CN110579602B

CN110579602B - Method for in vitro assessment of the presence or absence of rheumatoid arthritis by biomarker joint detection

Info

Publication number: CN110579602B
Application number: CN201810821257.1A
Authority: CN
Inventors: 饶星; 廖智星; 刘宇卉; 李临
Original assignee: Chemclin Diagnostics Corp
Current assignee: Chemclin Diagnostics Corp
Priority date: 2018-05-24
Filing date: 2018-07-24
Publication date: 2024-03-15
Anticipated expiration: 2038-07-24
Also published as: CN110579602A

Abstract

The present invention relates to a method for assessing the presence or absence of Rheumatoid Arthritis (RA) in vitro by biochemical markers. The method can remarkably improve the detection accuracy of RA positive of RA inflammatory joint lesion patients by jointly detecting the levels of serum 14-3-3 eta protein, anti-CCP antibody and anti-Carp antibody and correlating the detection results with RA.

Description

Method for in vitro assessment of the presence or absence of rheumatoid arthritis by biomarker joint detection

Technical Field

The invention belongs to the technical field of immunoassay, and particularly relates to a kit for in-vitro evaluation of the existence of rheumatoid arthritis through biomarker joint detection, a preparation method and a use method.

Background

Biomarkers (biomarks) refer to biochemical markers that can mark alterations or changes in the structure or function of systems, organs, tissues, cells and subcells, and have very broad uses.

Rheumatoid Arthritis (RA) is a systemic autoimmune disease characterized mainly by synovitis of the joints, and is manifested mainly by chronic polyarthritis, which ultimately leads to joint deformity. The biomarker is used for comprehensively judging the characteristics of the course and the disease condition of the RA patient, genetic background, epigenetic and the like, so that the accurate diagnosis and treatment of RA can be realized, and the life quality of the RA patient is improved. Therefore, how to improve the accuracy of RA positive detection in RA inflammatory arthropathy patients is an urgent technical problem to be solved.

Disclosure of Invention

To solve the technical problems, the invention provides a method for in vitro assessment of the presence or absence of rheumatoid arthritis by biomarker joint inspection. The method can remarkably improve the detection accuracy of RA positive of RA inflammatory joint lesion patients by jointly detecting the level of at least 2 of serum 14-3-3 eta protein, anti-CCP antibody and anti-Carp antibody and correlating the detection result with RA.

To this end, a first aspect of the invention provides a use of a homogeneous immunoassay to detect the concentration of each biomarker in a group of Rheumatoid Arthritis (RA) biomarkers in the preparation of a reagent for assessing the presence or absence of Rheumatoid Arthritis (RA) in vitro by biochemical markers, comprising:

a) Detecting the concentration of each biomarker in the biomarker panel in the sample to be tested;

b) Combining the measured concentration values of each biomarker of a) to obtain a combined concentration value of each biomarker in the biomarker panel; and

c) Correlating the combined concentration value obtained in step b) with the presence or absence of RA, wherein an increased combined value compared to the truncated combined concentration value of each marker in the corresponding biomarker panel measured from the reference population indicates the presence of RA;

Wherein the biomarker panel comprises an Anti-CCP antibody, a 14-3-3eta protein, and an Anti-card antibody.

In some embodiments of the invention, the combined concentration value of step b) is compared to a cutoff value derived from a reference population other than RA positive patients, the reference population comprising apparently healthy persons and patients selected from Osteoarthritis (OA) patients and other autoimmune disease patients.

In a second aspect, the invention provides a use of a homogeneous immunoassay to detect the concentration of each biomarker in a group of Rheumatoid Arthritis (RA) biomarkers in the manufacture of a reagent for assessing the severity of Rheumatoid Arthritis (RA) by means of off-biochemical markers, comprising:

b) Combining the measured concentration values of each biomarker of a), obtaining a combined concentration value of each biomarker; and

c) Correlating the combined concentration value obtained in step b) with the severity of RA, wherein an increased combined value compared to the truncated combined concentration value of each marker in the corresponding biomarker panel measured from the reference population indicates the severity of RA in the patient;

In a third aspect, the present invention provides a use of a homogeneous immunoassay for detecting the concentration of each biomarker in a group of Rheumatoid Arthritis (RA) biomarkers for the preparation of a formulation for classifying Rheumatoid Arthritis (RA) and other autoimmune diseases by biochemical labeling of an in vitro region, comprising:

c) Distinguishing RA from other autoimmune diseases from the combined concentration values obtained from step b), wherein an increased combined value compared to the truncated combined concentration value of each marker in the corresponding biomarker panel measured from the reference population indicates the presence of RA;

In some embodiments of the invention, the other autoimmune diseases include other joint diseases; the other joint disease is Osteoarthritis (OA).

In a fourth aspect the invention provides the use of a set of Rheumatoid Arthritis (RA) biomarkers for the preparation of a reagent for assessing in vitro the presence or absence of Rheumatoid Arthritis (RA) in a sample to be tested, wherein an increase in the combined concentration value measured for each biomarker in the set of Rheumatoid Arthritis (RA) biomarkers compared to the truncated combined concentration value for each marker in the corresponding set of biomarkers from a reference population measurement using homogeneous immunoassay is indicative of the presence of RA;

In the invention, the sample to be measured is selected from the group consisting of blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema effusion and tissue.

According to the use of the first to fourth aspects of the invention, the set of biomarkers comprises Anti-CCP antibodies, 14-3-eta proteins and Anti-map antibodies and other biomarkers, preferably RA.

According to some embodiments of the invention, the step further comprises measuring the 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 some embodiments of the invention, the amount of 14-3-3eta protein in the test sample is determined based on a 14-3-3eta protein standard working curve.

According to some embodiments of the invention, the step further comprises comparing the measured amount of 14-3-3eta protein or fragment thereof or the immune complex formed by the 14-3-3eta protein or fragment thereof with at least one antibody to the amount of the 14-3-3eta protein or fragment thereof or the immune complex formed by the 14-3-3eta protein or fragment thereof with at least one antibody in a normal control sample, a rheumatoid arthritis control sample or a pre-treatment sample from the same subject.

According to some embodiments of the invention, the step comprises contacting the sample with a sample 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 antibodies comprise a first antibody capable of specifically binding to a first epitope of 14-3-3eta protein and a second antibody capable of specifically binding to a second epitope of 14-3-3eta protein, wherein the second epitope and the first epitope do not overlap.

In some embodiments of the invention, the first antibody binds to a receptor that is capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal.

In some embodiments of the invention, the acceptor comprises an olefin compound and a metal chelate, which are in non-particulate form and are soluble in an aqueous medium; and/or the acceptor is a polymeric microparticle filled with a luminescent compound and a lanthanide.

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

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 further embodiments of the invention, the epitope is selected from the group consisting of a relatively specific fragment of the amino acid fragment of the sequence of a 14-3-3eta protein: 1-6aa, 27-38aa, 71-83aa, 112-119aa and 141-154aa.

In a fifth aspect, the invention provides a kit for assessing the presence or absence of Rheumatoid Arthritis (RA) by biochemical markers in vitro based on the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers, comprising reagents for detecting the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers using a homogeneous immunoassay, wherein the set of biomarkers comprises an Anti-CCP antibody, a 14-3-eta protein and an Anti-carp antibody.

In a sixth aspect, the invention provides a kit for assessing the severity of Rheumatoid Arthritis (RA) by biochemical markers ex vivo based on the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers, comprising reagents for detecting the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers, wherein the set of biomarkers comprises an Anti-CCP antibody, a 14-3-eta protein and an Anti-map antibody.

In a seventh aspect, the invention provides a kit for classifying Rheumatoid Arthritis (RA) and other autoimmune diseases by biochemically labeling an off-body region based on the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers, comprising reagents for detecting the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers, wherein the set of biomarkers comprises an Anti-CCP antibody, a 14-3-eta protein, and an Anti-carp antibody.

In an eighth aspect, the invention provides a kit for assessing the presence or absence of Rheumatoid Arthritis (RA) in a sample to be tested in vitro using a set of Rheumatoid Arthritis (RA) biomarkers, wherein an increase in the combined concentration value measured for each biomarker in the set of Rheumatoid Arthritis (RA) biomarkers compared to the truncated combined concentration value for each marker in the corresponding set of biomarkers from a reference population measurement using homogeneous immunoassay is indicative of the presence of RA; wherein the biomarker panel comprises an Anti-CCP antibody, a 14-3-3eta protein, and an Anti-card antibody.

In some preferred embodiments of the invention, the biomarker panel comprises Anti-CCP antibodies, 14-3-3eta proteins and Anti-map antibodies and other biomarkers, preferably RA.

According to a fifth to eighth aspect of the invention, the kit comprises reagents for detecting the concentration of Anti-CCP antibodies, 14-3-3eta proteins and Anti-card antibodies in a biomarker panel using a homogeneous immunoassay.

According to some embodiments of the invention, a homogeneous immunoassay reagent for detecting an anti-cyclic citrullinated peptide antibody comprises:

component a1 comprising a first antigen capable of specifically binding to an epitope binding site of an anti-CCP antibody;

component b1 comprising an anti-immune complex antibody that is capable of specifically recognizing and binding to an anti-CCP antibody in a first immune complex formed with a first antigen, and does not recognize a free, non-antigen-bound anti-CCP antibody.

In some embodiments of the invention, the first antigen or the anti-immune complex antibody is bound to a receptor capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal; preferably, the acceptor comprises an olefin compound and a metal chelate, which are in non-particulate form and are soluble in an aqueous medium; and/or the acceptor is a polymeric microparticle filled with a luminescent compound and a lanthanide.

In some embodiments of the invention, the kit further comprises component c1 comprising a donor capable of generating singlet oxygen in an excited state; preferably, the donor binds to one of the members of the specific binding pair, and the other member of the specific binding pair binds to the first antigen or the anti-immune complex antibody; further preferably, the donor binds to streptavidin, and the first antigen or the anti-immune complex antibody, respectively, binds to biotin.

In some specific embodiments of the invention, the reagent further comprises an anti-CCP antibody neat as a calibrator that is diluted in a ratio gradient by a calibrator dilution to working calibrator solutions of different concentrations.

According to some embodiments of the invention, a homogeneous immunoassay reagent for detecting 14-3-3eta proteins comprises:

a component a2 comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a first antibody or binding fragment thereof bound thereto, said first antibody or binding fragment thereof being capable of specifically binding to a first epitope of a 14-3-3eta protein;

a component b2 comprising a second antibody or binding fragment thereof capable of specifically binding to a second epitope of the 14-3-3eta protein, said second epitope and said first epitope do not overlap;

Component c2, which comprises a donor capable of generating singlet oxygen in the excited state.

In some embodiments of the invention, the reagent further comprises a pure 14-3-3eta protein as a calibrator, which is diluted in a ratio gradient by a calibrator diluent to working calibrator solutions of different concentrations.

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

According to some embodiments of the invention, a homogeneous immunoassay reagent for detecting an anti-Carp antibody comprises:

a component a3 comprising an antigen capable of specifically binding to the anti-Carp antibody of interest and a second anti-Carp antibody;

component b3, comprising a second anti-card antibody.

In some embodiments of the invention, the antigen or the second anti-Carp antibody binds to a receptor; preferably, the antigen is bound to a receptor; the acceptor is capable of reacting with the singlet oxygen to generate a detectable chemiluminescent signal.

In some embodiments of the invention, the kit further comprises a component c3 comprising a donor capable of generating singlet oxygen in an excited state; preferably, the donor binds to one of the specific binding partners and the other member of the specific binding partner binds to the antigen or the second anti-card antibody; further preferably, the donor binds to streptavidin and correspondingly the second anti-Carp antibody binds to biotin.

In some embodiments of the invention, the donor is a photoactivated or chemically activated sensitizer, which is in non-particulate form and is soluble in an aqueous medium; and/or the donor is polymer particles filled with photosensitive compounds, and can generate singlet oxygen under the excitation of light.

In some embodiments of the invention, a homogeneous sandwich immunoassay reagent for detecting an anti-Carp antibody comprises:

a component a4 comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a first antigen bound thereto, an epitope of the first antigen being capable of specifically binding to an epitope first binding site of an anti-card antibody;

Component b4 comprising a second antigen capable of specifically binding to an epitope second binding site of an anti-Carp antibody, the epitope first binding site and the epitope second binding site of the anti-Carp antibody not overlapping;

component c4, which comprises a donor capable of generating singlet oxygen in the excited state.

In some embodiments of the invention, the reagent further comprises an anti-card antibody neat product as a calibrator, which is diluted in a proportional gradient by a calibrator diluent to working calibrator solutions of different concentrations.

In some embodiments of the invention, the second antigen is bound to one member of the specific binding pair and the donor is bound to the other member of the specific binding pair, preferably the second antigen is bound to biotin and the donor is bound to streptavidin.

In some specific embodiments of the invention, the kit comprises reagents for detecting Anti-CCP antibodies in a biomarker panel by an indirect method using a homogeneous immunoassay, reagents for detecting 14-3-3eta proteins in a biomarker panel by a sandwich method using a homogeneous immunoassay, and reagents for detecting concentrations of Anti-Carp antibodies in a biomarker panel by a competitive method using a homogeneous immunoassay as described above.

In other specific embodiments of the present invention, the kit comprises reagents for detecting antibodies to Anti-CCP antibodies in a biomarker panel by electrochemical detection using an electrochemical immunoassay, reagents for detecting 14-3-eta proteins in a biomarker panel by sandwich detection using a homogeneous immunoassay, and reagents for detecting concentrations of Anti-card antibodies in a biomarker panel by sandwich detection using a homogeneous immunoassay as described above.

In a ninth aspect, the invention provides a kit for assessing the presence or absence of Rheumatoid Arthritis (RA) by biochemical labelling in vitro based on the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers comprising a kit according to the fifth to eighth aspects of the invention.

In a tenth aspect the invention provides a kit for assessing the severity of Rheumatoid Arthritis (RA) by biochemical markers out of a group of biomarkers based on the concentration of each biomarker in the group of Rheumatoid Arthritis (RA) biomarkers comprising a kit according to the fifth to eighth aspects of the invention.

According to an eleventh aspect of the present invention there is provided a kit for classifying Rheumatoid Arthritis (RA) and other autoimmune diseases by biochemically labeling off-body regions based on the concentration of each biomarker in a group of Rheumatoid Arthritis (RA) biomarkers, comprising a kit according to the fifth to eighth aspects of the invention.

In a twelfth aspect, the invention provides a kit for in vitro assessment of the presence or absence of Rheumatoid Arthritis (RA) in a sample to be tested using a set of Rheumatoid Arthritis (RA) biomarkers, comprising a kit according to the fifth to eighth aspects of the invention.

In a thirteenth aspect the invention provides a method of assessing the presence or absence of Rheumatoid Arthritis (RA) in vitro by biochemical markers comprising detecting the concentration of each biomarker in a group of Rheumatoid Arthritis (RA) biomarkers using a kit according to the fifth to eighth aspects of the invention or using a kit according to the ninth to twelfth aspects of the invention and assessing the presence or absence of Rheumatoid Arthritis (RA) in vitro by biochemical markers.

In some embodiments of the invention, the method comprises:

In some further embodiments of the invention, the combined concentration value of step b) is compared to a cutoff value derived from a reference population other than RA positive patients, said reference population comprising apparently healthy persons and patients selected from Osteoarthritis (OA) patients and other autoimmune disease patients.

In a fourteenth aspect the invention provides a method of assessing the severity of Rheumatoid Arthritis (RA) by means of biochemical markers ex vivo comprising detecting the concentration of each biomarker in a group of Rheumatoid Arthritis (RA) biomarkers and assessing the severity of Rheumatoid Arthritis (RA) by means of biochemical markers ex vivo using a kit according to the fifth to eighth aspects of the invention or using a kit according to the ninth to twelfth aspects of the invention.

In some embodiments of the invention, the method comprises:

In a fifteenth aspect the invention provides a method of classifying Rheumatoid Arthritis (RA) and other autoimmune diseases by biochemically labeling an off-body region, comprising detecting the concentration of each biomarker in a group of Rheumatoid Arthritis (RA) biomarkers and classifying Rheumatoid Arthritis (RA) and other autoimmune diseases by biochemically labeling an off-body region using a kit as described in the fifth to eighth aspects of the invention or using a kit as described in the ninth to twelfth aspects of the invention.

In some embodiments of the invention, the method comprises:

In some further embodiments of the invention, the other autoimmune diseases include other joint diseases; the other joint disease is Osteoarthritis (OA).

A sixteenth aspect of the invention provides a method for assessing in vitro the presence or absence of Rheumatoid Arthritis (RA) in a test sample using a kit according to the fifth to eighth aspects of the invention or using a kit according to the ninth to twelfth aspects of the invention, wherein an increase in the combined concentration value measured for each biomarker in the set of Rheumatoid Arthritis (RA) biomarkers compared to the truncated combined concentration value for each marker in the corresponding set of biomarkers from the reference population indicates the presence of RA;

According to the method described in thirteenth to sixteenth of the present invention, the concentration of Anti-CCP is detected by homogeneous immunoassay using a homogeneous immunoassay kit containing an antibody for detection of Anti-CCP, 14-3-eta protein and Anti-card antibody, which comprises:

r1, mixing a sample to be tested with the component a1 to obtain a first mixture;

step R2, mixing the first mixture with the component b1 to obtain a second mixture;

step R3, mixing the second mixture with component c1 to obtain a third mixture that produces a detectable chemiluminescent signal;

and step R4, detecting the intensity of the chemiluminescent signal in the step R3, thereby determining the content of the Anti-CCP antibody.

In some embodiments of the invention, the method further comprises the step of creating an Anti-CCP antibody standard working curve prior to step R1.

In some embodiments of the invention, in step R4, the intensity of the chemiluminescent signal in step R3 is detected and the amount of Anti-CCP antibody in the sample to be tested is determined based on an Anti-CCP antibody standard operating curve.

According to the method described in thirteenth to sixteenth of the present invention, the concentration of 14-3-3eta protein is detected by a homogeneous immunoassay using a homogeneous immunoassay kit containing an antibody for detecting Anti-CCP, 14-3-3eta protein and Anti-card antibody, which comprises:

r1, mixing a sample to be tested with the component a2 and the combination b2 to obtain a third mixture;

step R2, mixing the third mixture with the component c2 to obtain a fourth mixture;

step R3, contacting energy or an active compound with the fourth mixture to excite the donor to generate singlet oxygen, the acceptor being capable of reacting with the received singlet oxygen to generate a detectable chemiluminescent signal;

and R4, detecting the existence and/or intensity of the chemiluminescent signal in the step R3, so as to judge whether 14-3-3eta protein exists in the sample to be detected and/or determine the content of the 14-3-3eta protein.

In some embodiments of the invention, the method further comprises the step of creating a 14-3-3eta protein standard work curve prior to step R1.

In some embodiments of the invention, in step R4, the intensity of the chemiluminescent signal in step R3 is detected and the amount of 14-3-3eta protein in the test sample is determined based on a 14-3-3eta protein standard operating curve.

According to the method described in thirteenth to sixteenth of the present invention, the concentration of the Anti-Carp antibody is detected by a homogeneous immunoassay using a homogeneous immunoassay kit containing an Anti-CCP antibody, a 14-3-3eta protein and an Anti-Carp antibody, which comprises the steps of:

r1, mixing a second anti-Carp antibody combined with biotin with a sample to be tested to form a 2 nd mixture;

r2, mixing the antigen combined with the receptor with the mixture 2, and enabling the second anti-Carp antibody combined with biotin and the target anti-Carp antibody in the sample to be tested to compete for combining with the antigen combined with the receptor to form a third immune complex formed by the receptor-antigen-second anti-Carp antibody-biotin and a fourth immune complex formed by the receptor-antigen-anti-Carp antibody respectively, so as to form a mixture 3;

R3, mixing the donor combined with streptavidin with the 3 rd mixture, so that the donor combined with streptavidin is combined with biotin in the third immune complex to form a fifth immune complex consisting of an acceptor-antigen-second anti-Carp antibody-biotin-streptavidin-donor, thereby forming a 4 th mixture;

r4, exciting the donor with energy or an active compound to produce singlet oxygen, the acceptor reacting with the singlet oxygen to produce a detectable chemiluminescent signal; detecting the presence or absence of a fourth immune complex; if a fourth immune complex is present, the anti-Carp antibody of interest is present in the test sample.

In some embodiments of the invention, the method further comprises the step of creating a standard working curve for the target anti-Carp antibody prior to step R1.

In some embodiments of the invention, at step R4, the intensity of the chemiluminescent signal is detected and the amount of the target anti-Carp antibody in the sample to be tested is determined based on an anti-Carp antibody standard operating curve.

In some embodiments of the invention, the signal value of the emitted light of the 4 th mixture is detected and compared with the chemiluminescent signal value of a control immune complex formed by the antigen and the second anti-Carp antibody in equal amounts in step R2, thereby determining whether the target anti-Carp antibody and/or the concentration of the target anti-Carp antibody is present in the test sample.

r1, mixing a sample to be tested with the component a4 and the combination b4 to obtain a third mixture;

step R2, mixing the third mixture with the component c4 to obtain a fourth mixture;

and R4, detecting the existence and/or intensity of the chemiluminescent signal in the step R3, thereby determining the content of the anti-Carp antibody in the sample to be detected.

In some embodiments of the invention, the method further comprises the step of creating an anti-card antibody standard working curve prior to step R1.

In some embodiments of the invention, in step R4, the intensity of the chemiluminescent signal in step R3 is detected and the amount of anti-Carp antibody in the test sample is determined based on an anti-Carp antibody standard working curve.

In a seventeenth aspect the invention provides a chemiluminescent immunoassay system for detecting each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers, comprising detecting the presence or absence of each biomarker in the set of Rheumatoid Arthritis (RA) biomarkers and/or determining the amount of each biomarker in the set of Rheumatoid Arthritis (RA) biomarkers using a kit according to the fifth to eighth aspects of the invention or using a kit according to the ninth to twelfth aspects of the invention or a method according to the thirteenth to sixteenth aspects of the invention.

According to some embodiments of the invention, the system comprises:

a reaction device for chemically reacting a sample to be tested with the reagent set according to the fifth to eighth aspects of the present invention or with the reagents in the kit according to the ninth to twelfth aspects of the present invention;

excitation and reading means for exciting the donor microspheres with excitation light having a wavelength of 600-700nm to generate active oxygen, and the acceptor microspheres react with the received active oxygen to generate light emitted at 520-620nm, and recording the optical signal of the emitted light;

and the processor is used for judging whether the target molecule to be detected exists in the sample to be detected or not and/or determining the content of the target molecule to be detected according to the recorded existence and/or intensity of the light signal of the emitted light.

In some embodiments of the invention, the processor uses a cubic spline interpolation fit to perform the fitting, directly giving the concentration value of the target molecule to be measured in the sample to be measured.

In an eighteenth aspect, the present invention provides a homogeneous immunoassay method for detecting an Anti-CCP using the chemiluminescent immunoassay system of the seventeenth aspect of the invention and a homogeneous immunoassay kit or kit comprising antibodies for detecting Anti-CCP antibodies, 14-3-3eta proteins and Anti-carp antibodies according to the invention for detecting the concentration of Anti-CCP by homogeneous immunoassay comprising:

In a nineteenth aspect the present invention provides a homogeneous immunoassay for detecting 14-3-3eta protein using a chemiluminescent immunoassay system of the seventeenth aspect of the invention and a homogeneous immunoassay kit or kit comprising antibodies for detecting Anti-CCP antibodies, 14-3-3eta protein and Anti-card antibodies according to the invention for detecting the concentration of 14-3-3eta protein by homogeneous immunoassay comprising:

In a twentieth aspect the present invention provides a homogeneous immunoassay method for detecting Anti-Carp antibodies employing a chemiluminescent immunoassay system of the seventeenth aspect of the invention and a homogeneous immunoassay kit or kit comprising detection of Anti-CCP antibodies, 14-3-3eta proteins and Anti-Carp antibodies as described herein for detecting the concentration of Anti-Carp antibodies by homogeneous immunoassay comprising:

In a twenty-first aspect the present invention provides a homogeneous immunoassay method for detecting Anti-Carp antibodies using a chemiluminescent immunoassay system of the seventeenth aspect of the invention and a homogeneous immunoassay kit or kit comprising detection of Anti-CCP antibodies, 14-3-3eta proteins and Anti-Carp antibodies as described herein for detecting the concentration of Anti-Carp antibodies by homogeneous immunoassay comprising:

The method for assessing whether the Rheumatoid Arthritis (RA) exists in vitro through the biochemical markers can remarkably improve the detection accuracy of RA positive of RA inflammatory joint lesion patients by jointly detecting the level of at least 2 of serum 14-3-3 eta protein, anti-CCP antibody and anti-Carp antibody and correlating the detection result with RA.

Detailed Description

In order that the invention may be readily understood, the invention will be described in detail. Before the present 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, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

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

I terminology

"subject to be tested", "subject" and "patient" are used interchangeably and refer to mammals, such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs and other mammalian species, unless otherwise specified or limited.

The term "homogeneous" as used herein is defined as "homogeneous" and refers to a method that allows detection without the need to separate the bound antigen-antibody complex from the remaining free antigen or antibody.

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 samples to be tested 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 effusion, and tissues, and the like.

The term "citrullinated peptide" as used herein refers to a specific antigen that has a positive response to RA serum: the modified peptide fragments such as silk fibroin fragments, precursor of pre-silk fibroin, synthetic polypeptide, recombinant polypeptide, polypeptide coupled with a marker and the like are characterized by containing citrulline, wherein citrulline residues are the essential components of a substrate identified by an anti-CCP antibody.

The term "citrulline epitope" as used herein refers to a region of an antigen surface that is capable of specific binding by an anti-cyclic citrulline peptide antibody, including citrulline residues and the surrounding amino acid sequences in which they reside.

The term "epitope recognition site of an anti-cyclic citrullinated peptide antibody" also referred to as "epitope recognition site" herein refers to a region of an anti-cyclic citrullinated peptide antibody that recognizes and binds to an "citrullinated epitope", for example, the first epitope recognition site of an anti-cyclic citrullinated peptide antibody and the second epitope recognition site of an anti-cyclic citrullinated peptide antibody do not overlap, that is, they belong to different positions of epitope recognition sites having the same binding properties.

The term "mixture of citrulline peptide fragments" as used herein refers to a mixture of at least 2 single citrulline-containing peptide fragments, which may be cyclic or linear citrulline-containing peptide fragments.

The terms "14-3-3" and "14-3-3 protein" are used interchangeably herein to refer to at least one member of the 14-3-3 family of conserved intracellular regulatory molecules commonly expressed in eukaryotic cells. 14-3-3 proteins have the ability to bind to a number of functionally diverse signaling 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 an evolving member of the tetra-trico peptide repeat superfamily. They typically have 9 or 10 alpha helices, typically along their amino terminal helices, to form homodimer and/or heterodimer interactions. 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 are known to be expressed in mammals, each isoform comprising 242-255 amino acids. Seven 14-3-3 protein isoforms are designated 14-3-3 alpha/beta (alpha/beta), 14-3-3 delta/zeta, 14-3-3 epsilon (epsilon), 14-3-3 gamma (gamma), 14-3-3 eta (eta), 14-3-3 tau/theta (tau/theta), and 14-3-3 sigma/stratifin. 14-3-3 proteins have a high degree of sequence similarity and are 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 isotype, or may specifically bind to and/or recognize only one isotype (e.g., 14-3-3 eta). In addition, anti-14-3-3 antibodies can bind and/or recognize 14-3-proteins that have been modified, e.g., naturally (e.g., post-translationally) or chemically.

The term "relative specificity fragment" as used herein refers to a 14-3-3 protein of 7 isoforms of the 14-3-3 family, and the present inventors found by studying the amino acid SEQUENCEs of the 14-3-3eta protein or fragments thereof as shown in SEQ ID NO.1 that fragments 1-6aa, 27-38aa, 71-83aa, 112-119aa and 141-154aa are specific epitopes belonging only to the 14-3-3eta (eta) protein, which do not have any crossover with the amino acid SEQUENCEs of the 14-3-3 proteins of the other 6 isoforms of the 14-3-3 family, and monoclonal antibodies generated therefrom recognize or bind only the 14-3-3eta (eta) protein, and do not recognize or bind the 14-3-3 proteins of the other 6 isoforms of the 14-3-3 family.

The term "arthritis" as used herein is used interchangeably with "arthritic condition" and "joint pain" and refers generally, unless indicated otherwise, to inflammatory conditions of the joints of the human body. Pain, swelling, stiffness, and difficulty in moving are often associated with arthritic conditions. Arthritis consists of more than 100 different cases. These conditions may be anything from a relatively mild form to a severely compromised system form. The arthritic condition may be caused by any of a variety of causes, including infection, trauma, degenerative disease, metabolic disorder or interference, or other unknown etiology. The arthritic condition may be more specifically described in terms of subtypes such as rheumatoid arthritis, mixed Connective Tissue Disease (MCTD), crystalline 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. Well-defined arthritic conditions refer to knowledge about the type of arthritis and its stage, e.g., onset, remission, recurrence, etc.

The terms "antibody" and "immunoglobulin" as used herein are used in the broadest sense and include antibodies or immunoglobulins of any isotype that retain an antibody fragment that specifically binds to an antigen; including, but not limited to, fragments Fab, fv, scFv, fd, 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 desired case, the antibody may be further conjugated to other moieties, such as specific binding pair members, e.g., biotin or streptavidin (one of the biotin-streptavidin specific binding pair members), and the like.

The term "immune complex" (immunecomplex), i.e. antigen-antibody complex, according to the present invention; the term "human immune complex" refers to an immune complex existing in a human body, and may be an immune complex existing in blood circulation or an immune complex deposited in tissues.

The term "anti-immune complex antibody" as used herein refers to a substance capable of specifically recognizing and binding to an antigen-antibody immune complex, which does not recognize free, unbound antibodies and free human IgG antibodies. Specifically, the specific antibody in the sample is combined with the corresponding antigen to form an antigen-antibody-immune complex, and the antibody conformation or epitope in the immune complex state is changed to show a difference from other free nonspecific antibody conformations or epitopes, and the difference is specifically recognized by the anti-immune complex antibody. The anti-immune complex antibody can be used for distinguishing an antibody in an immune complex state from a nonspecific antibody and a free specific antibody which is not bound with an antigen.

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

The term "polyclonal antibody" as used herein refers to a collection of immunoglobulins derived from 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 binds to antibodies and sensitized lymphocytes, which are the products of the immune response, in vivo and in vitro, resulting in an immune effect.

The term "binding" as used herein refers to the 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 from a steric perspective, corresponds to the conformational correspondence between the corresponding reactants.

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 found 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 primary site of binding to streptavidin. Activated biotin can be coupled to almost all known biomacromolecules, including proteins, nucleic acids, polysaccharides, lipids, etc., mediated by protein cross-linking agents; and "streptavidin" is a protein secreted by Streptomyces and has a molecular weight of 65kD. The "streptavidin" molecule consists of 4 identical peptide chains, each of which is capable of binding to a biotin. Thus, each antigen or antibody can be conjugated to multiple biotin molecules simultaneously, thereby producing a "tentacle effect" that enhances assay sensitivity. In any case where desired, any agent used in the present invention, including antigen, antibody, receptor or donor, may be conjugated to any member of the biotin-streptavidin specific binding pair member as desired.

The term "donor" as used herein refers to a sensitizer that upon activation of energy or an active compound is capable of generating an active intermediate such as singlet oxygen that reacts with the acceptor. The donor may be photoactivated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.). In some embodiments of the invention, the donor 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, porphyrin, phthalocyanine, and chlorophyll as disclosed in U.S. Pat. No. 5709994 (which is incorporated herein by reference in its entirety), and derivatives of these compounds having 1-50 atom substituents for making these compounds more lipophilic or hydrophilic, and/or as linking groups to specific binding pair members. Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, such as described in U.S. patent No. 6406913, which is incorporated herein by reference. In other embodiments of the invention, the donor is a chemically activated other sensitizer, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Examples of other donors include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, and the like, and singlet oxygen is released by heating these compounds or by direct absorption of light by these compounds.

The term "acceptor" as used herein refers to a compound that is capable of reacting with singlet oxygen to produce a detectable signal. The donor is induced to activate by energy or an active compound and releases singlet oxygen in a high energy state which is captured by the acceptor in close proximity, thereby transferring energy to activate the acceptor. In some embodiments of the invention, the acceptor is a substance that undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate that may decompose while or subsequently emit light. Typical examples of such substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan, 9-alkylidene-N-alkyl acridines, arylvinyl ethers, bisoxyethylene, dimethylthiophene, aromatic imidazoles or gloss concentrates. In other embodiments of the invention, the acceptor is an olefin capable of reacting with singlet oxygen to form a hydroperoxide or dioxetane that can decompose to a ketone or carboxylic acid derivative; stable dioxetanes that can be decomposed by the action of light; acetylenes that can react with singlet oxygen to form diketones; hydrazones or hydrazides of azo compounds or azocarbonyl compounds, such as luminol, may be formed; and aromatic compounds which can form endoperoxides. A specific, non-limiting example of a receptor that can be utilized in accordance with the present disclosure and claimed invention is described in U.S. patent No. US5340716 (which is incorporated herein by reference in its entirety). In other embodiments of the present invention, the acceptor comprises an olefinic compound and a metal chelate that is non-particulated and soluble in an aqueous medium, as described in PCT/US2010/025433 (which is incorporated herein by reference in its entirety).

In the present invention, the "donor" may be polymer particles filled with a photosensitive compound formed by coating a functional group on a substrate, and capable of generating singlet oxygen under light excitation; and/or the "acceptor" may be a polymer particle filled with a luminescent compound and a lanthanoid element formed by coating a functional group on a substrate.

The "matrix" according to the invention is a microsphere or microparticle, known to the person skilled in the art, which may be of any size, which may be organic or inorganic, which may be expandable or non-expandable, which may be porous or non-porous, which has any density, but preferably has a density close to that of water, preferably is floatable in water, and is composed of a transparent, partially transparent or opaque material. The matrix may or may not be charged 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 a donor or acceptor by 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, hydroxyl, 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. Pat. nos. 5709994 and 5780646 (both of which are incorporated herein by reference in their entirety).

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

The term "homogeneous immunoassay kit" as used herein refers to a combination of all reagents or agents that must be used for a homogeneous immunoassay.

The term "detecting the concentration of each biomarker in a group of Rheumatoid Arthritis (RA) biomarkers" as used herein refers to "biomarker co-detection".

II. Embodiment

As previously mentioned, patients with early stage and/or seronegative symptoms, which are partially atypical, are often misdiagnosed and missed due to the diversity of RA manifestations. In order to improve the existing diagnostic strategy of RA and to improve the diagnostic level, the present inventors have made a great deal of studies on RA diagnostic methods.

The inventor researches find that 14-3-3 eta protein is obviously increased in RA serum and joint synovial fluid, and can up-regulate the expression of a plurality of inflammatory factors related to RA, which suggests that the protein possibly participates in RA diseases. The inventor further detects the level of at least 2 of the serum 14-3-3 eta protein, the anti-CCP antibody and the anti-Carp antibody of the RA, the non-RA inflammatory joint lesion patient and the synchronous health physical examination patient, analyzes and compares the levels, and correlates the levels with RA, discovers that the level of at least 2 of the serum 14-3-3 eta protein, the anti-CCP antibody and the anti-Carp antibody is detected jointly, correlates the detection result with RA, and can obviously improve the detection accuracy of RA positive of the RA inflammatory joint lesion patient. The present invention has been made based on the above findings.

Accordingly, a first aspect of the present invention relates to the use of a homogeneous immunoassay to detect the concentration of each biomarker in a group of Rheumatoid Arthritis (RA) biomarkers for the preparation of a reagent for assessing the presence or absence of Rheumatoid Arthritis (RA) in vitro by biochemical markers, comprising:

The second aspect of the invention relates to the use of a homogeneous immunoassay for detecting the concentration of each biomarker in a group of Rheumatoid Arthritis (RA) biomarkers for the manufacture of a reagent for assessing the severity of Rheumatoid Arthritis (RA) by means of off-biochemical markers, comprising:

A third aspect of the invention relates to the use of a homogeneous immunoassay to detect the concentration of each biomarker in a group of Rheumatoid Arthritis (RA) biomarkers for the preparation of a formulation for classifying Rheumatoid Arthritis (RA) and other autoimmune diseases by biochemical labeling of an in vitro region, comprising:

The fourth aspect of the invention relates to the use of a set of Rheumatoid Arthritis (RA) biomarkers for the preparation of a reagent for in vitro assessment of the presence or absence of Rheumatoid Arthritis (RA) in a sample to be tested, wherein an increase in the combined concentration value measured for each biomarker in the set of Rheumatoid Arthritis (RA) biomarkers compared to the truncated combined concentration value for each marker in the corresponding set of biomarkers from a reference population measurement using a homogeneous immunoassay indicates the presence of RA;

In the present invention, the sample to be measured is selected from the group consisting of blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, semen, saliva, synovial fluid, emphysema effusion and tissue, preferably the sample to be measured is selected from the group consisting of blood, plasma, serum, synovial fluid and tissue, more preferably the sample to be measured is selected from the group consisting of blood, plasma and serum, still more preferably the sample to be measured is serum.

In the present invention, anti-CCP antibodies are captured by one or more CCPs that act as antigens.

In the present invention, anti-carps antibodies are captured by one or more carps as antigens.

In the present invention, the 14-3-3eta protein is captured by one or more antibodies to the 14-3-3eta protein.

In some specific embodiments of the invention, the concentration of each biomarker in a biomarker panel is detected using a homogeneous immunoassay.

Specific embodiments for carrying out the invention are further provided below in the fifth to twenty-first aspects.

A fifth aspect of the invention relates to a kit for assessing the presence or absence of Rheumatoid Arthritis (RA) by biochemical markers in vitro based on the concentration of each biomarker in a group of Rheumatoid Arthritis (RA) biomarkers, comprising reagents for detecting the concentration of each biomarker in a group of Rheumatoid Arthritis (RA) biomarkers using a homogeneous immunoassay, wherein the group of biomarkers comprises an Anti-CCP antibody, a 14-3-eta protein and an Anti-carp antibody.

A sixth aspect of the invention relates to a kit for assessing the severity of Rheumatoid Arthritis (RA) by biochemical markers ex vivo based on the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers, comprising reagents for detecting the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers, wherein the set of biomarkers comprises an Anti-CCP antibody, a 14-3-eta protein and an Anti-carp antibody.

A seventh aspect of the invention relates to a kit for classifying Rheumatoid Arthritis (RA) and other autoimmune diseases by biochemically labeling an off-body region based on the concentration of each biomarker in a group of Rheumatoid Arthritis (RA) biomarkers, comprising reagents for detecting the concentration of each biomarker in a group of Rheumatoid Arthritis (RA) biomarkers, wherein the group of biomarkers comprises an Anti-CCP antibody, a 14-3-eta protein, and an Anti-map antibody.

An eighth aspect of the invention relates to a kit for in vitro assessment of the presence or absence of Rheumatoid Arthritis (RA) in a sample to be tested using a set of Rheumatoid Arthritis (RA) biomarkers, wherein an increase in the combined concentration value measured for each biomarker in the set of Rheumatoid Arthritis (RA) biomarkers compared to the truncated combined concentration value for each marker in the corresponding set of biomarkers from a reference population using a homogeneous immunoassay indicates the presence of RA; wherein the biomarker panel comprises an Anti-CCP antibody, a 14-3-3eta protein, and an Anti-card antibody.

In some embodiments of the invention, the method comprises:

In some preferred embodiments of the invention, the biomarker panel comprises Anti-CCP antibodies, 14-3-3eta proteins, and Anti-map antibodies, and other biomarkers.

In some particularly preferred embodiments of the invention, the other biomarker is RA.

The kit according to the fifth to eighth aspects of the present invention comprises the above-described reagent for detecting Anti-CCP antibodies in a biomarker panel by an indirect method using a homogeneous immunoassay, a reagent for detecting 14-3-eta proteins in a biomarker panel by a sandwich method using a homogeneous immunoassay, and a reagent for detecting the concentration of Anti-card antibodies in a biomarker panel by a competitive method using a homogeneous immunoassay.

In some embodiments of the invention, the first antigen or the anti-immune complex antibody is bound to a receptor that is capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal.

In other embodiments of the invention, the acceptor comprises an olefin compound and a metal chelate, which are in non-particulate form and are soluble in an aqueous medium; and/or the acceptor is a polymeric microparticle filled with a luminescent compound and a lanthanide.

According to the invention, the kit further comprises a component c1 comprising a donor capable of generating singlet oxygen in the excited state; preferably, the donor binds to one of the members of the specific binding pair, and the other member of the specific binding pair binds to the first antigen or the anti-immune complex antibody; further preferably, the donor binds to streptavidin, and the first antigen or the anti-immune complex antibody, respectively, binds to biotin.

In the present invention, the donor is a photoactivated or chemically activated sensitizer, which is in non-particulate form and is soluble in an aqueous medium; and/or the donor is polymer particles filled with photosensitive compounds, and can generate singlet oxygen under the excitation of light.

In the present invention, the anti-immune complex antibody binds to the anti-CCP antibody in the first immune complex through a recognition epitope, which is a conformational epitope and/or a linear epitope.

It will be appreciated by those skilled in the art that the anti-immune complex antibodies recognize the constant region portion of the anti-CCP antibody in the first immune complex. The anti-immune complex antibody does not recognize the light chain portion of the anti-CCP antibody in the first immune complex. The anti-immune complex antibody specifically recognizes the Fc segment of the anti-CCP antibody in the first immune complex.

In the invention, the anti-immune complex antibody is a polyclonal antibody and/or a monoclonal antibody; preferably, the anti-immune complex antibody is a monoclonal antibody.

In some embodiments, the method of preparing a polyclonal antibody comprises: immunizing an animal with a human immune complex to obtain animal serum containing the polyclonal antibody; the animal serum is purified by affinity chromatography to obtain the polyclonal antibody which specifically recognizes the human immune complex.

In some embodiments, the method of preparing the monoclonal antibody comprises: spleen cells of mice immunized with the human immune complex are fused with myeloma cells of the mice and then cultured, cell culture supernatant is detected, and positive cell strains are reserved.

According to the invention, the first antigen is a citrullinated antigen.

In some preferred embodiments of the invention, the first antigen is selected from the group consisting of a synthetic cyclic peptide comprising citrulline, a linear peptide comprising citrulline, a polypeptide formed on one peptide chain from at least 2 single citrulline-comprising peptide fragments, a mixture of citrulline-comprising peptide fragments comprising at least 2 single citrulline-comprising peptide fragments, and a citrullinated protein.

In some more preferred embodiments of the invention, the first antigen is selected from the group consisting of a synthetic cyclic peptide comprising cyclic citrulline, a linear peptide comprising citrulline, a polypeptide formed on one peptide chain from at least 2 single citrulline-comprising peptide fragments, and a mixture of citrulline peptide fragments comprising at least 2 single citrulline-comprising peptide fragments.

In some even more preferred embodiments of the invention, the first antigen is a polypeptide formed on one peptide chain from 2-4 citrulline-containing peptide fragments or a mixture of citrulline peptide fragments comprising 2-4 single citrulline-containing peptide fragments; preferably the citrulline-containing peptide fragment is selected from the group consisting of SEQ ID Nos. 2-5.

TABLE 1

Sequence number	Sequence(s)
		SEQ ID No.2	Ring- (HQCHQEST-Cit-GRSRGRCGRSGS)
SEQ ID No.3	ARGGSRERARGRGRG-Cit-GEKR
		SEQ ID No.4	GGSKTSLYNLR-Cit-GTALAIPQ
SEQ ID No.5	APPPISGGGY-cit-A-cit-PAKAAAT

In some preferred embodiments, the first antigen is bound to the receptor via an intermediate that is a hydrophilic polymer.

Preferably, the intermediate is a protein, preferably selected from the group consisting of hemocyanin, ovalbumin, bovine serum albumin or bovine thyroglobulin.

Preferably, the intermediate is selected from the group consisting of dendrimers, polycarboxylates, polythiols and polyethylene glycols.

In some embodiments, the total concentration of the receptor and the first antigen bound thereto is 10-200 μg/mL, preferably 20-150 μg/mL, more preferably 25-100 μg/mL; and/or the total concentration of the anti-immune complex antibody and one of the specific binding pair members bound thereto is from 0.1 to 10. Mu.g/mL, preferably from 0.5 to 5. Mu.g/mL, more preferably from 1 to 3. Mu.g/mL.

In some further particularly preferred embodiments of the present invention, the concentration of Anti-CCP is detected by homogeneous immunoassay using a homogeneous immunoassay kit comprising an antibody to detect Anti-CCP, a 14-3-3eta protein, and an Anti-card antibody, comprising:

In some preferred embodiments of the invention, the method further comprises the step of creating an Anti-CCP antibody standard working curve prior to step 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 amount of Anti-CCP antibody in the sample to be tested is determined based on an Anti-CCP antibody standard operating curve.

The above method for detecting the concentration of Anti-CCP by homogeneous immunoassay using a homogeneous immunoassay kit comprising an antibody for detecting Anti-CCP, a 14-3-eta protein and an Anti-card antibody may be understood as comprising the steps of:

s1, combining a first antigen and an anti-CCP antibody in a sample to be tested to form a first immune complex consisting of the first antigen-anti-CCP antibody;

s2, combining an anti-immune complex antibody with the first immune complex to form a second immune complex consisting of a first antigen-anti-CCP antibody-anti-immune complex antibody;

S3, detecting whether a second immune complex exists; if the second immune complex is present, it is indicative of the presence of anti-CCP antibodies in the test sample.

In some embodiments of the invention, the presence or absence of the second immunocomplexes is detected in step S3 by means of chemiluminescence.

In the present invention, the first immunocomplex binds to a donor via a first antigen, and the corresponding second immunocomplex binds to an acceptor via an anti-immunocomplex antibody, the acceptor being capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal, and the donor being capable of generating singlet oxygen in an excited state.

In the present invention, the first immunocomplex binds to a donor via a first antigen, and the corresponding second immunocomplex binds to a donor via an anti-immunocomplex antibody, the acceptor being capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal, and the donor being capable of generating singlet oxygen in an excited state.

In other embodiments of the present invention, the above-described method for detecting the concentration of Anti-CCP by homogeneous immunoassay using a homogeneous immunoassay kit comprising an antibody for detecting Anti-CCP, 14-3-eta protein and Anti-Carp antibody may be understood as comprising the steps of:

T1, combining a first antigen combined with biotin with an anti-CCP antibody in a sample to be detected to form a third immune complex consisting of biotin-first antigen-anti-CCP antibody;

t2, binding an anti-immune complex antibody that specifically recognizes an anti-CCP antibody in the third immune complex, which binds to the receptor, to the third immune complex to form a fourth immune complex composed of biotin-first antigen-anti-CCP antibody-anti-immune complex antibody-receptor;

t3, combining the donor combined with streptavidin with the fourth immunocomplex to form a fifth immunocomplex consisting of donor-streptavidin-biotin-first antigen-anti-CCP antibody-anti-immunocomplex antibody-acceptor;

t4, detecting the presence or absence of a fifth immune complex; if the fifth immune complex is present, an anti-CCP antibody is present in the test sample;

or,

r1, combining a first antigen combined with a receptor with an anti-CCP antibody in a sample to be detected to form a sixth immune complex formed by the receptor-first antigen-anti-CCP antibody;

r2, combining an anti-immune complex antibody which specifically recognizes an anti-CCP antibody in the sixth immune complex combined with biotin with the sixth immune complex to form a seventh immune complex composed of a receptor-first antigen-anti-CCP antibody-anti-immune complex antibody-biotin;

R3, combining the donor combined with streptavidin with biotin in the seventh immune complex to form an eighth immune complex consisting of the acceptor-first antigen-anti-CCP antibody-anti-immune complex antibody-biotin-streptavidin-donor;

r4, detecting the presence or absence of an eighth immune complex; if the eighth immune complex is present, an anti-CCP antibody is present in the test sample;

wherein, when the fifth immune complex or the eighth immune complex is present, the donor is excited with energy or an active compound to produce singlet oxygen and the acceptor reacts with the singlet oxygen to produce a detectable chemiluminescent signal.

In some embodiments, the method further comprises the step of creating an anti-CCP antibody standard working curve prior to step T1 or step R1.

In some further embodiments, in step T4 or step R4, the intensity of the chemiluminescent signal is detected and the amount of anti-CCP antibody in the test sample is determined based on an anti-CCP antibody standard operating curve.

In some further specific embodiments, the fifth immune complex or the eighth immune complex is irradiated with excitation light having a wavelength of 600-700nm, the excited donor generates singlet oxygen, the acceptor reacts with the contacted singlet oxygen to generate light of 520-620nm, and the signal value of the light is detected, thereby determining whether the anti-CCP antibody and/or the concentration of the anti-CCP antibody is present in the sample to be measured.

In some particularly preferred embodiments of the present invention, homogeneous immunoassay reagents for detecting 14-3-3eta proteins comprise:

In some embodiments of the invention, the amino acid SEQUENCE of the 14-3-3eta protein is as shown in SEQUENCE No. 1. Preferably, the second epitope and the first epitope are each independently selected from the group consisting of amino acid fragments that are relatively specific fragments of the sequence of a 14-3-3eta protein: 1-6aa, 27-38aa, 71-83aa, 112-119aa and 141-154aa.

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

In some preferred embodiments of the invention, the second antibody or binding fragment thereof binds to one member of the specific binding pair member and the donor binds to the other member of the specific binding pair member. Preferably, the second antibody or binding fragment thereof binds to biotin and the donor binds to streptavidin.

In some embodiments of the invention, the concentration of the receptor in component a2 and the primary antibody or binding fragment thereof bound thereto is 10-200 μg/mL, preferably 20-150 μg/mL, more preferably 30-100 μg/mL, most preferably 40-80 μg/mL; and/or the concentration of the second antibody or binding fragment thereof in component b2 is 0.1-8 μg/mL, preferably 0.2-6 μg/mL, more preferably 0.4-4 μg/mL, most preferably 0.6-2 μg/mL; and/or the concentration of said donor in component c2 is 5-20. Mu.g/mL, preferably 8-15. Mu.g/mL, more preferably 10-12. Mu.g/mL.

In the present invention, the acceptor comprises an olefin compound and a metal chelate compound, which are in a non-particulate form and are soluble in an aqueous medium; and/or the acceptor is a polymeric microparticle filled with a luminescent compound and a lanthanide.

In some further particularly preferred embodiments of the present invention, the concentration of 14-3-3eta protein is detected by a homogeneous immunoassay using a homogeneous immunoassay kit comprising an antibody to detect Anti-CCP, 14-3-3eta protein, and an Anti-card antibody, comprising:

In some further embodiments of the invention, in step R4, the intensity of the chemiluminescent signal in step R3 is detected and the amount of 14-3-eta protein in the test sample is determined based on a 14-3-3eta protein standard operating curve.

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

In some embodiments, in step R3, the fourth mixture is irradiated with excitation light having a wavelength of 600-700nm, which excites the donor to produce singlet oxygen, and the acceptor reacts with the contacted singlet oxygen to produce emitted light at 520-620 nm.

According to some preferred embodiments, the kit of parts according to the invention comprises a homogeneous immunoassay reagent for detecting Anti-carp antibodies, comprising:

component b3, comprising a second anti-card antibody.

In the present invention, the antigen or the second anti-Carp antibody is bound to a receptor; preferably, the antigen is bound to a receptor; the acceptor is capable of reacting with the singlet oxygen to generate a detectable chemiluminescent signal.

In the invention, the second anti-Carp antibody is a polyclonal antibody and/or a monoclonal antibody; preferably, the second anti-Carp antibody is a monoclonal antibody.

In some embodiments of the invention, the antigen is selected from the group consisting of a synthetic carbamylated peptide, a polypeptide formed on one peptide chain from at least 2 single carbamylated peptide, a carbamylated peptide mixture containing at least 2 single carbamylated peptide fragments, and a carbamylated protein.

Preferably, the antigen is selected from the group consisting of a synthetic carbamylated peptide fragment, a polypeptide formed on one peptide chain from at least 2 single carbamylated peptide fragments, and a carbamylated peptide fragment mixture containing at least 2 single carbamylated peptide fragments.

Further preferably, the antigen is a polypeptide formed by synthesis of 2-4 carbamylated peptide fragments on one peptide chain or a carbamylated peptide fragment mixture containing 2-4 single carbamylated peptide fragments; preferably the carbamylated peptide stretch is selected from SEQ ID No.6-9.

TABLE 2

Sequence number	Sequence(s)
		SEQ ID No.6	HQCHQEST-Hcit-GKSKGKCGKSGS
SEQ ID No.7	CKAAATQ-Hcit-KVERCARRR
		SEQ ID No.8	NEAN-Hcit-YQISVN-Hcit-YRG
SEQ ID No.9	NEEGFFSA-Hcit-GHRPLDKK

In some preferred embodiments of the invention, the antigen is bound to the receptor via an intermediate that is a hydrophilic polymer. Preferably, the intermediate is a protein, preferably selected from the group consisting of hemocyanin, ovalbumin, bovine serum albumin or bovine thyroglobulin. More preferably, the intermediate is selected from the group consisting of dendrimers, polycarboxylates, polythiols and polyethylene glycols.

In some embodiments of the invention, the total concentration of the antigen and the receptor bound thereto is 0.005-0.1 μg/mL; and/or the total concentration of one member of the second anti-card antibody and the specific binding pair bound thereto is 0.025-0.1 μg/mL; and/or the total concentration of the donor and the other member of the specific binding pair bound thereto is 5-20 μg/mL.

In some further preferred embodiments of the invention, the method for detecting the concentration of Anti-Carp by homogeneous immunoassay using a homogeneous immunoassay kit comprising an antibody for detecting Anti-Carp, corresponding to the above-described reagents in the kit of the invention, comprises the steps of:

m1, enabling the second anti-Carp antibody and a target anti-Carp antibody in a sample to be tested to compete for binding with an epitope of an antigen, and forming a first immune complex formed by the antigen-second anti-Carp antibody and a second immune complex formed by the antigen-target anti-Carp antibody through the epitope of the antigen and the antigen respectively;

M2, detecting the presence or absence of a second immune complex; if a second immune complex is present, it is indicative of the presence of anti-Carp antibodies in the test sample.

In some embodiments of the invention, the step M1 includes uniformly mixing the second anti-Carp antibody, the sample to be tested and the antigen, reacting, so that the second anti-Carp antibody and the target anti-Carp antibody in the sample to be tested compete for binding to the epitope of the antigen, and forming a first immune complex composed of the antigen-second anti-Carp antibody and a second immune complex composed of the antigen-anti-Carp antibody through the epitope of the antigen and the antigen, respectively.

In some further embodiments of the invention, the step of mixing the second anti-card antibody, the test sample, and the antigen uniformly comprises mixing the second anti-card antibody, the test sample, and the antigen simultaneously uniformly; or mixing the second anti-Carp antibody with the sample to be tested to form a 1 st mixture, and then mixing the antigen with the 1 st mixture.

In some embodiments of the invention, the step M1 includes the steps of:

p1, mixing an antigen with a sample to be detected, reacting to enable a target anti-Carp in the sample to be detected to be combined with an epitope of the antigen to form a second immune complex formed by an antigen-target anti-Carp antibody, and obtaining a 11 th mixture;

And P2, mixing the second anti-Carp antibody with the 11 th mixture, and reacting to enable the second anti-Carp antibody to be combined with an epitope of an antigen which is not combined with the anti-Carp in the sample to be tested in the 11 th mixture, so as to form a first immune complex formed by the antigen-second anti-Carp antibody.

In some embodiments, step M2 is performed by detecting the presence or absence of a second immune complex by means of chemiluminescence.

In some embodiments, step M2 comprises comparing the chemiluminescent signal value of the 1 st mixture to the chemiluminescent signal value of a control immune complex formed from an equivalent amount of antigen and second anti-Carp antibody as in step M1, thereby determining the presence or absence of the second immune complex.

In some embodiments of the invention, the antigen binds to a receptor, and the second anti-Carp antibody binds to biotin, and the receptor is capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal.

In some embodiments of the present invention, step M0 is further included before step M1, and the sample to be tested is diluted by the sample diluent according to the volume ratio of 1 (4-20); preferably, the sample to be tested is diluted with a sample diluent in a volume ratio of 1 (6-16); more preferably, the sample to be measured is diluted with a sample diluent at a volume ratio of 1 (8-12).

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

r2, mixing the antigen combined with the receptor with the mixture 2, enabling the second anti-Carp antibody combined with biotin and the target anti-Carp antibody in the sample to be tested to compete for combining with the epitope combined with the antigen combined with the receptor, and forming a third immune complex formed by the receptor-antigen-second anti-Carp antibody-biotin and a fourth immune complex formed by the receptor-antigen-anti-Carp antibody through the epitope and the antigen combined with the receptor respectively, so as to form a mixture 3;

r4, exciting the donor with energy or an active compound to produce singlet oxygen, the acceptor reacting with the singlet oxygen to produce a detectable chemiluminescent signal; detecting the presence or absence of a fourth immune complex; if a fourth immune complex is present, an anti-Carp antibody is present in the test sample.

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

t1, mixing an antigen combined with a receptor with a sample to be tested, reacting to enable a target anti-Carp in the sample to be tested and an epitope combined with the antigen of the receptor to be combined to form a fourth immune complex formed by the receptor-antigen-target anti-Carp antibody, and obtaining a 12 th mixture;

t2, mixing the second anti-Carp antibody combined with biotin with the 12 th mixture, reacting, and allowing the second anti-Carp antibody combined with biotin to compete for combining with the epitope of the antigen combined with the receptor of the anti-Carp in the 12 th mixture which is not combined with the sample to be tested, so as to form a third immune complex consisting of the antigen combined with the receptor, the second anti-Carp antibody and the biotin, thereby forming a 13 th mixture;

t3, mixing the streptavidin-conjugated donor with the 13 th mixture, and allowing the streptavidin-conjugated donor to bind to biotin in the third immunocomplex to form a fifth immunocomplex consisting of an acceptor-antigen-second anti-card antibody-biotin-streptavidin-donor, thereby forming the 14 th mixture;

t4, exciting the donor with energy or an active compound to generate singlet oxygen, the acceptor reacting with the singlet oxygen to generate a detectable chemiluminescent signal; detecting the presence or absence of a fourth immune complex; if a fourth immune complex is present, an anti-Carp antibody is present in the test sample.

In some embodiments of the invention, the method further comprises the step of making a standard working curve of the target anti-card antibody prior to step R1 or step T1.

In some embodiments of the invention, in step R4 or step T4, the intensity of the chemiluminescent signal is detected and the amount of the target anti-Carp antibody in the test sample is determined based on an anti-Carp antibody standard working curve.

In some embodiments of the invention, the donor in the fifth immunocomplexes is excited to generate singlet oxygen by irradiation of the 4 th mixture or the 14 th mixture with excitation light having a wavelength of 600-700nm, the acceptor reacts with the contacted singlet oxygen to generate light emitted at 520-620nm, the signal value of the light emitted from the 4 th mixture or the 14 th mixture is detected, and compared with the chemiluminescent signal value of the control immunocomplexes formed by the antigen and the second anti-Carp antibody in the medium amount in the step R2 or the steps T1 and T2, thereby determining whether the target anti-Carp antibody and/or the concentration of the target anti-Carp antibody is present in the sample to be measured.

The kit according to the fifth to eighth aspects of the present invention comprises the above-described reagent for detecting an Anti-CCP antibody in a biomarker panel by a homogeneous immunoassay using a homogeneous immunoassay, a reagent for detecting a 14-3-3eta protein in a biomarker panel by a sandwich method using a homogeneous immunoassay, and a reagent for detecting an Anti-card antibody concentration in a biomarker panel by a sandwich method using a homogeneous immunoassay.

or,

In the invention, the first antigen and the second antigen are carbamylated antigens; preferably the carbamylated antigen is carbamylated human serum albumin; it is further preferred that the human serum albumin is shown in SEQ ID No. 6.

In some preferred embodiments of the invention, the second antigen binds to one member of the specific binding pair and the donor binds to the other member of the specific binding pair. Preferably, the second antigen is bound to biotin and the donor is bound to streptavidin.

In some embodiments of the invention: the total concentration of the receptor and the first antigen bound thereto is 10-200. Mu.g/mL, preferably 20-150. Mu.g/mL, more preferably 25-100. Mu.g/mL; and/or the total concentration of the second antigen and one of the specific binding partners bound thereto is 0.1-10. Mu.g/mL, preferably 0.5-5. Mu.g/mL, more preferably 1-3. Mu.g/mL.

In some further preferred embodiments of the invention, corresponding to the above reagents in the kit of the invention, the concentration of Anti-Carp is detected by a homogeneous immunoassay using a homogeneous immunoassay kit comprising an antibody to detect Anti-Carp, comprising the steps of:

and R4, detecting the existence and/or intensity of the chemiluminescent signal in the step R3, so as to judge whether the anti-Carp antibody exists in the sample to be detected and/or determine the content of the anti-Carp antibody.

In some further embodiments of the invention, in step R4, the intensity of the chemiluminescent signal in step R3 is detected and the amount of anti-Carp antibody in the test sample is determined based on an anti-Carp antibody standard operating curve.

In some embodiments of the invention, in step R3, the fourth mixture is irradiated with excitation light of 600-700nm wavelength, which excites the donor to produce singlet oxygen and the acceptor reacts with the singlet oxygen in contact to produce light emissions of 520-620 nm.

According to some embodiments of the invention, the system comprises:

According to some embodiments of the invention, other reagents may also be included in the kit of the invention.

According to some preferred embodiments of the invention, the kit comprises reagents for detecting the concentrations of Anti-CCP antibodies, 14-3-eta proteins and Anti-card antibodies in a biomarker panel using a homogeneous immunoassay.

According to some preferred embodiments, the kit of parts according to the invention comprises homogeneous immunoassay reagents for detecting anti-CCP antibodies comprising:

A first composition comprising component a5, said component a5 consisting of a receptor capable of reacting with singlet oxygen to generate a detectable signal and a first antigen bound thereto, wherein said first antigen is capable of specifically binding to an epitope of an anti-cyclic citrullinated peptide antibody at a first recognition site;

a second composition comprising component b5, said component b5 consisting of a second antigen capable of binding specifically to a second recognition site of an epitope of an anti-cyclic citrullinated peptide antibody and one member of a specific counterpart bound thereto;

a third composition comprising a component c5, said component c being constituted by a donor capable of generating singlet oxygen in an excited state and another member of a specific pair bound thereto.

In the present invention, the first antigen and the second antigen are the same or different and are independently selected from the group consisting of a synthetic cyclic peptide containing citrulline, a linear peptide containing citrulline, a polypeptide formed on one peptide chain from at least 2 single citrulline-containing peptide fragments, a mixture of citrulline-containing peptide fragments containing at least 2 single citrulline-containing peptide fragments, and a citrullinated protein.

In some embodiments of the invention, the first antigen and the second antigen are each independently selected from the group consisting of a synthetic cyclic citrulline-containing cyclic peptide, a citrulline-containing linear peptide, a polypeptide formed on one peptide chain from at least 2 single citrulline-containing peptide fragments, and a citrulline-containing peptide fragment mixture comprising at least 2 single citrulline-containing peptide fragments.

Preferably, the first antigen and the second antigen are each independently a polypeptide formed on one peptide chain from 2-4 citrulline-containing peptide fragments synthesized or a citrulline-containing peptide fragment mixture comprising 2-4 single citrulline-containing peptide fragments; it is further preferred that the citrulline-containing peptide stretch is selected from the group consisting of SEQ ID Nos. 2-5.

In some preferred embodiments of the invention, the first antigen is bound to the receptor via an intermediate, which is a hydrophilic polymer.

In some embodiments, the intermediate is a protein, preferably selected from the group consisting of hemocyanin, ovalbumin, bovine serum albumin, or bovine thyroglobulin.

In some preferred examples, the second antigen is bound to biotin and the donor is bound to streptavidin.

In some embodiments of the invention, the total concentration of component a5 in the first composition is from 10 to 200 μg/mL, preferably from 20 to 150 μg/mL, more preferably from 25 to 100 μg/mL; and/or the concentration of said component b5 in the second composition is 0.1-10 μg/mL, preferably 0.5-5 μg/mL, more preferably 1-3 μg/mL.

In some further preferred embodiments of the invention, corresponding to the above reagents in the kit of the invention, the concentration of Anti-CCP is detected by a homogeneous immunoassay using a homogeneous immunoassay kit comprising a reagent for detecting Anti-CCP antibodies, comprising:

r1, mixing a sample to be tested with a first composition and a second composition, and reacting to obtain a first mixture;

Step R2, mixing the first mixture with the third composition and then reacting to obtain a second mixture;

step R3, contacting energy or an active compound with the second mixture to excite the donor to generate singlet oxygen, the acceptor being capable of reacting with the received singlet oxygen to generate a detectable chemiluminescent signal;

and R4, detecting the existence and/or intensity of the chemiluminescent signal in the step R3, so as to judge whether the anti-cyclic citrullinated peptide antibody exists in the sample to be detected and/or determine the content of the anti-cyclic citrullinated peptide antibody.

In some embodiments of the invention, the method further comprises the step of making an anti-cyclic citrullinated peptide antibody standard working curve prior to step R1.

In some further embodiments of the invention, in step R4, the intensity of the chemiluminescent signal in step R3 is detected and the amount of anti-cyclic citrullinated peptide antibody in the sample to be tested is determined based on the anti-cyclic citrullinated peptide antibody standard working curve.

In some embodiments of the invention, the second mixture is irradiated with excitation light having a wavelength of 600-700nm, the donor is excited to generate singlet oxygen, the acceptor reacts with the contacted singlet oxygen to generate light of 520-620nm, and the signal intensity of the light is detected, thereby judging whether the anti-cyclic citrullinated peptide antibody exists in the sample to be detected and/or determining the content of the anti-cyclic citrullinated peptide antibody.

component a6 comprising a first antigen capable of specifically binding to an epitope binding site of an anti-Carp antibody;

component b6 comprising an anti-immune complex antibody capable of specifically recognizing and binding to an anti-Carp antibody in forming a first immune complex with a first antigen, without recognizing a free, non-antigen-binding anti-Carp antibody.

In the present invention, the first antigen or the anti-immune complex antibody is bound to a receptor capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal.

In some embodiments of the invention, the kit further comprises a component c6 comprising a donor capable of generating singlet oxygen in an excited state; preferably, the donor binds to one of the members of the specific binding pair, and the other member of the specific binding pair binds to the first antigen or the anti-immune complex antibody; further preferably, the donor binds to streptavidin, and the first antigen or the anti-immune complex antibody, respectively, binds to biotin.

It will be appreciated by those skilled in the art that the anti-immune complex antibodies bind to the anti-card antibodies in the first immune complex via recognition epitopes, which are conformational epitopes and/or linear epitopes. The anti-immune complex antibody recognizes a constant region portion of the anti-card antibody in the first immune complex. The anti-immune complex antibody does not recognize the light chain portion of the anti-card antibody in the first immune complex. The anti-immune complex antibody specifically recognizes the Fc segment of the anti-Carp antibody in the first immune complex.

In some embodiments of the invention, the method of preparing the polyclonal antibody comprises: immunizing an animal with a human immune complex to obtain animal serum containing the polyclonal antibody; the animal serum is purified by affinity chromatography to obtain the polyclonal antibody which specifically recognizes the human immune complex.

In some embodiments of the invention, the method of preparing the monoclonal antibody comprises: spleen cells of mice immunized with the human immune complex are fused with myeloma cells of the mice and then cultured, cell culture supernatant is detected, and positive cell strains are reserved.

In the present invention, the first antigen is selected from the group consisting of a synthetic carbamylated peptide, a polypeptide formed on one peptide chain from at least 2 single carbamylated peptide, a carbamylated peptide mixture containing at least 2 single carbamylated peptide and a carbamylated protein.

Preferably, the first antigen is selected from the group consisting of a synthetic carbamylated peptide fragment, a polypeptide formed on one peptide chain from at least 2 single carbamylated peptide fragments, and a carbamylated peptide fragment mixture containing at least 2 single carbamylated peptide fragments.

Further preferred, the first antigen is a polypeptide formed by synthesis of 2-4 carbamylated peptide fragments on one peptide chain or a carbamylated peptide fragment mixture containing 2-4 single carbamylated peptide fragments; preferably, the carbamylated peptide is selected from Sequence No.6-9.

In the present invention, the intermediate is a protein, preferably selected from the group consisting of hemocyanin, ovalbumin, bovine serum albumin, and bovine thyroglobulin. Preferably, the intermediate is selected from the group consisting of dendrimers, polycarboxylates, polythiols and polyethylene glycols.

In some embodiments of the invention, the concentration of the receptor and anti-immunocomplex antibody bound thereto is 10-200 μg/mL, preferably 20-150 μg/mL, more preferably 25-100 μg/mL; and/or the concentration of the first antigen and one of the specific binding pair members bound thereto is in the range of 0.1-10. Mu.g/mL, preferably 0.5-5. Mu.g/mL, more preferably 1-3. Mu.g/mL.

In some further preferred embodiments of the invention, corresponding to the above reagents in the kit of the invention, the concentration of Anti-CCP is detected by a homogeneous immunoassay using a homogeneous immunoassay kit comprising a reagent for detecting Anti-CCP antibodies, comprising the steps of:

s1, combining a first antigen and an anti-Carp antibody in a sample to be tested to form a first immune complex consisting of the first antigen-anti-Carp antibody;

s2, combining an anti-immune complex antibody with the first immune complex to form a second immune complex consisting of a first antigen-anti-card antibody-anti-immune complex antibody;

S3, detecting whether a second immune complex exists; if a second immune complex is present, it is indicative of the presence of anti-Carp antibodies in the test sample.

In some embodiments, the presence or absence of the second immunocomplexes is detected in step S3 by a chemiluminescent method.

In some embodiments, the first immunocomplex binds to a donor via a first antigen, and the corresponding second immunocomplex binds to a donor via an anti-immunocomplex antibody, the acceptor being capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal, the donor being capable of generating singlet oxygen in an excited state.

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

t1, combining a first antigen combined with biotin with an anti-Carp antibody in a sample to be detected to form a third immune complex consisting of biotin-first antigen-anti-Carp antibody;

T2, binding an anti-immune complex antibody that specifically recognizes an anti-Carp antibody in the third immune complex, which binds to the receptor, to the third immune complex to form a fourth immune complex composed of biotin-first antigen-anti-Carp antibody-anti-immune complex antibody-receptor;

t3, combining the donor combined with streptavidin with the fourth immunocomplex to form a fifth immunocomplex consisting of donor-streptavidin-biotin-first antigen-anti-Carp antibody-anti-immunocomplex antibody-acceptor;

t4, detecting the presence or absence of a fifth immune complex; if the fifth immune complex exists, an anti-Carp antibody exists in the sample to be tested;

or,

r1, combining a first antigen combined with a receptor with an anti-Carp antibody in a sample to be detected to form a sixth immune complex formed by the receptor-first antigen-anti-Carp antibody;

r2, combining an anti-immune complex antibody which is combined with biotin and specifically recognizes an anti-Carp antibody in the sixth immune complex with the sixth immune complex to form a seventh immune complex formed by a receptor-first antigen-anti-Carp antibody-anti-immune complex antibody-biotin;

r3, combining the donor combined with streptavidin with biotin in the seventh immune complex to form an eighth immune complex consisting of an acceptor-first antigen-anti-Carp antibody-anti-immune complex antibody-biotin-streptavidin-donor;

R4, detecting the presence or absence of an eighth immune complex; if the eighth immune complex exists, an anti-Carp antibody exists in the sample to be tested;

In some embodiments, the method further comprises the step of creating an anti-card antibody standard working curve prior to step T1 or step R1.

In some further embodiments, in step T4 or step R4, the intensity of the chemiluminescent signal is detected and the amount of anti-Carp antibody in the test sample is determined based on an anti-Carp antibody standard operating curve.

In some embodiments of the present invention, the fifth immune complex or the eighth immune complex is irradiated with excitation light having a wavelength of 600-700nm, the excited donor generates singlet oxygen, the acceptor reacts with the contacted singlet oxygen to generate light with a wavelength of 520-620nm, and a signal value of the light is detected, so as to determine whether the anti-Carp antibody and/or the concentration of the anti-Carp antibody is present in the sample to be measured.

In some particularly preferred embodiments of the present invention, a heterogeneous chemiluminescent immunoassay reagent for detecting an Anti-CCP antibody comprises:

A component a7 comprising a solid support and a first antigen directly or indirectly bound thereto, said first antigen being capable of specifically binding to an epitope binding site of an anti-CCP antibody;

component b7 comprising a label capable of reacting with a substrate or capable of catalyzing the generation of a detectable signal by the substrate and an anti-immune complex antibody directly or indirectly bound thereto, said anti-immune complex antibody being capable of specifically recognizing and binding to an anti-CCP antibody in a first immune complex formed with a first antigen, and not recognizing free, non-antigen-bound anti-CCP antibodies.

In some embodiments of the invention, the reagent further comprises an Anti-CCP antibody pure as a calibrator, which is diluted in a ratio gradient by a calibrator diluent to working calibrator solutions of different concentrations.

In some preferred embodiments of the invention, the first antigen binds to one member of the specific binding pair member and the solid support binds to the other member of the specific binding pair member; preferably, the first antigen is bound to biotin and the solid support is bound to streptavidin.

In other preferred embodiments of the invention, the anti-immunocomplex antibody binds to one of the specific binding pair members, and the label binds to the other of the specific binding pair members; preferably, the anti-immune complex antibody binds to biotin and the label binds to streptavidin.

In some preferred embodiments of the invention, component c7 is also included, a substrate solution comprising A1 solution and a B1 solution, preferably the A1 solution is hydrogen peroxide solution, preferably the B1 solution is sodium hydroxide solution.

In some embodiments of the invention, the anti-immune complex antibody recognizes a constant region portion of an anti-CCP antibody in the first immune complex.

In some embodiments of the invention, the anti-immune complex antibody does not recognize the light chain portion of the anti-CCP antibody in the first immune complex.

In other embodiments of the invention, the anti-immune complex antibody specifically recognizes the Fc segment of the anti-CCP antibody in the first immune complex.

In some embodiments of the invention, the anti-immune complex antibody is a polyclonal antibody and/or a monoclonal antibody.

In some preferred embodiments of the invention, the anti-immune complex antibody is a monoclonal antibody.

In the present invention, the method for preparing the polyclonal antibody comprises: immunizing an animal with a human immune complex to obtain animal serum containing the polyclonal antibody; the animal serum is purified by affinity chromatography to obtain the polyclonal antibody which specifically recognizes the human immune complex.

In the present invention, the method for preparing the monoclonal antibody comprises: spleen cells of mice immunized with the human immune complex are fused with myeloma cells of the mice and then cultured, cell culture supernatant is detected, and positive cell strains are reserved.

According to the invention, the first antigen is a citrullinated antigen.

In some embodiments of the invention, the first antigen is selected from the group consisting of a synthetic cyclic peptide comprising citrulline, a linear peptide comprising citrulline, a polypeptide formed on one peptide chain from at least 2 single citrulline-comprising peptide fragments, a mixture of citrulline peptide fragments comprising at least 2 single citrulline-comprising peptide fragments, and a citrullinated protein.

In some preferred embodiments of the invention, the first antigen is selected from the group consisting of a synthetic cyclic citrulline-containing cyclic peptide, a citrulline-containing linear peptide, a polypeptide formed on one peptide chain from at least 2 single citrulline-containing peptide fragments, and a citrulline-containing peptide fragment mixture comprising at least 2 single citrulline-containing peptide fragments; preferably the first antigen is a polypeptide formed on one peptide chain from 2-4 citrulline-containing peptide fragments or a mixture of citrulline peptide fragments comprising 2-4 single citrulline-containing peptide fragments.

In some preferred embodiments of the invention, the citrulline-containing peptide fragment is selected from the group consisting of SEQ ID Nos. 2-5.

In some preferred embodiments, the first antigen is indirectly linked to a solid support.

In some embodiments of the invention, the concentration of the solid support and the first antigen bound thereto in component a7 is 1-100mg/mL, preferably 10-50mg/mL.

In some embodiments of the invention, the concentration of the first label in component b7 and the second antibody or binding fragment thereof bound thereto is 1-100mg/mL, preferably 10-50mg/mL.

In some further particularly preferred embodiments of the present invention, a heterogeneous chemiluminescent immunoassay kit comprising an antibody to detect an Anti-CCP antibody is employed to detect the concentration of the Anti-CCP antibody by a heterogeneous chemiluminescent immunoassay method comprising:

r1, mixing a sample to be tested with a component a7 to obtain a first mixture;

step R2, mixing the first mixture with the component b7 to obtain a second mixture;

step R3, mixing the second mixture with component c7 to obtain a third mixture that produces a detectable chemiluminescent signal;

III. Examples

In order that the invention may be more readily understood, the invention will be further described in detail with reference to the following examples, which are given by way of illustration only and are not limiting in scope of application. The starting materials or components used in the present invention may be prepared by commercial or conventional methods unless specifically indicated.

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

Example 1:

53 samples of confirmed rheumatoid arthritis were collected and assayed for Anti-CCP antibodies, 14-3-3eta protein and Anti-card antibodies, respectively, using homogeneous immunoassay.

1. And irradiating the fifth immune complex with excitation light with the wavelength of 600-700nm, exciting the donor to generate singlet oxygen, reacting the acceptor with the contacted singlet oxygen to generate 520-620nm emitted light, and detecting the signal value of the emitted light, thereby judging whether the anti-CCP antibody and/or the concentration of the anti-CCP antibody exist in the sample to be detected. The first antigen coating is taken as a luminous particle (reagent 1) of a receptor, biotin labeling anti-immune complex antibody (reagent 2) is assembled into an anti-citrullinated peptide antibody determination kit, then the kit is loaded on a full-automatic light excitation chemiluminescence immunoassay analyzer, and a detection step is set:

1) The sample adding Tip head sucks 20 mu L of calibrator into the reaction microwell plate;

2) The sample adding Tip head sucks 25 mu L of the reagent 1 into the reaction microwell plate;

3) The sample adding Tip head sucks 25 mu L of reagent 2 into the reaction microwell plate;

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

5) The sample adding Tip head absorbs 175 mu L of photosensitive liquid serving as a donor into the reaction micro-pore plate;

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

7) Under the irradiation of excitation light generated by the instrument, the donor (photosensitive particles) is induced to activate and release active oxygen ions in a high energy state. The active oxygen ions in the high energy state are trapped by the acceptor (luminescent particles) at a short distance, thereby transferring energy to activate the luminescent compound in the acceptor (luminescent particles). After a few microseconds, the luminescent compounds in the acceptor (luminescent particles) will release high-level red light, which is measured with a single photon counter;

8) 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 anti-CCP antibody;

9) And also detecting the sample to be detected according to the steps 1) -7), and calculating the concentration of the anti-CCP antibody in the sample to be detected according to the equation in the step 8).

2. The concentration of 14-3-3 was detected by a sandwich method using a homogeneous immunoassay.

Judging whether 14-3-3eta protein exists in the sample to be detected, wherein the homogeneous immunodetection method for detecting the 14-3-3eta protein in the sample to be detected comprises the following steps:

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

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

(3) Illuminating the fourth mixture with excitation light having a wavelength of 600-700nm capable of exciting the donor to produce singlet oxygen, and reacting the acceptor with the contacted singlet oxygen to produce an emitted light of 520-620nm as a detectable chemiluminescent signal;

(4) Detecting the presence or absence of a chemiluminescent signal in step (4).

In other embodiments of the present invention, the method for determining the content of 14-3-3eta protein in a sample to be tested comprises the steps of:

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

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

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

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

(4) Irradiating the fourth mixture with excitation light of 600-700nm wavelength to excite the donor to generate singlet oxygen, and reacting the acceptor with the singlet oxygen to generate 520-620nm emitted light as detectable chemiluminescent signal;

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

(6) And (3) repeating the steps (2) - (5) to detect the chemiluminescent signal values (intensities) of the working calibrator solution containing the 14-3-3eta proteins 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 proteins and the chemiluminescent signal values.

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

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

(3) Irradiating the fourth mixture with excitation light of 600-700nm wavelength to excite the donor to generate singlet oxygen, and reacting the acceptor with the singlet oxygen to generate 520-620nm emitted light as detectable chemiluminescent signal;

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

4. Experimental operation:

after the components are assembled into a 14-3-3eta protein measuring box, the 14-3 eta protein measuring box is loaded on a full-automatic light-activated chemiluminescence immunoassay analyzer, and a detection step is set:

2) The sample adding Tip head sucks 25 mu L of the reagent I into the reaction microwell plate;

3) The sample adding Tip head sucks 25 mu L of reagent II into the reaction microwell plate;

5) The sample adding Tip head sucks 175 mu L of mixed solution (matched with an instrument) containing a streptavidin modified donor into a reaction microplate;

7) Under 680nm excitation light generated by the instrument, the donor is induced to activate and release active oxygen ions in a high energy state. The high-energy active oxygen ions are trapped by the luminescent particles at close distances, thereby transferring energy to activate the luminescent compounds in the luminescent particles. After a few microseconds, the luminescent compound in the acceptor will release high-level red light of 612nm, which is measured with a single photon counter;

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 14-3-3eta protein concentration;

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

3. The concentration of anti-card antibodies was detected by a competition method using a homogeneous immunoassay.

The kit is applied to the detection step of a full-automatic light-activated chemiluminescence immunoassay analyzer LICA500 (manufactured by Shanghai Boyang).

1) Diluting the sample at the pre-dilution hole site according to the ratio of 1:10, and uniformly mixing for 20 seconds;

2) The sample loading Tip head sucks 10 mu L of diluted sample or calibrator into the reaction microwell plate;

3) The reagent sample-adding Tip absorbs 25 mu L of carbamylated human serum albumin coating receptor (luminescent particles) into the reaction microwell plate;

4) The reagent sample-adding Tip absorbs 25 mu L of biotinylated rabbit anti-carbamylated protein antibody into the reaction microwell plate;

5) Incubating for 17min at 37 ℃ after uniformly mixing for 20 seconds;

6) The reagent sample adding Tip head sucks 175 mu L of donor (photosensitive liquid, working concentration is 20 mu g/mL) into the reaction microwell plate;

7) Incubating for 15min at 37 ℃ after uniformly mixing for 20 seconds;

8) Under the irradiation of excitation light generated by the instrument, the photosensitive particles in the donor are induced to activate and release active oxygen ions in a high energy state. The active oxygen ions in the high energy state are trapped by the acceptor (luminescent particles) at a short distance, thereby transferring energy to activate the luminescent compound in the acceptor (luminescent particles). After a few microseconds, the luminescent compounds in the acceptor (luminescent particles) will release high-level red light, which is measured with a single photon counter;

9) Respectively testing the luminescence values of the standard substances with different concentrations according to the steps 1) -8), and drawing a standard curve according to a five-parameter fitting method to obtain a relational expression between the luminescence values and the anti-Carp Ab concentration; and then respectively testing the luminous values of the sample to be tested according to the steps 1) -8), and calculating the concentration of the anti-card Ab in the sample to be tested according to the relation.

4. Experimental data

The experimental data are shown in tables 3 and 4.

TABLE 3 Table 3

TABLE 4 Table 4

The experimental results show that: 14-3-3 individual positive rate 66%, anti-map individual positive rate 68%, anti-CCP individual positive rate 79%, combined three detection positive rate 88%.

Example 2:

collecting 40 samples of the confirmed rheumatoid arthritis, respectively detecting the Anti-CCP antibody, the 14-3-eta protein and the Anti-card antibody by adopting a homogeneous phase and/or heterogeneous phase immunoassay method, and then counting three detection results of the same sample together so as to improve the accurate diagnosis rate of the sample.

2. The concentration of 14-3-3 is detected by a sandwich method by adopting a 14-3-3eta detection kit (light excitation chemiluminescence method) (developed by Beijing Korea) and adopting a homogeneous immunoassay method

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

(4) Detecting the presence or absence of a chemiluminescent signal in step (4).

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

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

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

4. Experimental operation:

3. Detection of Anti-map antibody by Anti-map detection kit (photo-activated chemiluminescence method) (developed in Beijing Korea)

The kit is applied to the detection step of a full-automatic light excitation chemiluminescence immunoassay analyzer LICA400 (manufactured by Shanghai Boyang).

5) Incubating for 17min at 37 ℃ after uniformly mixing for 20 seconds;

7) Incubating for 15min at 37 ℃ after uniformly mixing for 20 seconds;

4. Results and conclusions

5.1, judging the negative and positive of the clinical sample according to the judgment value specified in the kit instruction.

The results of the three kits for 40 samples are shown in Table 5.

TABLE 5

In 40 samples of confirmed rheumatoid arthritis, the single CCP test item detected positive rate was 68%, the single 14-3-3eta test item detected positive rate was 50%, and the single card test item detected positive rate was 60%.

4.2 statistical analysis of three detection results

Statistical analysis of the three test results is shown in Table 6.

TABLE 6

When one of the CCP detection item and the 1433eta detection item is positive, the detected positive rate is increased to 75%; when one of the CCP detection item and the map detection item is positive and is judged to be positive, the detected positive rate is increased to 80%; when one of the CCP detection items or 1433eta or the card detection item is positive, the detected positive rate is increased to 82.5%. The study shows that the three serological indexes have no correlation, which indicates that certain complementarity exists, and the joint detection of two or three indexes can improve the sensitivity of the clinical diagnosis of the rheumatoid disease.

It should be noted that the above-described embodiments are only for explaining the present invention and do not constitute any limitation of the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.

Sequence listing

<110> Beijing Komei biotechnology Co., ltd

<120> method for in vitro evaluation of the presence or absence of rheumatoid arthritis by biomarker profiling

<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

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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. Use of a reagent required to detect the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers using a homogeneous immunoassay for the preparation of a kit for assessing the presence or absence of Rheumatoid Arthritis (RA) by biochemical markers in vitro comprising:

wherein the biomarker panel comprises an Anti-CCP antibody, a 14-3-3 eta protein, and an Anti-map antibody; homogeneous immunoassay reagents for detecting anti-cyclic citrullinated peptide antibodies include:

2. The use according to claim 1, characterized in that the combined concentration value of step b) is compared with a cut-off value derived from a reference population other than RA positive patients, said reference population comprising apparently healthy persons and patients selected from Osteoarthritis (OA) patients and other autoimmune disease patients.

3. Use of a reagent required to detect the concentration of each biomarker in a group of Rheumatoid Arthritis (RA) biomarkers using a homogeneous immunoassay in the preparation of a kit for assessing the severity of Rheumatoid Arthritis (RA) in vitro by biochemical markers, comprising:

4. Use of a reagent required to detect the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers using a homogeneous immunoassay for the preparation of a kit for classifying Rheumatoid Arthritis (RA) and other autoimmune diseases by biochemically labeling off-body regions, comprising:

5. The use according to claim 4, wherein the other autoimmune diseases comprise other joint diseases; the other joint disease is Osteoarthritis (OA).

6. Use of a set of Rheumatoid Arthritis (RA) biomarkers for the preparation of a reagent for assessing in vitro the presence or absence of Rheumatoid Arthritis (RA) in a sample to be tested, wherein an increase in the combined concentration value measured for each biomarker in the set of Rheumatoid Arthritis (RA) biomarkers compared to the truncated combined concentration value for each marker in the corresponding set of biomarkers measured from a reference population using a homogeneous immunoassay is indicative of the presence of RA;

7. The use according to any one of claims 1-6, wherein the biomarker panel comprises Anti-CCP antibodies, 14-3-3eta proteins and Anti-map antibodies and RA.

8. The use according to any one of claims 1 to 6, wherein the step further comprises measuring the amount of 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.

9. The use according to claim 8, wherein the amount of 14-3-3eta protein in the sample to be tested is determined based on a 14-3-3eta protein standard working curve.

10. The use according to claim 8, wherein the step further comprises comparing the amount of the 14-3-3eta protein or fragment thereof or the immunocomplex formed by the 14-3-3eta protein or fragment thereof with at least one antibody to the amount of the 14-3-3eta protein or fragment thereof or the immunocomplex formed by at least one antibody in a normal control sample, a rheumatoid arthritis control sample or a pre-treatment sample from the same subject.

11. The use according to claim 8, wherein the step comprises specifically binding the sample to an antibody comprising a polypeptide capable of forming an immune complex with at least one specific epitope of the 14-3-3eta protein or fragment thereof.

12. The use of claim 8, wherein 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.

13. The use of claim 12, wherein the first antibody binds to a receptor capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal.

14. Use according to claim 13, characterized in that the acceptor comprises an olefin compound and a metal chelate, which are in non-particulate form and are soluble in an aqueous medium; and/or the acceptor is a polymeric microparticle filled with a luminescent compound and a lanthanide.

15. The use according to claim 12, wherein the first and second antibodies are each independently selected from monoclonal and/or polyclonal antibodies.

16. The use according to claim 15, wherein the first and second antibodies are each independently selected from monoclonal antibodies.

17. The use according to claim 8, wherein the amino acid SEQUENCE of the 14-3-3eta protein or fragment thereof is as shown in SEQUENCE No. 1.

18. The use according to claim 12, wherein the epitope is selected from the group consisting of a relatively specific fragment of the amino acid fragment of the sequence of the 14-3-3 eta protein: 1-6aa, 27-38aa, 71-83aa, 112-119aa and 141-154aa.

19. A kit for in vitro assessment of the presence or absence of Rheumatoid Arthritis (RA) based on the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers, comprising reagents for detecting the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers using a homogeneous immunoassay, wherein the set of biomarkers comprises an Anti-CCP antibody, a 14-3-eta protein, and an Anti-carp antibody; homogeneous immunoassay reagents for detecting anti-cyclic citrullinated peptide antibodies include:

20. A kit for in vitro assessment of the severity of Rheumatoid Arthritis (RA) based on the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers, comprising reagents for detecting the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers, wherein the set of biomarkers comprises an Anti-CCP antibody, a 14-3-eta protein, and an Anti-card antibody; homogeneous immunoassay reagents for detecting anti-cyclic citrullinated peptide antibodies include:

21. A kit for differentiating Rheumatoid Arthritis (RA) from other autoimmune diseases in vitro based on the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers, comprising reagents for detecting the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers, wherein the set of biomarkers comprises an Anti-CCP antibody, a 14-3 eta protein, and an Anti-carp antibody; homogeneous immunoassay reagents for detecting anti-cyclic citrullinated peptide antibodies include:

22. A kit for in vitro assessing the presence or absence of Rheumatoid Arthritis (RA) in a sample to be tested using a set of Rheumatoid Arthritis (RA) biomarkers, wherein an increase in the combined concentration value measured for each biomarker in the set of Rheumatoid Arthritis (RA) biomarkers compared to the truncated combined concentration value for each marker in the corresponding set of biomarkers measured from a reference population using a homogeneous immunoassay indicates the presence of RA; wherein the biomarker panel comprises an Anti-CCP antibody, a 14-3-3 eta protein, and an Anti-map antibody;

homogeneous immunoassay reagents for detecting anti-cyclic citrullinated peptide antibodies include:

23. The kit of any one of claims 19-22, wherein the biomarker panel comprises Anti-CCP antibodies, 14-3-3 eta proteins and Anti-map antibodies, and RA.

24. The kit of any one of claims 19-22, wherein the kit comprises reagents for detecting the concentrations of Anti-CCP antibodies, 14-3-3 eta proteins, and Anti-Carp antibodies in a biomarker panel using a homogeneous immunoassay.

25. The kit of any one of claims 19-22, wherein the first antigen or the anti-immune complex antibody is conjugated to a receptor capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal.

26. The kit of claim 25, wherein the acceptor comprises an alkene compound and a metal chelate, which are in non-particulate form and are soluble in an aqueous medium; and/or the acceptor is a polymeric microparticle filled with a luminescent compound and a lanthanide.

27. The kit of any one of claims 19-22, further comprising component c1 comprising a donor capable of generating singlet oxygen in an excited state.

28. The kit of claim 27, wherein the donor binds to one member of a specific binding pair member and the other member of a specific binding pair member binds to the first antigen or the anti-immune complex antibody.

29. The kit of claim 27, wherein the donor binds to streptavidin and the first antigen or the anti-immune complex antibody, respectively, binds to biotin.

30. The kit of any one of claims 19-22, wherein the reagent further comprises pure anti-CCP antibody as a calibrator which is diluted in a ratio gradient to working calibrator solutions of different concentrations by a calibrator diluent.

31. The kit of claim 24, wherein the homogeneous immunoassay reagents for detecting 14-3-3 eta protein comprise:

a component a2 comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a first antibody or binding fragment thereof bound thereto, said first antibody or binding fragment thereof being capable of specifically binding to a first epitope of a 14-3-3 eta protein;

a component b2 comprising a second antibody or binding fragment thereof capable of specifically binding to a second epitope of the 14-3-3 eta protein, said second epitope and said first epitope do not overlap;

32. The kit of claim 31, wherein the reagent further comprises a pure 14-3-3 eta protein as a calibrator which is diluted in a proportional gradient by a calibrator diluent to working calibrator solutions of different concentrations.

33. The kit of claim 31, wherein the second antibody or binding fragment thereof binds to one member of a specific binding pair member and the donor binds to the other member of the specific binding pair member.

34. The kit of claim 33, wherein the second antibody or binding fragment thereof binds biotin and the donor binds streptavidin.

35. The kit of claim 24, wherein the homogeneous immunoassay reagents for detecting anti-Carp antibodies comprise:

component b3, comprising a second anti-card antibody.

36. The kit of claim 35, wherein the antigen or the second anti-Carp antibody binds to a receptor.

37. The kit of claim 36, wherein the antigen is conjugated to a receptor; the acceptor is capable of reacting with the singlet oxygen to generate a detectable chemiluminescent signal.

38. The kit of claim 36, wherein the acceptor comprises an alkene compound and a metal chelate, which are in non-particulate form and are soluble in an aqueous medium; and/or the acceptor is a polymeric microparticle filled with a luminescent compound and a lanthanide.

39. The kit of claim 35, further comprising a component c3 comprising a donor capable of generating singlet oxygen in an excited state.

40. The kit of claim 39, wherein the donor binds to one member of a specific binding pair and the other member of the specific binding pair binds to the antigen or the second anti-card antibody.

41. The kit of claim 39, wherein the donor binds to streptavidin and, correspondingly, the second anti-card antibody binds to biotin.

42. The kit of claim 39, wherein the donor is a photoactivated or chemically activated sensitizer, which is in a non-particulate form and is soluble in an aqueous medium; and/or the donor is polymer particles filled with photosensitive compounds, and can generate singlet oxygen under the excitation of light.

43. The kit of claim 24, wherein the homogeneous immunoassay reagents for detecting anti-Carp antibodies comprise:

44. The kit of claim 43, wherein the reagent further comprises an anti-Carp antibody neat product as a calibrator, which is diluted by a calibrator diluent in a proportional gradient to work calibrator solutions of different concentrations.

45. The kit of claim 43, wherein the second antigen binds to one member of the specific binding pair and the donor binds to the other member of the specific binding pair.

46. The kit of claim 45, wherein the second antigen is bound to biotin and the donor is bound to streptavidin.

47. The kit of claim 43, wherein the acceptor comprises an olefin compound and a metal chelate compound, which are in non-particulate form and are soluble in an aqueous medium; and/or the acceptor is a polymeric microparticle filled with a luminescent compound and a lanthanide.

48. The kit of claim 43, wherein the donor is a photoactivated or chemically activated sensitizer, which is in a non-particulate form and is soluble in an aqueous medium; and/or the donor is polymer particles filled with photosensitive compounds, and can generate singlet oxygen under the excitation of light.

49. A kit for in vitro assessment of the presence or absence of Rheumatoid Arthritis (RA) based on the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers comprising the kit of any one of claims 19-48.

50. A kit for in vitro assessment of the severity of Rheumatoid Arthritis (RA) based on the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers comprising the kit of any one of claims 19-48.

51. A kit for differentiating Rheumatoid Arthritis (RA) from other autoimmune diseases in vitro based on the concentration of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers comprising the kit of any one of claims 19-48.

52. A chemiluminescent immunoassay system for detecting each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers comprising detecting the presence of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers and/or determining the level of each biomarker in a set of Rheumatoid Arthritis (RA) biomarkers in a sample to be tested using a kit according to any one of claims 19-48 or using a kit according to any one of claims 49-51.

53. The detection system of claim 52, wherein the system comprises:

a reaction device for chemically reacting a sample to be tested with a reagent in a kit according to any one of claims 19 to 48 or a kit according to any one of claims 49 to 51;

54. The test system of claim 53, wherein the processor uses a cubic spline interpolation fit to directly give a concentration value of the test target molecule in the test sample.