CN110579597A

CN110579597A - Heterogeneous chemiluminescence immunoassay kit for detecting 14-3-3eta protein and application thereof

Info

Publication number: CN110579597A
Application number: CN201810812808.8A
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: 2018-05-23
Filing date: 2018-07-23
Publication date: 2019-12-17

Abstract

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

Description

Heterogeneous 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 heterogeneous 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 heterogeneous immunoassay method for detecting 14-3-3 protein has low sensitivity and low accuracy. 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 the sensitivity is low and the specificity is poor.

therefore, research and development of a 14-3-3eta protein heterogeneous immunoassay kit with high sensitivity and good specificity are urgently needed.

disclosure of Invention

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

To this end, the present invention provides in a first aspect a heterogeneous chemiluminescent immunoassay kit for the detection of 14-3-3eta protein comprising:

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

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

A component d comprising a second label capable of reacting with a substrate or capable of catalyzing the substrate to generate a detectable signal and a third antibody or binding fragment thereof directly or indirectly bound thereto, said third antibody or binding fragment thereof being capable of specifically binding to a second epitope of the 14-3-3eta protein, and said second epitope and said first epitope are non-overlapping.

In the present invention, the third antibody directly or indirectly binding to the second label and the second antibody directly or indirectly binding to the first label are the same antibodies binding to the same epitope of 14-3-3eta protein.

in the present invention, the first antibody, the second antibody and the third antibody are each independently selected from a monoclonal antibody and/or a polyclonal antibody, preferably a monoclonal antibody.

According to some embodiments of the invention, the kit further comprises 14-3-3eta protein as a calibrator diluted by a calibrator diluent in a proportional gradient to working calibrator solutions of different concentrations.

In some embodiments of the invention, the first antibody or binding fragment thereof is bound to one member of a specific binding pair member and the solid support is bound to the other member of the specific binding pair member; preferably, the first antibody or binding fragment thereof is bound to biotin and the solid support is bound to streptavidin.

In some embodiments of the invention, the second antibody or binding fragment thereof binds to one member of a specific binding pair member and the first label binds to the other member of the specific binding pair member; preferably, the second antibody or binding fragment thereof is bound to biotin and the first label is bound to streptavidin.

In some embodiments of the invention, the third antibody or binding fragment thereof binds to one member of the specific binding pair member and the second label binds to the other member of the specific binding pair member; preferably, the third antibody or binding fragment thereof binds to biotin and the second label binds to streptavidin.

According to some embodiments of the invention, the first label and the second label are each independently a chemiluminescent label, and the first label and the second label are each independently selected from luminol and derivatives thereof, isoluminol and derivatives thereof, acridinium ester and derivatives thereof, adamantane, rare earth elements, and ruthenium bipyridine complexes.

according to further embodiments of the present invention, the first label and the second label are each independently a chemiluminescent catalyst, and the first label and the second label are each independently selected from horseradish peroxidase and/or alkaline phosphatase.

in the invention, the solid phase carrier is selected from magnetic microspheres, plastic microparticles, microporous plates, glass, capillaries and nylon; magnetic microspheres are preferred.

in some embodiments of the invention, the magnetic microspheres have a particle size of 0.05 to 50 microns; preferably 0.1 to 40 microns; more preferably 5-20 microns.

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

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

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

According to the invention, the reagent kit further comprises component c, a substrate solution.

According to some embodiments of the invention, the substrate solution comprises a solution a and a solution B.

In some embodiments of the invention, the a solution is a hydrogen peroxide solution.

In some embodiments of the invention, the B solution is a sodium hydroxide solution.

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

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

In a second aspect, the present invention provides a heterogeneous chemiluminescent immunoassay kit for the detection of 14-3-3eta protein comprising a kit of heterogeneous chemiluminescent immunoassay reagents according to the first aspect of the present invention.

According to the invention, the kit comprises the following components:

a component a: comprising a magnetic microsphere directly or indirectly linked to a first antibody;

And (b) component b: comprising a second antibody directly linked or indirectly linked to a first label or a second label.

In some embodiments of the invention, the kit further comprises component d: comprising a third antibody directly linked or indirectly linked to a first label or a second label.

in some preferred embodiments of the invention, the first and second labels are the same.

in other preferred embodiments of the invention, the third antibody is the same as the second antibody.

in some embodiments of the invention, the kit further comprises component c: which comprises a substrate solution comprising a hydrogen peroxide solution and a sodium hydroxide solution.

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

According to the invention, the method comprises:

Step T1, mixing the sample to be tested with the component a and the combination b to obtain a third mixture;

step T2, mixing the third mixture with component d to obtain a fifth mixture;

Step T3, mixing the fifth mixture with component c to obtain a sixth mixture which generates a detectable signal;

And step T4, detecting the existence and/or the intensity of the chemiluminescence signal in the step T3, 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 embodiments of the invention, the method further comprises the step of preparing a standard working curve for the 14-3-3eta protein prior to step T1.

In some further embodiments of the present invention, in step T4, the intensity of the chemiluminescent signal of step T3 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.

the fourth aspect of the invention provides a method for detecting 14-3-3eta protein, which is characterized in that the kit of the second aspect is adopted, and the method comprises the following steps:

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

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

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

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

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

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

In a fifth aspect, the present invention provides a use of the kit of reagents for heterogeneous chemiluminescent immunoassay according to any one of the first aspect or the kit of reagents for heterogeneous chemiluminescent immunoassay according to the second aspect of the present invention or the method according to the third aspect of the present invention for detecting the presence and/or amount of 14-3-3eta protein in a sample selected from the group consisting of blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema fluid and tissue.

In a sixth aspect, the present invention provides a kit 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 specific embodiments of the invention, the relative amount of 14-3-3eta protein in the test sample compared to a pre-treatment sample from the same test subject is indicative of the efficacy of the treatment regimen.

in some specific embodiments of the present invention, in step M2, the method according to the third 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.

In a seventh aspect, the present invention provides a use of the heterogeneous chemiluminescent immunoassay kit according to the first aspect of the present invention or the heterogeneous chemiluminescent immunoassay kit according to the second aspect of the present invention or the heterogeneous chemiluminescent immunoassay method according to the third or fourth aspect of the present invention in a chemiluminescent analyzer.

an eighth aspect of the present invention provides the chemiluminescent immunoassay analyzer in the above application, which comprises:

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

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

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

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

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

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

A ninth aspect of the present invention provides a method of controlling a chemiluminescent immunoassay analyzer according to the eighth aspect of the present invention comprising the steps of:

Step T2, mixing the third mixture with component d to obtain a fifth mixture;

The tenth aspect of the present invention provides a method for detecting 14-3-3eta protein, which method employs the kit according to the second aspect of the present invention and the chemiluminescent immunoassay analyzer according to the eighth aspect of the present invention, comprising the steps of:

In an eleventh aspect, the present invention provides a use of the kit of reagents for heterogeneous chemiluminescent immunoassay according to the first aspect of the present invention or the kit of reagents for heterogeneous chemiluminescent immunoassay according to the second aspect of the present invention or the method according to the third or fourth aspect of the present invention or the chemiluminescent immunoassay according to the eighth aspect of the present invention or the method according to the ninth or tenth 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.

The heterogeneous chemiluminescence immunoassay kit for detecting 14-3-3eta protein provided by the invention is prepared by adopting a first antibody and a second antibody which can be specifically combined with 14-3-3eta protein, and a third antibody which is an isogenous antibody combined with the second antibody and has the same epitope of 14-3-3eta protein, compared with the prior art, the heterogeneous chemiluminescence immunoassay kit has the following beneficial effects:

According to the kit for detecting 14-3-3eta protein, the first marker system and the second marker system are arranged, when the kit is used for detecting 14-3-3eta protein, firstly, sample adding is carried out for one time, a sample to be detected is mixed with the first marker system and the magnetic microsphere system, the mixture is washed after incubation reaction, then, sample adding is carried out for the second time, the second marker system is added again, the double-antibody sandwich compound combined by the antibody-antigen-antibody is thoroughly formed, and then detection is carried out, so that the sensitivity of sample detection can be improved, and the sample with extremely high antigen concentration cannot be accurately detected.

according to the method for detecting the 14-3-3eta protein, the marker system is added in two steps of sample adding, so that a double-antibody sandwich compound is formed in the first step of reaction, the problem of antigen loss in the cleaning process is solved, and all antigens can react with the antibody to form a sandwich compound, so that the detection sensitivity of a low-value sample can be ensured; and the label system is continuously added in the second step of reaction, if the content of the antigen in the sample is extremely high, and the antigen which is not combined with the label system exists on the surface of the magnetic microsphere, the antigen which is combined with the magnetic microsphere system in the second step of reaction and the newly added label system are fully reacted and combined, so that the antigen which is combined with the magnetic microsphere system in the sample can form a double-antibody sandwich compound, and the HOOK effect is avoided. The method has the advantages of high sensitivity and no HOOK effect.

The kit for detecting 14-3-3eta protein disclosed by the invention is in a double-antibody sandwich mode, and utilizes the advantages of high sensitivity, wide linear range, good precision and the like of a heterogeneous chemiluminescence immunoassay platform, so that the diagnosis rate is improved for clinically diagnosing rheumatoid diseases, and auxiliary detection is provided for preventing rheumatoid diseases and finding the rheumatoid diseases in advance.

the application of the kit of the invention to a heterogeneous homogeneous immunoassay analyzer 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 is suitable for heterogeneous chemiluminescence immunoassay analyzers, and 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.

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 "heterogeneous" is used herein in the english language to define "heterology" and means that the bound antigen-antibody complex and the remaining free antigen or antibody must be separated for detection.

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, acceptors or donors, can be conjugated to any of the members 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 term "heterogeneous chemiluminescent immunoassay kit" as used herein refers to all reagents or combinations of reagents necessary for a heterogeneous chemiluminescent immunoassay.

The term "HOOK effect" as used herein means a phenomenon of false negative due to an inappropriate antigen-antibody ratio, wherein antibody excess is called prozone effect; antigen overdose is called the postzone effect.

Embodiments II

the present inventors have found that inaccurate detection occurs when the 14-3-3eta (eta) protein antigen concentration is too high by a one-step heterogeneous chemiluminescence immunoassay, for example, when a sandwich assay is used, a second antibody linked to a label is added simultaneously with a solid support linked to a first antibody, and a "sandwich" complex of the first antibody-antigen (14-3-eta protein) -second antibody is formed after the incubation reaction is completed. If the concentration of the antigen (14-3-3 eta protein) in the sample is extremely high, the obvious HOOK effect can be produced, namely, when the concentration of the antigen to be detected in the sample is too high, the excessive antigen (14-3-3 eta protein) is respectively combined with the solid phase carrier connected with the first antibody and the second antibody connected with the first marker, and no sandwich compound is formed any more, so that the detection result is lower than the actual content of the 14-3-3eta protein, and the HOOK effect is produced. However, the problem of low sensitivity is caused by adopting a two-step method, namely, in the first step of sample application, only the solid phase carrier connected with the first antibody is added, so that the antigen (14-3-3 eta protein) is fully reacted and combined with the solid phase carrier connected with the first antibody, and then the second step of adding the second antibody connected with the label after cleaning is carried out, so as to form a ' sandwich ' compound of the first antibody-antigen (14-3-3 eta protein) -second antibody, although the possibility of HOOK effect is avoided, in the first step of sample application reaction of the two-step method, only the antigen (14-3-3 eta protein) in the sample is reacted with the solid phase carrier connected with the first antibody, and the ' sandwich ' compound ' combined by the first antibody-antigen (14-3-3 eta protein) -second antibody is not formed, therefore, a certain amount of antigen (14-3-3. eta. protein) is lost during the washing process, resulting in a decrease in detection sensitivity.

On the basis of the research findings, the invention is improved on the basis of the one-step method and the two-step method, the first marker system and the second marker system are arranged in the kit, when the kit is used for detecting 14-3-3eta protein antigen, sample adding is firstly carried out for one time, a sample to be detected is mixed with the first marker system and the solid phase carrier system, the sample is washed after incubation reaction, then sample adding is carried out for the second time, the second marker system is added again, a double-antibody sandwich compound combined by antibody-antigen-antibody is thoroughly formed, and then detection is carried out, so that the problem of HOOK effect is avoided, and the problem of sensitivity reduction caused by certain antigen loss in the washing process is also avoided.

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

In some embodiments of the invention, the steps further comprise measuring the amount of 14-3-3eta protein or fragment thereof or an immune complex formed by the 14-3-3eta protein or fragment thereof and at least one antibody; and further comparing the measured amount of the 14-3-3eta protein or fragment thereof or the 14-3-3eta protein or fragment thereof and at least one antibody forming immune complexes with the amount of the 14-3-3eta protein or fragment thereof or the 14-3-3eta protein or fragment thereof and at least one antibody forming immune complexes in a normal control sample, a rheumatoid arthritis control sample or a pre-treatment sample from the same subject.

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

In some embodiments, the amount of immunocomplexes formed by the 14-3-3eta protein or fragment thereof or the 14-3-3eta protein or fragment thereof and at least one antibody measured is compared to the amount of immunocomplexes formed by the 14-3-3eta protein or fragment thereof or the 14-3-3eta protein or fragment thereof and at least one antibody in a normal control sample. For example, an increase in the content of the immune complex formed by the 14-3-3eta protein or fragment thereof or the 14-3-3eta protein or fragment thereof and at least one antibody in the test sample compared to a normal control sample is a diagnostic indicator of rheumatoid arthritis in the test subject. In some embodiments, the amount of immunocomplexes formed by the 14-3-3eta protein or fragment thereof or the 14-3-3eta protein or fragment thereof and at least one antibody measured is compared to the amount of immunocomplexes formed by the 14-3-3eta protein or fragment thereof or the 14-3-3eta protein or fragment thereof and at least one antibody in a rheumatoid arthritis control sample. For example, the relative amount of the immune complex formed by the 14-3-3eta protein or fragment thereof or the 14-3-3eta protein or fragment thereof and at least one antibody in the test sample as compared to a rheumatoid arthritis control sample is a prognostic indicator of rheumatoid arthritis in the test subject.

In some embodiments, the amount of 14-3-3eta protein or fragment thereof or the amount of immune complexes formed by the 14-3-3eta protein or fragment thereof and at least one antibody measured is compared to the amount of immune complexes formed by the 14-3-3eta protein or fragment thereof or the 14-3-3eta protein or fragment thereof and at least one antibody in a pre-treatment sample from the same test subject. For example, the relative amount of the 14-3-3eta protein or fragment thereof or the immune complex formed by the 14-3-3eta protein or fragment thereof and at least one antibody 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.

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

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

In some embodiments of the invention, one of the first and second antibodies is bound directly or indirectly to a first label and the other is bound directly or indirectly to a solid support; the first label is capable of reacting with the substrate to generate a chemiluminescent signal or capable of catalyzing the reaction of the substrate to generate a chemiluminescent signal.

In some embodiments of the invention, the antibodies further comprise a third antibody capable of specifically binding to the first or second epitope of the 14-3-3eta protein; the third antibody is directly or indirectly bound to a second label.

It will be appreciated by those skilled in the art that the third antibody of the present invention, which directly or indirectly binds to the second label, and the first or second antibody, which directly or indirectly binds to the first label, are monoclonal antibodies that bind to the same epitope of the 14-3-3eta protein.

In the present invention, the second marker may be the same as or different from the first marker.

In the invention, the first marker and the second luminescent marker are luminescent markers which are respectively and independently selected from luminol and derivatives thereof, isoluminol and derivatives thereof, acridinium ester and derivatives thereof, adamantane, rare earth elements and bipyridine ruthenium complexes. Preferably, the acridinium ester and derivatives thereof comprise acridinium ester, acridinium ester sulfonamide, acridinium ester p-methyl sulfonamide, acridinium ester trifluoromethyl sulfonamide.

In the present invention, the first label and the second luminescent label are chemiluminescent catalysts, each independently selected from horseradish peroxidase and/or alkaline phosphatase.

In some preferred embodiments of the present invention, the surface of the solid phase carrier may be surface-modified to introduce a specific functional group, and further, the surface of the solid phase carrier is modified by an active group such as aldehyde group (-CHO), carboxyl group (-COOH), amino group (-NH2), hydroxyl group (-OH), thiol group (-SH), etc., so as to be effectively and stably bonded and connected to the biomolecule.

in some embodiments of the invention, the first, second and third antibodies are each independently selected from monoclonal and/or polyclonal antibodies, preferably monoclonal antibodies.

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

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

The first to eleventh aspects below further provide specific embodiments for implementing the present invention.

The heterogeneous chemiluminescent immunoassay kit for detecting 14-3-3eta protein according to the first aspect of the invention comprises:

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

A component b comprising a label capable of reacting with a substrate to generate a detectable signal and a second antibody or binding fragment thereof bound thereto, said second antibody or binding fragment thereof being capable of specifically binding to a second epitope of the 14-3-3eta protein, and said second epitope and said first epitope being non-overlapping;

It will be appreciated by those skilled in the art that the third antibody, which directly or indirectly binds to the second label, and the second antibody, which directly or indirectly binds to the first label, are monoclonal antibodies that bind to the same epitope of the 14-3-3eta protein.

the inventor researches and discovers that the Hook effect in the detection process can be avoided or overcome to the maximum extent by adding the homologous antibody which is combined with the 14-3-3eta protein and the second marker combined with the homologous antibody, wherein the homologous antibody is combined with the second antibody combined with the first marker and the second marker, and the accuracy of the detection result is greatly improved.

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).

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.

2 healthy rams were selected and injected subcutaneously in multiple spots, each with 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 CNBr activated sepharose 4B (GE) dry powder according to the fact that each gram of dry powder can swell into 3mL of gel, each mL of gel is coupled with 5mg of antigen, swelling the dry powder with 1mM HCl and continuously washing the dry powder in a filter flask, generally washing 1mL of gel with 100mL of 1mM HCl, and washing the gel dry powder with a protective agent;

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 preferred embodiments of the present invention, the kit further comprises 14-3-3eta protein as a calibrator diluted by a calibrator diluent in a proportional gradient to working calibrator solutions of different concentrations.

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

It will be appreciated that the specific binding pair members described herein act as bridges and are therefore also referred to as bridging systems. For example, in the present invention, the first antibody or binding fragment thereof is bound to one member of a specific binding pair member and the solid support is bound to the other member of the specific binding pair member; in some preferred embodiments, the first antibody or binding fragment thereof is bound to biotin and the solid support is bound to streptavidin.

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

also for example, in some preferred embodiments of the invention, the third antibody or binding fragment thereof binds to one member of the specific binding pair member and the second label binds to the other member of the specific binding pair member; preferably, the third antibody or binding fragment thereof binds to biotin and the second label binds to streptavidin.

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

in the invention, the first marker and the second luminescent marker are chemiluminescent markers and are respectively and independently selected from luminol and derivatives thereof, isoluminol and derivatives thereof, acridinium ester and derivatives thereof, adamantane, rare earth elements and bipyridine ruthenium complexes.

In the present invention, the label and the second luminescent label are chemiluminescent catalysts, each independently selected from horseradish peroxidase and alkaline phosphatase.

in the present invention, the solid phase carrier is selected from a microplate, a magnetic bead, a plastic particle and a plastic microsphere, and preferably the solid phase carrier is a magnetic bead.

In some preferred embodiments of the invention, the kit further comprises component c, a substrate solution.

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

In some embodiments of the present invention, the method for preparing the heterogeneous chemiluminescent immunoassay reagent kit for detecting 14-3-3eta protein of the present invention comprises essentially the steps of preparing reagent I (component a), preparing reagent II (component b), and preparing reagent III (component d), wherein component a comprises a solid support and a first antibody or binding fragment thereof bound thereto, wherein the first antibody or binding fragment thereof is capable of specifically binding to a first epitope of 14-3-3eta protein; component b comprises a first label capable of reacting with a substrate to produce a detectable signal or catalyzing a substrate to produce a detectable signal and a second antibody or binding fragment thereof bound thereto, said second antibody or binding fragment thereof being capable of specifically binding to a second epitope of the 14-3-3eta protein, and said second epitope and said first epitope being non-overlapping; a component d comprising a second label capable of reacting with a substrate or capable of catalyzing the substrate to generate a detectable signal and a third antibody or binding fragment thereof directly or indirectly bound thereto, said third antibody or binding fragment thereof being capable of specifically binding to a second epitope of the 14-3-3eta protein, and said second epitope and said first epitope are non-overlapping.

The heterogeneous chemiluminescence immunoassay kit for detecting 14-3-3eta protein in the second aspect of the invention comprises the heterogeneous chemiluminescence immunoassay kit for detecting 14-3-3eta protein in the first aspect of the invention. The kit can be prepared by putting the heterogeneous chemiluminescence immunoassay reagent for detecting 14-3-3eta protein into a kit.

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

In some embodiments of the invention, the first and second labels are the same.

in other embodiments of the invention, the third antibody is the same as the second antibody.

In a third aspect of the present invention, the heterogeneous chemiluminescence immunoassay method for detecting 14-3-3eta protein in a sample to be detected comprises using the heterogeneous chemiluminescence immunoassay reagent set 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 heterogeneous chemiluminescence immunoassay method for detecting 14-3-3eta protein in a sample to be detected in the invention also comprises 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 heterogeneous chemiluminescence immunoassay kit in the third aspect of the invention.

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

Step T2, mixing the third mixture with component d to obtain a fifth mixture;

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 T1-T4, 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 the present invention, the sample to be tested is reacted under heterogeneous conditions, and the process needs to be separated and washed, namely, the separation and washing steps are also included between the steps T1 and T2, between the steps T2 and T3, and between the steps T3 and T4.

In some embodiments of the present invention, in step T3, the sixth mixture produces emission light at a wavelength of 470 nm.

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

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

(2) Mixing the third mixture with component d to obtain a fifth mixture;

(3) mixing the fifth mixture with component c to provide a sixth mixture that produces a detectable signal;

(4) Detecting whether the chemiluminescence signal in the step (3) exists.

in other embodiments of the present invention, the content of 14-3-3eta protein is determined, and the heterogeneous chemiluminescence immunoassay method for detecting 14-3-3eta protein in a sample to be detected comprises:

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

(1) Diluting a 14-3-3eta protein pure product serving as a calibrator into working calibrator solutions with different concentrations according to a proportional gradient by using a calibrator diluent;

(2) mixing the working calibrator solution with the component a and the composition b to obtain a third mixture;

(3) Mixing the third mixture with component d to obtain a fifth mixture;

(4) Mixing the fifth mixture with component c to provide a sixth mixture that produces a detectable signal;

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

(6) and (3) repeating the steps (2) to (5) to detect the chemiluminescence signal values (intensities) of the working calibrator solution containing the 14-3-3eta protein with different concentrations, 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 the functional relation between the concentrations of the 14-3-3eta protein and the chemiluminescence signal values.

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

(2) Mixing the third mixture with component d to obtain a fifth mixture;

(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.

In a fourth aspect, the method for detecting 14-3-3eta protein provided by the invention adopts the kit for detection, and specifically comprises the following steps:

In a fifth aspect of the present invention, the use of the heterogeneous chemiluminescent immunoassay reagent kit according to the first aspect of the present invention for 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 14-3-3eta protein in a sample to be tested by using the heterogeneous chemiluminescent immunoassay reagent kit according to the first aspect of the present invention, 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 tissues, preferably the sample to be tested is selected from the group consisting of blood, plasma, serum, synovial fluid and tissues, further preferably the sample to be tested is selected from the group consisting of blood, plasma, serum, synovial fluid and tissues, Plasma and serum, and more preferably the sample to be tested is serum.

Similarly, the use of the heterogeneous chemiluminescent immunoassay kit according to the second aspect of the present invention for 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 using the heterogeneous chemiluminescent immunoassay kit according to the second aspect of the present invention, 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, preferably the sample to be tested is selected from the group consisting of blood, plasma, serum, synovial fluid and tissue, further preferably the sample to be tested is selected from the group consisting of blood, plasma and serum, still more preferably, the sample to be tested is serum.

Similarly, the use of the heterogeneous chemiluminescence immunoassay method provided by the invention in detecting the presence and/or amount of 14-3-3eta protein in a sample to be detected can be understood as a method for determining the presence and/or amount of 14-3-3eta protein in a sample to be detected by using the heterogeneous chemiluminescence immunoassay kit provided by the first aspect of the invention and using the heterogeneous chemiluminescence immunoassay method provided by the third aspect of the invention, wherein the sample to be detected is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema fluid and tissues, preferably the sample to be detected is selected from blood, plasma, serum, Synovial fluid and tissue, further preferably said sample to be tested is selected from the group consisting of blood, plasma and serum, and still further preferably said sample to be tested is serum.

Similarly, the use of the heterogeneous chemiluminescence immunoassay method provided by the invention according to the third aspect of the invention in detecting the presence and/or amount of 14-3-3eta protein in a sample to be detected can be understood as a method for determining the presence and/or amount of 14-3-3eta protein in a sample to be detected by using the heterogeneous chemiluminescence immunoassay kit provided by the second aspect of the invention and using the heterogeneous chemiluminescence immunoassay method according to the third aspect of the invention, wherein the sample to be detected is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema fluid and tissues, preferably the sample to be detected is selected from blood, plasma, serum, synovial fluid and tissues, further preferably, the sample to be tested is selected from the group consisting of blood, plasma and serum, and still further preferably, the sample to be tested is serum.

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

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

step M2, adopting the heterogeneous chemiluminescence immunoassay method of the second 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;

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, serum, synovial fluid and tissue, further 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.

in a seventh aspect, the present invention relates to the use of a heterogeneous chemiluminescent immunoassay kit according to the first aspect of the present invention or a heterogeneous chemiluminescent immunoassay kit according to the second aspect of the present invention or a heterogeneous chemiluminescent immunoassay according to the third or fourth aspects of the present invention in a chemiluminescent analyzer.

Step T2, mixing the third mixture with component d to obtain a fifth mixture;

example III

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 reagent I (magnetic microspheres coated with anti-14-3-3 eta protein antibody):

In the embodiment, a 10-micron carboxyl magnetic microsphere one-step coating process is adopted, wherein the magnetic particles are formed by taking ferric oxide as a core and coating a thin layer of polystyrene outside. The preparation process comprises the following steps:

(1) Cleaning of

Adding 10mg of magnetic microspheres into a centrifuge tube, adding 0.1M coating buffer solution (pH5.0 MES), mixing uniformly, placing the mixture in a magnetic separator, standing for 15 seconds, removing supernatant, adding the coating buffer solution into the centrifuge tube again, and cleaning the magnetic microspheres twice.

(2) Activation of

Adding 2000 μ L coating buffer to resuspend the particles to a particle concentration of 5mg/ml, taking 50 μ L of 10mg/ml EDC ((1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) which is a water-soluble carbodiimide, used as a carboxyl group activating reagent in amide synthesis, and also used for activating phosphate groups, protein-nucleic acid crosslinking and immune conjugate preparation.) solution, immediately adding into a centrifuge tube and mixing, reacting at room temperature on a vertical rotary mixer at 25-40rpm for 15min, and fully activating carboxyl groups on the surface of the magnetic microspheres.

(3) Coating quilt

adding 0.1mg of protein raw material to be coated, uniformly mixing, placing the centrifugal tube at 37 ℃, vertically rotating the mixer for 3 hours, and enabling the amino group of the protein to be fully coupled with the activated carboxyl group.

(4) Cleaning and preserving

placing the centrifuge tube in a magnetic separator for standing for 15 seconds, removing supernatant, adding a washing buffer solution of 0.1M PH 7.4PBST into the centrifuge tube, uniformly mixing, placing in the magnetic separator for standing for 15 seconds, removing supernatant, repeating twice to fully wash and remove uncoupled protein.

Add 1000. mu.L of 0.1M pH 7.4PBS buffer for magnetic microsphere storage to a storage concentration of 10mg/ml and record the lot number.

2. Reagent II and reagent III (containing antibiotics)14-3-3eta proteinAntibody-labeled acridinium ester) preparation:

The SAE labeling process used in this example is specifically as follows:

(1) SAE formulation

10mg of SAE was weighed and dissolved in Dimethylformamide (DMF) as an organic solvent to prepare a 5mM solution, which was stored at a low temperature of-20 ℃ in the dark for use.

(2) Marking

adding 500 mu L of labeled buffer solution 0.1M of PBS labeled buffer solution with the pH value of 8.0 into a centrifuge tube, then adding 0.1mg of protein raw material to be labeled, adding 10 mu L of SAE solution, immediately mixing uniformly, placing on a vertical rotary mixer, reacting at the room temperature of 25-40rpm for 30 min.

(3) sealing of

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

(4) purification by dialysis

Selecting a dialysis bag with 14KD, dialyzing with 0.1M PBS (pH6.3PBS) dialysis buffer solution, changing the solution once every 4 hours for five times, collecting the marker in the dialysis bag, adding glycerol protectant, and storing at-20 deg.C in dark place for later use.

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

1.1 preparation of a calibrator diluent: weighing 4.77g of HEPES and 1.7g of NaCl, adding 160g of purified water, uniformly mixing for 30min, adjusting the pH value to 7.4 +/-0.2 by using 1M concentrated hydrochloric acid and 1M NaOH solution, continuously adding 3000.1g of Proclin, 30g of BSA, 20.5ml of 1M MgCl20.1ml of Proclin and 0.1M MgCl20.1ml, stirring for 30min, adding the purified water to weigh to 200g, repeatedly measuring the pH value, and keeping the pH value at 2-8 ℃ for later use.

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:

After the components are assembled into a 14-3-3eta protein determination box, the box is loaded on a full-automatic heterogeneous chemiluminescence immunoassay analyzer, and the detection steps are set as follows:

the reaction steps are as follows: (full-automatic analyzer)

1) adding a sample of 50 muL +50 muL reagent I into a reaction cup, reacting for 15min at 37 ℃, carrying out magnetic separation, and washing for five times;

2) Adding 100 μ L reagent II, reacting at 37 deg.C for 10min, magnetically separating, and washing for five times;

3) adding 100 μ L reagent III, reacting at 37 deg.C for 10min, magnetically separating, and washing for five times;

4) 200. mu.L of substrate solution was added and the signal was immediately measured.

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

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

7) and detecting the sample to be detected according to the steps 1) to 5), and calculating the concentration of the 14-3-3eta protein in the sample to be detected according to the equation in the step 6).

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

From the data in Table 1, it can be derived that the standard curve fit equation R2 > 0.99. Very good linearity is obtained in the measurement range of 0.2-20 ng/mL.

5.2 evaluation results

the results of evaluation of 96 samples of the rheumatoid case group and 102 samples of the normal control group are shown in Table 2.

TABLE 2

and (4) conclusion: as can be seen from the data in Table 2, the concentrations of 14-3-3eta in the two groups of samples are significantly different, the specificity of the normal control group is 95%, and the sensitivity of the rheumatoid arthritis disease case group is 36%, which indicates that the 14-3-3eta subtype protein is highly expressed in arthritis patients and is significantly increased in the serum of RA patients.

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> heterogeneous 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 heterogeneous chemiluminescent immunoassay kit for detecting 14-3-3eta protein comprising:

2. The kit of claim 1, wherein the third antibody that directly or indirectly binds to the second label and the second antibody that directly or indirectly binds to the first label are monoclonal antibodies that bind to the same epitope of the 14-3-3eta protein.

3. the kit of claim 1, wherein the first antibody, the second antibody and the third antibody are each independently selected from a monoclonal antibody and/or a polyclonal antibody, preferably a monoclonal antibody.

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

5. the kit of any one of claims 1 to 4, wherein the first antibody or binding fragment thereof is bound to one member of a specific binding pair member and the solid support is bound to the other member of the specific binding pair member; preferably, the first antibody or binding fragment thereof is bound to biotin and the solid support is bound to streptavidin.

6. the kit of any one of claims 1 to 4, wherein the second antibody or binding fragment thereof binds to one member of a specific binding pair member and the first label binds to the other member of the specific binding pair member; preferably, the second antibody or binding fragment thereof is bound to biotin and the first label is bound to streptavidin.

7. The kit of any one of claims 1 to 4, wherein the third antibody or binding fragment thereof is bound to one member of a specific binding pair member and the second label is bound to the other member of the specific binding pair member; preferably, the third antibody or binding fragment thereof binds to biotin and the second label binds to streptavidin.

8. the kit of any one of claims 1 to 7, wherein the first label and the second label are each independently a chemiluminescent label selected from the group consisting of luminol and its derivatives, isoluminol and its derivatives, acridinium ester and its derivatives, adamantane, rare earth elements, and ruthenium bipyridine complexes.

9. The kit of any one of claims 1 to 7, wherein the first label and the second label are each independently a chemiluminescent catalyst selected from horseradish peroxidase and/or alkaline phosphatase.

10. The reagent set of any one of claims 1 to 7, wherein the solid support is selected from the group consisting of magnetic microspheres, plastic microparticles, microwell plates, glass, capillaries, and nylon; magnetic microspheres are preferred.

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

12. the kit of any one of claims 1 to 11, wherein the concentration of the solid support and the first antibody or binding fragment thereof bound thereto in component a is 1 to 100mg/mL, preferably 10 to 50 mg/mL; and/or the concentration of the first label and the second antibody or binding fragment thereof bound thereto in component b is 1-100mg/mL, preferably 10-50 mg/mL; and/or the concentration of the second label and the third antibody or binding fragment thereof bound thereto in said fraction d is 1-100mg/mL, preferably 10-50 mg/mL.

13. The kit of any one of claims 1 to 12, wherein the kit further comprises component c, a substrate solution; preferably, the substrate solution comprises a solution A and a solution B; more preferably, the solution A is hydrogen peroxide solution, and the solution B is sodium hydroxide solution.

14. The kit according to any one of claims 1 to 13, wherein the amino acid SEQUENCE of the 14-3-3eta protein is as shown in SEQUENCE No. 1.

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

16. A heterogeneous chemiluminescent immunoassay kit for detecting 14-3-3eta protein comprising the heterogeneous chemiluminescent immunoassay kit of any one of claims 1-15.

17. the kit according to claim 16, characterized in that it comprises the following components:

18. The kit of claim 16, further comprising a component d: comprising a third antibody directly linked or indirectly linked to a first label or a second label.

19. the kit of claim 17 or 18, wherein the first and second labels are the same; and/or, the third antibody is the same as the second antibody.

20. The kit according to any one of claims 17 to 19, wherein the kit further comprises component c: which comprises a substrate solution comprising a hydrogen peroxide solution and a sodium hydroxide solution.

21. A heterogeneous chemiluminescent immunoassay method for detecting 14-3-3eta protein in a sample to be detected, which comprises using the heterogeneous chemiluminescent immunoassay kit according to any one of claims 1 to 15 or the heterogeneous chemiluminescent immunoassay kit according to any one of claims 16 to 20 to determine the presence or absence of 14-3-3eta protein in the sample to be detected and/or to determine the content of 14-3-3eta protein.

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

step T2, mixing the third mixture with component d to obtain a fifth mixture;

23. The method according to claim 22, further comprising the step of preparing a standard working curve of 14-3-3eta protein before step T1.

24. The method of claim 23, wherein in step T4, the intensity of the chemiluminescent signal of step T3 is detected and the amount of 14-3-3eta protein in the test sample is determined based on a 14-3-3eta protein standard working curve.

25. A method for detecting 14-3-3eta protein, which comprises the steps of using the kit of claims 16-20:

26. use of the heterogeneous chemiluminescent immunoassay kit of any one of claims 1 to 15 or the heterogeneous chemiluminescent immunoassay kit of any one of claims 16 to 20 or the method of any one of claims 21 to 25 for the detection of the presence and/or amount of 14-3-3eta protein in a sample selected from the group consisting of blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema fluid and tissue.

27. Use of a kit according to any one of claims 1 to 15 in the preparation of a kit for the detection of rheumatoid arthritis, comprising:

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

28. The use according to claim 27, 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.

29. The use according to claim 27, 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.

30. The use according to claim 27, 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.

31. The use of claim 27, 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.

32. The use of claim 27, 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.

33. the use according to any of claims 27 to 32, wherein in step M2, the method according to any of claims 21 to 25 is used to determine the presence of 14-3-3eta protein in the test sample and/or to determine the content of 14-3-3eta protein.

34. use of the heterogeneous chemiluminescent immunoassay kit of any one of claims 1 to 15 or the heterogeneous chemiluminescent immunoassay kit of any one of claims 16 to 20 or the heterogeneous chemiluminescent immunoassay method of any one of claims 21 to 25 in a chemiluminescent immunoassay analyzer.

35. The chemiluminescent immunoassay analyzer in use of claim 34 comprising:

36. A method of controlling the chemiluminescent immunoassay analyzer of claim 35 comprising the steps of:

Step T2, mixing the third mixture with component d to obtain a fifth mixture;

37. The method according to claim 36, further comprising the step of preparing a standard working curve for 14-3-3eta protein prior to step T1.

38. the method of claim 37, wherein in step T4, the intensity of the chemiluminescent signal of step T3 is detected, and the amount of 14-3-3eta protein in the test sample is determined based on a 14-3-3eta protein standard working curve.

39. a method for detecting 14-3-3eta protein, which comprises the steps of using the kit of claims 16-20 and the chemiluminescent immunoassay of claim 35, comprising:

40. a heterogeneous chemiluminescent immunoassay kit according to any one of claims 1 to 15 or a heterogeneous chemiluminescent immunoassay kit according to any one of claims 16 to 20 or a method according to any one of claims 21 to 25 or a chemiluminescent immunoassay according to claim 35 or a use according to any one of claims 36 to 39 for detecting the presence and/or amount of 14-3-3eta protein in a test sample selected from the group consisting of blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema fluid and tissue.