CN110726843A

CN110726843A - Chemiluminescence immunoassay kit for detecting 14-3-3eta protein and application thereof

Info

Publication number: CN110726843A
Application number: CN201810779909.XA
Authority: CN
Inventors: 饶星; 廖智星; 刘宇卉; 李临; 其他发明人请求不公开姓名
Original assignee: Chemclin Diagnostics Corp
Current assignee: Chemclin Diagnostics Corp
Priority date: 2018-07-16
Filing date: 2018-07-16
Publication date: 2020-01-24

Abstract

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

Description

Chemiluminescence immunoassay kit for detecting 14-3-3eta protein and application thereof

Technical Field

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

Background

The 14-3-3eta (eta) protein is a novel serum/plasma protein marker that induces inflammatory factors, such as Interleukins (IL) -1 and-6, and is linked to joint damage. Only 14-3-3eta protein in 7 subtypes is highly expressed in RA active inflammation synovium and serum, and is positively correlated with RA pro-inflammatory factor expression and synovium inflammation, and the level of synovial fluid 14-3-3eta protein is at least 5 times higher than that of serum, which indicates that the synovium is the main source of 14-3-3eta protein. The 14-3-3eta subtype is expressed at high levels in arthritis patients compared to healthy humans, which is believed to be directly related to the ability of 14-3-3eta protein to induce associated inflammatory factors and joint damage.

The existing methods for detecting 14-3-3 protein are all heterogeneous immunoassay methods which have low sensitivity, low accuracy and complex operation. In addition, no commercial 14-3-3eta protein detection kit is found in domestic and foreign markets. Enzyme-linked immunosorbent assay (ELISA) has been reported to detect 14-3-3eta protein in patient serum, but it has low sensitivity, poor specificity and complicated operation.

Therefore, research and development of a 14-3-3eta protein detection kit with high sensitivity, good specificity and simple and convenient operation method are urgently needed.

Disclosure of Invention

In order to solve the technical problems, the invention provides a chemiluminescence immunoassay kit for detecting 14-3-3eta protein. The kit is prepared by adopting a first anti-14-3-3 eta protein antibody and a second anti-14-3-3 eta protein antibody which can be specifically combined with 14-3-3eta protein; the advantages of high sensitivity, wide linear range, good precision and the like of the photo-induced chemiluminescence immunoassay platform are utilized, the diagnosis rate is improved for the clinical diagnosis of rheumatoid diseases, and auxiliary detection is provided for preventing rheumatoid diseases and finding out the rheumatoid diseases in advance, so that the photo-induced chemiluminescence immunoassay platform becomes a novel marker for the rheumatoid diseases.

The invention provides in a first aspect a chemiluminescent immunoassay kit for the detection of 14-3-3eta protein comprising: a first reagent, a second reagent, a calibrator, a low level quality control, and a high level quality control, wherein:

the first reagent comprises a luminescent microparticle coated with a first anti-14-3-3 eta protein antibody, the first anti-14-3-3 eta protein antibody being capable of specifically binding to a first epitope of 14-3-3eta protein;

the second reagent comprises a second anti-14-3-3 eta protein antibody containing a biotin label capable of specifically binding to a second epitope of the 14-3-3eta protein, which second epitope is non-overlapping with the first epitope.

In the invention, the calibrator, the low-level quality control product and the high-level quality control product all comprise recombinant antigen 14-3-3eta protein.

According to the invention, the calibrant is diluted by a calibrant diluent in a proportional gradient to 6 bottles of working calibrant solution at six concentration levels.

In some embodiments of the invention, the calibrator is diluted by the calibrator diluent in a proportional gradient to a working calibrator solution at six concentration levels as follows: 0ng/mL, 0.2ng/mL, 0.5ng/mL, 2ng/mL, 5ng/mL, 20 ng/mL.

In some preferred embodiments of the invention, the calibrator further comprises neonatal bovine serum and a lyoprotectant.

In some embodiments of the invention, the low level quality control product has a quality control range of 10% to 40%, preferably 10% to 30%, more preferably 10% to 20%.

In some embodiments of the invention, the target concentration of the low level quality control agent is 0.20 ± 0.05 ng/mL.

In some embodiments of the invention, the high level of quality control product has a quality control range of 10% to 40%, preferably 10% to 30%, more preferably 10% to 20%.

In some embodiments of the invention, the high level of quality control has a target concentration of 5.0 ± 1.0 ng/mL.

According to the invention, the kit further comprises a third reagent comprising avidin and/or streptavidin coated photosensitive microparticles.

According to the invention, the kit further comprises a microwell plate.

In the invention, the calibrator, the low-level quality control product and/or the high-level quality control product are all freeze-dried powder.

In some preferred embodiments of the invention, the low-level quality control and/or the high-level quality control comprises newborn bovine serum.

In other preferred embodiments of the invention, the first agent and/or the second agent comprises a protein stabilizing agent.

In some particularly preferred embodiments of the invention, the protein stabilizing agent is bovine serum albumin.

In the present invention, the luminescent particles in the first reagent are polymer particles filled with luminescent compounds and lanthanide elements, which can react with singlet oxygen to generate a detectable chemiluminescent signal.

In the present invention, the photosensitive particles in the third reagent are polymer particles filled with a photosensitive compound, and are capable of generating singlet oxygen in an excited state.

In some embodiments of the invention, the concentration of luminescent particles coating the first anti-14-3-3 eta protein antibody in the first reagent is 10-200. mu.g/mL, preferably 20-150. mu.g/mL, more preferably 30-100. mu.g/mL, most preferably 40-80. mu.g/mL.

In some embodiments of the invention, the concentration of the biotin-labeled secondary anti-14-3-3 eta protein antibody in the second reagent is 0.1-8. mu.g/mL, preferably 0.2-6. mu.g/mL, more preferably 0.4-4. mu.g/mL, and most preferably 0.6-2. mu.g/mL.

In some embodiments of the invention, the concentration of said avidin and/or streptavidin-coated photosensitive microparticles in said third reagent is 5-20 μ g/mL, preferably 8-15 μ g/mL, more preferably 10-12 μ g/mL.

In some embodiments of the invention, the amino acid SEQUENCE of the 14-3-3eta protein is shown in SEQUENCE No. 1.

In other embodiments of the invention, the kit has a dose-response curve linear correlation coefficient (r) within the range of 0.1ng/mL to 20.0ng/mL of not less than 0.990.

In a second aspect, the invention provides a chemiluminescent immunoassay method for detecting 14-3-3eta protein in a sample to be detected, which comprises using the chemiluminescent immunoassay kit according to the first aspect of the invention to judge whether 14-3-3eta protein exists in the sample to be detected and/or determine the content of 14-3-3eta protein.

In some embodiments of the invention, the method comprises:

step R1, mixing a sample to be detected with a first reagent and a second reagent to obtain a first mixture;

step R2, mixing the first mixture with a third reagent to obtain a second mixture;

step R3 of contacting an energy or reactive compound with said second mixture to excite said photosensitive particles to produce singlet oxygen, said luminescent particles being capable of reacting with said singlet oxygen received to produce a detectable chemiluminescent signal;

and step R4, detecting the existence and/or the intensity of the chemiluminescence signal in the step R3, 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.

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

In some further preferred embodiments of the present invention, in step R4, the intensity of the chemiluminescent signal in step R3 is detected, and the content of 14-3-3eta protein in the sample to be tested is determined based on the 14-3-3eta protein standard working curve.

In the present invention, there is no separation and/or washing step between steps R1 and R2 and between steps R2 and R3.

In some embodiments of the invention, in step R3, the second mixture is irradiated with excitation light with a wavelength of 600-.

In some preferred embodiments of the invention, the method further comprises a calibration step: duplicate wells were assayed and calibrated using working calibrator solutions at six concentration levels of 0ng/mL, 0.2ng/mL, 0.5ng/mL, 2ng/mL, 5ng/mL, 20 ng/mL.

In other preferred embodiments of the present invention, the method further comprises a quality control step of fitting using a cubic spline interpolation fit to give concentration values of the sample.

In still other embodiments of the present invention, the chemiluminescence immunoassay kit according to the first aspect of the present invention is used to determine whether the upper limit of the normal range of the 14-3-3eta protein in the test sample is 0.2ng/mL, and the chemiluminescence immunoassay kit is determined to be positive when the concentration of the 14-3-3eta protein in the test sample is greater than or equal to 0.2ng/mL, and determined to be negative when the concentration of the 14-3-3eta protein in the test sample is less than 0.2 ng/mL.

In a third aspect, the invention provides a chemiluminescent immunoassay system for detecting 14-3-3eta protein, which uses the kit of the first aspect of the invention and/or the method of the second aspect of the invention to detect the presence of 14-3-3eta protein in a sample to be detected and/or determine the content of 14-3-3eta protein.

In some embodiments of the invention, the system comprises:

the reaction device is used for performing chemiluminescence immune reaction on a sample to be detected, a first reagent and a second reagent;

an excitation and reading device for exciting and recording a chemiluminescent signal of a chemiluminescent immune response;

and the processor judges whether the 14-3-3eta protein exists in the sample to be detected and/or determines the content of the 14-3-3eta protein according to the existence and/or the intensity of the chemiluminescence signal.

In some embodiments of the invention, the processor uses a cubic spline interpolation fit for fitting, directly giving the concentration values of the sample.

In a fourth aspect, the invention provides a chemiluminescent immunoassay method for detecting 14-3-3eta protein, which uses the detection system of the third aspect and the chemiluminescent immunoassay kit of the first aspect of the invention to detect the presence of 14-3-3eta protein in a sample to be detected and/or determine the content of 14-3-3eta protein by the method of the first aspect of the invention.

In some embodiments of the invention, the method comprises the steps of:

1) respectively adding a sample to be detected or a calibrator and a quality control product into the reaction hole;

2) sequentially adding a first reagent and a second reagent into the reaction hole;

3) incubating the mixture obtained in step 2);

4) adding a third reagent into the reaction hole;

5) incubating the mixture obtained in step 4);

6) irradiating the reaction holes by laser and calculating the quantity of light-emitting photons of each hole;

7) and calculating the concentration of the target molecules to be detected in the sample to be detected according to the standard curve.

In some embodiments of the invention, the method comprises the steps of:

1) respectively adding 25 mu L of sample to be detected or calibrator and quality control material into the reaction hole;

2) adding 25 mu L of first reagent and 25 mu L of second reagent into the reaction hole in sequence;

3) incubation at 37 ℃ for 15 minutes;

4) adding 175 mu L of a third reagent into the reaction hole;

5) incubation at 37 ℃ for 10 min;

6) irradiating the micropores by laser and calculating the quantity of light photons emitted by each hole;

7) the sample concentration was calculated from the standard curve.

In some preferred embodiments of the invention, the method further comprises a calibration procedure comprising the steps of:

1) operating software of a chemiluminescence analysis instrument, entering a calibration program, and performing multi-hole measurement and calibration by using six calibration products;

2) submitting a calibration task;

3) automatically operated by the detection system.

In some preferred embodiments of the invention, the method further comprises a quality control program comprising: and fitting by adopting cubic spline interpolation fitting through a processor of the detection system, and automatically giving the concentration value of the sample.

In some embodiments of the present invention, the upper limit of the normal range for determining whether 14-3-3eta protein exists in the test sample is 0.2ng/mL by using the detection system according to the fourth aspect of the present invention and the chemiluminescent immunoassay kit according to the first aspect of the present invention, and the test sample is determined to be positive when the concentration of 14-3-3eta protein in the test sample is greater than or equal to 0.2ng/mL, and determined to be negative when the concentration of 14-3-3eta protein in the test sample is less than 0.2 ng/mL.

In a fifth aspect, the present invention provides a use of a detection system according to the third aspect of the present invention and a method according to the first aspect of the present invention or according to the second or fourth aspect of the present invention for detecting the presence and/or amount of 14-3-3eta protein in a sample to be tested, wherein the sample to be tested is selected from the group consisting of blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema fluid and tissue.

In a sixth aspect, the present invention provides a use of the kit according to the first aspect of the present invention for preparing a kit for detecting 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.

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 invention, an increase 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.

In some embodiments of the invention, 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.

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

In some embodiments of the invention, 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.

In some embodiments of the present invention, in step M2, the detection system according to the third aspect of the present invention and/or the method according to the second or fourth aspect of the present invention is used to determine whether 14-3-3eta protein is present in the test sample and/or to determine the content of 14-3-3eta protein.

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

The application of the kit and the detection system of the invention to the chemiluminescence immunoassay for detecting 14-3-3eta protein has the following advantages: (1) the signal value range is wide, and the quantitative test standard is achieved; (2) the sensitivity is high, and the sensitivity to early RA and RA-confirmed patients is high; (3) the stability is good, and the precision is good; (4) the kit becomes the first domestic 14-3-3eta protein detection kit. Promotes the progress of clinical diagnosis of rheumatoid diseases and establishes a new standard for the industry.

Drawings

For the present invention to be readily understood, the following description is made with reference to the accompanying drawings.

FIG. 1 shows the results of the detection of 14-3-3eta (eta) protein in the different sample groups of example 1.

Detailed Description

In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the invention is described in detail, it is to be understood that this invention is not limited to 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, to the extent that there is no stated or intervening value in that stated range, to the extent that there is no such intervening value, 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 specifically excluded limit in the stated range. Where a specified 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.

Term (I)

"subject", "subject" and "patient" are used interchangeably and, without particular reference or limitation, refer to mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs and other mammalian species.

The term "homogeneous" as used herein is defined in english as "homogeneous" and means that the bound antigen-antibody complex and the remaining free antigen or antibody are detected without separation.

The term "test sample" as used herein refers to a mixture that may contain an analyte, including but not limited to a protein, hormone, antibody or antigen. Typical test samples that can be used in the disclosed methods include body fluids and tissues such as blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema fluid and tissues, and the like.

The terms "14-3-3" and "14-3-3 protein" are used interchangeably herein and refer to at least one member of the 14-3-3 family of conserved intracellular regulatory molecules that are ubiquitously expressed in eukaryotic cells. The 14-3-3 protein has the ability to bind a number of functionally diverse signal transduction proteins, including kinases, phosphatases and transmembrane receptors. Indeed, more than 100 signal transduction proteins have been reported as ligands for 14-3-3. The 14-3-3 protein can be considered as an evolved member of the tetrico peptide repeat superfamily. They typically have 9 or 10 alpha helices, often forming homodimer and/or heterodimer interactions along their amino terminal helices. These proteins contain a number of 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 designated 14-3-3 α/β (alpha/beta), 14-3-3 δ/ξ (delta/zeta), 14-3-3 ε (epsilon), 14-3-3 γ (gamma), 14-3-3 η (eta), 14-3-3 τ/θ (tau/theta) and 14-3-3 σ (sigma/stratfin). The 14-3-3 protein has a high degree of sequence similarity and is known to undergo post-translational processing such as phosphorylation, citrullination, and the like. See, e.g., 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., 14-3-3 η). In addition, anti-14-3-3 antibodies can bind to and/or recognize 14-3-3-protein that has been modified, e.g., naturally (e.g., post-translationally) or chemically.

The term "relatively specific fragment" as used herein means that, with respect to 7 isoforms of 14-3-3 protein of the 14-3-3 family, the present inventors found through studies that fragments 1-6aa, 27-38aa, 71-83aa, 112-154 aa and 141-154aa in the amino acid SEQUENCE of 14-3-3eta protein or a fragment thereof as represented by SEQ ID NO.1 are specific epitopes belonging only to the 14-3-3eta (eta) protein, it does not have any cross-over with the amino acid sequences of the other 6 isoforms of the 14-3-3 family 14-3-3 protein, and the monoclonal antibodies produced therefrom only recognize or bind to the 14-3-3eta (eta) protein and do not recognize or bind to the other 6 isoforms of the 14-3-3 family 14-3-3 protein.

In the present invention, the term "arthritis" is used interchangeably with "arthritic conditions" and "joint pain", and generally refers to inflammatory conditions of human joints, unless otherwise indicated. Pain, swelling, stiffness and difficulty moving are often associated with arthritic conditions. Arthritis consists of more than 100 different cases. These conditions can be anything from a relatively mild form to a severely compromised system form. Arthritic conditions can be caused by any of a variety of causes, including infection, trauma, degenerative disease, metabolic disorder or disturbance, or other unknown etiology. Arthritic conditions can be more particularly described in terms of subtypes such as rheumatoid arthritis, Mixed Connective Tissue Disease (MCTD), crystal arthritis, reactive arthritis, spondyloarthropathies, osteoarthritis, sarcoidosis, recurrent rheumatism, post-traumatic arthritis, malignancy-associated arthritis, septic arthritis, lyme arthritis, osteoarthritis, bacterial infectious arthritis, and the like. Arthritis may also be accompanied by other identified diseases including gout, ankylosing spondylitis, systemic lupus erythematosus, inflammatory bowel disease, psoriasis, and the like. A well-defined arthritic condition refers to the knowledge about the type of arthritis and its stage, e.g., onset, remission, relapse, and the like.

The terms "antibody" and "immunoglobulin" are used in the broadest sense of the invention, and include antibodies or immunoglobulins of any isotype, 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 an antigen-binding portion of an antibody and a non-antibody protein. In any case desired, the antibody may be further conjugated to other moieties, such as a specific binding pair member, e.g., biotin or streptavidin (a member of a biotin-streptavidin specific binding pair member), and the like.

The term "monoclonal antibody" as used herein refers to an immunoglobulin secreted from a monoclonal B lymphocyte, which can be prepared by methods known to those skilled in the art.

The term "polyclonal antibody" as used herein refers to a collection of immunoglobulins produced by more than one B lymphocyte clone, which may be prepared by methods well known to those skilled in the art.

The term "antigen" as used herein refers to a substance that stimulates the body to produce an immune response and that binds to the immune response product antibodies and sensitized lymphocytes in vitro and in vivo to produce an immune effect.

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

The term "specific binding" or "specific binding" as used herein refers to the mutual discrimination and selective binding reaction between two substances, and is the conformational correspondence between the corresponding reactants from the perspective of the three-dimensional structure.

The term "specific binding pair member" as used herein refers to a pair of molecules that are capable of specifically binding to each other, e.g., enzyme-substrate, antigen-antibody, ligand-receptor. An example of a specific binding pair member pair is the biotin-streptavidin system, where "biotin" is widely present in animal and plant tissues and has two cyclic structures on the molecule, an imidazolone ring and a thiophene ring, respectively, where the imidazolone ring is the main site for binding to streptavidin. Activated biotin can be conjugated to almost any biological macromolecule known, including proteins, nucleic acids, polysaccharides, lipids, and the like, mediated by a protein cross-linking agent; "streptavidin" is a protein secreted by Streptomyces and has a molecular weight of 65 kD. The "streptavidin" molecule consists of 4 identical peptide chains, each of which is capable of binding a biotin. Thus, each antigen or antibody can be conjugated to multiple biotin molecules simultaneously, thereby creating a "tentacle effect" that increases assay sensitivity. Any reagent used in the present invention, including antigens, antibodies, luminescent or photosensitive particles, can be conjugated to any member of the biotin-streptavidin specific binding pair as desired.

The term "epitope" as used herein refers to any protein determinant capable of specifically binding to an immunoglobulin or T cell receptor. In some embodiments of the invention, an epitope is a region of the antigen surface that can be specifically assembled by an antibody. Epitope determinants may generally include chemically active surface groups of the molecule such as, but not limited to: amino acids, sugar side chains, phosphoryl groups and/or sulfonyl groups. In other embodiments of the invention, epitopes may be characterized by specific three-dimensional structural features as well as specific charge characteristics.

The photosensitive particles are high molecular particles filled with photosensitive compounds formed by coating functional groups on a substrate, and can generate singlet oxygen under the excitation of light. In some embodiments of the invention, the photoactive compound is a photosensitizer, which may be a photosensitizer known in the art, preferably a compound that is relatively light stable and does not react efficiently with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrins, phthalocyanines, and chlorophylls disclosed in, for example, U.S. Pat. No. 5,5709994, which is incorporated herein by reference in its entirety, and derivatives of these compounds having 1 to 50 atom substituents that serve to render these compounds more lipophilic or more hydrophilic, and/or as a linker group to a member of a specific binding pair. Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, such as those described in US patent No. US6406913, which is incorporated herein by reference. In some embodiments of the invention, the photoactive compound is a chemically activated additional sensitizer, non-limiting examples of which are compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Other examples of photosensitive particles include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, etc., which are heated or directly absorb light to release singlet oxygen.

The term "luminescent particle" as used herein refers to a polymeric particle that is capable of reacting with singlet oxygen to produce a detectable signal. The photosensitive particles are induced by energy or an active compound to activate and release singlet oxygen in a high energy state that is captured by the nearby light emitting particles, thereby transferring energy to activate the light emitting particles. In some embodiments of the invention, the luminescent particles comprise a species that undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate that can decompose with or subsequently emit light. Typical examples of such substances include, but are not limited to: enol ether, enamine, 9-alkylidene xanthan gum, 9-alkylidene-N-alkyl acridin, aromatic vinyl ether, diepoxy ethylene, dimethyl thiophene, aromatic imidazole or lucigenin. In other embodiments of the present invention, the luminescent particles are olefinic compounds containing hydroperoxides or dioxetanes capable of reacting with singlet oxygen to form compounds that can decompose into ketones or carboxylic acid derivatives; a stable dioxetane which can be decomposed by the action of light; acetylenes which can react with singlet oxygen to form diketones; hydrazones or hydrazides which can form azo compounds or azocarbonyl compounds, such as luminol; and aromatic compounds that can form endoperoxides. Specific, non-limiting examples of luminescent particles that may be utilized in accordance with the disclosed and claimed invention are described in U.S. patent No. US5340716, which is hereby incorporated by reference in its entirety. Preferably, the "luminescent particles" may be polymer particles filled with luminescent compounds and lanthanides, which are formed by coating functional groups on a substrate.

The "matrix" according to the present invention is microspheres or microparticles known to the skilled person, of any size, which may be organic or inorganic, which may be expandable or non-expandable, which may be porous or non-porous, which have any density, but preferably have a density close to that of water, preferably are capable of floating in water, and which are made of a transparent, partially transparent or opaque material. The substrate may or may not have a charge, and when charged, is preferably negatively charged. The matrix may be a solid (e.g., polymers, metals, glass, organic and inorganic substances such as minerals, salts and diatoms), oil droplets (e.g., hydrocarbons, fluorocarbons, siliceous fluids), vesicles (e.g., synthetic such as phospholipids, or natural such as cells, and organelles). The matrix may be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silica particles, metal sols, cells and microcrystalline dyes. The matrix is generally multifunctional or capable of binding to photosensitive or luminescent particles through specific or non-specific covalent or non-covalent interactions. Many functional groups are available or incorporated. Typical functional groups include carboxylic acid, acetaldehyde, amino, cyano, vinyl, hydroxy, mercapto, and the like. One non-limiting example of a matrix suitable for use in the present invention is a carboxyl modified latex particle. Details of such substrates can be found in U.S. patent nos. US5709994 and US5780646 (both of which are incorporated herein by reference in their entirety).

The term "chemiluminescent immunoassay kit" as used herein refers to all reagents or combinations of reagents necessary for a chemiluminescent immunoassay.

The term "chemiluminescent immunoassay kit" as used herein refers to a kit or container containing all the reagents or agents necessary for chemiluminescent immunoassay; the chemiluminescence immunoassay kit can be prepared by directly filling all reagents or medicaments which are necessary to be used for chemiluminescence immunoassay into a proper box or container, and can also be prepared by filling the corresponding chemiluminescence immunoassay kit into a proper box or container; the chemiluminescent immunoassay kit may further be used to construct or prepare kits for specific uses, such as for the detection of rheumatoid arthritis.

Embodiments II

As mentioned above, the existing methods for detecting 14-3-3 protein are heterogeneous immunoassays, which have low sensitivity, low accuracy and complicated operation. The inventor finds that the 14-3-3 protein is high in sensitivity, wide in linear range and good in precision by adopting a homogeneous chemiluminescence immunoassay method, washing and other steps are omitted in the detection process, and the operation is simple and convenient. The present invention has been made based on the above findings.

Accordingly, the present invention relates to a chemiluminescent immunoassay kit for detecting 14-3-3eta protein comprising: a first reagent, a second reagent, a calibrator, a low level quality control, and a high level quality control, wherein: the first reagent comprises a luminescent microparticle coated with a first anti-14-3-3 eta protein antibody, the first anti-14-3-3 eta protein antibody being capable of specifically binding to a first epitope of 14-3-3eta protein;

According to the invention, the reagent set further comprises a third reagent comprising avidin and/or streptavidin coated photosensitive microparticles.

According to the invention, the reagent kit further comprises a microplate strip.

In other embodiments of the invention, the reagent kit is in the range of 0.1ng/mL to 20.0ng/mL and the linear correlation coefficient (r) of the reagent kit dose-response curve is not less than 0.990.

The chemiluminescent immunoassay kit for detecting 14-3-3eta protein according to the first aspect of the present invention is a homogeneous chemiluminescent immunoassay kit, which can be understood as being made by packaging the above reagents in a suitable kit or container, or as being made by packaging all reagents or agents necessary for carrying out a chemiluminescent immunoassay in a suitable kit or container; the kit comprises: a first reagent, a second reagent, a calibrator, a low level quality control, and a high level quality control, wherein:

In the invention, the Sequence of the 14-3-3eta protein is shown in Sequence NO. 1. And the second epitope and the first epitope are respectively and independently selected from relative specificity fragments of amino acid fragment 14-3-3eta protein sequences: amino acids 1-6 (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 (141-154 aa).

In the present invention, the first anti-14-3-3 eta protein antibody and the second anti-14-3-3 eta protein antibody are each independently selected from a monoclonal antibody and/or a polyclonal antibody, preferably a monoclonal antibody.

The monoclonal antibody of the invention is capable of specifically binding to an epitope of the 14-3-3eta protein. For example, the monoclonal antibody of the invention is a fragment (epitope) capable of associating with the sequence of the 14-3-3eta protein with relative specificity: 1-6aa, 27-38aa, 71-83aa, 112-154 aa and 141-154 aa.

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

step one, animal immunization

1. First immunization

Selecting 3-5 Balb/c male mice about 8 weeks, immunizing for the first time, mixing and emulsifying 14-3-3eta antigen and Freund's complete adjuvant in equal volume, and injecting the emulsified antigen into the abdominal cavity of each mouse, wherein the immunizing dose is 100 mu g/mouse.

2. Boosting immunity

After the first immunization, the immunization is carried out every 2 weeks, 14-3-3eta antigen and Freund's incomplete adjuvant are mixed and emulsified in equal volume, each mouse is injected with the emulsified antigen in the abdominal cavity, and the immunization dose is 50 mu g/mouse for 3 times. After 2 weeks, a final boost was performed by tail vein injection of 50 μ g of non-emulsified antigen per mouse.

Step two, cell fusion

On the third day after the end of the last booster immunization, mice were sacrificed in a sterile environment and their spleens were removed and the splenocytes were evenly dispersed by an appropriate method. Fusion was performed with mouse myeloma cell SP2/0 under PEG mediation, and the cells were cultured by dropping into a 96-well cell culture plate by limiting dilution.

Step three, antibody detection

After fusion is completed for about 10 days, cell culture supernatants of each well are examined until the clones grow to a suitable size. The specific detection scheme is as follows:

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

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

3) adding an anti-mouse secondary antibody marked by HRP, and fully washing after reaction;

4) TMB substrate was added for reaction for 15min for color development, 2M sulfuric acid was added to stop the reaction and OD450 readings were taken and the well number corresponding to the positive clone was determined.

Step four, cloning

Positive clones were cloned 2-3 times to stabilize the cell lines.

Step five, expanding culture and preparing monoclonal antibody

The monoclonal antibody is prepared by in vitro culture or ascites preparation and other modes.

The method for producing the polyclonal antibody of the present invention is not particularly limited, and the polyclonal antibody can be produced by a method known to those skilled in the art. For example, in some embodiments, 14-3-3eta sheep polyclonal antibody is prepared, comprising:

step one, animal immunization and blood collection

1. First immunization

Regulating the concentration of 14-3-3eta recombinant protein to 2mg/ml, respectively sucking 2ml of complete adjuvant and 2ml of 14-3-3eta recombinant protein into two 5ml syringes, inserting into a three-way tube, alternately pushing the needle tubes to mix uniformly, and performing reciprocating operation until a viscous emulsion is formed. And (3) taking a small amount of emulsified antigen, and dripping the emulsified antigen onto the surface of clear water, wherein the emulsion antigen does not diffuse, which indicates that the emulsification is successful.

Selecting 2 healthy rams, injecting subcutaneously at multiple points, each ram injecting 2ml of emulsified antigen (i.e. 2mg14-3-3eta antigen)

2. Boosting immunity

The first immunization was followed by a booster immunization every 2 weeks for a total of 4 booster immunizations

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 in the same manner.

After the first immunization, the sheep will have lymph node enlargement due to the stimulation of BCG and antigen in the complete adjuvant, and at this time, lymph node injection is carried out by emulsified antigen, and as many lymph nodes as possible are injected.

3. Collecting blood, and separating serum

One week after the last boosting immunization, bleeding is carried out on carotid artery, the whole blood is coagulated, cut into blocks, placed at 37 ℃ for incubation for 1-2 hours, serum is sucked, and centrifugation is carried out at 12000rpm for 5min to remove solids such as blood cells and the like.

Step two, polyclonal antibody purification

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

Dialyzing 14-3-3eta antigen (with different affinity tag from antigen for immunization, such as His tag for immunogen and GST tag for affinity purification) to 0.1M NaHCO₃0.5M NaCl, pH8.3 buffer;

weighing appropriate amount of CNBractivated sepharose 4B (GE) dry powder according to the swelling capacity of each gram of dry powder to be 3ml of gel, coupling 5mg of antigen to each ml of gel, swelling the dry powder by using 1mM HCl, continuously washing the dry powder in a filter flask, generally washing 1ml of gel by using 100ml of 1mM HCl, and washing the protective agent in the gel dry powder;

filtering residual liquid on the gel by using a filter flask, adding the swelled gel into the dialyzed 14-3-3eta antigen solution, slowly stirring, and reacting at 2-8 ℃ overnight to enable the antigen to be combined on the gel through a covalent bond;

filtering the solution supernatant after reaction, adding 10 times of 0.1M Tris.HCl and pH8.0 into the coupled gel, and respectively using the free amino of Tris to separate the unreacted active groups on the gel;

the column was packed with gel, washed with two buffers, 0.1M Tris.HCl, 0.5M NaCl, pH8.5 and 0.1M HAc, 0.5M NaCl, pH4.0, in turn for 10 column volumes, and washed repeatedly 5 times to remove non-covalently bound antigen, to the completion of the affinity column preparation.

2. Polyclonal antibody affinity chromatography purification

Diluting 14-3-3eta antiserum with 3 times volume of physiological saline, and gradually adding saturated ammonium sulfate solution with the same volume as the diluted antiserum under stirring to generate precipitate;

centrifuging the above mixture at 12000rpm for 10min, discarding supernatant, dissolving precipitate with 3-5 times volume of PBS of original antiserum, and filtering with 0.22 μm filter;

balancing 14-3-3eta antigen affinity column with PBS, loading the filtrate onto the affinity column, and washing with PBS until no protein is washed out;

eluting the column with 0.1M glycine, pH3.0 buffer solution, collecting eluate and neutralizing with 3M Tris.HCl, pH 8.5;

dialyzing the eluent by using PBS with the volume of 20 times, changing the dialyzate once every 4 hours, and changing the dialyzate for 2 times in total to obtain the affinity-purified 14-3-3eta polyclonal antibody.

In some particularly preferred embodiments of the invention, the calibrator comprises recombinant antigen 14-3-3eta protein, neonatal bovine serum, and a lyoprotectant. In some preferred embodiments of the present invention, for example, the calibrator is diluted by the calibrator diluent in a proportional gradient to 6 working calibrator solutions at six concentration levels: 0ng/mL, 0.2ng/mL, 0.5ng/mL, 2ng/mL, 5ng/mL, 20 ng/mL.

In some preferred embodiments of the present invention, the low-level quality control product and/or the high-level quality control product further comprises newborn bovine serum.

In some preferred embodiments of the present invention, the low-level quality control product and/or the high-level quality control product further comprises a preservative.

In some embodiments of the invention, the low-level quality control product (lyophilized powder) comprises recombinant antigen 14-3-3eta protein, newborn bovine serum and preservative.

In some embodiments of the invention, the low level quality control product has a quality control range of 10% to 40%; preferably 10% -30%; more preferably 10% to 20%.

In some embodiments of the invention, the high-level quality control product (lyophilized powder) comprises recombinant antigen 14-3-3eta protein, newborn bovine serum and preservative.

In some embodiments of the invention, the high level of quality control is in the range of 10% to 40%; preferably 10% -30%; more preferably 10% to 20%.

In some preferred embodiments of the invention, the first agent and/or the second agent comprises a protein stabilizing agent.

In some preferred embodiments of the invention, the first reagent and/or the second reagent further comprises a phosphate buffer.

In some preferred embodiments of the invention, the first agent and/or the second agent further comprises a preservative.

In some particularly preferred embodiments of the invention, the first reagent comprises luminescent microparticles coated with the first anti-14-3-3 eta protein antibody, a phosphate buffer, a protein stabilizing agent, and a preservative.

In other particularly preferred embodiments of the invention, the second reagent comprises a second anti-14-3-3 eta protein antibody comprising a biotin label capable of specifically binding to a second epitope of 14-3-3eta protein, a phosphate buffer, a protein stabilizer, a preservative.

In some embodiments of the invention, the kit has a dose-response curve linear correlation coefficient (r) within the range of 0.1ng/mL to 20.0ng/mL of not less than 0.990.

According to the invention, the kit further comprises a microwell plate.

In some embodiments of the present invention, the method for preparing the chemiluminescent immunoassay kit for detecting 14-3-3eta protein of the present invention mainly comprises the steps of preparing a first reagent and preparing the first reagent; wherein the first reagent comprises a luminescent microparticle coated with a first anti-14-3-3 eta protein antibody, the first anti-14-3-3 eta protein antibody being capable of specifically binding to a first epitope of 14-3-3eta protein; the second reagent comprises a second anti-14-3-3 eta protein antibody comprising a biotin label capable of specifically binding to a second epitope of the 14-3-3eta protein, said second epitope not overlapping said first epitope.

In some embodiments of the invention, the step of preparing the first agent (first anti-14-3-3 eta protein antibody-coated luminescent particles) comprises:

step S1, mixing a luminescent microsphere solution with a certain concentration and a first anti-14-3-3 eta protein antibody in a coating buffer solution to obtain a first mixture;

and step S2, adding a stop solution into the mixture I to stop the reaction, then adding a sealing solution to seal, and washing to obtain the first reagent.

In some preferred embodiments of the present invention, the stop solution is a sodium borohydride solution, and the concentration of the sodium borohydride solution is 2-40mg/ml, preferably 4-30mg/ml, and more preferably 5-10 mg/ml.

In some preferred embodiments of the invention, the blocking solution is a glycine solution having a concentration of 10-200mg/ml, preferably 20-150mg/ml, more preferably 50-100 mg/ml.

In some embodiments of the invention, the step of the second reagent (second anti-14-3-3 eta protein antibody-labeled biotin) comprises:

step T1, mixing a biotin solution with a certain concentration and a second anti-14-3-eta protein antibody in a labeled buffer solution to obtain a second mixture;

and step T2, dialyzing the second mixture, and diluting the second mixture by using a buffer solution to obtain the second reagent.

In some preferred embodiments of the present invention, the coating buffer solution and the labeling buffer solution are both sodium bicarbonate buffer solutions.

In some embodiments of the present invention, the method for preparing the chemiluminescent immunoassay kit for detecting 14-3-3eta protein of the present invention mainly comprises the steps of preparing a first reagent and preparing the first reagent, wherein the first reagent comprises luminescent particles coated with a first anti-14-3-3 eta protein antibody, a protein stabilizing agent and a preservative, and the first anti-14-3-3 eta protein antibody can be specifically combined with a first epitope of 14-3-3eta protein; the second reagent comprises a second anti-14-3-3 eta protein antibody containing a biotin label capable of specifically binding to a second epitope of the 14-3-3eta protein, which does not overlap with the first epitope, a protein stabilizing agent and a preservative.

In some embodiments of the invention, the step of preparing the first agent (the first anti-14-3-3 eta protein antibody-coated luminescent particles, the protein stabilizing agent, and the preservative) comprises:

and step S2, adding a stop solution into the mixture I to stop the reaction, then adding a sealing solution to seal, and adding a buffer solution, a protein stabilizer and a preservative after cleaning to obtain a first reagent.

In some embodiments of the invention, the step of preparing the second reagent (second anti-14-3-3 eta protein antibody-labeled biotin, protein stabilizer, and preservative) comprises:

and step T2, dialyzing the second mixture, diluting the second mixture by using a buffer solution, and adding a protein stabilizer and a preservative to obtain the second reagent.

In a second aspect of the present invention, the chemiluminescent immunoassay method for detecting 14-3-3eta protein in a sample to be detected according to the present invention is a homogeneous chemiluminescent immunoassay method, which comprises using the chemiluminescent immunoassay kit according to the first aspect of the present invention to determine whether 14-3-3eta protein exists in the sample to be detected and/or determine the content of 14-3-3eta protein.

Similarly, the chemiluminescence immunoassay method for detecting 14-3-3eta protein in a sample to be detected can also comprise the step of judging whether 14-3-3eta protein exists in the sample to be detected and/or determining the content of 14-3-3eta protein by using the chemiluminescence immunoassay reagent kit.

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

In some embodiments of the invention, the method further comprises the step of preparing a 14-3-3eta protein standard working curve before step R1. In some embodiments of the present invention, the step of preparing the 14-3-3eta protein standard working curve comprises: firstly, detecting chemiluminescence signal values of working calibrator solutions containing 14-3-3eta proteins with different concentrations according to steps R1-R4, and then fitting a 14-3-3eta protein standard working curve according to the corresponding relation between the concentrations and the signal values to obtain a functional relation between the concentrations of the 14-3-3eta proteins and the chemiluminescence signal values.

In some further embodiments of the present invention, in step R4, the intensity of the chemiluminescent signal of step R3 is detected, and 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 the present invention, the sample to be tested is reacted under homogeneous chemiluminescence conditions without washing, i.e., without separation and/or washing steps between steps R1 and R2 and between steps R2 and R3.

In some preferred embodiments of the invention, the method further comprises a calibration step: measurements and calibrations were performed using six concentration levels of working calibrator solutions of 0ng/mL, 0.2ng/mL, 0.5ng/mL, 2ng/mL, 5ng/mL, 20 ng/mL.

In still other embodiments of the present invention, the upper limit of the normal range for determining whether 14-3-3eta protein exists in the test sample is 0.2ng/mL by using the kit of the present invention or the chemiluminescent immunoassay kit according to the first aspect of the present invention, and the test sample is determined to be positive if the concentration of 14-3-3eta protein in the test sample is greater than or equal to 0.2ng/mL, and determined to be negative if the concentration of 14-3-3eta protein in the test sample is less than 0.2 ng/mL.

In some embodiments of the present invention, the method for determining whether 14-3-3eta protein exists in a sample to be tested comprises:

(1) mixing a sample to be detected with a first reagent and a second reagent to obtain a first mixture;

(2) mixing the first mixture with a third reagent to obtain a second mixture;

(3) irradiating the second mixture with excitation light with the wavelength of 600-700nm, wherein the excitation light can excite the photosensitive particles to generate singlet oxygen, and the luminescent particles react with the contacted singlet oxygen to generate emitted light with the wavelength of 520-620nm as a detectable chemiluminescence signal;

(4) detecting whether the chemiluminescence signal in the step (4) exists.

In some further specific embodiments of the present invention, the method for determining the content of 14-3-3eta protein comprises the following steps:

step one, making a 14-3-3eta protein standard working curve.

(1) Taking working calibrator solution (six concentration levels are 0ng/mL, 0.2ng/mL, 0.5ng/mL, respectively,

2ng/mL, 5ng/mL, 20ng/mL) with the first reagent and the second reagent to obtain a third mixture;

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

(3) irradiating the fourth mixture by using excitation light with the wavelength of 600-700nm to excite the photosensitive particles to generate singlet oxygen, and reacting the luminescent particles with the contacted singlet oxygen to generate emitted light with the wavelength of 520-620nm as a detectable chemiluminescence signal;

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

(5) and detecting the chemiluminescence signal value (intensity) of the working calibrator solution containing 14-3-3eta protein with different concentrations, and fitting a 14-3-3eta protein standard working curve according to the corresponding relation between the concentration and the signal value to obtain the functional relation between the concentration of the 14-3-3eta protein and the chemiluminescence signal value.

And step two, detecting the content of the 14-3-3eta protein in the sample to be detected.

(2) mixing the first mixture with a third reagent to obtain a second mixture;

(3) irradiating the second mixture by using excitation light with the wavelength of 600-700nm to excite the photosensitive particles to generate singlet oxygen, and reacting the luminescent particles with the contacted singlet oxygen to generate emitted light with the wavelength of 520-620nm as a detectable chemiluminescence signal;

(4) and (4) detecting the intensity of the chemiluminescence signal generated in the step (3), and determining the content of the 14-3-3eta protein in the sample to be detected based on the 14-3-3eta protein standard working curve.

(5) The upper limit of the normal range for judging whether 14-3-3eta protein exists in a sample to be detected by using the reagent kit or the chemiluminescence immunoassay kit according to the first aspect of the invention is 0.2ng/mL, the sample to be detected is judged to be positive when the concentration of 14-3-3eta protein in the sample to be detected is more than or equal to 0.2ng/mL, and the sample to be detected is judged to be negative when the concentration of 14-3-3eta protein in the sample to be detected is less than 0.2 ng/mL.

In a third aspect, the invention provides a chemiluminescent immunoassay system for detecting 14-3-3eta protein, which uses the aforementioned kit of reagents of the invention or the kit of the first aspect of the invention to detect the presence of 14-3-3eta protein in a sample to be detected and/or determine the content of 14-3-3eta protein by the method of the second aspect of the invention.

In some embodiments of the invention, the detection system comprises:

In some embodiments of the invention, the detection system of the invention comprises a sample introduction module, a sample addition arm module, an incubation tray module, a reagent arm module, an excitation and reading module, and a processor module; the sample in the sample feeding module is transferred to a reaction cup on the incubation disc module through the sample adding arm module, the reaction cup rotates to a designated position along with the incubation disc, the reagent in the reagent disc module is transferred to the reaction cup through the reagent arm module, the sample and the reagent in the reaction cup are mixed and incubated in the incubation disc module, and after the incubation is finished, the reaction cup is transferred to the excitation and reading module for optical excitation and reading detection on a luminescent signal generated after the excitation; and finally, the processor module judges whether the 14-3-3eta protein exists in the sample to be detected and/or determines the content of the 14-3-3eta protein according to the existence and/or the intensity of the generated luminescent signal.

In a fourth aspect, the invention provides a chemiluminescent immunoassay method for detecting 14-3-3eta protein, which uses the detection system of the third aspect of the invention and the aforementioned kit of reagents of the invention or the kit of the first aspect of the invention to detect the presence of 14-3-3eta protein in a sample to be detected and/or determine the content of 14-3-3eta protein.

In some embodiments of the invention, the method comprises the steps of:

3) incubating the mixture obtained in step 2);

4) adding a third reagent into the reaction hole;

5) incubating the mixture obtained in step 4);

In some embodiments of the invention, the method comprises the steps of:

3) incubation at 37 ℃ for 15 minutes;

4) adding 175 mu L of a third reagent into the reaction hole;

5) incubation at 37 ℃ for 10 min;

7) the sample concentration was calculated from the standard curve.

2) submitting a calibration task;

3) automatically operated by the detection system.

In other preferred embodiments of the present invention, the method further comprises a quality control program comprising: and fitting by adopting cubic spline interpolation fitting through a processor of the detection system, and automatically giving the concentration value of the sample.

In some embodiments of the present invention, the upper limit of the normal range for determining whether 14-3-3eta protein exists in the test sample is 0.2ng/mL by using the detection system of the third aspect of the present invention and the aforementioned reagent kit of the present invention or the kit of the first aspect of the present invention, and the test sample is determined as positive when the concentration of 14-3-3eta protein in the test sample is greater than or equal to 0.2ng/mL, and determined as negative when the concentration of 14-3-3eta protein in the test sample is less than 0.2 ng/mL.

In a fifth aspect of the present invention, the use of the detection system according to the third aspect of the present invention in detecting the presence and/or amount of 14-3-eta protein in a sample to be tested is understood as a method for determining the presence and/or amount of 14-3-eta protein in a sample to be tested by the chemiluminescence immunoassay method according to the second or fourth aspect of the present invention using the detection system according to the third aspect of the present invention and the aforementioned kit according to the first aspect of the present invention, wherein the sample to be tested is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema fluid and tissues, preferably the sample to be tested is selected from blood, lung fluid, emphysema fluid and tissues, Plasma and serum, and more preferably the sample to be tested is serum.

Similarly, the invention provides the use of a kit according to the invention as described above or a kit according to the first aspect of the invention for detecting the presence and/or amount of 14-3-3eta protein in a test sample, it is understood that the method for determining the presence or absence of the 14-3-3eta protein in the sample to be tested and/or determining the content of the 14-3-3eta protein by the chemiluminescence immunoassay method provided by the second or fourth aspect of the invention using the detection system according to the third aspect of the invention, the sample to be detected is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema effusion and tissue, preferably the sample to be detected is selected from blood, plasma and serum, and further preferably the sample to be detected is serum.

Similarly, the use of the chemiluminescence immunoassay method provided by the invention in the second or fourth aspect of the invention for detecting the presence and/or content of 14-3-3eta protein in a sample to be detected can be understood as a method for determining whether 14-3-3eta protein is present in the sample to be detected and/or determining the content of 14-3-3eta protein by using the detection system provided by the third aspect of the invention and the aforementioned reagent kit of the invention or the kit provided by the first aspect of the invention and by using the chemiluminescence immunoassay method provided by the second or fourth aspect of the invention, wherein the sample to be detected is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, salivary fluid, synovial fluid, emphysema fluid and tissues, preferably the sample to be detected is selected from blood, Plasma and serum, and more preferably the sample to be tested is serum.

The use of the kit according to the invention according to the sixth aspect of the invention or according to the first aspect of the invention for the preparation of a kit for the detection of rheumatoid arthritis is understood to be a method for the preparation of a kit for the detection of rheumatoid arthritis according to the kit according to the invention according to the first aspect of the invention or according to the first aspect of the invention, comprising:

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

step M2, determining whether 14-3-3eta protein exists in the sample to be detected and/or determining the content of 14-3-3eta protein by using the detection system of the third aspect of the invention and/or the method of the second or fourth aspect of the invention;

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

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

In the present invention, an increase 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 subject.

In some preferred embodiments, 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.

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

In the present invention, 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.

Detailed description of the preferred embodiments

In order that the present invention may be more readily understood, the following detailed description will proceed with reference being made to examples, which are intended to be illustrative only and are not intended to limit the scope of the invention. The starting materials or components used in the present invention may be commercially or conventionally prepared unless otherwise specified.

In the method of the present invention, all reagents may be mixed or mixed, and then mixed and/or incubated according to actual needs. Specifically, the temperature of the incubation can be any temperature in the temperature range of 25-45 ℃, and the incubation time can be overnight or 10-20 min.

Example 1:

1. preparation of the first reagent (anti-14-3-3 eta protein antibody coated luminescent particles):

(1) preparation of luminescent and photosensitive particles

The preparation method, composition structure and content of the luminescent particles and photosensitive particles used in the present invention can be found in example 1 of chinese patent CN100429197C (which is incorporated herein by reference in its entirety).

(2) Pretreatment of

Adding 0.2mg of antibody raw material to be treated into dialysis bag (molecular weight cut-off is 14KD), placing dialysis bag into beaker, adding 100 times volume of 0.05M pH9.6 CB dialysis buffer solution into beaker, placing on magnetic stirrer, and dialyzing at 2-8 deg.C. The dialysate is changed at least 1 time, and dialyzed for at least 4-5 hours each time. The dialyzed protein was aspirated and transferred to a clean centrifuge tube, and a sample was taken to determine the protein concentration.

(3) Coating process

3.1 taking 2mg of luminescent particles, adding 0.05M of pH9.6 CB crosslinking buffer solution into a centrifuge tube, centrifuging at 7500rpm for 15min, discarding the supernatant, adding 400ul of crosslinking buffer solution into the centrifuge tube, ultrasonically cleaning the particles, and centrifuging again.

3.2 adding 200ul cross-linking buffer solution to resuspend the particles to make the particle concentration 10mg/ml, adding 0.1mg antibody raw material, mixing, placing the centrifugal tube at 37 ℃, mixing evenly on a vertical rotary mixer at 25-40rpm overnight.

3.3 placing the centrifuge tube into the container to be cooled for 10min at the temperature of 2-8 ℃, and taking 4 mu L of 8mg/ml NaBH₄The solution, immediately added to the centrifuge tube and mixed well, reacted at room temperature for 2 hours on a vertical rotary mixer at 25-40 rpm.

3.4 Add 32. mu.L of 75mg/mL Gly solution to mix well in the centrifuge tube, and react for 1 hour at 25-40rpm on a vertical rotary mixer.

(4) Cleaning of

After the centrifuge tube was weighed and balanced, it was centrifuged at 7500rpm for 15min, the supernatant was discarded, and 0.1M PBST wash buffer pH7.4 was added to perform ultrasonic washing of the microparticles. This was repeated twice and washed once with the microparticle storage buffer.

(5) Preparation of

4mL of working dilution was added to make the working concentration 50. mu.g/mL to prepare the first reagent for use.

2. Preparation of the second reagent (anti-14-3-3 eta protein antibody-labeled biotin):

(1) pretreatment of

Taking 0.2mg of antibody raw material to be treated, filling into a dialysis bag (molecular weight cut-off is 14KD), putting the dialysis bag into a beaker, adding 100 times of 0.1M NaHCO with pH of 8.0 into the beaker₃Dialyzing the buffer solution on a magnetic stirrer at 2-8 deg.C. The dialysate is changed at least 1 time, and dialyzed for at least 4-5 hours each time. The dialyzed protein was aspirated and transferred to a clean centrifuge tube, and a sample was taken to determine the protein concentration.

(2) Marking process

200 μ L0.1M pH8.0 NaHCO was taken₃Adding the labeled buffer solution into a centrifuge tube, adding 0.1mg of the raw material of the anti-14-3-eta protein antibody, and uniformly mixing. Then 8 mul of prepared biotin solution with the concentration of 5mg/mL is added and mixed quickly. The reaction was carried out overnight at 25-40rpm on a vertical rotary mixer at 2-8 ℃.

(3) Dialysis

Putting the biotin labeling solution to be treated into a dialysis bag (the molecular weight cut-off is 14KD), putting the dialysis bag into a beaker, adding 100 times of 0.1M PBS (phosphate buffer solution) with pH value of 7.4 into the beaker, putting the beaker on a magnetic stirrer, and dialyzing at the temperature of 2-8 ℃. The dialysate is changed at least 1 time, and dialyzed for at least 4-5 hours each time.

(4) Preparation of

Add 20mL of working dilution to a working concentration of 1. mu.g/mL to make a second reagent ready for use.

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

1.1 preparation of a calibrator diluent: weighing 4.77g HEPES and 1.7g NaCl, adding 160g purified water, mixing for 30min, adjusting pH to 7.4 + -0.2 with 1M concentrated hydrochloric acid and 1M NaOH solution, and adding proclin 3000.1g, BSA 30g and 1M MgCl₂0.5mL、0.1M MgCl₂0.1mL, stirring for 30min, adding purified water to weight of 200g, re-measuring pH, and keeping at 2-8 deg.C.

1.2 preparation of calibrator: diluting the product calibration substance into a working calibration substance according to a proportional gradient, and calibrating the antibody concentration of the product calibration substance by the working calibration substance to finish the preparation of the calibration substance.

4. And (3) experimental operation:

the components are assembled into a 14-3-3eta protein measuring box, and then loaded on a LiCA500 full-automatic light-activated chemiluminescence immunoassay analyzer produced by Boyang Biotechnology (Shanghai) Limited, and the detection steps are set as follows:

1) a sample adding Tip sucks 20 mu L of calibrator to a reaction micropore plate;

2) a sample adding Tip sucks 25 mu L of reagent I into a reaction micropore plate;

3) a sample adding Tip sucks 25 mu L of reagent II into a reaction micropore plate;

4) horizontally oscillating and uniformly mixing for 20 seconds, and then incubating for 17min at 37 ℃;

5) the sample adding Tip sucks 175 mu L of mixed liquid (matched with an instrument) containing the streptavidin modified photosensitive particles into a reaction microporous plate;

6) horizontally oscillating and uniformly mixing for 20 seconds, and then incubating for 15min at 37 ℃;

7) under the irradiation of 680nm exciting light generated by the instrument, the photosensitive particles are induced to be activated and release active oxygen ions in a high energy state. The active oxygen ions in the high energy state are captured by the luminescent particles at a close distance, thereby transferring energy to activate the luminescent compounds in the luminescent particles. After several microseconds, the luminescent compound in the luminescent particles releases 612nm high-energy level red light, and a single photon counter is used for measuring the high-energy level photons;

8) fitting a standard curve according to the signal value of the calibrator and a five-parameter fitting method to obtain an equation between the signal value and the concentration of the 14-3-3eta protein;

9) and detecting the sample to be detected according to the steps 1) to 7), and calculating the concentration of the 14-3-3eta protein in the sample to be detected according to the equation in 8).

5. Results of the experiment

5.1 testing the Linear Range of the working calibrators

The linear range of the working calibrators was checked and the results are shown in table 1.

TABLE 1

As can be seen from Table 1, the standard curve degree fit equation R2 > 0.99. Very good linearity is obtained in the measurement range of 0.2-20 ng/mL.

5.2 sensitivity

The measurement was repeated 20 times with the low concentration sample, and the results are shown in Table 2. As can be seen from Table 2, the precision of the detection result was 7.42%, and the functional sensitivity was 0.2 ng/mL. When the sample contains 0.2ng/mL detection target substance, a signal value different from that of a normal population (a sample with zero concentration) can be generated, and the sensitivity is high.

TABLE 2

5.3 evaluation results

The prepared kit is used for evaluating 46 cases of rheumatoid cases and 50 cases of normal control group samples, and the results are shown in table 3 and figure 1, and meanwhile, the results are shown in table 4 and figure 1 when other arthritis samples are detected.

TABLE 3

TABLE 4

And (4) conclusion: the concentration of 14-3-3eta protein in samples of the health examination group is lower than 0.2ng/mL, and the concentration of 14-3-3eta protein detected by 19 samples of the rheumatoid arthritis group is higher than 0.2ng/mL, which indicates that 14-3-3eta subtype protein is highly expressed in arthritis patients and is detected to be remarkably increased in serum of RA patients. Meanwhile, when other arthritis samples are detected, the concentration of the 14-3-3eta protein is lower than 0.2ng/mL, so that the detection result of the 14-3-3eta protein is considered to be related to clinical diagnosis of rheumatoid arthritis, and the detection of the protein is prompted to improve the diagnosis of the rheumatoid arthritis and help the clinical diagnosis of RA.

Example 2: the kit for detecting 14-3-3eta protein in a sample to be detected is adopted to detect and detect the 14-3-3eta protein in the sample to be detected

1. The kit for detecting the 14-3-3eta protein in the sample to be detected mainly comprises the following components:

a first reagent: 2 × 50 test/box, 1.5 mL/vial × 2 vial; 2X 100 test/box, 3.0 mL/vial X2 vial. Contains luminescent particles coated by an anti-14-3-3 eta protein antibody, phosphate buffer solution, protein stabilizer and preservative, and is stored at the temperature of 2-8 ℃ in a dark place.

A second reagent: 2 × 50 test/box, 1.5 mL/vial × 2 vial; 2X 100 test/box, 3.0 mL/vial X2 vial. Contains biotin-labeled anti-14-3-3 eta protein antibody, phosphate buffer solution, protein stabilizer and preservative, and is stored at the temperature of 2-8 ℃ in a dark place.

Calibrator (lyophilized powder): 2 × 50 test/box, 0.5 mL/vial, total 6 vials; 2X 100 test/box, 0.5 mL/vial, total 6 vials. Contains recombinant antigen 14-3-3eta protein, newborn calf serum and freeze-drying protective agent. The six concentration levels were: 0ng/mL, 0.2ng/mL, 0.5ng/mL, 2ng/mL, 5ng/mL, 20ng/mL, the specific concentration is fixed, 2-8 ℃ storage.

Low level quality control (lyophilized powder): 2 × 50 test/box, 0.5 mL/vial, 2 vials in total; 2X 100 test/box, 0.5 mL/vial, 2 vials in total. Contains recombinant antigen 14-3-3eta protein, newborn calf serum and preservative, and has the target value concentration: (0.20 +/-0.05) ng/mL, batch specificity, quality control range of quality control see fixed value list, and preservation at 2-8 ℃.

High-level quality control (lyophilized powder): 2 × 50 test/box, 0.5 mL/vial, 2 vials in total; 2X 100 test/box, 0.5 mL/vial, 2 vials in total. Contains recombinant antigen 14-3-3eta protein, newborn calf serum and preservative, and has the target value concentration: (5.0 +/-1.0) ng/mL, batch specificity, quality control range of quality control see fixed value list, and preservation at 2-8 ℃.

Other essential materials (see Table 5)

TABLE 5

2. Preparing a sample to be tested:

(1) the sample to be detected is serum;

(2) collecting all blood samples according to a universal venipuncture measure;

(3) ensuring that the sample has been sufficiently agglutinated before centrifugation;

(4) if at room temperature, the sample is preserved in the plugged test tube for no more than 8 hours; the product can be used for a short time and can be stored for 3 days at the temperature of 2-8 ℃; samples needing to be stored for a long time can be stabilized at-20 ℃ for 3 months, repeated freeze thawing is avoided, and the samples are fully mixed after being thawed;

(5) the lipemic sample is preferably centrifuged by ultracentrifugation.

3. Inspection method

The kit in the embodiment can carry out quantitative determination on the 14-3-3eta protein, and during detection, a calibrator is used for calibration, and a quality control material is used for quality control.

4. Reagent preparation

The reagent can be used after opening the bottle; the calibrator and the quality control material need to be dissolved by 0.5mL of distilled water or deionized water, balanced to the ambient temperature and uniformly mixed for use.

5. Conditions of the experiment

The detection process is completed by a LiCA500 automatic light-activated chemiluminescence analysis system produced by Boyang Biotechnology (Shanghai) Limited company in a full-automatic manner, and a detection result is output. The specific process steps are as follows:

(1) respectively adding 25 mu L of sample or calibrator and quality control material into the reaction hole;

(2) adding 25 mu L of first reagent and 25 mu L of second reagent into the reaction hole in sequence;

(3) incubation at 37 ℃ for 15 minutes;

(4) adding 175 mu L of the third reagent;

(5) incubation at 37 ℃ for 10 min;

(6) irradiating the micropores by laser and calculating the quantity of light photons emitted by each hole;

(7) the sample concentration was calculated from the standard curve.

6. Calibration procedure

In the initial experiment, the calibrator in the human 14-3-3eta protein assay kit is used for calibrating the instrument, and the calibration period is 28 days.

The calibration method comprises the following steps:

(1) the LiCA500 is used for activating the operation software of the chemiluminescence analysis system automatically, the calibration program of the human 14-3-3eta protein assay kit is entered, and the calibrator 1-calibrator 6 is used for multi-well assay and calibration.

(2) And submitting a calibration task.

(3) Operated automatically by the instrument.

7. Quality control program

The kit of this example was used as a control. The test of quality control material should be carried out during the experiment, single-hole measurement is carried out, the test result should be in the required quality control range, if the test result exceeds the range, the test result is not credible, and the test is necessary to be carried out again, and if necessary, the test is carried out again.

8. Calculation of the results of the experiment

The concentration value of the sample is directly given by adopting a data processing program (fitting type: cubic spline interpolation fitting) of LiCA500 automatic light excitation chemiluminescence detector software.

9. Reference interval

The upper limit of the normal range of the kit of this example is 0.2 ng/mL.

The results are explained according to the following principles: more than or equal to 0.2ng/mL is positive, and less than 0.2ng/mL is negative.

10. Results of the experiment

Through experiments, the performance indexes of the kit of the embodiment are as follows:

blank limit: not higher than 0.1 ng/mL.

Accuracy: adding antigen with known concentration into serum sample to obtain high and low concentration (in the linear range of kit measurement) recovered sample with recovery rate in the range of 85-115%.

Linearity: the reagent kit is in the range of 0.1ng/mL-20.0ng/mL, and the linear correlation coefficient (r) of the reagent kit dose-response curve is not lower than 0.990.

Precision: internal precision of analysis: coefficient of Variation (CV) is not more than 10.0%;

batch precision: the Coefficient of Variation (CV) is not more than 15.0%.

Difference between the calibrator and the quality control bottle: the Coefficient of Variation (CV) is not more than 10.0%.

Quality control material assignment validity: the measurement results are all within the range specified in the kit.

Specificity: the calculated cross-reactivity of the results of the measurement of six proteins 14-3-3. alpha./beta., 14-3-3. gamma., 14-3-3. delta./zeta., 14-3-3. epsilon., 14-3. theta./tau., 14-3-3. sigma. on the kit at a concentration of 200ng/mL was less than 0.1%, and the results are shown in Table 6.

TABLE 6

It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.

Sequence listing

<110> Beijing Koume Biotechnology Ltd

<120> chemiluminescence immunoassay kit for detecting 14-3-3eta protein and application thereof

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>246

<212>PRT

<213> (14-3-3 eta 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

1. A chemiluminescent immunoassay kit for detecting 14-3-3eta protein comprising: a first reagent, a second reagent, a calibrator, a low level quality control, and a high level quality control, wherein:

2. The kit of claim 1, wherein the calibrator, the low-level quality control, and the high-level quality control each comprise recombinant antigen 14-3-3eta protein.

3. The kit of claim 1 or 2, wherein the calibrator is diluted by the calibrator diluent to a working calibrator solution at six concentration levels according to a proportional gradient, preferably wherein the calibrator is diluted by the calibrator diluent to a working calibrator solution at six concentration levels according to a proportional gradient: 0ng/mL, 0.2ng/mL, 0.5ng/mL, 2ng/mL, 5ng/mL, 20 ng/mL.

4. The kit of any one of claims 1 to 3, wherein the calibrator further comprises neonatal bovine serum and a lyoprotectant.

5. The kit according to any one of claims 1 to 4, wherein the low level quality control substance has a quality control range of 10% to 40%, preferably 10% to 30%, more preferably 10% to 20%.

6. The kit of any one of claims 1-5, wherein the low level quality control has a target value concentration of 0.20 ± 0.05 ng/mL.

7. The kit according to any one of claims 1 to 6, wherein the high level quality control product has a quality control range of 10% to 40%, preferably 10% to 30%, more preferably 10% to 20%.

8. The kit of any one of claims 1 to 7, wherein the high level quality control substance has a target value concentration of 5.0 ± 1.0 ng/mL.

9. The kit according to any one of claims 1 to 8, wherein the concentration of the luminescent particles coated with the first anti-14-3-3 eta protein antibody in the first reagent is 10 to 200 μ g/mL, preferably 20 to 150 μ g/mL, more preferably 30 to 100 μ g/mL, most preferably 40 to 80 μ g/mL; and/or the concentration of the biotin-labeled second anti-14-3-3 eta protein antibody in the second reagent is 0.1-8 μ g/mL, preferably 0.2-6 μ g/mL, more preferably 0.4-4 μ g/mL, and most preferably 0.6-2 μ g/mL.

10. The kit of any one of claims 1 to 9, wherein the kit further comprises a third reagent comprising an avidin and/or streptavidin coated photosensitive microparticle.

11. The kit of any one of claims 1-10, wherein the kit further comprises a microwell plate.

12. The kit of any one of claims 1 to 11, wherein the calibrator, the low-level quality control, and/or the high-level quality control are lyophilized powders.

13. The kit of any one of claims 1 to 12, wherein the low level and/or high level quality control comprises neonatal bovine serum.

14. The kit of any one of claims 1-13, wherein the first reagent and/or the second reagent comprises a protein stabilizing agent; preferably, the protein stabilizing agent is bovine serum albumin.

15. The kit of any one of claims 1-14, wherein the luminescent particles in the first reagent are polymeric particles filled with a luminescent compound and a lanthanide that is capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal.

16. The kit according to any one of claims 10 to 15, wherein the photosensitive particles in the third reagent are polymer particles filled with a photosensitive compound capable of generating singlet oxygen in an excited state.

17. The kit according to any one of claims 10 to 16, wherein the concentration of the avidin and/or streptavidin-coated photosensitive microparticles in the third reagent is 5 to 20 μ g/mL, preferably 8 to 15 μ g/mL, more preferably 10 to 12 μ g/mL.

18. The kit according to claims 1 to 17, wherein the amino acid SEQUENCE of the 14-3-3eta protein is shown as SEQUENCE No. 1.

19. The kit of claims 1-18, wherein the kit has a kit dose-response curve linear correlation coefficient (r) of not less than 0.990 in the range of 0.1ng/mL to 20.0 ng/mL.

20. A chemiluminescent immunoassay method for detecting 14-3-3eta protein in a test sample, which comprises using the chemiluminescent immunoassay kit according to any one of claims 1 to 19 to determine the presence of 14-3-3eta protein in the test sample and/or to determine the content of 14-3-3eta protein.

21. The method of claim 20, wherein the method comprises:

22. The method according to claim 21, further comprising the step of preparing a 14-3-3eta protein standard working curve before step R1.

23. The method according to claim 22, wherein in step R4, the intensity of the chemiluminescent signal of step R3 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.

24. The method according to any one of claims 21 to 23, wherein there is no separation and/or washing step between steps R1 and R2 and between steps R2 and R3.

25. The method as claimed in any one of claims 21 to 24, wherein in step R3, the second mixture is irradiated with excitation light with a wavelength of 600-700nm to excite the photosensitive particles to generate singlet oxygen, and the luminescent particles react with the singlet oxygen to generate emission light with a wavelength of 520-620 nm.

26. A method according to any of claims 21-25, characterized in that the method further comprises a calibration step: duplicate wells were assayed and calibrated using working calibrator solutions at six concentration levels of 0ng/mL, 0.2ng/mL, 0.5ng/mL, 2ng/mL, 5ng/mL, 20 ng/mL.

27. A method according to any one of claims 21 to 26, further comprising a quality control step of fitting using a cubic spline interpolation fit to give the concentration values of the sample.

28. The method according to any one of claims 21 to 27, wherein the chemiluminescence immunoassay kit according to any one of claims 1 to 19 is used for determining whether the upper limit of the normal range of the 14-3-3eta protein in the sample to be detected exists is 0.2ng/mL, and the chemiluminescence immunoassay kit is determined to be positive when the concentration of the 14-3-3eta protein in the sample to be detected is greater than or equal to 0.2ng/mL, and determined to be negative when the concentration of the 14-3-3eta protein in the sample to be detected is less than 0.2 ng/mL.

29. A chemiluminescent immunoassay system for detecting 14-3-3eta protein, which uses the kit of any one of claims 1 to 19 and/or the method of any one of claims 20 to 28 to detect the presence of 14-3-3eta protein in a sample to be tested and/or to determine the content of 14-3-3eta protein.

30. The detection system according to claim 29, wherein the system comprises:

31. The detection system of claim 30, wherein the processor fits using a cubic spline interpolation fit to directly give the concentration values of the sample.

32. A chemiluminescent immunoassay for detecting 14-3-3eta protein using the detection system of any one of claims 29 to 31 and the chemiluminescent immunoassay kit of any one of claims 1 to 19 to detect the presence of 14-3-3eta protein and/or determine the content of 14-3-3eta protein in a test sample by the method of any one of claims 20 to 28.

33. The method of claim 32, comprising the steps of:

3) incubating the mixture obtained in step 2);

4) adding a third reagent into the reaction hole;

5) incubating the mixture obtained in step 4);

34. The method of claim 33, comprising the steps of:

3) incubation at 37 ℃ for 15 minutes;

4) adding 175 mu L of a third reagent into the reaction hole;

5) incubation at 37 ℃ for 10 min;

7) the sample concentration was calculated from the standard curve.

35. The method according to any of claims 32-34, further comprising a calibration procedure comprising the steps of:

2) submitting a calibration task;

3) automatically operated by the detection system.

36. The method of any one of claims 32-35, further comprising a quality control program comprising: and fitting by adopting cubic spline interpolation fitting through a processor of the detection system, and automatically giving the concentration value of the sample.

37. The method according to any one of claims 32 to 36, wherein the upper limit of the normal range for determining whether 14-3-3eta protein exists in the sample to be tested is 0.2ng/mL by using the detection system according to any one of claims 29 to 31 and the chemiluminescence immunoassay kit according to any one of claims 1 to 19, and the method is determined to be positive when the concentration of 14-3-3eta protein in the sample to be tested is greater than or equal to 0.2ng/mL, and determined to be negative when the concentration of 14-3-3eta protein in the sample to be tested is less than 0.2 ng/mL.

38. Use of a test system according to any one of claims 29 to 31 and a kit according to any one of claims 1 to 19 or a method according to any one of claims 20 to 28 or 32 to 37 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 and tissue.

39. Use of a kit according to any one of claims 1 to 19 for 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;

40. The use according to claim 39, 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.

41. The use according to claim 39, wherein an increase 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.

42. The use according to claim 39, 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.

43. The use of claim 39, 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.

44. The use of claim 39, 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.

45. The use according to any one of claims 39 to 44, wherein in step M2, the presence or absence of 14-3-3eta protein in a test sample is determined and/or the content of 14-3-3eta protein is determined using the test system according to any one of claims 29 to 31 and/or by the method according to any one of claims 20 to 28 or claims 32 to 37.