45. Thus, an anti-14-3-3 autoantibody may specifically bind to and/or recognize more than one 14-3-3 protein isoform, or may specifically bind to and/or recognize only one isoform (e.g., 14-3-3η). In addition, anti-14-3-3 antibodies can bind and/or recognize 14-3-3-proteins that have been modified, eg, naturally (eg, post-translationally) or chemically.

The term "relatively specific fragment" in the present invention refers to the seven isoform 14-3-3 proteins of the 14-3-3 family. The inventors found through research that the 14- Fragments 1-6aa, 27-38aa, 71-83aa, 112-119aa and 141-154aa in the amino acid sequence of the 3-3eta protein or its fragments are specific epitopes only belonging to the 14-3-3eta (eta) protein, which There is no intersection with the amino acid sequences of the other six isoform 14-3-3 proteins of the 14-3-3 family, and the monoclonal antibodies produced therefrom only recognize or bind to the 14-3-3η(eta) protein, not Recognizes or binds the other six isoforms of the 14-3-3 family of 14-3-3 proteins.

"Arthritis" in the present invention is used interchangeably with "arthritic disease" and "joint pain", and generally refers to an inflammatory disease of human joints unless otherwise specified. Pain, swelling, stiffness, and difficulty moving are often associated with arthritic conditions. Arthritis consists of more than 100 different conditions. These conditions can be anything from relatively minor forms to severely compromised systems. Arthritic conditions can result from any of a variety of causes, including infection, trauma, degenerative disease, metabolic disorder or disturbance, or other unknown etiology. Arthritic disorders can be more specifically described by subtype, eg, rheumatoid arthritis, mixed connective tissue disease (MCTD), crystal arthritis, reactive arthritis, spondyloarthropathies, osteoarthritis, sarcoidosis , recurrent rheumatism, posttraumatic arthritis, malignancy-associated arthritis, septic arthritis, Lyme arthritis, osteoarthritis, bacterial infectious arthritis, etc. Arthritis may also be associated with other identified diseases, including gout, ankylosing spondylitis, systemic lupus erythematosus, inflammatory bowel disease, psoriasis, and the like. A well-defined arthritic disorder refers to knowledge about the type of arthritis and its stages, eg, onset, remission, relapse, and the like.

The terms "antibody" and "immunoglobulin" used herein are used in the broadest sense and include any isotype of antibody or immunoglobulin, antibody fragments that retain specific binding to an antigen; including but not limited to Fab, Fv , scFv, Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. Where desired, antibodies may be further combined with other moieties, such as members of a specific binding pair, for example biotin or streptavidin (a member of a biotin-streptavidin specific binding pair), etc. conjugate.

The term "monoclonal antibody" in the present invention refers to immunoglobulin secreted by monoclonal B lymphocytes, which can be prepared by methods known to those skilled in the art.

The term "polyclonal antibody" in the present invention refers to a collection of immunoglobulins produced by more than one B lymphocyte clone, which can be prepared by methods known to those skilled in the art.

The term "antigen" in the present invention refers to a substance that can stimulate the body to produce an immune response, and can combine with immune response product antibodies and sensitized lymphocytes in vivo and in vitro to produce an immune effect.

The term "binding" in the present invention refers to the direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and/or hydrogen bonds, including but not limited to interactions such as salt bridges and water bridges. .

The term "specific binding" or "specific binding" in the present invention refers to the mutual discrimination and selective binding reaction between two substances, which means the conformational correspondence between the corresponding reactants from the perspective of three-dimensional structure.

The term "specific binding pair member" in the present invention refers to a pair of molecules that can specifically bind to each other, for example, enzyme-substrate, antigen-antibody, ligand-receptor. An example of a specific specific binding pair member pair is the biotin-streptavidin system, in which "biotin" widely exists in animal and plant tissues, and there are two ring structures on its molecule, which are respectively imidazolone ring And the thiophene ring, of which the imidazolone ring is the main site for binding to streptavidin. Activated biotin can be coupled with almost all known biological macromolecules, including proteins, nucleic acids, polysaccharides and lipids, under the mediation of protein cross-linking agents; while "streptavidin" is produced by Streptomyces A secreted protein with a molecular weight of 65kD. The "streptavidin" molecule consists of four identical peptide chains, each of which is capable of binding a biotin. Therefore, each antigen or antibody can be coupled with multiple biotin molecules at the same time, thereby producing a "tentacle effect" and improving analytical sensitivity. Wherever desired, any reagent used in the present invention, including antigens, antibodies, acceptors or donors, can be conjugated to any member of the biotin-streptavidin specific binding pair according to actual needs.

The term "epitope" in the present invention refers to any protein determinant capable of specifically binding to immunoglobulin or T cell receptor. In some embodiments of the present invention, an epitope is a region on the surface of an antigen that can be specifically assembled by an antibody. Epitopic determinants may generally include chemically active surface groups of molecules such as, but not limited to, amino acids, sugar side chains, phosphoryl and/or sulfonyl groups. In some other specific embodiments of the present invention, the epitope can have specific three-position structure characteristics and specific charge characteristics.

The term "heterogeneous chemiluminescent immunoassay reagent set" in the present invention refers to the combination of all reagents or agents necessary for heterogeneous chemiluminescent immunoassay.

Ⅱ. Implementation plan

A first aspect of the present invention relates to the detection of the presence of a 14-3-3eta protein or a fragment thereof or an immune complex formed by said 14-3-3eta protein or a fragment thereof and at least one antibody in the preparation of a method using heterogeneous chemistry The luminescent immunoassay method is used in a reagent for assessing rheumatoid arthritis in a subject by the following steps: a) providing a test sample from a subject suspected of having rheumatoid arthritis; b) detecting the test sample 14-3-3eta protein or its fragment or the immune complex formed by said 14-3-3eta protein or its fragment and at least one antibody in the test sample; wherein said 14-3-3eta protein or its fragment or immune complex The presence of the substance indicates rheumatoid arthritis in the subject; wherein, the 14-3-3eta protein or fragment thereof comprises at least one 14-3-3eta epitope, and the test sample is selected from blood, blood derivatives , serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema effusion and tissue, preferably the sample to be tested is selected from blood, plasma, serum, synovial fluid and tissue, more preferably The test sample is selected from blood, plasma and serum, and it is further preferred that the test sample is serum.

In some embodiments of the present invention, the step further comprises measuring the content of the 14-3-3eta protein or its fragment or the immune complex formed by the 14-3-3eta protein or its fragment and at least one antibody; and Further, the content of the measured 14-3-3eta protein or its fragments or the immune complex formed by the 14-3-3eta protein or its fragments and at least one antibody was compared with the normal control sample, rheumatoid arthritis comparing the amount of said 14-3-3eta protein or fragment thereof or of an immune complex formed by said 14-3-3eta protein or fragment thereof with at least one antibody in a control sample or a pre-treatment sample from the same subject .

In some preferred embodiments of the present invention, the content of 14-3-3eta protein in the sample to be tested is determined based on the standard working curve of 14-3-3eta protein.

In some embodiments, the measured content of the 14-3-3eta protein or its fragment or the immune complex formed by the 14-3-3eta protein or its fragment and at least one antibody is compared with that in the normal control sample The 14-3-3eta protein or fragment thereof or the content of immune complexes formed by the 14-3-3eta protein or fragment thereof and at least one antibody is compared. For example, compared with a normal control sample, the 14-3-3eta protein or its fragments or the immune complex formed by the 14-3-3eta protein or its fragments and at least one antibody in the test sample An increase in the level is a diagnostic indicator of rheumatoid arthritis in the subject to be tested. In some embodiments, the measured content of the 14-3-3eta protein or its fragment or the immune complex formed by the 14-3-3eta protein or its fragment and at least one antibody is correlated with the rheumatoid arthritis The content of the 14-3-3eta protein or its fragment or the immune complex formed by the 14-3-3eta protein or its fragment and at least one antibody in the inflammation control sample is compared. For example, compared with a rheumatoid arthritis control sample, the 14-3-3eta protein or its fragments or the 14-3-3eta protein or its fragments in the test sample are immune to at least one antibody The relative amount of the complex is a prognostic indicator of rheumatoid arthritis in said test subject.

In some embodiments, the measured content of the 14-3-3eta protein or its fragment or the immune complex formed by the 14-3-3eta protein or its fragment and at least one antibody is compared with that from the same test sample The amount of said 14-3-3eta protein or fragment thereof or of an immune complex formed by said 14-3-3eta protein or fragment thereof and at least one antibody in a pre-treatment sample of the subject is compared. For example, compared with a sample before treatment from the same subject to be tested, the 14-3-3eta protein or its fragments or the 14-3-3eta protein or its fragments in the test sample and at least one antibody The relative amount of immune complexes formed is indicative of the efficacy of the treatment regimen.

According to the present invention, the step includes contacting the sample with an antibody capable of specifically binding to at least one specific epitope of the 14-3-3eta protein or a fragment thereof to form an immune complex.

In some embodiments of the present invention, the antibody comprises a first antibody capable of specifically binding to the first epitope of the 14-3-3eta protein and a second antibody capable of specifically binding to the second epitope of the 14-3-3eta protein The second antibody, wherein said second epitope and said first epitope do not overlap.

In some embodiments of the present invention, one of the first antibody and the second antibody is directly or indirectly bound to the label, and the other is directly or indirectly bound to the solid phase carrier; the label can be combined with the substrate react with a substrate to generate a chemiluminescent signal or can catalyze a substrate reaction to generate a chemiluminescent signal.

In the present invention, the marker is a luminescent marker, and the luminescent marker is selected from luminol and its derivatives, isoluminol and its derivatives, acridinium esters and its derivatives, adamantane, rare earth element And bipyridyl ruthenium complexes. Preferably, the acridinium ester and its derivatives include acridinium ester, acridinium ester sulfonamide, acridinium ester p-methylsulfonamide, and acridinium ester trifluoromethylsulfonamide.

In the present invention, the marker is a chemiluminescent catalyst selected from horseradish peroxidase and/or alkaline phosphatase.

In some preferred embodiments of the present invention, the surface of the solid phase support can be surface modified to introduce specific functional groups, and then aldehyde groups (-CHO), carboxyl groups (-COOH), amino groups (- NH2), hydroxyl (-OH), sulfhydryl (-SH) and other active groups can be effectively and stably bonded to biomolecules.

In the present invention, the solid phase carrier is selected from magnetic microspheres, plastic microspheres, plastic particles, microporous plates, glass, capillaries and nylon; preferably magnetic microspheres.

In some embodiments of the present invention, the particle size of the magnetic microspheres is 0.05-50 microns; preferably 0.1-40 microns; more preferably 5-20 microns.

In some embodiments of the present invention, the first antibody and the second antibody are independently selected from monoclonal antibodies and/or polyclonal antibodies, preferably monoclonal antibodies.

In some embodiments of the present invention, the amino acid sequence of the 14-3-3eta protein or a fragment thereof is shown in SEQUENCE NO.1.

In some embodiments of the present invention, the epitope is selected from relatively specific fragments of the amino acid fragments of the 14-3-3eta protein sequence: 1-6aa, 27-38aa, 71-83aa, 112-119aa and 141- 154aa.

The second to twelfth aspects below further provide specific embodiments for realizing the present invention.

The heterogeneous chemiluminescent immunoassay kit for detecting 14-3-3eta protein involved in the second aspect of the present invention includes:

Component a, which comprises a solid phase carrier and a first antibody or its binding fragment bound thereto, the first antibody or its binding fragment can specifically bind to the first epitope of the 14-3-3eta protein;

Component b, which comprises a label capable of reacting with a substrate to generate a detectable signal and a second antibody or a binding fragment thereof capable of binding to the second antibody of the 14-3-3eta protein The two epitopes specifically bind, and the second epitope does not overlap with the first epitope.

In the present invention, the sequence of the 14-3-3eta protein is shown in Sequence NO.1. The second epitope and the first epitope are respectively independently selected from relatively specific fragments whose amino acid fragments are 14-3-3eta protein sequences: the 1st-6th amino acid (1-6aa), Amino acids 27-38 (27-38aa), amino acids 71-83 (71-83aa), amino acids 112-119 (112-119aa) and amino acids 141-154 (141-154aa).

In the present invention, the first antibody and the second antibody are independently selected from monoclonal antibodies and/or polyclonal antibodies, preferably monoclonal antibodies.

The monoclonal antibody in the present invention can specifically bind to the epitope of 14-3-3eta protein. For example, the monoclonal antibody described in the present invention is a relatively specific fragment (epitope) capable of binding to the sequence of the 14-3-3eta protein: 1-6aa, 27-38aa, 71-83aa, 112-119aa and 141-154aa specific binding antibody.

The preparation method of the monoclonal antibody in the present invention is not particularly limited, and it can be prepared by methods known to those skilled in the art. For example, in some embodiments, a 14-3-3eta monoclonal antibody is prepared comprising:

Step 1. Animal immunization

1. First immunization

Select 3-5 male Balb/c mice about 8 weeks old, mix and emulsify the 14-3-3eta antigen and Freund's complete adjuvant in equal volumes for the first immunization, and inject the emulsified antigen intraperitoneally into each mouse, and the immunization dose is 100 μg/mouse.

2. Strengthen immunity

After the first immunization, immunize once every 2 weeks, mix and emulsify with equal volumes of 14-3-3eta antigen and Freund’s incomplete adjuvant, and inject the emulsified antigen intraperitoneally into each mouse, and the immunization dose is 50 μg/mouse , a total of 3 times of immunization. After 2 weeks, inject unemulsified antigen 50 μg/mouse via tail vein for the last booster immunization.

Step 2. Cell Fusion

On the third day after the last booster immunization, the mice were sacrificed under a sterile environment, and the spleens of the mice were taken, and splenocytes were evenly dispersed by an appropriate method. Fusion with mouse myeloma cell SP2/0 was mediated by PEG, and was added dropwise to 96-well cell culture plate by limiting dilution method for culture.

Step 3. Antibody detection

About 10 days after the end of the fusion, the cell culture supernatant of each well was detected after the clones had grown to an appropriate size. The specific detection scheme is as follows:

1) Coating with 14-3-3eta antigen and blocking with 2% BSA;

2) adding the cell culture supernatant of each well in the cell culture plate, fully washing after the reaction;

3) Add HRP-labeled anti-mouse secondary antibody, wash thoroughly after reaction;

4) Add TMB substrate to react for 15 minutes to develop color, add 2M sulfuric acid to terminate the reaction and perform OD450 reading, and determine the well number corresponding to the positive clone.

Step 4. Cloning

The positive clones were cloned 2-3 times to stabilize the cell line.

Step 5. Expand culture and prepare monoclonal antibody

Preparation of monoclonal antibody by in vitro culture or preparation of ascites.

The preparation method of the polyclonal antibody in the present invention is not particularly limited, and it can be prepared by methods known to those skilled in the art. For example, in some embodiments, a 14-3-3eta goat polyclonal antibody is prepared, which includes:

Step 1. Animal immunization and blood collection

1. First immunization

Adjust the concentration of 14-3-3eta recombinant protein to 2mg/mL, draw 2mL of complete adjuvant and 2mL of 14-3-3eta recombinant protein into two 5mL syringes respectively, then insert into the three-way tube, alternately push the needle tube to mix, Repeat until a thick emulsion is formed. Take a small amount of emulsified antigen and drop it on the surface of clear water. If the emulsified antigen does not spread, it means that the emulsification is successful.

Select 2 healthy rams, inject subcutaneously at multiple points, and inject 2mL emulsified antigen (ie 2mg 14-3-3eta antigen) into each sheep

2. Strengthen immunity

A booster immunization was given every 2 weeks after the first immunization, and a total of 4 booster immunizations were carried out

The concentration of 14-3-3eta recombinant protein was adjusted to 1mg/mL, and 2mL of incomplete adjuvant and 2mL of 14-3-3eta recombinant protein were emulsified by the same method.

After the first immunization, due to the stimulation of BCG in the complete adjuvant and the antigen, the sheep will have enlarged lymph nodes. At this time, the emulsified antigen is used for lymph node injection, and as many lymph nodes as possible are injected.

3. Take blood and separate serum

One week after the last booster immunization, the carotid artery was bled, the whole blood was clotted, cut into blocks and incubated at 37 degrees for 1-2 hours, the serum was drawn and centrifuged at 12000rpm for 5min to remove solids such as blood cells.

Step 2: Antibody purification

1. Preparation of 14-3-3eta antigen immunoaffinity column

Dialyze the 14-3-3eta antigen (with a different affinity tag from the antigen used for immunization, such as the His tag for the immunogen, and the GST tag for the antigen for affinity purification) into 0.1M NaHCO3, 0.5M Nacl, PH8.3 buffer ;

According to the fact that each gram of dry powder can be swelled into 3mL of gel, each mL of gel is coupled with 5mg of antigen, weigh an appropriate amount of CNBractivated sepharose 4B (GE) dry powder, swell with 1mM HCl and wash continuously in a suction filter bottle, usually 1mL of gel Wash with 100mL1mM HCl to clean the protective agent in the dry gel powder;

Use a suction filter bottle to filter the residual liquid on the gel, add the swollen gel to the dialyzed 14-3-3eta antigen solution, and stir slowly, react overnight at 2-8 degrees, so that the antigen can be combined by covalent bonds onto the gel;

Filter off the supernatant of the solution after the reaction, add 10 times the volume of 0.1M Tris.HCl, pH 8.0 to the coupled gel, and use the free amino group of Tris to separate the unreacted active groups on the gel;

Pack the gel column, wash 10 times the column volume alternately with 0.1M Tris. The covalently bound antigen, so far the preparation of the affinity column is completed.

2. Polyantibody affinity chromatography purification

Dilute the 14-3-3eta antiserum with 3 times the volume of normal saline, and gradually add an equal volume of saturated ammonium sulfate solution to the diluted antiserum while stirring to make it precipitate;

Centrifuge the above mixture at 12000rpm for 10min, discard the supernatant, dissolve the precipitate with 3-5 times the volume of PBS of the original antiserum, and filter it with a 0.22μm filter;

Equilibrate the 14-3-3eta antigen affinity column with PBS, load the above filtrate to the affinity column, wash with PBS after loading, until no protein is washed out;

Use 0.1M glycine, PH3.0 buffer to elute the above column, collect the eluate and neutralize it with 3MTris.HCl, PH8.5 in time;

The eluate was dialyzed with 20 times the volume of PBS, and the dialysate was changed every 4 hours or more, and the dialysate was changed twice in total to obtain the affinity-purified 14-3-3eta polyclonal antibody.

In some preferred embodiments of the present invention, the kit also includes a pure 14-3-3eta protein as a calibrator, and the calibrator is diluted into working calibrators of different concentrations by the calibrator diluent according to a proportional gradient solution.

In some preferred embodiments of the present invention, one of the first antibody and the second antibody is indirectly bound to the label through a specific binding pair member, and the other is indirectly bound to the solid phase support through a specific binding pair member .

It should be understood that the specific binding pair members described in the present invention function as a bridge, and are therefore also referred to as a bridge system. For example, in the present invention, the first antibody or its binding fragment binds to one member of the specific binding pair, and the solid support binds to the other member of the specific binding pair; in some preferred In an embodiment, the first antibody or its binding fragment is bound to biotin, and the solid phase carrier is bound to streptavidin.

For another example, in the present invention, the second antibody or binding fragment thereof binds to one member of the specific binding pair, and the label binds to the other member of the specific binding pair. In some embodiments, for example, the second antibody or binding fragment thereof is bound to biotin and the label is bound to streptavidin.

In some embodiments of the present invention, the concentration of the solid phase carrier in component a and the first antibody or its binding fragment bound thereto is 1-100 mg/mL, preferably 10-50 mg/mL; more preferably 10 mg /mL.

In some embodiments of the present invention, the concentration of the label and the second antibody or binding fragment thereof in the component b is 1-100 mg/mL, preferably 10-50 mg/mL.

In the present invention, the marker is a chemiluminescent marker selected from luminol and its derivatives, isoluminol and its derivatives, acridinium esters and its derivatives, adamantane, rare earth elements and bipyridyl ruthenium Complexes.

In the present invention, the label is a chemiluminescent catalyst selected from horseradish peroxidase and/or alkaline phosphatase.

In the present invention, the solid phase carrier is selected from microwell plates, magnetic beads, plastic particles and plastic microspheres, preferably the solid phase carrier is magnetic beads.

In some preferred embodiments of the present invention, the reagent set further includes component c, a substrate solution.

In the present invention, the substrate solution includes A solution and B solution. In some embodiments, for example, the A solution is a hydrogen peroxide solution, and the B solution is a sodium hydroxide solution.

In some embodiments of the present invention, the preparation method of the heterogeneous chemiluminescent immunoassay reagent set for detecting 14-3-3eta protein mainly includes the steps of preparing reagent I (component a) and preparing The step of reagent II (component b), wherein component a comprises a solid phase carrier and a first antibody or a binding fragment thereof bound thereto, and the first antibody or a binding fragment thereof is capable of binding to the 14-3-3eta protein The first epitope specifically binds; component b comprises a label capable of reacting with the substrate to generate a detectable signal or catalyzing the reaction of the substrate to generate a detectable signal and a second antibody or binding fragment thereof that binds thereto, the second The antibody or its binding fragment can specifically bind to the second epitope of the 14-3-3eta protein, and the second epitope does not overlap with the first epitope.

The heterogeneous chemiluminescence immunoassay kit for detecting 14-3-3eta protein involved in the third aspect of the present invention, which comprises the heterogeneous chemiluminescence immunoassay kit for detecting 14-3-3eta protein described in the second aspect of the present invention Phase chemiluminescent immunoassay kit. It can be prepared by packing the heterogeneous chemiluminescence immunoassay reagent set for detecting 14-3-3eta protein into a kit.

In some embodiments of the invention, the kit includes the following components:

Component a: it comprises magnetic microspheres directly or indirectly linked to the first antibody.

Component b: it comprises a second antibody directly or indirectly linked to a label or a second label;

In some embodiments of the present invention, the kit further includes component c, which includes a substrate solution containing hydrogen peroxide solution and sodium hydroxide solution.

In the fourth aspect of the present invention, the heterogeneous chemiluminescent immunoassay method for detecting 14-3-3eta protein in the sample to be tested according to the present invention includes using the heterogeneous chemiluminescent immunoassay as described in the second aspect of the present invention The detection reagent kit is used to determine whether there is 14-3-3eta protein in the sample to be tested and/or determine the content of 14-3-3eta protein.

Similarly, the heterogeneous chemiluminescence immunoassay method for detecting 14-3-3eta protein in the test sample involved in the present invention also includes the use of the heterogeneous chemiluminescence immunoassay kit as described in the third aspect of the present invention To judge whether there is 14-3-3eta protein in the test sample and/or determine the content of 14-3-3eta protein.

In some embodiments of the present invention, the heterogeneous chemiluminescence immunoassay method for detecting the 14-3-3eta protein in the test sample comprises:

Step R1, mixing the sample to be tested with component a and combination b to obtain a third mixture;

Step R2, mixing the third mixture with component c to obtain a fourth mixture that produces a detectable signal;

Step R3, detecting the presence and/or intensity of the chemiluminescent signal in step R2, so as to determine whether there is 14-3-3eta protein in the sample to be tested and/or determine the content of 14-3-3eta protein.

In some embodiments of the present invention, the method further includes the step of preparing a standard working curve of 14-3-3eta protein before step R1. In some specific embodiments of the present invention, the step of making the 14-3-3eta protein standard working curve includes: first, according to steps R1-R3, detect the working calibrator solution containing 14-3-3eta protein in different concentrations The chemiluminescence signal value, and then according to the corresponding relationship between the concentration and the signal value, the 14-3-3eta protein standard working curve was fitted to obtain the functional relationship between the concentration of the 14-3-3eta protein and the chemiluminescence signal value.

In some further embodiments of the present invention, in step R3, the intensity of the chemiluminescence signal described in step R2 is detected, and the 14-3-3eta protein in the test sample is determined based on the 14-3-3eta protein standard working curve. protein content.

In the present invention, the sample to be tested is reacted under heterogeneous conditions, and the process requires separation and washing, that is, there are steps of separation and washing between steps R1 and R2 and between steps R2 and R3.

In some embodiments of the present invention, in step R2, the fourth mixture produces emission light with a wavelength of 470 nm.

In some specific embodiments of the present invention, to determine whether there is 14-3-3eta protein in the test sample, the heterogeneous chemiluminescence immunoassay method for detecting the 14-3-3eta protein in the test sample comprises:

(1) mixing the sample to be tested with component a and combination b to obtain a third mixture;

(2) mixing the third mixture with component c to obtain a fourth mixture;

(3) Detecting whether the chemiluminescent signal in step (2) exists.

In other specific embodiments of the present invention, the content of 14-3-3eta protein is determined, and the heterogeneous chemiluminescence immunoassay method for detecting 14-3-3eta protein in the test sample comprises:

Step 1, making a 14-3-3eta protein standard working curve.

(1) Dilute the pure 14-3-3eta protein as a calibrator with a calibrator diluent according to a proportional gradient into working calibrator solutions of different concentrations;

(2) get working calibrator solution and mix with component a and combination b, obtain the 3rd mixture;

(3) mixing the third mixture with component c to obtain a fourth mixture;

(4) detecting the intensity of the chemiluminescent signal generated in step (3);

(5) Repeat steps (2)-(4) to detect the chemiluminescence signal value (intensity) of the working calibrator solution containing different concentrations of 14-3-3eta protein, and then according to the corresponding relationship between concentration and signal value, fitting The standard working curve of 14-3-3eta protein was obtained, and the functional relationship between the concentration of 14-3-3eta protein and the value of chemiluminescent signal was obtained.

Step 2, detecting the content of 14-3-3eta protein in the sample to be tested.

(1) mixing the sample to be tested with component a and combination b to obtain a third mixture;

(2) mixing the third mixture with component c to obtain a fourth mixture;

(3) Detect the intensity of the chemiluminescent signal generated in step (2), and determine the content of the 14-3-3eta protein in the sample to be tested based on the 14-3-3eta protein standard working curve.

In the fifth aspect, the method for detecting 14-3-3eta protein provided by the present invention uses the aforementioned kit for detection, specifically comprising:

1) Adding the sample for the first time: mix the sample to be tested with component a and incubate to form a complex;

2) Cleaning: apply an external magnetic field to precipitate the above reaction product, remove the supernatant, and wash with buffer;

3) The second sample addition: add component b to the above precipitate, mix evenly, and incubate to form a double-antibody sandwich complex;

4) Detection: Precipitate the above double-antibody sandwich complex by applying an external magnetic field, remove the supernatant, add a luminescent substrate after washing, detect the relative light intensity emitted, and calculate the content of 14-3-3eta protein.

In the sixth aspect of the present invention, the heterogeneous chemiluminescence immunoassay kit provided by the present invention as provided in the second aspect of the present invention is used in detecting the presence and/or content of 14-3-3eta protein in the sample to be tested. Application can be understood as using the heterogeneous chemiluminescence immunoassay kit provided by the second aspect of the present invention to determine whether there is 14-3-3eta protein in the sample to be tested and/or to determine the content of 14-3-3eta protein method, wherein the test sample is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema effusion and tissue, preferably the test sample The sample is selected from blood, plasma, serum, synovial fluid and tissue, more preferably, the sample to be tested is selected from blood, plasma and serum, and even more preferably, the sample to be tested is serum.

Similarly, the application of the heterogeneous chemiluminescent immunoassay kit provided by the present invention as provided in the third aspect of the present invention in detecting the presence and/or content of 14-3-3eta protein in the sample to be tested can be understood In order to use the heterogeneous chemiluminescent immunoassay kit provided by the third aspect of the present invention to determine whether there is 14-3-3eta protein in the sample to be tested and/or to determine the content of 14-3-3eta protein, wherein , the sample to be tested is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema effusion and tissue, preferably the sample to be tested is selected from blood , plasma, serum, synovial fluid and tissue, further preferably the sample to be tested is selected from blood, plasma and serum, more preferably the sample to be tested is serum.

Similarly, the application of the heterogeneous chemiluminescent immunoassay method provided by the present invention as provided in the fourth aspect of the present invention in detecting the presence and/or content of 14-3-3eta protein in the sample to be tested can be understood as Utilize the heterogeneous chemiluminescent immunoassay kit provided in the second aspect of the present invention, and use the heterogeneous chemiluminescent immunoassay method as described in the fourth aspect of the present invention to determine whether 14-3 exists in the sample to be tested -3eta protein and/or the method for determining the content of 14-3-3eta protein, wherein, the sample to be tested is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovium Fluid, emphysema effusion and tissue, preferably said sample to be tested is selected from blood, plasma, serum, synovial fluid and tissue, more preferably said sample to be tested is selected from blood, plasma and serum, more preferably said The sample to be tested is serum.

Similarly, the application of the heterogeneous chemiluminescent immunoassay method provided by the present invention as provided in the fourth aspect of the present invention in detecting the presence and/or content of 14-3-3eta protein in the sample to be tested can be understood as Use the heterogeneous chemiluminescence immunoassay kit provided in the third aspect of the present invention, and use the heterogeneous chemiluminescence immunoassay method as described in the fourth aspect of the present invention to determine whether 14-3 exists in the sample to be tested -3eta protein and/or the method for determining the content of 14-3-3eta protein, wherein, the sample to be tested is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovium Fluid, emphysema effusion and tissue, preferably said sample to be tested is selected from blood, plasma, serum, synovial fluid and tissue, more preferably said sample to be tested is selected from blood, plasma and serum, more preferably said The sample to be tested is serum.

The application of the reagent set as described in the second aspect of the present invention involved in the seventh aspect of the present invention in the preparation of a kit for detecting rheumatoid arthritis can be understood as using the reagent as described in the second aspect of the present invention Set up to prepare the method in the kit for detecting rheumatoid arthritis, it comprises:

Step M1, providing a sample to be tested from a subject to be tested;

Step M2, using the heterogeneous chemiluminescence immunoassay method described in the second aspect of the present invention to determine whether 14-3-3eta protein exists in the sample to be tested and/or determine the content of 14-3-3eta protein;

Step M3, comparing it with the content of the 14-3-3eta protein in a normal control sample, a rheumatoid arthritis control sample, or a sample from the same subject before treatment;

Wherein, the sample to be tested is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema effusion and tissue, preferably the sample to be tested is selected from Blood, plasma, serum, synovial fluid and tissue, it is further preferred that the sample to be tested is selected from blood, plasma and serum, and it is even more preferred that the sample to be tested is serum.

In the present invention, compared with the normal control sample, the presence of 14-3-3eta protein in the test sample is a diagnostic indicator of rheumatoid arthritis in the test subject.

In the present invention, compared with the normal control sample, the increase in the content of 14-3-3eta protein in the test sample is a diagnostic indicator of rheumatoid arthritis in the test subject.

In some preferred embodiments, compared with the normal control sample, the 0.2ng/ml increase in the content of 14-3-3eta protein in the test sample is a diagnostic test for rheumatoid arthritis in the test subject. indicator.

In the present invention, compared with the rheumatoid arthritis control sample, the relative content of 14-3-3eta protein in the test sample is a prognostic indicator of rheumatoid arthritis in the test subject.

In the present invention, the relative content of 14-3-3eta protein in the test sample indicates the effectiveness of the treatment regimen compared with the pre-treatment sample from the same test subject.

In the eighth aspect, the present invention relates to the heterogeneous chemiluminescent immunoassay kit as described in the second aspect of the present invention or the heterogeneous chemiluminescent immunoassay kit as described in the third aspect of the present invention or as described in the fourth or Application of the heterogeneous chemiluminescence immunoassay method described in the fifth aspect in a chemiluminescence analyzer.

The ninth aspect of the present invention provides the chemiluminescence immunoassay analyzer described in the above application, which includes:

A sample filling module, which is used to fill the preset position of the chemiluminescence analyzer with the sample to be tested of the subject suspected of suffering from rheumatoid arthritis;

Reagent filling module, which is used to fill the pipetting of various reagents to the preset position of the chemiluminescence analyzer;

The incubation module is used to provide a suitable incubation reaction environment for the immune reaction of the sample to be tested and the reagent;

A magnetic separation module, which is used to clean the magnetic particles in the reaction mixture, and discharge the reaction solution after the incubation reaction, leaving the cleaned magnetic particles;

A detection module, which is used to detect a chemiluminescent signal and determine the concentration of the 14-3-3eta protein in the sample to be tested;

The electrical control module is used to coordinate and control the incubation module, the sample filling module and the reagent filling module, and the magnetic separation module and the detection module operate according to the set program.

The tenth aspect of the present invention provides a method for controlling the chemiluminescence immunoassay analyzer as described in the ninth aspect of the present invention, which includes the following steps:

Step R1, mixing the sample to be tested with component a and combination b to obtain a third mixture;

Step R2, mixing the third mixture with component c to obtain a fourth mixture that produces a detectable signal;

Step R3, detecting the presence and/or intensity of the chemiluminescent signal in step R2, so as to determine whether there is 14-3-3eta protein in the sample to be tested and/or determine the content of 14-3-3eta protein.

In some embodiments of the present invention, the method further includes the step of preparing a standard working curve of 14-3-3eta protein before step R1.

In some further embodiments of the present invention, in step R3, the intensity of the chemiluminescence signal described in step R2 is detected, and the 14-3-3eta protein in the test sample is determined based on the 14-3-3eta protein standard working curve. protein content.

The eleventh aspect of the present invention provides a method for detecting 14-3-3eta protein, which uses the kit described in the third aspect of the present invention and the chemiluminescent immunoassay analyzer described in the ninth aspect of the present invention, Include the following steps:

1) Adding the sample for the first time: mix the sample to be tested with component a and incubate to form a complex;

2) Cleaning: apply an external magnetic field to precipitate the above reaction product, remove the supernatant, and wash with buffer;

3) The second sample addition: add component b to the above precipitate, mix evenly, and incubate to form a double-antibody sandwich complex;

4) Detection: Precipitate the above double-antibody sandwich complex by applying an external magnetic field, remove the supernatant, add a luminescent substrate after washing, detect the relative light intensity emitted, and calculate the content of 14-3-3eta protein.

The twelfth aspect of the present invention provides the heterogeneous chemiluminescent immunoassay kit as described in the second aspect of the present invention or the heterogeneous chemiluminescent immunoassay kit as described in the third aspect of the present invention or the heterogeneous chemiluminescent immunoassay kit as described in the third aspect of the present invention The method described in the fourth or fifth aspect or the chemiluminescence immunoassay analyzer described in the ninth aspect of the present invention or the method described in the tenth or eleventh aspect is used to detect the presence and /or application in content, wherein, the sample to be tested is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema effusion and tissue.

Ⅲ.Example

In order to make the present invention easier to understand, the present invention will be further described in detail below in conjunction with examples, and these examples are for illustrative purposes only, and do not limit the scope of application of the present invention. The raw materials or components used in the present invention can be prepared by commercial channels or conventional methods unless otherwise specified.

In the method of the present invention, after all reagents are combined or mixed, they can be uniformly mixed and/or incubated according to actual needs. Specifically, the incubation temperature may be any temperature within the temperature range of 25-45° C., and the incubation time may be overnight or 10-20 minutes.

Example 1:

1. Preparation of reagent Ⅰ (magnetic particles coated with anti-14-3-3eta protein antibody):

In this embodiment, a one-step coating process of 10 μm carboxyl magnetic microspheres is adopted, in which the magnetic particles are composed of ferric oxide as the core and a thin layer of polystyrene. Concrete preparation process is as follows:

(1) cleaning

Take 10 mg of magnetic particles and add them to the centrifuge tube, add coating buffer 0.1M PH5.0MES, mix well and place them in the magnetic separator for 15 seconds, discard the supernatant, add coating buffer to the centrifuge tube again, and wash the magnet. particles twice.

(2) activation

Add 2000 μL of coating buffer to resuspend the microparticles, so that the concentration of the microparticles is 5 mg/ml, take 50 μL of 10 mg/ml EDC ((1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Salt) is a water-soluble carbodiimide, which is used as an activating reagent for carboxyl groups in amide synthesis, and is also used for activating phosphate groups, cross-linking of proteins and nucleic acids, and preparation of immunoconjugates.) solution , immediately added to the centrifuge tube and mixed evenly, at room temperature, reacted for 15 minutes on a vertical rotary mixer at 25-40rpm to fully activate the carboxyl groups on the surface of the magnetic particles.

(3) Coated

Add 0.1 mg of the protein material to be coated, mix well, place the centrifuge tube at 37°C, and place it on a vertical rotary mixer for 3 hours, so that the amino group of the protein is fully coupled with the activated carboxyl group.

(4) Cleaning and preservation

Place the centrifuge tube in the magnetic separator for 15 seconds, discard the supernatant, add washing buffer 0.1MpH 7.4PBST to the centrifuge tube, mix well and place it in the magnetic separator for 15 seconds, discard the supernatant, repeat Twice for extensive washing to remove uncoupled protein.

Add 1000 μL magnetic particle preservation buffer 0.1M pH 7.4PBS to make the preservation concentration 10mg/mL, and record the batch number.

2. Preparation of reagent II (acridine ester labeled with anti-14-3-3eta protein antibody):

Adopt the technology of SAE mark in the present embodiment, specifically as follows:

(1) SAE preparation

Weigh 10 mg of SAE, dissolve it with the organic solvent dimethylformamide (DMF) to form a 5 mM solution, and store it in the dark at -20°C for future use.

(2) mark

Add 500 μL of labeling buffer 0.1M pH 8.0 PBS labeling buffer to the centrifuge tube, then add 0.1 mg of the protein material to be labeled, add 10 μL of SAE solution, mix immediately, and place it on a vertical rotary mixer at 25-40 rpm for 30 minutes at room temperature.

(3) closed

Add 50 µL of 10% lysine solution to the centrifuge tube to block unreacted active groups.

(4) Dialysis purification

Select a 14KD dialysis bag, dialyze against 0.1M pH6.3PBS dialysis buffer, change the solution every 4 hours, a total of five times, collect the markers in the dialysis bag, add glycerol protective agent, and store at -20°C in a dark place spare.

3. Preparation of 14-3-3eta protein calibrator:

1.1 Preparation of calibrator dilution: Weigh 4.77g of HEPES and 1.7g of NaCl, add 160g of purified water and mix for 30min, use 1M concentrated hydrochloric acid and 1M NaOH solution to adjust the pH value to 7.4±0.2, continue to add Proclin300 0.1g, BSA 30g, 1M MgCl2 0.5ml, 0.1M MgCl2 0.1ml, after stirring for 30min, add purified water to set the weight to 200g, retest the pH value, and set aside at 2-8°C.

1.2 Preparation of calibrator: Use the diluent of the calibrator to dilute into a working calibrator according to the proportional gradient, and then use the working calibrator to calibrate the antibody concentration of the product calibrator to complete the preparation of the calibrator.

4. Experimental operation:

After assembling the above components into a 14-3-3eta protein assay box, load it on a fully automatic chemiluminescence immunoassay analyzer, and set the detection steps and reaction steps as follows:

1) Add 50 μL of sample + 50 μL of reagent I to the cuvette, react at 37 degrees for 15 minutes, magnetically separate, and wash five times;

2) Add 100 μL reagent II, react at 37°C for 10 minutes, magnetically separate, and wash five times

3) Add 200 μL of substrate solution and immediately measure the signal value.

4) The substrate liquid is a mixture of sodium hydroxide, hydrogen peroxide and a surfactant.

5) According to the signal value of the calibrator, the standard curve is fitted according to the four-parameter fitting method, and the equation between the signal value and the 14-3-3eta protein concentration is obtained;

6) The sample to be tested is also detected according to steps 1)-4), and the concentration of 14-3-3eta protein in the sample to be tested is calculated from the equation in 5).

5. Experimental results

5.1 Check the linear range of the working calibrator

Check the linear range of the working calibrator, the results are shown in Table 1.

Table 1

It can be seen from Table 1 and Figure 1 that the standard curve fitting equation R2>0.99. It has very good linearity in the measurement range of 0.2-20ng/mL.

5.2 Evaluation Results

96 samples from the rheumatoid case group and 102 samples from the normal control group were evaluated, and the results are shown in Table 2 and Figure 2.

Table 2

Conclusion: The concentration of 14-3-3eta in the two groups of samples is significantly different, the specificity of the normal control group is 96%, and the sensitivity of the rheumatoid arthritis case group is 35%, indicating that the 14-3-3eta subtype protein is high in arthritis patients. The level of expression was significantly increased in the serum of RA patients.

It should be noted that the above-mentioned embodiments are only used to explain the present invention, and do not constitute any limitation to the present invention. The invention has been described with reference to typical embodiments, but the words which have been used therein are words of description and explanation rather than words of limitation. The present invention can be modified within the scope of the claims of the present invention as prescribed, and the present invention can be revised without departing from the scope and spirit of the present invention. Although the invention described therein refers to specific methods, materials and examples, it is not intended that the invention be limited to the specific examples disclosed therein, but rather, the invention extends to all other methods and applications having the same function.

sequence listing

<110> Beijing Kemei Biotechnology Co., Ltd.

<120> Heterogeneous chemiluminescent immunoassay kit for detecting 14-3-3 eta protein and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 246

<212> PRT

<213> (14-3-3eta protein)

<400> 1

Met Thr Met Asp Lys Ser Glu Leu Val Gln Lys Ala Lys Leu Ala Glu

1 5 10 15

Gln Ala Glu Arg Tyr Asp Asp Met Ala Ala Ala Met Lys Ala Val Thr

20 25 30

Glu Gln Gly His Glu Leu Ser Asn Glu Glu Arg Asn Leu Leu Ser Val

35 40 45

Ala Tyr Lys Asn Val Val Gly Ala Arg Arg Ser Ser Trp Arg Val Ile

50 55 60

Ser Ser Ile Glu Gln Lys Thr Glu Arg Asn Glu Lys Lys Gln Gln Met

65 70 75 80

Gly Lys Glu Tyr Arg Glu Lys Ile Glu Ala Glu Leu Gln Asp Ile Cys

85 90 95

Asn Asp Val Leu Glu Leu Leu Asp Lys Tyr Leu Ile Pro Asn Ala Thr

100 105 110

Gln Pro Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly Asp Tyr Phe

115 120 125

Arg Tyr Leu Ser Glu Val Ala Ser Gly Asp Asn Lys Gln Thr Thr Val

130 135 140

Ser Asn Ser Gln Gln Ala Tyr Gln Glu Ala Phe Glu Ile Ser Lys Lys

145 150 155 160

Glu Met Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala Leu Asn Phe

165 170 175

Ser Val Phe Tyr Tyr Glu Ile Leu Asn Ser Pro Glu Lys Ala Cys Ser

180 185 190

Leu Ala Lys Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu

195 200 205

Asn Glu Glu Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu Arg

210 215 220

Asp Asn Leu Thr Leu Trp Thr Ser Glu Asn Gln Gly Asp Glu Gly Asp

225 230 235 240

Ala Gly Glu Gly Glu Asn

245

Claims (48)

1. use of detecting the presence of 14-3-3eta protein or fragment thereof or an immune complex formed by said 14-3-3eta protein or fragment thereof and at least one antibody for the preparation of a reagent for assessing rheumatoid arthritis in a subject by a heterogeneous chemiluminescent immunoassay method by: a) providing a test sample from a subject suspected of having rheumatoid arthritis; b) detecting an immune complex formed by the 14-3-3eta protein or the fragment thereof or the 14-3-3eta protein or the fragment thereof and at least one antibody in the sample to be detected; wherein the presence of the 14-3-3eta protein or fragment thereof or immune complex is indicative of rheumatoid arthritis in the subject; wherein the 14-3-3eta protein or the fragment thereof comprises at least one 14-3-3eta epitope, and the sample to be tested is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema effusion and tissue.

2. The use according to claim 1, characterized in that said step further comprises measuring the content of 14-3-3eta protein or fragment thereof or of immune complexes formed by said 14-3-3eta protein or fragment thereof with at least one antibody.

3. the use according to claim 2, characterized in that the content of 14-3-3eta protein in the sample to be tested is determined based on a 14-3-3eta protein standard working curve.

4. The use according to claim 2, wherein said step further comprises comparing the amount of 14-3-3eta protein or fragment thereof or immune complex formed by said 14-3-3eta protein or fragment thereof and at least one antibody measured with the amount of immune complex formed by said 14-3-3eta protein or fragment thereof or said 14-3-3eta protein or fragment thereof and at least one antibody in a normal control sample, a rheumatoid arthritis control sample or a pre-treatment sample from the same subject.

5. Use according to claim 1, characterized in that said step comprises contacting said sample with an antibody comprising an antibody capable of specifically binding to at least one specific epitope of the 14-3-3eta protein or fragment thereof to form an immune complex.

6. The use according to any one of claims 1 to 5, wherein the antibodies comprise a first antibody capable of specifically binding to a first epitope of 14-3-3eta protein and a second antibody capable of specifically binding to a second epitope of 14-3-3eta protein, wherein the second epitope and the first epitope do not overlap.

7. The use of claim 6, wherein one of the first and second antibodies is directly or indirectly bound to a label and the other is directly or indirectly bound to a solid support.

8. use according to claim 7, characterized in that the label is a luminescent label selected from luminol and its derivatives, isoluminol and its derivatives, acridinium esters and its derivatives, adamantane, rare earth elements and ruthenium bipyridine complexes.

9. Use according to claim 7, wherein the label is a chemiluminescent catalyst selected from horseradish peroxidase and/or alkaline phosphatase.

10. The use according to claim 7, wherein the solid support is selected from the group consisting of magnetic microspheres, plastic microparticles, microplates, glass, capillaries, and nylon; magnetic microspheres are preferred.

11. use according to claim 10, wherein the magnetic microspheres have a particle size of 0.05-50 μm; preferably 0.1 to 40 microns; more preferably 5-20 microns.

12. Use according to claim 6, wherein the first and second antibodies are each independently selected from monoclonal and/or polyclonal antibodies, preferably monoclonal antibodies.

13. the use according to claim 1, wherein the amino acid SEQUENCE of said 14-3-3eta protein or fragment thereof is as shown in SEQUENCE No. 1.

14. Use according to claim 13, characterized in that said epitope is selected from the relatively specific fragments of the sequence of amino acid fragments 14-3-3eta protein: 1-6aa, 27-38aa, 71-83aa, 112-154 aa and 141-154 aa.

15. A heterogeneous chemiluminescent immunoassay kit for detecting 14-3-3eta protein comprising:

A component a comprising a solid support and, directly or indirectly bound thereto, a first antibody or binding fragment thereof capable of specifically binding to a first epitope of a 14-3-3eta protein;

A component b comprising a second antibody or binding fragment thereof capable of reacting with a substrate or capable of catalyzing the substrate to produce a detectable signal label and bound directly or indirectly thereto, said second antibody or binding fragment thereof being capable of specifically binding to a second epitope of the 14-3-3eta protein, and said second epitope and said first epitope do not overlap.

16. The kit according to claim 15, wherein the amino acid SEQUENCE of the 14-3-3eta protein is shown as SEQUENCE No. 1.

17. The kit of claim 16, wherein the second epitope and the first epitope are each independently selected from the group consisting of relatively specific fragments whose amino acid fragments are the sequences of 14-3-3eta protein: 1-6aa, 27-38aa, 71-83aa, 112-154 aa and 141-154 aa.

18. the kit of claim 1, wherein the first and second antibodies are each independently selected from monoclonal and/or polyclonal antibodies, preferably monoclonal antibodies.

19. the kit of any one of claims 1 to 18, further comprising 14-3-3eta protein as a calibrator diluted by a calibrator diluent in a proportional gradient to working calibrator solutions of different concentrations.

20. The kit of any one of claims 1 to 19, wherein the first antibody or binding fragment thereof is bound to one member of a specific binding pair member and the solid support is bound to the other member of the specific binding pair member; preferably, the first antibody or binding fragment thereof is bound to biotin and the solid support is bound to streptavidin.

21. The kit of any one of claims 1 to 20, wherein the second antibody or binding fragment thereof binds to one member of a specific binding pair member and the label binds to the other member of the specific binding pair member; preferably, the second antibody or binding fragment thereof is bound to biotin and the label is bound to streptavidin.

22. The kit of any one of claims 1 to 21, wherein the concentration of the solid support and the first antibody or binding fragment thereof bound thereto in component a is 1 to 100mg/mL, preferably 10 to 50 mg/mL; and/or the concentration of the label and the second antibody or binding fragment thereof bound thereto in component b is 1-100mg/mL, preferably 10-50 mg/mL.

23. the kit of any one of claims 1 to 22, wherein the label is a chemiluminescent label selected from the group consisting of luminol and its derivatives, isoluminol and its derivatives, acridinium ester and its derivatives, adamantane, rare earth elements, and ruthenium bipyridine complexes.

24. the kit of any one of claims 1 to 22, wherein the label is a chemiluminescent catalyst selected from the group consisting of horseradish peroxidase and alkaline phosphatase.

25. the kit of any one of claims 1 to 24, further comprising component c, a substrate solution; preferably, the substrate solution comprises a solution A and a solution B; more preferably, the solution A is hydrogen peroxide solution, and the solution B is sodium hydroxide solution.

26. A heterogeneous chemiluminescent immunoassay kit for detecting 14-3-3eta protein comprising the heterogeneous chemiluminescent immunoassay kit of any one of claims 15-25.

27. the kit of claim 26, wherein the kit comprises the following components:

Component a comprising magnetic microspheres directly or indirectly linked to a first antibody;

A component b comprising a second antibody directly or indirectly linked to a label or a second label.

28. The kit of claim 27, further comprising component c comprising a substrate solution comprising a hydrogen peroxide solution and a sodium hydroxide solution.

29. a heterogeneous chemiluminescent immunoassay for detecting 14-3-3eta protein in a test sample, comprising using the heterogeneous chemiluminescent immunoassay kit according to any one of claims 15 to 25 or the heterogeneous chemiluminescent immunoassay kit according to claim 26 or 27 to determine the presence of 14-3-3eta protein in the test sample and/or to determine the content of 14-3-3eta protein.

30. the method of claim 29, comprising:

Step R1, mixing the sample to be tested with the component a and the combination b to obtain a third mixture;

Step R2, mixing the third mixture with component c to obtain a fourth mixture which generates a detectable signal;

and step R3, detecting the existence and/or the intensity of the chemiluminescence signal in the step R2, thereby judging whether the 14-3-3eta protein exists in the sample to be detected and/or determining the content of the 14-3-3eta protein.

31. The method according to claim 30, further comprising the step of preparing a standard working curve for 14-3-3eta protein prior to step R1.

32. The method according to claim 31, wherein in step R3, the intensity of the chemiluminescent signal of step R2 is detected and the content of 14-3-3eta protein in the sample to be tested is determined based on a 14-3-3eta protein standard working curve.

33. a method for detecting 14-3-3eta protein, which comprises the steps of using the kit of any one of claims 26 to 29:

1) First sample adding: mixing a sample to be tested with the component a, and incubating to form a complex;

2) Cleaning: adding a magnetic field to precipitate the reaction product, removing supernatant, and washing with a buffer solution;

3) And (3) second sample adding: adding the component b into the precipitate, uniformly mixing, and incubating to form a double-antibody sandwich compound;

4) and (3) detection: and (3) precipitating the double-antibody sandwich compound by an external magnetic field, removing supernatant, cleaning, adding a luminescent substrate, detecting the relative light intensity emitted, and calculating to obtain the content of the 14-3-3eta protein.

34. Use of the heterogeneous chemiluminescent immunoassay kit of any one of claims 1 to 25 or the heterogeneous chemiluminescent immunoassay kit of any one of claims 26 to 28 or the method of any one of claims 29 to 33 for detecting the presence and/or amount of 14-3-3eta protein in a sample selected from the group consisting of blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema fluid and tissue.

35. use of a kit according to any one of claims 15 to 25 in the preparation of a kit for the detection of rheumatoid arthritis, comprising:

step M1, providing a sample to be tested from a main body to be tested;

Step M2, judging whether 14-3-3eta protein exists in the sample to be detected and/or determining the content of the 14-3-3eta protein;

Step M3, comparing the content of the 14-3-3eta protein in a normal control sample, a rheumatoid arthritis control sample or a sample from the same subject before treatment;

wherein the sample to be tested is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema effusion and tissues.

36. the use of claim 35, wherein the presence of 14-3-3eta protein in the test sample compared to a normal control sample is a diagnostic indicator of rheumatoid arthritis in the test subject.

37. The use according to claim 35, wherein an increase in the amount of 14-3-3eta protein in the test sample, as compared to a normal control sample, is a diagnostic indicator of rheumatoid arthritis in the test subject.

38. The use according to claim 35, wherein an increase of 0.2ng/ml in the amount of 14-3-3eta protein in the test sample compared to a normal control sample is a diagnostic indicator of rheumatoid arthritis in the test subject.

39. the use of claim 35, wherein the relative amount of 14-3-3eta protein in the test sample compared to a rheumatoid arthritis control sample is a prognostic indicator of rheumatoid arthritis in the test subject.

40. The use of claim 35, wherein the relative amount of 14-3-3eta protein in the test sample, as compared to a pre-treatment sample from the same test subject, is indicative of the efficacy of the treatment regimen.

41. the use according to any of claims 35 to 40, wherein in step M2, the method according to any of claims 28 to 36 is used to determine the presence of 14-3-3eta protein in the test sample and/or to determine the content of 14-3-3eta protein.

42. use of the heterogeneous chemiluminescent immunoassay kit of any one of claims 15 to 25 or the heterogeneous chemiluminescent immunoassay kit of any one of claims 26 to 28 or the heterogeneous chemiluminescent immunoassay method of any one of claims 28 to 32 in a chemiluminescent immunoassay analyzer.

43. The chemiluminescent immunoassay analyzer of use of claim 42 comprising:

A sample filling module for filling a sample to be tested of a subject suspected of having rheumatoid arthritis to a preset position of a chemiluminescence analyzer;

The reagent filling module is used for filling the pipettes of various reagents to the preset position of the chemiluminescence analyzer;

the incubation module is used for providing a proper incubation reaction environment for immunoreaction of a sample to be detected and a reagent;

The magnetic separation module is used for cleaning the magnetic particles in the reaction mixed liquid, discharging the reaction liquid after incubation reaction and leaving the cleaned magnetic particles;

The detection module is used for detecting the chemiluminescence signal and judging the concentration of the 14-3-3eta protein in the sample to be detected;

And the electric control module is used for coordinating and controlling the incubation module, the sample filling module and the reagent filling module, and the magnetic separation module and the detection module act according to a set program.

44. a method of controlling the chemiluminescent immunoassay analyzer of claim 43 comprising the steps of:

step R1, mixing the sample to be tested with the component a and the combination b to obtain a third mixture;

Step R2, mixing the third mixture with component c to obtain a fourth mixture which generates a detectable signal;

And step R3, detecting the existence and/or the intensity of the chemiluminescence signal in the step R2, thereby judging whether the 14-3-3eta protein exists in the sample to be detected and/or determining the content of the 14-3-3eta protein.

45. the method according to claim 44, further comprising a step of preparing a standard working curve of 14-3-3eta protein before step R1.

46. the method according to claim 45, wherein in step R3, the intensity of the chemiluminescent signal of step R2 is detected and the content of 14-3-3eta protein in the sample to be tested is determined based on a 14-3-3eta protein standard working curve.

47. A method for detecting 14-3-3eta protein, which comprises the steps of using the kit of any one of claims 26 to 28 and the chemiluminescent immunoassay analyzer of claim 43, comprising:

1) First sample adding: mixing a sample to be tested with the component a, and incubating to form a complex;

2) cleaning: adding a magnetic field to precipitate the reaction product, removing supernatant, and washing with a buffer solution;

3) And (3) second sample adding: adding the component b into the precipitate, uniformly mixing, and incubating to form a double-antibody sandwich compound;

4) And (3) detection: and (3) precipitating the double-antibody sandwich compound by an external magnetic field, removing supernatant, cleaning, adding a luminescent substrate, detecting the relative light intensity emitted, and calculating to obtain the content of the 14-3-3eta protein.

48. use of the heterogeneous chemiluminescent immunoassay kit of any one of claims 1 to 25 or the heterogeneous chemiluminescent immunoassay kit of any one of claims 26 to 28 or the method of any one of claims 29 to 33 or the chemiluminescent immunoassay analyzer of claim 43 or the method of any one of claims 44 to 47 for the detection of the presence and/or amount of 14-3-eta protein in a sample selected from the group consisting of blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema fluid and tissue.