CN110579602A

CN110579602A - Method for assessing whether rheumatoid arthritis exists in vitro through biomarker-associated sample

Info

Publication number: CN110579602A
Application number: CN201810821257.1A
Authority: CN
Inventors: 饶星; 廖智星; 刘宇卉; 李临; 其他发明人请求不公开姓名
Original assignee: Chemclin Diagnostics Corp
Current assignee: Chemclin Diagnostics Corp
Priority date: 2018-05-24
Filing date: 2018-07-24
Publication date: 2019-12-17
Anticipated expiration: 2038-07-24
Also published as: CN110579602B

Abstract

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

Description

Method for assessing whether rheumatoid arthritis exists in vitro through biomarker-associated sample

Technical Field

The invention belongs to the technical field of immunoassay, and particularly relates to a kit for evaluating whether rheumatoid arthritis exists in vitro through a biomarker-associated sample, a preparation method and a use method.

Background

Biomarkers (biomarkers) are biochemical markers that can mark changes or changes that may occur in the structure or function of systems, organs, tissues, cells, and subcellular structures and functions, and have a wide range of uses.

Rheumatoid Arthritis (RA) is a systemic autoimmune disease characterized primarily by synovitis, with clinical manifestations dominated by chronic polyarthritis, which ultimately leads to joint deformity. The biomarker is used for comprehensively judging the disease course, the illness state, the genetic background, the epigenetics and other characteristics of the RA patient, so that the accurate diagnosis and treatment of RA can be realized, and the survival quality of the RA patient is improved. Therefore, how to improve the detection accuracy of RA positivity of RA inflammatory arthropathy patients is an urgent technical problem to be solved.

disclosure of Invention

To solve the above technical problems, the present invention provides a method for assessing the presence or absence of rheumatoid arthritis in vitro by biomarker-linked samples. The method can remarkably improve the detection accuracy of RA positivity of RA inflammatory arthropathy patients by jointly detecting the levels of at least 2 of serum 14-3-3eta protein, anti-CCP antibody and anti-Carp antibody and correlating the detection result with RA.

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

a) Respectively detecting the concentration of each biomarker in the biomarker group in a sample to be detected;

b) combining a) the measured concentration values of the biomarkers to obtain a combined concentration value of the biomarkers in the biomarker group; and

c) correlating the combined concentration values 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 respective biomarker panel measured from the reference population is indicative of the presence of RA;

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

In some embodiments of the invention, the combined concentration values of step b) are compared to cut-off values derived from a reference population other than RA positive patients, said reference population comprising apparently healthy patients and patients selected from Osteoarthritis (OA) patients and other autoimmune disease patients.

In a second aspect, the present invention provides a use of a homogeneous immunoassay method for detecting the concentration of each biomarker in a Rheumatoid Arthritis (RA) biomarker panel in the preparation of a reagent for evaluating the severity of Rheumatoid Arthritis (RA) by biochemical marker, comprising:

b) Combining a) the measured concentration values of the biomarkers to obtain a combined concentration value of the biomarkers; and

c) correlating the combined concentration values 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 respective biomarker panel measured from the reference population is indicative of 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 Rheumatoid Arthritis (RA) biomarker panel in the preparation of a formulation for classifying Rheumatoid Arthritis (RA) and other autoimmune diseases by biochemical labeling of extracellular regions, 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 is indicative for 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 Rheumatoid Arthritis (RA) biomarker panel 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 Rheumatoid Arthritis (RA) biomarker panel using a homogeneous immunoassay compared to the truncated combined concentration value for each marker in the corresponding biomarker panel measured from a reference population is indicative of the presence of RA;

In the present invention, the sample to be tested is selected from 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 biomarker panel comprises Anti-CCP antibody, 14-3-3eta protein and Anti-carp antibody and a further biomarker, preferably the further biomarker is RA.

According to some embodiments of the invention, the step further comprises measuring the content of 14-3-3eta protein or fragment thereof or of immune complexes formed by said 14-3-3eta protein or fragment thereof with at least one antibody.

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

According to some embodiments of the invention, the step comprises specifically binding the sample to an antibody comprising an epitope capable of forming an immune complex with at least one specific epitope of the 14-3-3eta protein or fragment thereof.

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

in some embodiments of the invention, 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 olefinic compound and a metal chelate, which is in non-particulate form and soluble in an aqueous medium; and/or the acceptor is polymer particles filled with a luminescent compound and 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 relatively specific fragments whose amino acid fragments are sequences of 14-3-3eta protein: 1-6aa, 27-38aa, 71-83aa, 112-154 aa and 141-154 aa.

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

The sixth aspect of the present invention provides a kit for assessing the severity of Rheumatoid Arthritis (RA) by biochemical marker in vitro based on the concentration of each biomarker in a Rheumatoid Arthritis (RA) biomarker panel, comprising reagents for detecting the concentration of each biomarker in the Rheumatoid Arthritis (RA) biomarker panel, wherein the biomarker panel comprises Anti-CCP antibody, 14-3-3eta protein, and Anti-carp antibody.

The seventh aspect of the present invention provides a kit of reagents for classifying Rheumatoid Arthritis (RA) from other autoimmune diseases by biochemical marker extracellular region based on the concentration of each biomarker in a Rheumatoid Arthritis (RA) biomarker panel, comprising reagents for detecting the concentration of each biomarker in a Rheumatoid Arthritis (RA) biomarker panel, wherein the biomarker panel comprises Anti-CCP antibody, 14-3-3eta protein, and Anti-carp antibody.

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

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

The kit according to the fifth to eighth aspects of the present invention, which comprises reagents for detecting the concentrations of Anti-CCP antibody, 14-3-3eta protein and Anti-Carp antibody in the biomarker panel by 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 anti-immune complex antibodies capable of specifically recognizing and binding to anti-CCP antibodies in a first immune complex formed with a first antigen, and not recognizing free, antigen-unbound anti-CCP antibodies.

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

in some embodiments of the invention, the reagent set further comprises component c1 comprising a donor capable of producing singlet oxygen in an excited state; preferably 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; further preferably, the donor is bound to streptavidin and the first antigen or the anti-immune complex antibody, respectively, is bound to biotin.

in some specific embodiments of the present invention, the reagent further comprises pure anti-CCP as a calibrator, and the calibrator is diluted by calibrator dilution to working calibrator solutions of different concentrations according to a proportional gradient.

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

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;

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

Component c2 comprising a donor capable of generating singlet oxygen in an excited state.

In some embodiments of the invention, the reagent further comprises 14-3-3eta protein pure product as a calibrator, and the calibrator is diluted by calibrator diluent according to a proportional gradient to working calibrator solutions with different concentrations.

In some embodiments of the invention, the second antibody or binding fragment thereof binds to one member of a specific binding pair member and the 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, the homogeneous immunoassay reagent for the detection of anti-Carp antibodies comprises:

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

Component b3 comprising a second anti-Carp antibody.

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

In some embodiments of the invention, the reagent set further comprises component c3 comprising a donor capable of producing singlet oxygen in an excited state; preferably the donor is bound to one member of a specific binding pair and the other member of the specific binding pair is bound to the antigen or the second anti-Carp antibody; further preferably, the donor is bound to streptavidin and the second anti-Carp antibody is bound to biotin, respectively.

In some embodiments of the invention, the donor is a photoactivated or chemically activated sensitizer, which is in non-particulate form and 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 the detection of an anti-Carp antibody comprises:

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

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

Component c4 comprising a donor capable of generating singlet oxygen in an excited state.

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

In some embodiments of the invention, the second antigen is bound to one member of a 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 present invention, the kit of reagents comprises the above-mentioned reagents for detecting Anti-CCP antibodies in the biomarker panel by indirect method using homogeneous immunoassay, 14-3-3eta protein in the biomarker panel by sandwich method using homogeneous immunoassay, and Anti-Carp antibody concentration in the biomarker panel by competitive method using homogeneous immunoassay.

in other specific embodiments of the present invention, the kit of reagents comprises the above-mentioned reagents for detecting Anti-CCP antibodies in the biomarker panel by electrochemical method using electrochemical immunoassay, 14-3-3eta protein in the biomarker panel by sandwich method using homogeneous immunoassay, and Anti-Carp antibody concentration in the biomarker panel by sandwich method using homogeneous immunoassay.

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

The eleventh aspect of the present invention provides a kit for classifying Rheumatoid Arthritis (RA) and other autoimmune diseases by biochemically labeling extra-corporeal regions based on the concentration of each biomarker in a Rheumatoid Arthritis (RA) biomarker panel, comprising the kit of reagents according to the fifth to eighth aspects of the present invention.

in a twelfth aspect, the invention provides a kit for assessing the presence of Rheumatoid Arthritis (RA) in a test sample in vitro using a Rheumatoid Arthritis (RA) biomarker panel, comprising the kit of parts according to the fifth to eighth aspects of the invention.

In a thirteenth aspect, the present invention provides a method for assessing the presence or absence of Rheumatoid Arthritis (RA) in vitro by biochemical markers, comprising detecting the concentration of each biomarker in a panel of Rheumatoid Arthritis (RA) biomarkers using a kit of reagents 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 values of step b) are compared to cut-off values 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 present invention provides a method for assessing the severity of Rheumatoid Arthritis (RA) by the presence of biochemical markers, comprising detecting the concentration of each biomarker in a panel of Rheumatoid Arthritis (RA) biomarkers using a kit of reagents 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 severity of Rheumatoid Arthritis (RA) by the presence of biochemical markers.

In some embodiments of the invention, the method comprises:

In a fifteenth aspect, the present invention provides a method for classifying Rheumatoid Arthritis (RA) and other autoimmune diseases by biochemical labeling of extracellular regions, comprising detecting the concentration of each biomarker in a Rheumatoid Arthritis (RA) biomarker panel using a kit of reagents as described in the fifth to eighth aspects of the present invention or using a kit as described in the ninth to twelfth aspects of the present invention and classifying Rheumatoid Arthritis (RA) and other autoimmune diseases by biochemical labeling of extracellular regions.

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 of reagents 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 Rheumatoid Arthritis (RA) biomarker panel compared to the truncated combined concentration value measured for each marker in the corresponding biomarker panel from a reference population is indicative of the presence of RA;

according to the method described in the thirteenth to sixteenth aspects of the present invention, the concentration of Anti-CCP is measured by a homogeneous immunoassay using a homogeneous immunoassay reagent set containing Anti-CCP antibody, 14-3-3eta protein and Anti-carp antibody, comprising:

step R1, mixing a sample to be tested with the component a1 to obtain a first mixture;

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

Step R3, mixing the second mixture with component c1 to obtain a third mixture which generates a detectable chemiluminescent signal;

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

In some embodiments of the invention, the method further comprises the step of preparing an Anti-CCP standard working curve before step R1.

in some embodiments of the present invention, in step R4, the intensity of the chemiluminescence signal in step R3 is detected, and the content of Anti-CCP antibody in the sample to be tested is determined based on the Anti-CCP antibody standard working curve.

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

Step 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 component c2 to obtain a fourth mixture;

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

and step R4, detecting the existence and/or the intensity of the chemiluminescence signal in the step R3, thereby judging whether the 14-3-3eta protein exists in the sample to be detected and/or determining the content of the 14-3-3eta protein.

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

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

according to the method described in the thirteenth to sixteenth aspects of the present invention, the concentration of the Anti-Carp antibody is measured by a homogeneous immunoassay using a homogeneous immunoassay reagent set containing Anti-CCP antibody, 14-3-3eta protein and Anti-Carp antibody, which comprises the steps of:

R1, mixing the second anti-Carp antibody combined with biotin with the sample to be tested to form a2 nd mixture;

r2, mixing the antigen bound with the receptor with the 2 nd mixture, allowing the second anti-Carp antibody bound with biotin and the target anti-Carp antibody in the sample to compete for binding with the antigen bound with the receptor, to form a third immune complex composed of receptor-antigen-second anti-Carp antibody-biotin and a fourth immune complex composed of receptor-antigen-anti-Carp antibody, respectively, thereby forming a3 rd mixture;

r3, mixing the streptavidin-bound donor with the 3 rd mixture, and allowing the streptavidin-bound donor to bind to biotin in the third immune complex to form a fifth immune complex consisting of receptor-antigen-second anti-Carp antibody-biotin-streptavidin-donor, thereby forming a4 th mixture;

r4, exciting the donor with energy or an active compound to produce singlet oxygen, said acceptor reacting with the singlet oxygen to generate a detectable chemiluminescent signal; detecting the presence or absence of a fourth immune complex; if the 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 preparing a standard working curve of the anti-Carp antibody of interest 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 the anti-Carp antibody standard working curve.

In some embodiments of the invention, the signal value of the emission light of the 4 th mixture is detected and compared to the chemiluminescence signal value of a control immunocomplex formed by equal amounts of antigen and second anti-Carp antibody in step R2, thereby determining the presence or absence of the target anti-Carp antibody and/or the concentration of the target anti-Carp antibody in the test sample.

Step 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 component c4 to obtain a fourth mixture;

and step R4, detecting the existence and/or the intensity of the chemiluminescence 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 preparing an anti-Carp antibody standard working curve prior to step R1.

In some embodiments of the present invention, in step R4, the intensity of the chemiluminescent signal of step R3 is detected, and the content of anti-Carp antibody in the sample to be tested is determined based on the anti-Carp antibody standard working curve.

A seventeenth aspect of the present invention provides a chemiluminescent immunoassay system for detecting each biomarker in a Rheumatoid Arthritis (RA) biomarker panel, comprising detecting the presence or absence of each biomarker in the Rheumatoid Arthritis (RA) biomarker panel in a sample to be tested and/or determining the content of each biomarker in the Rheumatoid Arthritis (RA) biomarker panel using a kit according to the fifth to eighth aspects of the present invention or using a kit according to the ninth to twelfth aspects of the present invention or a method according to the thirteenth to sixteenth aspects of the present invention.

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

a reaction device, which is used for the sample to be tested to perform chemical reaction with the reagent set according to the fifth to eighth aspects of the invention or the reagent in the kit according to the ninth to twelfth aspects of the invention;

The excitation and reading device excites the donor microsphere to generate active oxygen by using excitation light with the wavelength of 600-700nm, the acceptor microsphere reacts with the received active oxygen to generate emission light with the wavelength of 520-620nm, and the optical signal of the emission light is recorded;

And the processor judges whether the target molecules to be detected exist in the sample to be detected or not and/or determines the content of the target molecules to be detected according to the recorded existence and/or intensity of the optical signals of the emitted light.

In some embodiments of the present invention, the processor performs fitting by using cubic spline interpolation fitting, and directly gives the concentration value of the target molecule to be detected in the sample to be detected.

An eighteenth aspect of the present invention provides a homogeneous immunoassay method for detecting Anti-CCP, which employs the chemiluminescent immunoassay system of the seventeenth aspect of the present invention, and a homogeneous immunoassay kit or kit containing a homogeneous immunoassay reagent for detecting Anti-CCP antibody, 14-3-3eta protein and Anti-carp antibody as described in the present invention to detect the concentration of Anti-CCP by homogeneous immunoassay, comprising:

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

The nineteenth aspect of the present invention provides a homogeneous immunoassay method for detecting 14-3-3eta protein, which employs the chemiluminescent immunoassay system of the seventeenth aspect of the present invention, and a homogeneous immunoassay kit or kit containing the reagents for detecting Anti-CCP, 14-3-3eta protein and Anti-carp antibody according to the present invention to detect the concentration of 14-3-3eta protein by homogeneous immunoassay, comprising:

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

the twentieth aspect of the present invention provides a homogeneous immunoassay method for detecting an Anti-Carp antibody, which employs the chemiluminescent immunoassay system according to the seventeenth aspect of the present invention, and a homogeneous immunoassay kit or kit containing reagents for detecting Anti-CCP antibody, 14-3-3eta protein and Anti-Carp antibody according to the present invention to detect the concentration of the Anti-Carp antibody by homogeneous immunoassay, comprising:

according to a twenty-first aspect of the present invention, there is provided a homogeneous immunoassay method for detecting an Anti-Carp antibody, which employs the chemiluminescent immunoassay system according to the seventeenth aspect of the present invention and a homogeneous immunoassay kit or kit containing reagents for detecting Anti-CCP antibody, 14-3-3eta protein and Anti-Carp antibody according to the present invention to detect the concentration of the Anti-Carp antibody by homogeneous immunoassay, comprising:

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

the method for assessing whether the Rheumatoid Arthritis (RA) exists in vitro through biochemical markers provided by the invention can obviously improve the detection accuracy of RA positivity of RA inflammatory joint disease patients by jointly detecting the levels of at least 2 of serum 14-3-3eta 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, a detailed description of the invention is provided below. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

where a range of values is provided, it is understood that each intervening value, to the extent that there is no stated or intervening value in that stated range, to the extent that there is no such intervening value, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

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

Term (I)

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

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

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

the term "citrullinated peptide" as used herein refers to a specific antigen that reacts positively with RA serum: the modified polypeptide fragments include serine fragment, pre-serine precursor, synthesized polypeptide, recombinant polypeptide, marker-coupled polypeptide and other modified polypeptide fragments, and features that the modified polypeptide fragments contain citrulline, which is essential component for the recognition of CCP antibody.

The term "citrulline epitope" as used herein refers to a region on the surface of an antigen to which an anti-cyclic citrullinated peptide antibody can specifically bind, including citrulline residues and surrounding amino acid sequences in which the citrulline residues are located.

the term "epitope recognition site of an anti-cyclic citrullinated peptide antibody" also referred to as "epitope recognition site" as used herein means a region of the anti-cyclic citrullinated peptide antibody that recognizes and binds to a "citrullinated epitope", for example, a first recognition site of an epitope of an anti-cyclic citrullinated peptide antibody and a second recognition site of an epitope of an anti-cyclic citrullinated peptide antibody do not overlap, i.e., belong to different epitope recognition sites with the same binding property.

the term "citrulline peptide fragment mixture" as used herein refers to a mixture formed by mixing at least 2 single citrulline-containing peptide fragments, wherein the citrulline-containing peptide fragments can be cyclic peptide fragments containing citrulline or linear peptide fragments containing citrulline.

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

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

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

The terms "antibody" and "immunoglobulin" are used in the broadest sense of the invention, and include antibodies or immunoglobulins of any isotype, antibody fragments that retain specific binding to an antigen; including but not limited to Fab, Fv, scFv, Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In any case desired, the antibody may be further conjugated to other moieties, such as a specific binding pair member, e.g., biotin or streptavidin (a member of a biotin-streptavidin specific binding pair member), and the like.

the term "immune complex" (antigen-antibody complex) as used herein; the "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 a tissue.

the term "anti-immunocomplex antibody" as used herein refers to a substance that specifically recognizes and binds to an antigen-antibody immunocomplex, and does not recognize free, antigen-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 conformation or epitope of the antibody in the immune complex state is changed and shows a difference from the conformation or epitope of other free non-specific antibodies, and the difference is specifically recognized by the anti-immune complex antibody provided by the invention. The use of such anti-immune complex antibodies allows discrimination between antibodies in the immune complex state and non-specific antibodies, free specific antibodies not bound to the antigen.

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

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

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

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

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

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

The term "donor" as used herein refers to a sensitizer capable of generating a reactive intermediate such as singlet oxygen that reacts with an acceptor upon activation by energy or an active compound. 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, porphyrins, phthalocyanines, and chlorophylls disclosed in, for example, U.S. Pat. No. 5,5709994, which is incorporated herein by reference in its entirety, as well as derivatives of these compounds having 1 to 50 atom substituents that serve to render these compounds more lipophilic or more hydrophilic, and/or as a linker group to a member of a specific binding pair. Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, such as those described in US patent No. US6406913, which is incorporated herein by reference. In 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. Other examples of donors include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, etc., which are heated or directly absorb light to release singlet oxygen.

the term "acceptor" as used herein refers to a compound capable of reacting with singlet oxygen to produce a detectable signal. The donor is induced by energy or an active compound to activate and release singlet oxygen in a high energy state that is trapped by a close proximity acceptor, 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 can decompose with or subsequently emit light. Typical examples of such substances include, but are not limited to: enol ether, enamine, 9-alkylidene xanthan gum, 9-alkylidene-N-alkyl acridin, aromatic vinyl ether, diepoxy ethylene, dimethyl thiophene, aromatic imidazole or lucigenin. In other embodiments of the invention, the acceptor is an alkene capable of reacting with singlet oxygen to form a hydroperoxide or dioxetane that can be decomposed into ketones or carboxylic acid derivatives; a stable dioxetane which can be decomposed by the action of light; acetylenes which can react with singlet oxygen to form diketones; hydrazones or hydrazides which can form azo compounds or azocarbonyl compounds, such as luminol; and aromatic compounds that can form endoperoxides. Specific, non-limiting examples of receptors that can be utilized in accordance with the disclosed and claimed invention are described in U.S. patent No. US5340716, which is incorporated herein by reference in its entirety. In other embodiments of the invention, the receptor comprises an olefinic compound and a metal chelate, which is non-particulated and soluble in an aqueous medium, as in the case of the receptor described in patent PCT/US2010/025433 (which is incorporated herein by reference in its entirety).

the 'donor' can be polymer particles filled with photosensitive compounds formed by coating functional groups on a substrate, and can generate singlet oxygen under the excitation of light; and/or the acceptor can be polymer particles filled with the luminescent compound and the lanthanide element formed by coating the functional groups on the substrate.

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

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

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

in the present invention, the phrase "detecting the concentration of each biomarker in a Rheumatoid Arthritis (RA) biomarker panel" refers to "biomarker combination test".

embodiments II

As mentioned above, some atypical early stage and/or seronegative patients are often misdiagnosed and missed because RA is heterogeneous in appearance. In order to improve the existing diagnostic strategy for RA and improve the diagnostic level, the present inventors have conducted extensive studies on the RA diagnostic method.

the research of the inventor finds that the 14-3-3eta protein is remarkably increased in RA serum and joint synovial fluid and can up-regulate the expression of a plurality of inflammatory factors related to RA, which indicates that the protein is possibly involved in RA disease occurrence. The inventor further finds that the detection accuracy of RA positivity of RA inflammatory arthropathy patients can be remarkably improved by detecting the levels of at least 2 of serum 14-3-3eta protein, anti-CCP antibody and anti-Carp antibody of RA, non-RA inflammatory arthropathy patients and contemporary healthy examinees, analyzing and comparing the levels and correlating with RA to jointly detect the levels of at least 2 of serum 14-3-3eta protein, anti-CCP antibody and anti-Carp antibody and correlating the detection result with RA. The present invention has been made based on the above findings.

Accordingly, a first aspect of the present invention relates to a use of a homogeneous immunoassay for detecting the concentration of each biomarker in a panel 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:

The second aspect of the present invention relates to a use of a homogeneous immunoassay method for detecting the concentration of each biomarker in a Rheumatoid Arthritis (RA) biomarker panel in the preparation of a reagent for evaluating the severity of Rheumatoid Arthritis (RA) by biochemical marker, comprising:

in a third aspect, the present invention relates to a use of a homogeneous immunoassay for detecting the concentration of each biomarker in a Rheumatoid Arthritis (RA) biomarker panel for preparing a preparation for classifying Rheumatoid Arthritis (RA) and other autoimmune diseases by biochemical labeling of extracellular regions, comprising:

the fourth aspect of the invention relates to the use of a Rheumatoid Arthritis (RA) biomarker panel 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 values measured for each biomarker in the Rheumatoid Arthritis (RA) biomarker panel using a homogeneous immunoassay compared to the truncated combined concentration values for each marker in the corresponding biomarker panel measured from a reference population is indicative of the presence of RA;

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

in the present invention, Anti-CCP antibody is captured by one or more CCPs as antigens.

in the present invention, the Anti-carp antibody is 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 embodiments of the invention, the concentration of each biomarker in the biomarker panel is detected using a homogeneous immunoassay.

the fifth to twenty-first aspects below further provide specific embodiments for implementing the invention.

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

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

the seventh aspect of the present invention relates to a kit of reagents for classifying Rheumatoid Arthritis (RA) from other autoimmune diseases by biochemical marker extracorporeally based on the concentration of each biomarker in a Rheumatoid Arthritis (RA) biomarker panel, comprising reagents for detecting the concentration of each biomarker in a Rheumatoid Arthritis (RA) biomarker panel, wherein the biomarker panel comprises Anti-CCP antibody, 14-3-3eta protein, and Anti-carp antibody.

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

In some embodiments of the invention, the method comprises:

in some embodiments of the invention, the method comprises:

In some embodiments of the invention, the method comprises:

in some preferred embodiments of the invention, the set of biomarkers comprises Anti-CCP antibodies, 14-3-3eta protein and Anti-carp antibodies and other biomarkers.

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

The reagent set according to the fifth to eighth aspects of the present invention comprises the above-mentioned reagent for detecting Anti-CCP antibodies in the biomarker panel by an indirect method using a homogeneous immunoassay, 14-3-3eta protein in the biomarker panel by a sandwich method using a homogeneous immunoassay, and a reagent for detecting the concentration of Anti-Carp antibodies in the biomarker panel by a competition 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 olefinic compound and a metal chelate, which is in non-particulate form and soluble in an aqueous medium; and/or the acceptor is polymer particles filled with a luminescent compound and lanthanide.

According to the invention, the kit of parts further comprises a component c1 comprising a donor capable of generating singlet oxygen in the excited state; preferably 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; further preferably, the donor is bound to streptavidin and the first antigen or the anti-immune complex antibody, respectively, is bound to biotin.

In the present invention, the donor is a photoactivated or chemically activated sensitizer, which is in a non-particulate form and 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-immunocomplex antibody binds to the anti-CCP antibody in the first immunocomplex 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 antibodies 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 fragment 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 making the polyclonal antibody comprises: immunizing an animal by using a human immune complex to obtain animal serum containing the polyclonal antibody; and purifying the animal serum by affinity chromatography to obtain the polyclonal antibody for specifically recognizing the human immune complex.

In some embodiments, the monoclonal antibody is prepared by a method comprising: fusing spleen cells of mice immunized by the human immune complex with mouse myeloma cells, culturing, detecting cell culture supernatant, and reserving positive cell strains.

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

In some preferred embodiments of the present invention, the first antigen is selected from the group consisting of synthetic citrullinated cyclic peptides, citrullinated linear peptides, polypeptides formed by synthesizing at least 2 single citrullinated peptide fragments on one peptide chain, a mixture of citrullinated peptide fragments comprising at least 2 single citrullinated peptide fragments, and citrullinated proteins.

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

in some further more preferred embodiments of the present invention, the first antigen is a polypeptide synthesized on one peptide chain from 2 to 4 citrulline-containing peptide fragments or a citrulline peptide fragment mixture containing 2 to 4 single citrulline-containing peptide fragments; preferably, the peptide segment containing citrulline is selected from SEQ ID Nos. 2-5.

TABLE 1

Serial number	sequence of
		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 through an intermediate that is a hydrophilic polymeric substance.

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, polymercaptans, and polyethylene glycols.

in some embodiments, the total concentration of the receptor and the first antigen bound thereto is 10-200 μ g/mL, preferably 20-150 μ g/mL, more preferably 25-100 μ g/mL; and/or the total concentration of the anti-immunocomplex antibody and the member of the specific binding pair to which it binds 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 invention, the concentration of Anti-CCP is measured by homogeneous immunoassay using a homogeneous immunoassay kit comprising Anti-CCP antibody, 14-3-3eta protein and Anti-carp antibody, comprising:

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

In some preferred embodiments of the present invention, the method further comprises the step of preparing an Anti-CCP standard working curve before 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 content of Anti-CCP antibody in the sample to be tested is determined based on the Anti-CCP standard working curve.

The above method for detecting the concentration of Anti-CCP by homogeneous immunoassay using the homogeneous immunoassay kit containing the Anti-CCP antibody, the 14-3-3eta protein and the Anti-Carp antibody can be understood as comprising the following steps:

S1, combining the first antigen with the anti-CCP antibody in the sample to be detected to form a first immune complex composed of the first antigen and the anti-CCP antibody;

S2, combining the anti-immune complex antibody with the first immune complex to form a second immune complex composed of the first antigen-anti-CCP antibody-anti-immune complex antibody;

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

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

In the present invention, the first immune complex is bound to a donor by a first antigen, and the corresponding second immune complex is bound to a receptor by an anti-immune complex antibody, the receptor being capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal, and the donor being capable of producing singlet oxygen in an excited state.

in the present invention, the first immune complex is bound to a receptor via a first antigen, and the corresponding second immune complex is bound to a donor via an anti-immune complex antibody, the receptor being capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal, and the donor being capable of producing singlet oxygen in an excited state.

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

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

t2, combining the anti-immunocomplex antibody that specifically recognizes the anti-CCP antibody in the third immunocomplex, which is bound to the receptor, with the third immunocomplex to form a fourth immunocomplex consisting of biotin-first antigen-anti-CCP antibody-anti-immunocomplex antibody-receptor;

T3, combining the donor bound with streptavidin with the fourth immune complex to form a fifth immune complex composed of donor-streptavidin-biotin-first antigen-anti-CCP antibody-anti-immune complex antibody-receptor;

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

alternatively, the first and second electrodes may be,

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

r2, combining an anti-immunocomplex antibody that specifically recognizes the anti-CCP antibody in the sixth immunocomplex, which is bound to biotin, with the sixth immunocomplex to form a seventh immunocomplex consisting of receptor-first antigen-anti-CCP antibody-anti-immunocomplex antibody-biotin;

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

r4, detecting the presence or absence of the eighth immune complex; if the eighth immune complex exists, the anti-CCP antibody exists in the sample to be detected;

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 preparing a standard working curve for anti-CCP antibodies 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 antibodies in the test sample is determined based on an anti-CCP standard working curve.

In some further specific embodiments, the excitation light with the wavelength of 600-700nm is used to irradiate the fifth immune complex or the eighth immune complex, so as to excite the donor to generate singlet oxygen, the acceptor reacts with the contacted singlet oxygen to generate emission light with the wavelength of 520-620nm, and the signal value of the emission light is detected, thereby determining whether the anti-CCP antibody and/or the concentration of the anti-CCP antibody exists in the sample to be detected.

In some particularly preferred embodiments of the invention, the homogeneous immunoassay reagent for detecting 14-3-3eta protein comprises:

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

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 and the first antibody or binding fragment thereof bound thereto in component a2 is 10-200 μ g/mL, preferably 20-150 μ g/mL, more preferably 30-100 μ g/mL, most preferably 40-80 μ g/mL; and/or the concentration of the second antibody or binding fragment thereof in component b2 is 0.1-8 μ g/mL, preferably 0.2-6 μ g/mL, more preferably 0.4-4 μ g/mL, most preferably 0.6-2 μ g/mL; and/or the concentration of the 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 polymer particles filled with a luminescent compound and lanthanide.

in some further particularly preferred embodiments of the invention, the concentration of 14-3-3eta protein is detected by a homogeneous immunoassay using a homogeneous immunoassay kit containing Anti-CCP antibodies, 14-3-3eta protein and Anti-Carp antibodies, comprising:

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

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

in the present invention, there is no separation and/or washing step 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 with a wavelength of 600-700nm to excite the donor to generate singlet oxygen, and the acceptor reacts with the contacted singlet oxygen to generate emission light with a wavelength of 520-620 nm.

According to some preferred embodiments, the kit of reagents according to the invention comprises homogeneous immunodetection reagents for the detection of Anti-carp antibodies, comprising:

Component b3 comprising a second anti-Carp antibody.

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

In the present 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 a peptide chain synthesized from at least 2 single carbamylated peptide, a mixture of carbamylated peptide containing at least 2 single carbamylated peptide, and a carbamylated protein.

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

Further preferably, the antigen is a polypeptide synthesized on one peptide chain by 2-4 carbamylated peptide fragments or a carbamylated peptide fragment mixture containing 2-4 single carbamylated peptide fragments; preferably, the carbamylated peptide fragment is selected from SEQ ID Nos. 6-9.

TABLE 2

Serial number	sequence of
		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, which is a hydrophilic polymeric substance. 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, polymercaptans, 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 μ g/mL; and/or the total concentration of said second anti-Carp antibody and one of the members of the specific binding pair bound thereto is from 0.025 to 0.1. mu.g/mL; and/or the total concentration of the donor and the other member of the specific binding pair bound thereto is 5-20. mu.g/mL.

In some further preferred embodiments of the present invention, the method for detecting the concentration of Anti-Carp by homogeneous immunoassay using a homogeneous immunoassay kit containing Anti-Carp antibody according to the above reagents in the kit of the present invention comprises the following steps:

M1, allowing the second anti-Carp antibody and the target anti-Carp antibody in the sample to compete for binding with 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-target anti-Carp antibody through the epitope and the antigen, respectively;

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

In some embodiments of the present invention, the step M1 includes mixing the second anti-Carp antibody, the sample to be tested and the antigen uniformly, reacting, allowing the second anti-Carp antibody and the target anti-Carp antibody in the sample to compete for binding with 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 and the antigen, respectively.

In some further embodiments of the present invention, the step of uniformly mixing the second anti-Carp antibody, the sample to be tested and the antigen comprises uniformly mixing the second anti-Carp antibody, the sample to be tested and the antigen simultaneously; alternatively, the second anti-Carp antibody is first mixed with the sample to be tested to form a1 st mixture, and then the antigen is mixed with the 1 st mixture.

in some embodiments of the invention, said step M1 comprises the steps of:

p1, mixing and reacting the antigen with the sample to be detected, and combining the target anti-Carp in the sample to be detected with the epitope of the antigen to form a second immune complex formed by the antigen-target anti-Carp antibody, so as to obtain a 11 th mixture;

P2, mixing the second anti-Carp antibody with the 11 th mixture, reacting the mixture to allow the second anti-Carp antibody to bind to the epitope of the antigen in the 11 th mixture that is not bound to the anti-Carp antibody in the sample to be tested, thereby forming a first immune complex composed of the antigen-the second anti-Carp antibody.

in some embodiments, step M2 detects the presence or absence of the second immune complex by chemiluminescence.

In some embodiments, step M2 includes comparing the chemiluminescent signal value of the mixture of step 1 to the chemiluminescent signal value of a control immunocomplex formed from an equivalent amount of the antigen and the second anti-Carp antibody of step M1, thereby determining whether the second immunocomplex is present.

In some embodiments of the invention, the antigen is bound to a receptor and the second anti-Carp antibody is bound 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, in which the sample to be tested is diluted with the sample diluent in a volume ratio of 1 (4-20); preferably, the sample to be tested is diluted by the sample diluent according to the volume ratio of 1 (6-16); more preferably, the sample to be tested is diluted with the sample diluent according to the volume ratio of 1 (8-12).

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

r2, mixing the antigen bound with the receptor with the mixture of item 2, allowing the second anti-Carp antibody bound with biotin and the target anti-Carp antibody in the sample to compete for binding to the epitope of the antigen bound with the receptor, and forming a third immune complex composed of receptor-antigen-second anti-Carp antibody-biotin and a fourth immune complex composed of receptor-antigen-anti-Carp antibody with the antigen bound with the receptor through the epitope, respectively, thereby forming an item 3 mixture;

R4, exciting the donor with energy or an active compound to produce singlet oxygen, said acceptor reacting with the singlet oxygen to generate a detectable chemiluminescent signal; detecting the presence or absence of a fourth immune complex; if the fourth immune complex is present, then an anti-Carp antibody is present in the test sample.

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

T1, mixing the antigen bound with the receptor with the sample to be tested, reacting, and binding the target anti-Carp in the sample to be tested with the epitope of the antigen bound with the receptor to form a fourth immune complex composed of the receptor-antigen-target anti-Carp antibody, to obtain a 12 th mixture;

t2, mixing the biotin-bound second anti-Carp antibody with the 12 th mixture, reacting the mixture to compete with the biotin-bound second anti-Carp antibody for binding to an epitope of the receptor-bound antigen not bound to the anti-Carp in the sample to be tested in the 12 th mixture, and forming a third immune complex composed of the receptor-bound antigen-second anti-Carp antibody-biotin, thereby forming a 13 th mixture;

t3, mixing the streptavidin-bound donor with the 13 th mixture, and allowing the streptavidin-bound donor to bind to biotin in the third immune complex to form a fifth immune complex consisting of receptor-antigen-second anti-Carp antibody-biotin-streptavidin-donor, thereby forming a 14 th mixture;

T4, exciting the donor with energy or an active compound to produce 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 the fourth immune complex is present, then an anti-Carp antibody is present in the test sample.

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

in some embodiments of the present 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 sample to be tested is determined based on an anti-Carp antibody standard working curve.

In some embodiments of the present invention, the 4 th mixture or the 14 th mixture is irradiated with 600-700nm excitation light to excite the donor in the fifth immunocomplex to generate singlet oxygen, the acceptor reacts with the contacted singlet oxygen to generate 520-620nm emission light, and the signal value of the emission light of the 4 th mixture or the 14 th mixture is detected and compared with the chemiluminescence signal value of a control immunocomplex formed by the same amount of the antigen and the second anti-Carp antibody in the step R2 or the steps T1 and T2, thereby determining whether the concentration of the target anti-Carp antibody and/or the target anti-Carp antibody is present in the sample to be tested.

the reagent set according to the fifth to eighth aspects of the present invention comprises the above-mentioned reagent for detecting Anti-CCP antibodies in the biomarker panel by a homogeneous immunoassay, the 14-3-3eta protein in the biomarker panel by a sandwich method by a homogeneous immunoassay, and the reagent for detecting the concentration of Anti-Carp antibodies in the biomarker panel by a sandwich method by a homogeneous immunoassay.

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

alternatively, the first and second electrodes may be,

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

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

In some preferred embodiments of the invention, the second antigen is bound to one member of a 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 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 the member of the specific binding pair 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 preferred embodiments of the invention, the concentration of Anti-Carp is detected by homogeneous immunoassay using a homogeneous immunoassay kit containing Anti-Carp antibodies, which comprises the following steps:

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

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

In some further embodiments of the present invention, in step R4, the intensity of the chemiluminescent signal of 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 some embodiments of the present invention, in step R3, the fourth mixture is irradiated with excitation light with a wavelength of 600-700nm to excite the donor to generate singlet oxygen, and the acceptor reacts with the contacted singlet oxygen to generate emission light with a wavelength of 520-620 nm.

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

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

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

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

According to some embodiments of the invention, the kit of reagents according to the invention may further comprise other reagents.

according to some preferred embodiments of the invention, the kit of reagents comprises reagents for detecting the concentration of Anti-CCP antibody, 14-3-3eta protein and Anti-Carp antibody in the biomarker panel using a homogeneous immunoassay.

According to some preferred embodiments, the kit of reagents of the invention comprises a homogeneous immunoassay reagent for the detection of anti-CCP antibodies, comprising:

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

A second composition comprising component b5, said component b5 consisting of a second antigen capable of specifically binding 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 component c5, said component c consisting of a donor capable of generating singlet oxygen in an excited state and another member of the 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 synthetic cyclic peptides containing citrulline, linear peptides containing citrulline, polypeptides formed by synthesizing at least 2 single citrulline-containing peptide fragments on one peptide chain, a mixture of citrulline peptide fragments containing at least 2 single citrulline-containing peptide fragments, and citrullinated proteins.

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 citrullinated peptide, a citrullinated linear peptide, a polypeptide formed by synthesizing at least 2 single citrullinated peptide fragments on one peptide chain, and a mixture of citrullinated peptide fragments comprising at least 2 single citrullinated peptide fragments.

preferably, the first antigen and the second antigen are respectively and independently polypeptides formed by synthesizing 2-4 peptide fragments containing citrulline on one peptide chain or citrulline peptide fragment mixture containing 2-4 single peptide fragments containing citrulline; further preferably, the peptide fragment containing citrulline is selected from SEQ ID Nos. 2-5.

In some preferred embodiments of the present invention, the first antigen is bound to the receptor through an intermediate, which is a hydrophilic polymeric substance.

In some embodiments, the intermediate is a protein, preferably selected from 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 10-200 μ g/mL, preferably 20-150 μ g/mL, more preferably 25-100 μ g/mL; and/or the concentration of said component b5 in the second composition 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, the concentration of Anti-CCP is measured by a homogeneous immunoassay using a homogeneous immunoassay kit containing Anti-CCP antibodies, corresponding to the above-described reagents in the kit of the invention, comprising:

step R1, mixing the sample to be tested with the first composition and the second composition, and reacting to obtain a first mixture;

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

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

and step R4, detecting the existence and/or the intensity of the chemiluminescence 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 preparing a standard working curve for the anti-cyclic citrullinated peptide antibody prior to step R1.

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

In some embodiments of the present invention, the excitation light with the wavelength of 600-700nm is used to irradiate the second mixture, the donor is excited to generate singlet oxygen, the acceptor reacts with the contacted singlet oxygen to generate emission light with the wavelength of 520-620nm, and the signal intensity of the emission light is detected, so as to determine whether the anti-cyclic citrullinated peptide antibody exists in the sample to be tested and/or determine 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 an anti-Carp antibody in a first immune complex with a first antigen, not recognizing a free, unbound antigen anti-Carp antibody.

in the present invention, the first antigen or the anti-immunocomplex 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 reagent set further comprises component c6 comprising a donor capable of producing singlet oxygen in an excited state; preferably 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; further preferably, the donor is bound to streptavidin and the first antigen or the anti-immune complex antibody, respectively, is bound to biotin.

it will be appreciated by those skilled in the art that the anti-immune complex antibody binds to the anti-Carp antibody in the first immune complex via a recognition epitope, which is a conformational epitope and/or a linear epitope. The anti-immune complex antibody recognizes a constant region portion of an anti-Carp antibody in the first immune complex. The anti-immune complex antibody does not recognize the light chain portion of the anti-Carp antibody in the first immune complex. The anti-immune complex antibody specifically recognizes the Fc fragment of the anti-Carp antibody in the first immune complex.

In some embodiments of the invention, the polyclonal antibody is prepared by a method comprising: immunizing an animal by using a human immune complex to obtain animal serum containing the polyclonal antibody; and purifying the animal serum by affinity chromatography to obtain the polyclonal antibody for specifically recognizing the human immune complex.

in some embodiments of the invention, the monoclonal antibody is prepared by a method comprising: fusing spleen cells of mice immunized by the human immune complex with mouse myeloma cells, culturing, detecting cell culture supernatant, and reserving positive cell strains.

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

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

Further preferably, the first antigen is a polypeptide synthesized on one peptide chain by 2-4 carbamylated peptide fragments or a carbamylated peptide fragment mixture containing 2-4 single carbamylated peptide fragments; preferably the carbamylated peptide fragment 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, polymercaptans, and polyethylene glycols.

in some embodiments of the invention, the concentration of the receptor and anti-immune complex antibody bound thereto is 10-200. mu.g/mL, preferably 20-150. mu.g/mL, more preferably 25-100. mu.g/mL; and/or the concentration of the first antigen and the member of the specific binding pair to which it binds 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 preferred embodiments of the invention, the concentration of Anti-CCP is measured by a homogeneous immunoassay using a homogeneous immunoassay kit containing Anti-CCP antibodies for detection, corresponding to the above-described reagents in the kit of the invention, comprising the steps of:

s1, combining the first antigen with the anti-Carp antibody in the sample to be detected to form a first immune complex composed of the first antigen and the anti-Carp antibody;

S2, combining the anti-immune complex antibody with the first immune complex to form a second immune complex composed of the first antigen-anti-Carp antibody-anti-immune complex antibody;

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

in some embodiments, the presence or absence of the second immune complex is detected in step S3 by a method of chemiluminescence.

In some embodiments, the first immune complex is bound to a receptor via a first antigen, and the corresponding second immune complex is bound to a donor via an anti-immune complex antibody, the receptor being capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal, and the donor being capable of producing singlet oxygen in an excited state.

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

T1, combining the first antigen combined with biotin with the anti-Carp antibody in the sample to be detected to form a third immune complex composed of biotin-first antigen-anti-Carp antibody;

T2, combining an anti-immune complex antibody which is bound to the receptor and specifically recognizes the anti-Carp antibody in the third immune complex with the third immune complex to form a fourth immune complex consisting of biotin-first antigen-anti-Carp antibody-anti-immune complex antibody-receptor;

t3, combining the donor bound with streptavidin with the fourth immune complex to form a fifth immune complex composed of donor-streptavidin-biotin-first antigen-anti-Carp antibody-anti-immune complex antibody-receptor;

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 detected;

Alternatively, the first and second electrodes may be,

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

R2, combining an anti-immune complex antibody which specifically recognizes the anti-Carp antibody in the sixth immune complex and binds to biotin with the sixth immune complex to form a seventh immune complex consisting of receptor-first antigen-anti-Carp antibody-anti-immune complex antibody-biotin;

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

r4, detecting the presence or absence of the eighth immune complex; if the eighth immune complex exists, an anti-Carp antibody exists in the sample to be detected;

in some embodiments, the method further comprises the step of preparing an anti-Carp 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 working curve.

in some embodiments of the present invention, the excitation light with a wavelength of 600-.

In some particularly preferred embodiments of the present invention, the heterogeneous chemiluminescent immunoassay reagent for the detection of Anti-CCP antibodies comprises:

Component a7 comprising a solid support and, directly or indirectly bound thereto, a first antigen 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 substrate to generate a detectable signal and an anti-immune complex antibody directly or indirectly bound thereto, said anti-immune complex antibody being capable of specifically recognizing and binding to anti-CCP antibodies in a first immune complex formed with a first antigen and not recognizing free, antigen-unbound anti-CCP antibodies.

In some embodiments of the present invention, the reagent further comprises Anti-CCP pure product as a calibrator, and the calibrator is diluted by calibrator diluent to working calibrator solutions with different concentrations according to a proportional gradient.

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

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

in some preferred embodiments of the present invention, component c7, a substrate solution, said substrate solution comprising a1 solution and a B1 solution, preferably said a1 solution is a hydrogen peroxide solution, preferably said B1 solution is a sodium hydroxide solution.

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

in some embodiments of the invention, the anti-immune complex antibodies do not recognize the light chain portion of the anti-CCP antibodies in the first immune complex.

in other embodiments of the invention, the anti-immune complex antibodies specifically recognize the Fc fragment 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 by using a human immune complex to obtain animal serum containing the polyclonal antibody; and purifying the animal serum by affinity chromatography to obtain the polyclonal antibody for specifically recognizing the human immune complex.

in the present invention, the method for preparing the monoclonal antibody comprises: fusing spleen cells of mice immunized by the human immune complex with mouse myeloma cells, culturing, detecting cell culture supernatant, and reserving positive cell strains.

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 synthetic citrullinated cyclic peptides, citrullinated linear peptides, polypeptides formed by synthesizing at least 2 single citrullinated peptide fragments on one peptide chain, a mixture of citrullinated peptide fragments comprising at least 2 single citrullinated peptide fragments, and citrullinated proteins.

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 by synthesizing at least 2 single citrulline-containing peptide fragments on one peptide chain, and a citrulline peptide fragment mixture comprising at least 2 single citrulline-containing peptide fragments; preferably, the first antigen is a polypeptide synthesized on one peptide chain by 2-4 citrulline-containing peptide fragments or a citrulline peptide fragment mixture containing 2-4 single citrulline-containing peptide fragments.

In some preferred embodiments of the present invention, the citrullinated peptide fragment is selected from 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 10-50 mg/mL.

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

In some further particularly preferred embodiments of the present invention, the concentration of Anti-CCP antibody is measured by a heterogeneous chemiluminescent immunoassay using a heterogeneous chemiluminescent immunoassay kit comprising Anti-CCP antibody detection reagents, comprising:

step R1, mixing a sample to be tested with the component a7 to obtain a first mixture;

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

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

example III

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

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

example 1:

53 samples for confirmed diagnosis of rheumatoid arthritis were collected and Anti-CCP antibody, 14-3-3eta protein and Anti-carp antibody were detected by homogeneous immunoassay, respectively.

1. and irradiating the fifth immune complex by using excitation light with the wavelength of 600-700nm to excite the donor to generate singlet oxygen, reacting the acceptor with the contacted singlet oxygen to generate emission light with the wavelength of 520-620nm, and detecting a signal value of the emission light so as to judge whether the anti-CCP antibody and/or the concentration of the anti-CCP antibody exist in the sample to be detected. The method comprises the following steps that a first antigen is coated with luminescent particles (reagent 1) serving as a receptor, a biotin-labeled anti-immune complex antibody (reagent 2) is assembled into an anti-citrullinated peptide antibody determination kit, and then the kit is loaded on a full-automatic light-activated chemiluminescence immunoassay analyzer, and the detection steps are as follows:

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

2) a sample adding Tip sucks 25 mu L of reagent 1 into a reaction micropore plate;

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

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

5) the sample adding Tip absorbs 175 mu L of photosensitive solution as a donor to a reaction micropore plate;

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

7) Under the irradiation of the excitation light generated by the instrument, the donor (photosensitive particles) is induced to be activated and releases active oxygen ions in high energy state. The active oxygen ions in the high energy state are captured by the acceptor (luminescent particle) at a close distance, thereby transferring energy to activate the luminescent compound in the acceptor (luminescent particle). After a few microseconds, the luminescent compound in the receptor (luminescent particle) will release high-level red light, and the high-level photons are measured by 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 detecting the sample to be detected according to the steps 1) to 7), and calculating the concentration of the anti-CCP antibody in the sample to be detected by the equation in 8).

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

judging whether 14-3-3eta protein exists in a sample to be detected, wherein the homogeneous phase immunoassay method for detecting 14-3-3eta protein in the sample to be detected comprises the following steps:

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

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

(3) irradiating the fourth mixture with excitation light with a wavelength of 600-700nm, wherein the excitation light can excite the donor to generate singlet oxygen, and the acceptor reacts with the contacted singlet oxygen to generate emission light with a wavelength of 520-620nm as a detectable chemiluminescence signal;

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

in other embodiments of the present invention, the method for determining the content of 14-3-3eta protein comprises the following steps:

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

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

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

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

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

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

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

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

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

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

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

4. And (3) experimental operation:

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

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

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

5) the sample adding Tip sucks 175 mu L of mixed liquor (matched with an instrument) containing a streptavidin modified donor into a reaction micropore plate;

7) Under the irradiation of 680nm excitation light generated by the instrument, the donor is induced to be activated and releases active oxygen ions in a high energy state. The active oxygen ions in the high energy state are captured by the luminescent particles at a close distance, thereby transferring energy to activate the luminescent compounds in the luminescent particles. After several microseconds, the luminescent compound in the receptor releases 612nm high-level red light, and the high-level photons are measured by 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 concentration of the 14-3-3eta protein;

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

3. The concentration of anti-carp antibody is determined by a competition method using a homogeneous immunoassay.

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

1) diluting the sample in a pre-dilution hole position according to a ratio of 1:10, and uniformly mixing for 20 seconds;

2) a sample adding Tip sucks 10 mu L of diluted sample or calibrator into a reaction microplate;

3) A reagent sample adding Tip absorbs 25 mu L of carbamylated human serum albumin coated receptor (luminescent particles) to a reaction microporous plate;

4) Absorbing 25 mu L of biotinylated rabbit anti-carbamylation protein antibody into a reaction microplate by a reagent sample adding Tip;

5) Mixing for 20 s, and incubating at 37 deg.C for 17 min;

6) Absorbing 175 mu L of donor (photosensitive solution with working concentration of 20 mu g/mL) into a reaction micropore plate by a reagent sample adding Tip;

7) mixing for 20 s, and incubating at 37 deg.C for 15 min;

8) under the irradiation of the excitation light generated by the instrument, the photosensitive particles in the donor are induced to be activated, and active oxygen ions in high energy states are released. The active oxygen ions in the high energy state are captured by the acceptor (luminescent particle) at a close distance, thereby transferring energy to activate the luminescent compound in the acceptor (luminescent particle). After a few microseconds, the luminescent compound in the receptor (luminescent particle) will release high-level red light, and the high-level photons are measured by a single photon counter;

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

4. Experimental data

The experimental data are shown in tables 3 and 4.

TABLE 3

TABLE 4

The experimental results show that: the individual positive rate of 14-3-3 is 66%, the individual positive rate of anti-carp is 68%, the individual positive rate of anti-CCP is 79%, and the positive rate of the combined three tests is 88%.

example 2:

40 samples for diagnosing the rheumatoid arthritis are collected, the Anti-CCP antibody, the 14-3-3eta protein and the Anti-carp antibody are respectively detected by a homogeneous and/or heterogeneous immunoassay method, and then three detection results of the same sample are put together for statistics so as to improve the accurate diagnosis rate of the sample.

2. detecting the concentration of 14-3-3 by sandwich method with 14-3-3eta detection kit (light-activated chemiluminescence method) (developed by Beijing Koimei, Beijing) and homogeneous immunoassay

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

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

step one, making 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. and (3) experimental operation:

3. An Anti-carp antibody is detected by adopting an Anti-carp detection kit (a light-activated chemiluminescence method) (developed by Beijing Korea of origin)

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

5) mixing for 20 s, and incubating at 37 deg.C for 17 min;

7) mixing for 20 s, and incubating at 37 deg.C for 15 min;

4. Results and conclusions

And 5.1, judging whether the clinical sample is positive or negative according to a 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 for confirmed diagnosis of rheumatoid arthritis, the positive rate of CCP test item alone was 68%, the positive rate of 14-3-3eta test item alone was 50%, and the positive rate of carp test item alone was 60%.

4.2 statistical analysis of the three test results

the statistical analysis of the three test results is shown in table 6.

TABLE 6

when one of the CCP detection item or the 1433eta detection item is positive and determined to be positive, the detection positive rate is increased to 75 percent; when one of the CCP detection item or the carp detection item is positive and is judged to be positive, the positive rate is increased to 80%; when one of the CCP test item, the 1433eta test item and the carp test item is positive and determined to be positive, the positive rate is increased to 82.5%. Research shows that the three serological indexes have no correlation, which indicates that certain complementarity exists, and the joint detection of two or three serological indexes can improve the sensitivity of the rheumatoid clinical diagnosis.

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

Sequence listing

<110> Beijing Koume Biotechnology Ltd

<120> method for assessing the presence or absence of rheumatoid arthritis in vitro by biomarker-linked assay

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 246

<212> PRT

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

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Ala Gly Glu Gly Glu Asn

245

Claims

1. use of a homogeneous immunoassay for detecting the concentration of each biomarker in a panel 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:

2. Use according to claim 1, characterized in that the combined concentration values of step b) are compared with cut-off values 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 homogeneous immunoassay for detecting the concentration of each biomarker in a panel of Rheumatoid Arthritis (RA) biomarkers in the preparation of a reagent for assessing the severity of Rheumatoid Arthritis (RA) by means of biochemical markers in addition, comprising:

4. use of a homogeneous immunoassay for detecting the concentration of each biomarker in a panel of Rheumatoid Arthritis (RA) biomarkers in the preparation of a formulation for the classification of Rheumatoid Arthritis (RA) from other autoimmune diseases by biochemical labeling of the extra-corporeal region, comprising:

Wherein the biomarker panel comprises Anti-CCP antibodies, 14-3-3eta protein and Anti-carp antibodies; preferably the other autoimmune diseases include other joint diseases; the other joint disease is Osteoarthritis (OA).

5. use of a Rheumatoid Arthritis (RA) biomarker panel for the preparation of a reagent for assessing in vitro the presence or absence of Rheumatoid Arthritis (RA) in a test sample, wherein an increase in the combined concentration value measured for each biomarker in the Rheumatoid Arthritis (RA) biomarker panel as compared to the truncated combined concentration value for each marker in the corresponding biomarker panel measured from a reference population using a homogeneous immunoassay is indicative of the presence of RA;

6. use according to any one of claims 1 to 5, wherein said biomarker panel comprises Anti-CCP antibodies, 14-3-3eta protein and Anti-carp antibodies and other biomarkers, preferably said other biomarker is RA.

7. use according to any one of claims 1 to 6, characterized in that said step further comprises measuring the content of 14-3-3eta protein or fragment thereof or of immune complexes formed by said 14-3-3eta protein or fragment thereof with at least one antibody.

8. The use according to claim 7, wherein the content of 14-3-3eta protein in the sample to be tested is determined based on a 14-3-3eta protein standard working curve.

9. the use according to claim 7, wherein said step further comprises comparing the amount of 14-3-3eta protein or fragment thereof or immune complex formed by said 14-3-3eta protein or fragment thereof and at least one antibody measured with the amount of immune complex formed by said 14-3-3eta protein or fragment thereof or said 14-3-3eta protein or fragment thereof and at least one antibody in a normal control sample, a rheumatoid arthritis control sample or a pre-treatment sample from the same subject.

10. Use according to claim 7, characterized in that said step comprises contacting said sample with an antibody comprising an antibody capable of binding specifically to at least one specific epitope of the 14-3-3eta protein or fragment thereof to form an immune complex.

11. The use according to any one of claims 7 to 10, wherein the antibodies comprise a first antibody capable of specifically binding to a first epitope of 14-3-3eta protein and a second antibody capable of specifically binding to a second epitope of 14-3-3eta protein, wherein the second epitope and the first epitope do not overlap.

12. the use according to claim 11, wherein the first antibody binds to a receptor capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal.

13. use according to claim 12, characterized in that the receptor comprises an olefinic compound and a metal chelate, in non-particulate form and soluble in an aqueous medium; and/or the acceptor is polymer particles filled with a luminescent compound and lanthanide.

14. use according to claim 11, wherein the first and second antibodies are each independently selected from monoclonal and/or polyclonal antibodies, preferably monoclonal antibodies.

15. The use according to claim 7, wherein the amino acid SEQUENCE of the 14-3-3eta protein or fragment thereof is as shown in SEQUENCE No. 1.

16. Use according to claim 15, characterized in that said epitope is selected from the relatively specific fragments of the sequence of amino acid fragments 14-3-3eta protein: 1-6aa, 27-38aa, 71-83aa, 112-154 aa and 141-154 aa.

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

18. a kit of reagents for assessing the severity of Rheumatoid Arthritis (RA) by biochemical marker in vitro based on the concentration of each biomarker in a Rheumatoid Arthritis (RA) biomarker panel, comprising reagents for detecting the concentration of each biomarker in a Rheumatoid Arthritis (RA) biomarker panel, wherein the biomarker panel comprises an Anti-CCP antibody, a 14-3-3eta protein and an Anti-carp antibody.

19. a kit of reagents for classifying Rheumatoid Arthritis (RA) from other autoimmune diseases by biochemical marker extracorporal regions based on the concentration of each biomarker in a Rheumatoid Arthritis (RA) biomarker panel, comprising reagents for detecting the concentration of each biomarker in a Rheumatoid Arthritis (RA) biomarker panel, wherein the biomarker panel comprises Anti-CCP antibody, 14-3-3eta protein, and Anti-carp antibody.

20. A kit of reagents for assessing in vitro the presence or absence of Rheumatoid Arthritis (RA) in a test sample using a Rheumatoid Arthritis (RA) biomarker panel, wherein an increase in a combined concentration value measured using a homogeneous immunoassay for each biomarker in the Rheumatoid Arthritis (RA) biomarker panel as compared to a truncated combined concentration value for each marker in a corresponding biomarker panel measured from a reference population is indicative of the presence of RA; wherein the biomarker panel comprises Anti-CCP antibody, 14-3-3eta protein and Anti-carp antibody.

21. The kit of any one of claims 17 to 20, wherein the set of biomarkers comprises Anti-CCP antibody, 14-3-3eta protein and Anti-carp antibody and a further biomarker, preferably the further biomarker is RA.

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

23. The kit of claim 22, wherein the homogeneous immunoassay reagent for detecting anti-cyclic citrullinated peptide antibodies comprises:

24. the kit of claim 23, wherein the first antigen or the anti-immunocomplex antibody binds to a receptor capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal; preferably, the acceptor comprises an olefinic compound and a metal chelate, which is in non-particulate form and soluble in an aqueous medium; and/or the acceptor is polymer particles filled with a luminescent compound and lanthanide.

25. The kit according to claim 23 or 24, further comprising a component c1 comprising a donor capable of producing singlet oxygen in an excited state; preferably 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; further preferably, the donor is bound to streptavidin and the first antigen or the anti-immune complex antibody, respectively, is bound to biotin.

26. The reagent kit of any one of claims 23 to 25 wherein the reagents further comprise pure anti-CCP as a calibrator diluted by a calibrator diluent in a proportional gradient to working calibrator solutions of different concentrations.

27. The kit of claim 22, wherein the homogeneous immunoassay reagent for detecting 14-3-3eta protein comprises:

28. The kit of any one of claims 27, wherein the reagents further comprise 14-3-3eta protein as a calibrator diluted by a calibrator diluent in a proportional gradient to working calibrator solutions of different concentrations.

29. The kit of claim 27 or 28, 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; preferably, the second antibody or binding fragment thereof binds to biotin and the donor binds to streptavidin.

30. The kit of claim 22, wherein the homogeneous immunoassay reagent for the detection of anti-Carp antibodies comprises:

Component b3 comprising a second anti-Carp antibody.

31. The kit of claim 30, wherein the antigen or the second anti-Carp antibody binds to a receptor; preferably, the antigen binds to a receptor; the receptor is capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal.

32. The kit of claim 31, wherein the receptor comprises an olefinic compound and a metal chelate, in non-particulate form, and soluble in an aqueous medium; and/or the acceptor is polymer particles filled with a luminescent compound and lanthanide.

33. the kit according to any one of claims 30 to 32, wherein the kit further comprises component c3 comprising a donor capable of producing singlet oxygen in an excited state; preferably the donor is bound to one member of a specific binding pair and the other member of the specific binding pair is bound to the antigen or the second anti-Carp antibody; further preferably, the donor is bound to streptavidin and the second anti-Carp antibody is bound to biotin, respectively.

34. the kit of claim 33, wherein the donor is a photoactivated or chemically activated sensitizer, which is in a non-particulate form and 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.

35. The kit of claim 22, wherein the homogeneous immunoassay reagent for the detection of anti-Carp antibodies comprises:

36. The kit of claim 35, wherein the reagents further comprise an anti-Carp antibody purified product as a calibrator diluted by a calibrator diluent in a proportional gradient to working calibrator solutions of different concentrations.

37. The kit according to claim 35 or 36, wherein the second antigen is bound to one member of a specific binding pair and the donor is bound to the other member of the specific binding pair, preferably wherein the second antigen is bound to biotin and the donor is bound to streptavidin.

38. the kit of any one of claims 35 to 37, wherein the receptor comprises an olefinic compound and a metal chelate, in non-particulate form, and soluble in an aqueous medium; and/or the acceptor is polymer particles filled with a luminescent compound and lanthanide.

39. The kit of any one of claims 35 to 37, 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.

40. The kit of claim 22, wherein the kit comprises the reagents for indirect detection of Anti-CCP antibodies in the biomarker panel using a homogeneous immunoassay according to any of claims 23 to 26, the reagents for detection of 14-3-3eta proteins in the biomarker panel by sandwich method using a homogeneous immunoassay according to any of claims 27 to 29 and the reagents for detection of Anti-Carp antibody concentration in the biomarker panel by competition method using a homogeneous immunoassay according to any of claims 30 to 34.

41. The kit of claim 22, wherein the kit comprises the reagents for indirect detection of Anti-CCP antibodies in the biomarker panel using a homogeneous immunoassay according to any of claims 23 to 26, the reagents for detection of 14-3-3eta proteins in the biomarker panel by sandwich method using a homogeneous immunoassay according to any of claims 27 to 29 and the reagents for detection of Anti-Carp antibody concentration in the biomarker panel by sandwich method using a homogeneous immunoassay according to any of claims 35 to 39.

42. A kit for assessing the presence or absence of Rheumatoid Arthritis (RA) by biochemical marker ex vivo based on the concentration of each biomarker in a panel of Rheumatoid Arthritis (RA) biomarkers, comprising the kit of reagents of any one of claims 17-41.

43. A kit for assessing the severity of Rheumatoid Arthritis (RA) by biochemical marker-based on the concentration of each biomarker in a panel of RA biomarkers, comprising the kit of reagents of any one of claims 17-41.

44. A kit for classifying Rheumatoid Arthritis (RA) from other autoimmune diseases by biochemically labeling extra-corporeal regions based on the concentration of each biomarker in a panel of Rheumatoid Arthritis (RA) biomarkers, comprising the kit of reagents of any one of claims 17-41.

45. A reagent kit for assessing the presence of Rheumatoid Arthritis (RA) in a test sample in vitro using a Rheumatoid Arthritis (RA) biomarker panel comprising the reagent set of any one of claims 17-41.

46. A method for assessing the presence or absence of Rheumatoid Arthritis (RA) in vitro by biochemical markers, comprising detecting the concentration of each biomarker in a panel of Rheumatoid Arthritis (RA) biomarkers using the kit of reagents according to any one of claims 17-41 or using the kit of claims 42-45 and assessing the presence or absence of Rheumatoid Arthritis (RA) in vitro by biochemical markers.

47. The method of claim 46, comprising:

48. the method of claim 47 wherein the combined concentration values of step b) are compared to cutoff values 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.

49. A method for assessing the severity of Rheumatoid Arthritis (RA) by the external biochemical marker, comprising detecting the concentration of each biomarker in a panel of Rheumatoid Arthritis (RA) biomarkers using the kit of any one of claims 17-41 or using the kit of claims 42-45 and assessing the severity of Rheumatoid Arthritis (RA) by the external biochemical marker.

50. the method of claim 49, wherein the method comprises:

51. a method of classifying Rheumatoid Arthritis (RA) from other autoimmune diseases by biochemical labeling of extra-corporeal regions, comprising detecting the concentration of each biomarker in a panel of Rheumatoid Arthritis (RA) biomarkers using the kit of any one of claims 17 to 41 or using the kit of claims 42 to 45 and classifying Rheumatoid Arthritis (RA) from other autoimmune diseases by biochemical labeling of extra-corporeal regions.

52. The method of claim 51, wherein the method comprises:

53. a method for assessing in vitro the presence or absence of Rheumatoid Arthritis (RA) in a test sample using the kit of any one of claims 17-41 or using the kit of claims 42-45, wherein an increase in the combined concentration value measured for each biomarker in the Rheumatoid Arthritis (RA) biomarker panel as compared to the truncated combined concentration value for each marker in the corresponding biomarker panel measured from a reference population is indicative of the presence of RA;

54. The method according to any one of claims 46 to 53, wherein said set of biomarkers comprises Anti-CCP antibody, 14-3-3eta protein and Anti-carp antibody and a further biomarker, preferably said further biomarker is RF.

55. The method of any one of claims 46 to 54, wherein the concentration of Anti-CCP is detected by a homogeneous immunoassay using the kit of reagents of any one of claims 23 to 26, comprising:

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

56. The method of claim 55 further comprising the step of preparing an Anti-CCP standard working curve prior to step R1.

57. the method of claim 56, wherein in step R4, the intensity of said chemiluminescent signal of step R3 is detected and the amount of Anti-CCP antibody in the sample is determined based on the Anti-CCP standard working curve.

58. the method of any one of claims 46 to 54, wherein the kit of reagents of any one of claims 27 to 29 is used to detect the concentration of 14-3-3eta protein by a homogeneous immunoassay comprising:

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

59. the method according to claim 58, further comprising a step of preparing a standard working curve of 14-3-3eta protein before step R1.

60. The method according to claim 59, wherein in step R4, the intensity of the chemiluminescent signal of 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 working curve.

61. The method according to any one of claims 46 to 54, wherein the kit of reagents according to any one of claims 30 to 34 is used to detect the concentration of anti-Carp antibody by a homogeneous immunoassay comprising the steps of:

62. The method according to claim 61, further comprising the step of preparing a standard working curve of the target anti-Carp antibody prior to step R1.

63. the method according to claim 62, wherein in 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 working curve.

64. The method of claim 63, wherein the concentration of the target anti-Carp antibody in the test sample is determined by detecting the signal value of the emission light of the 4 th mixture and comparing it with the chemiluminescence signal value of a control immunocomplex formed between equal amounts of the antigen and the second anti-Carp antibody in step R2.

65. the method according to any one of claims 46 to 54, wherein the kit of reagents according to any one of claims 35 to 39 is used to detect the concentration of anti-Carp antibody by a homogeneous immunoassay comprising the steps of:

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

66. the method according to claim 65, further comprising the step of preparing a standard working curve for the anti-Carp antibody prior to step R1.

67. The method of claim 66, wherein in step R4, the intensity of the chemiluminescent signal of 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.

68. A chemiluminescent immunoassay system for detecting each biomarker in a Rheumatoid Arthritis (RA) biomarker panel comprising detecting the presence of each biomarker in a Rheumatoid Arthritis (RA) biomarker panel and/or determining the amount of each biomarker in a Rheumatoid Arthritis (RA) biomarker panel in a test sample using the kit of any one of claims 17 to 41 or using the kit of any one of claims 42 to 45 or using the method of any one of claims 46 to 67.

69. A detection system according to claim 68, characterised in that the system comprises:

a reaction device for the chemical reaction of a sample to be tested with the reagents of the reagent kit according to any one of claims 17 to 41 or the kit according to any one of claims 42 to 45;

70. the detection system according to claim 69, wherein the processor uses a cubic spline interpolation fit to perform the fitting to directly provide a concentration value of the target molecule to be detected in the sample to be detected.