CN108344865A

CN108344865A - Detect homogeneous immunological detection reagent box and its application of 14-3-3eta albumen

Info

Publication number: CN108344865A
Application number: CN201810011520.0A
Authority: CN
Inventors: 饶星; 廖智星; 陈英豪; 李会强; 刘宇卉; 李临
Original assignee: BEIJING KEMEI BIOLOGICAL TECHNOLOGY Co Ltd
Current assignee: BEIJING KEMEI BIOLOGICAL TECHNOLOGY Co Ltd
Priority date: 2018-01-05
Filing date: 2018-01-05
Publication date: 2018-07-31
Also published as: CN114397442A; CN114354919A; CN114354918A

Abstract

The present invention relates to a kind of homogeneous immunological detection reagent boxes for detecting 14 33 eta albumen.The kit is made of the first antibody and secondary antibody that can be combined with 14 33 eta protein-specifics；It, using the high sensitivity of light-induced chemiluminescent immunoassay platform, the advantages such as the range of linearity is wide, precision is good, improves diagnosis rate, to prevent rheumatoid and finding that rheumatoid provides auxiliary detection ahead of time in a manner of double-antibody sandwich for clinical diagnosis rheumatoid.

Description

Detect homogeneous immunological detection reagent box and its application of 14-3-3eta albumen

Technical field

The invention belongs to technical field of immunoassay, and in particular to a kind of homogeneous immune inspection of detection 14-3-3eta albumen Test agent box and preparation method thereof, application method.

Background technology

14-3-3eta (η) albumen is a kind of novel serum/plasma protein marker, induces inflammatory factor, such as leucocyte Interleukin (IL) -1 and -6, taken in conjunction to joint injury.Only have 14-3-3 η albumen in RA Active inflammations synovial membrane in 7 kinds of hypotypes and High expression in serum, and be proportionate with the expression of RA Pro-inflammatory Cytokines and synovial membrane inflammation, and synovial fluid 14-3-3 η protein levels are extremely It is higher than 5 times of serum levels or more less, it is the main source of 14-3-3 η albumen to prompt synovial membrane.Compared with the people of health, 14-3-3 η Hypotype high level expression in arthritic, this is considered damaging with the protein induced relevant inflammatory factors of 14-3-3 η and joint The ability of wound is directly related.

The method of existing detection 14-3-3 albumen is heterogeneous immunodetection, these methods are sensitive relatively low, accurately Degree is not high, and operation is also complex.Also, home and abroad is in the market still without finding commercialization 14-3-3eta protein assay reagents Box.Once report had enzyme linked immunosorbent assay (ELISA method) to detect 14-3-3eta albumen in patients serum, but its is sensitive relatively low, Specificity is also poor, and operation is also relatively complicated.

Therefore, there is an urgent need for a kind of high sensitivity of research and development, the 14-3-3eta eggs that specificity is good, operating method is easy at present White detection kit.

Invention content

In order to solve the above technical problems, the light activation hair that the present invention provides a kind of for detecting 14-3-3eta albumen Light immunity detection reagent.The kit is anti-using the first antibody that can be combined with 14-3-3eta protein-specifics and second System in conjunction with light-induced chemiluminescent technology at being made；Utilize the high sensitivity of light-induced chemiluminescent immunoassay platform, linear model The advantages such as wide, precision is good are enclosed, diagnosis rate is improved for clinical diagnosis rheumatoid, to prevent rheumatoid and finding that rheumatoid carries ahead of time It is detected for auxiliary, becomes rheumatoid Novel marker.

First aspect present invention detect 14-3-3eta albumen or its segment or the 14-3-3eta albumen or its segment with The existing of the immune complex that at least one antibody is formed is preparing for passing through following step using homogeneous immunologic detection method Purposes in the reagent of the rheumatoid arthritis of rapid detection subject：A) it provides and suffers from rheumatoid arthritis from suspection Subject sample to be tested；B) 14-3-3eta albumen or its segment or the 14-3-3eta in the sample to be tested are detected The immune complex that albumen or its segment are formed at least one antibody；The wherein described 14-3-3eta albumen or its segment are exempted from The rheumatoid arthritis of the presence instruction subject of epidemic disease compound；Wherein, the 14-3-3eta albumen or its segment include At least one 14-3-3eta epitopes, the sample to be tested are selected from blood, blood plasma, serum, synovial fluid and tissue.

In certain embodiments of the present invention, the step further includes measuring 14-3-3eta albumen or its segment or institute State the content for the immune complex that 14-3-3eta albumen or its segment are formed at least one antibody.

In some preferred embodiments of the present invention, determined based on 14-3-3eta protein standards working curve to be measured The content of 14-3-3eta albumen in sample.

In certain embodiments of the present invention, the step further includes by measured 14-3-3eta albumen or its piece The content for the immune complex that section or the 14-3-3eta albumen or its segment are formed at least one antibody, with normal control 14-3-3eta albumen described in sample before sample, rheumatoid arthritis control sample or treatment from same subject Or the content of immune complex that its segment or the 14-3-3eta albumen or its segment are formed at least one antibody is compared Compared with.

According to the present invention, the step include by the sample with comprising can be with 14-3-3eta albumen or its segment At least one specific epitopes specifically bind the antibody to form immune complex.

In certain embodiments of the present invention, the antibody include can be special with the first epitope of 14-3-3eta albumen The first antibody and the secondary antibody that can be combined with the second epitope specificity of 14-3-3eta albumen that the opposite sex combines, wherein Second epitope and the first epitope non-overlapping.

In certain embodiments of the present invention, the first antibody is combined with receptor, and the receptor can be with singlet Oxygen reaction generates detectable chemiluminescence signal.

In the present invention, the receptor includes olefin(e) compound and metallo-chelate, is non-particulate forms, and in aqueous Jie It is solvable in matter.

In the present invention, the receptor is the high molecular particle filled with luminophor and lanthanide series.

In certain embodiments of the present invention, the first antibody and secondary antibody are separately anti-selected from monoclonal Body and/or polyclonal antibody, preferably monoclonal antibody.

In some embodiments of the invention, the 14-3-3eta albumen or the amino acid sequence of its segment be such as Shown in SEQUENCE NO.1.

In some embodiments of the invention, the epitope is selected from the sequence that amino acid fragment is 14-3-3eta albumen Relative specificity segment：1-6aa, 27-38aa, 71-83aa, 112-119aa and 141-154aa.

For this purpose, second aspect of the present invention provides a kind of homogeneous immunological detection reagent for detecting 14-3-3eta albumen Suit comprising：

Component a, it includes the receptor and in combination first for generating detectable signal can be reacted with singlet oxygen Antibody or its binding fragment, the first antibody or its binding fragment can be with the first epitope specificities of 14-3-3eta albumen In conjunction with；

Component b, it includes the secondary antibody that can be combined with the second epitope specificity of 14-3-3eta albumen or its combinations Segment, second epitope and the first epitope non-overlapping；

Component c comprising the donor of singlet oxygen can be generated in excited state.

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

In certain embodiments of the present invention, second epitope and first epitope are separately selected from amino Acid fragment is the relative specificity segment of the sequence of 14-3-3eta albumen：1-6aa, 27-38aa, 71-83aa, 112-119aa and 141-154aa。

In the present invention, the first antibody and secondary antibody are separately selected from monoclonal antibody and/or polyclonal Antibody, preferably monoclonal antibody.

According to certain embodiments of the present invention, the reagent set further includes the 14-3-3eta albumen as calibration object Sterling, the calibration object be calibrated product dilution proportionally gradient dilution at various concentration working calibration product solution.

In the present invention, the secondary antibody or its binding fragment are combined with a member in specific binding pair member, and The donor and another Yuan combination in specific binding pair member.

In some embodiments of the invention, the secondary antibody or its binding fragment are combined with biotin, and the confession Body is combined with Streptavidin.

In some embodiments of the invention, receptor and first antibody in combination or its combination in the component a A concentration of 10-200 μ g/mL of segment, preferably 20-150 μ g/mL, more preferable 30-100 μ g/mL, most preferably 40-80 μ g/mL.

In other embodiments of the present invention, a concentration of 0.1-8 of secondary antibody or its binding fragment in the component b μ g/mL, preferably 0.2-6 μ g/mL, more preferable 0.4-4 μ g/mL, most preferably 0.6-2 μ g/mL.

In the other embodiment of the present invention, a concentration of 5-20 μ g/mL of donor described in component c, preferably 8-15 μ g/ ML, more preferable 10-12 μ g/mL.

In some embodiments of the invention, the receptor is that the macromolecule filled with luminophor and lanthanide series is micro- Grain.

In the present invention, the donor is photoactivation or chemical activation sensitizer, is non-particulate forms, and aqueous It is solvable in medium.

In some embodiments of the invention, the donor is the high molecular particle filled with Photoactive compounds, is swashed in light Singlet oxygen can be generated by giving.

Third aspect present invention provides a kind of homogeneous immunological detection reagent box for detecting 14-3-3eta albumen, Including the homogeneous immunological detection reagent suit described in second aspect of the present invention.

Fourth aspect present invention provides a kind of homogeneous immune detection side for detecting 14-3-3eta albumen in sample to be tested Method comprising use homogeneous immune described in homogeneous immunological detection reagent suit or the third aspect described in second aspect of the present invention Detection kit judges to survey in sample to be tested with the presence or absence of 14-3-3eta albumen and/or determines that 14-3-3eta albumen contains Amount.

According to the present invention, this method includes：

Step R1 by sample to be tested and component a and combines b and mixes, obtains third mixture；

Third mixture is mixed with component c, obtains the 4th mixture by step R2；

Step R3 makes energy or reactive compound be contacted with the 4th mixture, and the donor is excited to generate single line State oxygen, the receptor can be reacted with the singlet oxygen received generates detectable chemiluminescence signal；

Step R4, the presence of chemiluminescence signal and/or intensity described in detecting step R3, to judge to survey sample to be tested In with the presence or absence of 14-3-3eta albumen and/or determine the content of 14-3-3eta albumen.

According to certain embodiments of the present invention, the method further includes the making 14-3-3eta eggs before step R1 The step of white standard working curve.

In some further specific embodiments of the present invention, in step R4, chemistry hair described in detecting step R3 The intensity of optical signal, and containing for 14-3-3eta albumen in sample to be tested is determined based on 14-3-3eta protein standards working curve Amount.

In the present invention, the step of not detaching and/or wash between step R1 and R2 and between step R2 and R3.

In some embodiments of the invention, mixed using the exciting light irradiation the 4th of 600-700nm wavelength in step R3 Object is closed, excited donor generates singlet oxygen, and receptor reacts the transmitting light for generating 520-620nm with the singlet oxygen touched.

Fifth aspect present invention provide it is a kind of as described in respect of the second aspect of the invention homogeneous immunological detection reagent suit or The method as described in homogeneous immunological detection reagent box or fourth aspect present invention as described in third aspect present invention is to be measured in detection The presence and/or the application in content of 14-3-3eta albumen in sample, wherein the sample to be tested is selected from blood, blood plasma, blood Clearly, synovial fluid and tissue.

Sixth aspect present invention provides a kind of homogeneous immunological detection reagent as described in the first aspect of the invention and is sleeved on Prepare the application in the kit for detecting rheumatoid arthritis comprising：

Step M1 provides the sample to be tested from main body to be measured；

Step M2 judges to whether there is 14-3-3eta albumen in survey sample to be tested and/or determines 14-3-3eta albumen Content；

Step M3, by it with normal reference sample, rheumatoid arthritis control sample or from same main body to be measured The comparision contents of 14-3-3eta albumen described in sample before treatment；

Wherein, the sample to be tested is selected from blood, blood plasma, serum, synovial fluid and tissue.

In certain embodiments of the present invention, with all condition ratios of normal control, 14-3-3eta in the sample to be tested The presence of albumen is the diagnostic indicant of rheumatoid arthritis in the main body to be measured.

In certain embodiments of the present invention, with all condition ratios of normal control, 14-3-3eta in the sample to be tested The increase of the content of albumen is the diagnostic indicant of rheumatoid arthritis in the main body to be measured.

In some preferred embodiments of the present invention, and all condition ratios of normal control, 14- in the sample to be tested The content of 3-3eta albumen increases the diagnostic indicant that 0.2ng/ml is rheumatoid arthritis in the main body to be measured.

In certain embodiments of the present invention, compared with rheumatoid arthritis control sample, in the sample to be tested The relative amount of 14-3-3eta albumen is the prognostic indicant of rheumatoid arthritis in the main body to be measured.

It is described compared with the sample before the treatment from same main body to be measured in other embodiments of the present invention The effect of the relative amount instruction therapeutic scheme of 14-3-3eta albumen in sample to be tested.

According to the method for the present invention, in step M2, judge to survey using the method as described in fourth aspect present invention to be measured With the presence or absence of the content of 14-3-3eta albumen and/or determining 14-3-3eta albumen in sample.

Homogeneous immunological detection reagent box provided by the present invention for detecting 14-3-3eta albumen uses can be with 14- The first antibody and secondary antibody that 3-3eta protein-specifics combine are made；It utilizes light activation in a manner of double-antibody sandwich Advantages that high sensitivity, the range of linearity of learning luminescence immunoassay platform are wide, precision is good etc., are improved really for clinical diagnosis rheumatoid Rate is examined, to prevent rheumatoid and finding that rheumatoid provides auxiliary detection ahead of time.

By the present invention kit be applied to homogeneous immunoassay instrument have it is following a little：1, signal value range is wide, reaches Quantitative test standard；2, high sensitivity, to early stage RA and the susceptibility height for determining RA patient；3, stability is good, and precision is good；4、 This kit is suitable for light-induced chemiluminescent immunoassay instrument, becomes domestic first 14-3-3eta protein detection kits.It pushes The progress of rheumatoid clinical diagnosis establishes new standard for industry.

Description of the drawings

To make the present invention be readily appreciated that, illustrate the present invention below in conjunction with the accompanying drawings.

Fig. 1 shows the testing result of different sample sets 14-3-3eta (η) albumen in embodiment 1.

Specific implementation mode

To make the present invention be readily appreciated that, the present invention is described more detail below.But before describing the present invention in detail, it should be understood that The present invention is not limited to the specific implementation modes of description.It is also understood that term used herein is embodied only for description Mode, and be not offered as restrictive.

In the case where providing numberical range, it should be understood that in the upper and lower bound of the range and the prescribed limit Any other regulation or between two parties each of between numerical value numerical value is encompassed by the present invention between two parties.These small range of upper limits It can independently be included in smaller range with lower limit, and be also covered by within the present invention, obey any clear in prescribed limit The limit of exclusion.Defined range include one or two limit in the case of, exclude any of the limit that those include or The range of the two is also included in the present invention.

Unless otherwise defined, all terms used herein and those skilled in the art's is usual Understand meaning having the same.Although similar or equivalent any method and material also may be used with method described herein and material To be used in the implementation or test of the present invention, but preferred method and material will now be described.

I, terms

" main body to be measured ", " subject " and " patient " are used interchangeably, in the case where being not particularly illustrated or limiting, Refer to mammal, such as people and non-human primates and rabbit, rat, mouse, goat, pig and other food in one's mouth animal species.

English corresponding to term " homogeneous " of the present invention is defined as " homogeneous ", and referring to need not be to combining Antigen antibody complex and remaining free antigen or antibody detached and can be both detected.

Term " sample to be tested " of the present invention refers to that may include containing a kind of mixture of analyte, analyte But it is not limited to protein, hormone, antibody or antigen.The typical sample to be tested that can be used in method disclosed by the invention includes Body fluid, such as blood, blood plasma, serum, synovial fluid, urine, sperm, saliva.

Term " 14-3-3 " of the present invention and " 14-3-3 albumen " are used interchangeably, and refer to the universal table in eukaryocyte The conservative intracellular reached adjusts at least one member of the 14-3-3 families of molecule.14-3-3 albumen, which has, combines many Various Functions Signal transducer, include the ability of kinases, phosphatase and transmembrane receptor.In fact, more than 100 kinds of signal transducers It is reported as the ligand of 14-3-3.14-3-3 albumen can be considered as being evolved into for Tetratrico peptide repeated fragment superfamilies Member.They usually have 9 or 10 α spirals, generally along its amino terminal spiralization homodimer and/or heterodimer phase Interaction.These albumen include multiple known structure domains, and the structural domain includes for bivalent cation interaction, phosphoric acid Change the region of acetylation and proteolytic cleavage and other.The known 14- for expressing seven kinds of different genetic codings in mammals 3-3 protein isoforms, each isotype include 242-255 amino acid.Seven kinds of 14-3-3 protein isoforms are named as 14-3-3 α/β(alpha/beta)、14-3-3δ/ξ(delta/zeta)、14-3-3ε(epsilon)、14-3-3γ(gamma)、14-3-3 η (eta), 14-3-3 τ/θ (tau/theta) and 14-3-3 σ (sigma/stratifin).14-3-3 albumen has high level Sequence similarity, it is known that experience translation post-processing such as, phosphorylation, it is citrullinated, etc..Referring to such as, Megidish etc. (1998) J.Biol.Chem.273：21834-45.Therefore, anti-14-3-3 autoantibodies can specifically bind and/or identify and is more than one 14-3-3 protein isoforms, or can specifically bind and/or identify only a kind of isotype (e.g., 14-3-3 η).In addition, anti-14-3- 3 antibody are combinable and/or identification is by such as, the 14-3-3- albumen of natural (e.g., after translation) or chemical method modification.

Heretofore described term " relative specificity segment " refers to 7 isotype 14-3-3 eggs for 14-3-3 families In vain, the present inventor has found 14-3-3eta albumen or the amino acid sequence of its segment as shown in SEQUENCE NO.1 by research Middle segment 1-6aa, 27-38aa, 71-83aa, 112-119aa and 141-154aa are the spy for only belonging to 14-3-3 η (eta) albumen Anisotropic epitope is intersected with the amino acid sequence of other 6 isotype 14-3-3 albumen of 14-3-3 families without any, by its production Raw monoclonal antibody, only identifies or combines 14-3-3 η (eta) albumen, nonrecognition or combine other 6 of 14-3-3 families it is same Kind type 14-3-3 albumen.

Heretofore described " arthritis " is used interchangeably with " arthritis illness " and " arthralgia ", in addition to what is indicated sentences Outside, the inflammatory disease of human synovial is typically referred to.Pain, swelling, it is stiff and be difficult to movement it is usually related with arthritis illness. Arthritis is formed by being more than 100 kinds of different situations.These are damaged it may is that any situation from relatively slight form to serious Harmful system form.Arthritis illness can be caused by any reason of many reasons, including infection, wound, degenerative disease, generation Thank disorderly or interference or other unknown etiologies.Arthritis illness can be more specifically described according to hypotype, for example, rheumatoid joint Inflammation, mixed connective tissue disease (MCTD), crystal-induced arthritis, adjuvant arthritis, spondyloarthropathy, osteoarthritis, class meat Tumor disease, palindromic rheumatism, post-traumatic arthritis, the relevant arthritis of malignant tumour, septic arthritis, Lyme arthritis, Osteoarthritis, bacterial infectivity arthritis, etc..Arthritis can also be with the disease of other identifications, including gout, tatanic ridge Column inflammation, systemic loupus erythematosus, inflammatory bowel disease, psoriasis, etc..Clearly defined arthritis illness refers to knowing about pass The scorching type of section and its stage, e.g., break out, alleviate, recurring, etc..

Term " antibody " of the present invention and " immunoglobulin " are used with most wide meaning, include the antibody of any isotype Or immunoglobulin, retain the antibody fragment of the specific binding to antigen；Including but not limited to Fab, Fv, scFv, Fd segment, Chimeric antibody, humanized antibody, single-chain antibody, bispecific antibody, and the antigen-binding portion thereof comprising antibody and non-antibody The fusion protein of albumen.In the case of any need, antibody can further with other parts, such as specific binding pair Member, such as biotin or Streptavidin (a member in biotin-Streptavidin specific binding pair member) etc. are sewed It closes.

Term " monoclonal antibody " of the present invention refers to the immunoglobulin secreted by the bone-marrow-derived lymphocyte of monoclonal, It can be prepared by method known in those skilled in the art.

Term " polyclonal antibody " of the present invention refers to the immune globulin generated by more than one bone-marrow-derived lymphocyte clone White set, can be prepared by method known in those skilled in the art.

Term " antigen " of the present invention is to refer to stimulation body to generate immune response, and with immune response product can resist Body and sensitized lymphocyte combine in vivo and in vitro, and the substance of immunological effect occurs.

Term " in conjunction with " of the present invention refers to due to interactions such as example covalent, electrostatic, hydrophobic, ion and/or hydrogen bonds, Including but not limited to two intermolecular direct joints caused by such as salt bridge and the interaction of water bridge.

Term " specific binding " of the present invention or " specific bond " refer to mutual discrimination and choosing between two kinds of substances Selecting property association reaction, said from stereochemical structure angle be exactly conformation between corresponding reactant correspondence.

Term " specific binding pair member " of the present invention refers to so a pair of of molecule, they can be mutually specific In conjunction with for example, enzyme-substrate, Ag-Ab, ligand-receptor.The example of one specific specific binding pair member couple is Biotin-Streptavidin system, wherein " biotin " is widely present in animal vegetable tissue, there are two cyclic annular knots on molecule Structure, respectively imidazolone ring and thiphene ring, wherein imidazolone ring are the main portions combined with Streptavidin.The biology of activation Element can under the mediation of protein cross agent, with known almost all creatures macromolecular be coupled, including protein, nucleic acid, Polysaccharide and lipid etc.；And " Streptavidin " is a kind of protein secreted by streptomycete, molecular weight 65kD." strepto- is affine Molecule is made of element " 4 identical peptide chains, wherein every peptide chain can combine a biotin.Therefore each antigen or antibody It can be coupled multiple biotin molecules simultaneously, sensitivity for analysis is improved to generate " tentacle effect ".In the case of any need, Any reagent used in the present invention, including antigen, antibody, receptor or donor, can biotin-conjugated-strepto- according to actual needs Any member in Avidin specific binding pair member.

Term " donor " of the present invention refers to that can generate after the activation by energy or reactive compound and receptor The sensitizer of the reactive intermediate of such as singlet oxygen of reaction.Donor can be photoactivation (such as dyestuff and aromatic compound) Or (such as enzyme, metal salt) of chemical activation.In particular embodiments of the invention, the donor is photosensitizer, described Photosensitizer can be photosensitizer known in the art, preferably stable with respect to the light and not compound with singlet oxygen effecting reaction, Its non-limiting example includes sub- disclosed in such as United States Patent (USP) US5709994 (patent document is hereby incorporated by reference) The compounds such as methyl blue, rose-red, porphyrin, phthalocyanine and chlorophyll and these compounds have 1-50 replacing group Derivative, the substituent group for so that these compounds with more lipophilicity or with more hydrophily, and/or as connection To the linking group of specific binding pair member.The example of other photosensitizers well known by persons skilled in the art can also be at this It is used in invention, such as the content described in United States Patent (USP) US6406913, which is hereby expressly incorporated by reference.At this It invents in other specific embodiments, the donor is other sensitizers of chemical activation, and non-limiting example is certain Compound, their catalyzing hydrogen peroxides are converted into singlet oxygen and water.The example of some other donor includes：1,4- dicarboxyl second Base-Isosorbide-5-Nitrae-naphthalene endoperoxides object, 9,10- diphenylanthrancene -9,10- endoperoxides objects etc. heat these compounds or these changes Conjunction object, which directly absorbs light, can discharge singlet oxygen.

Term " receptor " of the present invention is to refer to react the compound that can generate detectable signal with singlet oxygen. For donor by energy or reactive compound induced activation and the singlet oxygen for discharging upper state, the singlet oxygen of the upper state is close The receptor of distance is captured, to transmit energy to activate the receptor.In some specific embodiments of the present invention, the receptor It is such substance, the chemical reaction of experience and singlet oxygen is to form unstable metastable state intermediate, the metastable state Intermediate can decompose, and subsequently or simultaneously shine.The exemplary of these substances includes but not limited to：Enol ether, enamine, 9- alkane Pitch xanthans, 9- alkylidene-N- alkyl Acridane, aromatic ethylene ether, bicyclic ethylene oxide, thioxene, armaticity imidazoles or gloss Essence.In other specific embodiments of the present invention, the receptor is can to react resolve into be formed with singlet oxygen The olefines of the hydroperoxides or dioxy cyclobutane of ketone or carboxylic acid derivates；Two can be stablized by what the effect of light was decomposed Oxygen cyclobutane；It can be reacted with singlet oxygen to form the acetylene class of diones；Azo-compound or azo carbonyl can be formed The hydrazone class or hydrazides of compound, such as luminol；With the aromatic compounds that can form endoperoxides species.It can be according to this Specific, the non-limiting examples for the receptor that the invention disclosed and claimed utilizes are recorded in U.S. Patent number US5340716 (patent document is hereby incorporated by reference).In other specific embodiments of the invention, the receptor includes olefinic compound The case where object and metallo-chelate are non-particlizeds and solvable in water-bearing media, this receptor can be found in patent PCT/US2010/025433 (patent document is hereby incorporated by reference).

" donor " can be coated on matrix to be formed filled with sensitized by functional group in the present invention The high molecular particle for closing object can generate singlet oxygen under light excitation；And/or " receptor " can be by function base Group, which is coated on matrix, forms the high molecular particle filled with luminophor and lanthanide series.

" matrix " of the present invention is microballoon or particle known in those skilled in the art, can be any size , it can be organic or inorganic, can be inflatable or nondistensible, can be porous or non-porous , with any density, but preferably have and the close density of water, can preferably float in water, and by transparent, partially transparent Or opaque material is constituted.Described matrix can be with or without charge, when with charge, preferably negative electrical charge.The base Body can be that solid (such as polymer, metal, glass, organic and inorganic matter such as mineral, salt and diatom), small oil droplet are (such as hydrocarbon Compound, fluorocarbon, silicic fluid), vesica (such as phosphatide such as synthesis or natural such as cell and organelle Official).Matrix can be latex particle or other particles, lipid bilayer such as liposome, phosphatide containing organic or inorganic polymer Vesica, small oil droplet, silicon particle, metal-sol, cell and crystallite dyestuff.Matrix usually has multifunctionality, or can pass through Special or non-specific covalently or non-covalently interaction and be attached on donor or receptor.Be available there are many functional group or Person is merged in.Typical functional group includes carboxylic acid, acetaldehyde, amino, cyano, vinyl, hydroxyl, sulfydryl etc..It is suitable for One unrestricted example of the matrix of the present invention is carboxy-modified latex particle.The details of this matrix can be found in United States Patent (USP) US5709994 and US5780646 (this two patents document is hereby incorporated by reference).

Term " epitope " of the present invention is any egg for referring to specific binding immunoglobulin or T cell receptor White determinant.In some specific embodiments of the present invention, epitope is that antigenic surface can be by the region of antibody specificity set. Epitopic determinants usually may include the chemically active surface group of molecule, such as, but not limited to：Amino acid, carbohydrate side chain, phosphinylidyne Base and/or sulfonyl.In some other specific embodiment of the present invention, epitope can specific specific three-valued structures feature and Specific charge feature.

Term " homogeneous immunological detection reagent suit " of the present invention refers to the whole that homogeneous immune detection must use The combination of reagent or medicament.

II, embodiments

As previously mentioned, the method for existing detection 14-3-3 albumen is heterogeneous immunodetection, these methods are sensitive Relatively low, accuracy is not high, and operation is also complex.The present inventor using homogeneous immunodetection the study found that examine 14-3-3 Albumen high sensitivity, the range of linearity are wide, precision is good, and remove from detection process washing and etc., operation it is relatively simple.

Therefore, first aspect present invention be related to detecting 14-3-3eta albumen or its segment or the 14-3-3eta albumen or The existing of the immune complex that its segment is formed at least one antibody is preparing for logical using homogeneous immunologic detection method Cross the purposes in the reagent of the rheumatoid arthritis of the following steps evaluation subject：A) it provides and suffers from rheumatoid from suspection The sample to be tested of the arthritic subject of property；B) 14-3-3eta albumen or its segment or described in the sample to be tested are detected The immune complex that 14-3-3eta albumen or its segment are formed at least one antibody；The wherein described 14-3-3eta albumen or its The rheumatoid arthritis of the presence instruction subject of segment or immune complex；Wherein, the 14-3-3eta albumen or its Segment includes at least one 14-3-3eta epitopes, and the sample to be tested is selected from blood, blood plasma, serum, synovial fluid and tissue, excellent The sample to be tested is selected to be selected from blood, blood plasma and serum, the further preferred sample to be tested is serum.

In certain embodiments of the present invention, the step further includes measuring 14-3-3eta albumen or its segment or institute State the content for the immune complex that 14-3-3eta albumen or its segment are formed at least one antibody；And it further will be measured 14-3-3eta albumen or its segment or the 14-3-3eta albumen or its segment formed at least one antibody it is immune multiple Sample before the content for closing object, with normal reference sample, rheumatoid arthritis control sample or treatment from same subject What 14-3-3eta albumen described in product or its segment or the 14-3-3eta albumen or its segment were formed at least one antibody The content of immune complex is compared.

In some embodiments, by measured 14-3-3eta albumen or its segment or the 14-3-3eta albumen or its The content for the immune complex that segment and at least one antibody are formed, with 14-3-3eta albumen described in normal reference sample or The content for the immune complex that its segment or the 14-3-3eta albumen or its segment are formed at least one antibody is compared Compared with.For example, with all condition ratios of normal control, 14-3-3eta albumen described in the sample to be tested or its segment or the 14- The increase of the content for the immune complex that 3-3eta albumen or its segment are formed at least one antibody is in the main body to be measured The diagnostic indicant of rheumatoid arthritis.In some embodiments, by measured 14-3-3eta albumen or its segment or The content for the immune complex that the 14-3-3eta albumen or its segment are formed at least one antibody, with rheumatoid joint 14-3-3eta albumen described in scorching control sample or its segment or the 14-3-3eta albumen or its segment and at least one are anti- The content for the immune complex that body is formed is compared.For example, compared with rheumatoid arthritis control sample, it is described to wait for test sample What 14-3-3eta albumen described in product or its segment or the 14-3-3eta albumen or its segment were formed at least one antibody The relative amount of immune complex is the prognostic indicant of rheumatoid arthritis in the main body to be measured.

In some embodiments, by measured 14-3-3eta albumen or its segment or the 14-3-3eta albumen or its The content for the immune complex that segment is formed at least one antibody, with institute in the sample before the treatment from same main body to be measured State 14-3-3eta albumen or its segment or the 14-3-3eta albumen or its segment formed at least one antibody it is immune multiple The content for closing object is compared.For example, compared with the sample before the treatment from same main body to be measured, institute in the sample to be tested State 14-3-3eta albumen or its segment or the 14-3-3eta albumen or its segment formed at least one antibody it is immune multiple Close the effect of the relative amount instruction therapeutic scheme of object.

Hereinafter the second to the 6th aspect further provides realization specific embodiments of the present invention.

The homogeneous immunological detection reagent suit packet for detecting 14-3-3eta albumen involved by second aspect of the present invention It includes：

In the present invention, the sequence of the 14-3-3eta albumen is as shown in Sequence NO.1.And second epitope and First epitope independently the relative specificity segment selected from the sequence that amino acid fragment is 14-3-3eta albumen：1st The-the 6 amino acids (1-6aa) of position, the 27th the-the 38 amino acids (27-38aa), the 71st the-the 83 amino acids (71- 83aa), the 112nd the-the 119 amino acids (112-119aa) and the 141st the-the 154 amino acids (141-154aa).

Heretofore described monoclonal antibody can be combined with the epitope specificity of 14-3-3eta albumen.For example, of the invention Described in monoclonal antibody be can be with the relative specificity segment (epitope) of the sequence of the 14-3-3eta albumen：1- The antibody of 6aa, 27-38aa, 71-83aa, 112-119aa and 141-154aa specific binding.

The preparation method of the monoclonal antibody is not particularly limited in the present invention, art technology can be passed through Method well known to personnel is prepared.For example, in some embodiments, preparing 14-3-3eta monoclonal antibodies comprising：

Step 1: animal immune

1, first immunisation

3-5 8 weeks or so Balb/c male mices are chosen, first immunisation helps 14-3-3eta antigens and Freund completely The isometric mixing and emulsifying of agent, every mouse antigen after intraperitoneal injection emulsifies, immunizing dose is 100 μ g/ mouse.

2, booster immunization

After first immunisation, primary immunization was carried out every 2 weeks, it is isometric with 14-3-3eta antigens and incomplete Freund's adjuvant Mixing and emulsifying, every mouse antigen after intraperitoneal injection emulsifies, immunizing dose is 50 μ g/ mouse, carries out 3 times altogether and is immunized.Every 2 Zhou Hou carries out last time booster immunization through the not emulsified 50 μ g/ mouse of antigen of tail vein injection.

Step 2: cell fusion

Third day after last time booster immunization puts to death mouse under gnotobasis and takes mouse spleen and with side appropriate The evenly dispersed splenocyte of method.It is merged with murine myeloma cell SP2/0 under PEG mediations, and is dripped using limiting dilution assay 96 porocyte culture plates are added to be cultivated.

Step 3: antibody test

Fusion terminates 10 days or so, waits for that clone is long to appropriately sized, is detected to the cells and supernatant in each hole.Specifically Detection scheme is as follows：

1) wrapper sheet is carried out with 14-3-3eta antigens, 2%BSA is used in combination to be closed；

2) cells and supernatant in each hole in tissue culture plate is added, is fully washed after reaction；

3) the anti-mouse secondary antibody of HRP labels is added, is fully washed after reaction；

4) tmb substrate reaction 15min colour developings are added, 2M sulfuric acid is added and terminates and reacts and carry out OD450 readings, and determines sun Property the corresponding hole number of clone.

Step 4: cloning

Positive colony is subjected to 2-3 time clonings, cell strain is made to stablize.

Step 5: expanding culture, monoclonal antibody is prepared

Monoclonal antibody preparation is carried out with the modes such as in vitro culture or preparation ascites.

The preparation method of the polyclonal antibody is not particularly limited in the present invention, art technology can be passed through Method well known to personnel is prepared.For example, in some embodiments, it is mostly anti-to prepare 14-3-3eta sheep comprising：

Step 1: animal immune and blood sampling

1, first immunisation

14-3-3eta recombinant protein concentration is adjusted to 2mg/ml, the Freund's complete adjuvant of 2ml and 2ml14-3-3eta are recombinated Albumen is inhaled respectively in two 5ml syringes of people, is inserted into three-way pipe, and needle tubing mixing is alternately pushed, and reciprocating operation is until shape Until sticky emulsion.Antigen after emulsifying on a small quantity is taken, clear water surface is dropped to, emulsification antigen indiffusion illustrates to emulsify successfully.

The healthy and strong ram of selection 2, carries out subcutaneous multi-point injection, injection 2ml emulsification antigens (the i.e. 2mg14-3-3eta per bellwether Antigen)

2, booster immunization

A booster immunization was carried out every 2 weeks after first immunisation, carries out 4 booster immunizations altogether

14-3-3eta recombinant protein concentration is adjusted to 1mg/ml, by the Freund's incomplete adjuvant of 2ml and 2ml14-3-3eta weights Histone is emulsified with same procedure.

After first immunisation, due to the stimulation of BCG vaccine and antigen in Freund's complete adjuvant, sheep will appear enlargement of lymph nodes, at this time Lymph node injection is carried out with the antigen of emulsification, the several lymph nodes of injection as more as possible.

3, blood is taken, serum is detached

Last time booster immunization latter week, arteria carotis bloodletting are cut into bulk and are positioned over 37 degree of incubation 1-2 after whole blood agglutination Hour, draw the solids such as serum and 12000rpm centrifugation 5min removal haemocytes.

Step 2: more antivenom purifications

1, prepared by 14-3-3eta antigens immune affinity column

14-3-3eta antigens (are used into antigen zone difference affinity tag, such as immunogene band His labels, affinity purification with immune With antigen zone GST labels) dialysis is to 0.1M NaHCO3, in 0.5M Nacl, PH8.3 buffer solutions；

3ml gels are expanded by every gram of dry powder is solvable, 5mg antigens is coupled per ml gels, weighs the CNBr of appropriate amount Activated sepharose 4B (GE) dry powder is swollen and is constantly washed in bottle,suction, usual 1ml gels with 1mM HCl It need to be washed with 100ml1mM HCl, by the protective agent washes clean in gel dry powder；

It is filtered dry residual liquid on gel with bottle,suction, it is molten that the gel being swollen is added to the 14-3-3eta antigens dialysed It in liquid, and is slowly stirred, the reaction of 2-8 degree overnight, makes antigen pass through covalently bonded and is bonded on gel；

The solution supernatant after reaction is filtered, 0.1M Tris.HCl, the PH8.0 of 10 times of volumes are added to the gel being coupled, With unreacted active group on the free amine group difference gel of Tris；

Gel fills column, with 0.1M Tris.HCl, 0.5M NaCl, pH8.5 and 0.1M HAc, 0.5M NaCl, PH4.0 two Kind buffer solution rinses 10 times of column volumes in turn, repeats to rinse 5 times, and to remove the antigen of Non-covalent binding, so far prepared by affinity column It completes.

2, how anti-affinitive layer purification

14-3-3eta antiserums are diluted with 3 times of mL normal salines, and are resisted after being gradually added into dilution in the case of stirring The isometric saturated ammonium sulfate solution of serum is allowed to generate precipitation；

12000rpm centrifuges the above mixed liquor 10min, abandons supernatant, and the PBS of precipitation 3-5 times of volume of primary antisera is molten Solution is used in combination 0.22um filters to filter；

Balance 14-3-3eta antigen affinity columns with PBS, by the above filtrate loading to affinity column, after loading with PBS into Row washing, until washed out without albumen；

With 0.1M glycine, PH3.0 buffer solutions with upper prop to being eluted, collecting eluent and using 3M in time Tris.HCl, PH8.5 are neutralized；

Eluent is dialysed with the PBS of 20 times of volumes, and it is primary to change within every 4 hours or more dialyzate, changes 2 dialyzates altogether, The 14-3-3eta for obtaining affinity purification is mostly anti-.

In some preferred embodiments of the present invention, the kit further includes the 14-3-3eta eggs as calibration object Bai Chunpin, the calibration object be calibrated product dilution proportionally gradient dilution at various concentration working calibration product solution.

It should be understood that heretofore described specific binding pair member plays the role of bridging, thus also by Referred to as bridging system.In the present invention, the secondary antibody or its binding fragment and a member knot in specific binding pair member It closes, and the donor and another Yuan combination in specific binding pair member.In some embodiments, for example, described second Antibody or its binding fragment are combined with biotin, and the donor is combined with Streptavidin.

In some embodiments of the invention, receptor and first antibody in combination or its combination in the component a A concentration of 10-200 μ g/mL of segment, preferably 20-150 μ g/mL, more preferable 30-100 μ g/mL, most preferably 40-80 μ g/mL；Institute State a concentration of 0.1-8 μ g/mL of secondary antibody or its binding fragment in component b, preferably 0.2-6 μ g/mL, more preferable 0.4-4 μ g/ ML, most preferably 0.6-2 μ g/mL；A concentration of 5-20 μ g/mL of donor described in component c, preferably 8-15 μ g/mL, more preferable 10- 12 μ g/mL, most preferably 10 μ g/mL.

Heretofore described receptor includes olefin(e) compound and metallo-chelate, is non-particulate forms, and in aqueous Jie It is solvable in matter.

In some embodiments, the receptor is the high molecular particle filled with luminophor and lanthanide series.

In some embodiments, the donor is the high molecular particle filled with Photoactive compounds, can under light excitation Generate singlet oxygen.

In certain embodiments of the present invention, the homogeneous immune inspection of the present invention for detecting 14-3-3eta albumen The preparation method of test agent suit includes mainly, reagent preparation I (the step of component a) and reagent preparation II (the step of component b), Wherein, component a include can react with singlet oxygen generation detectable signal receptor and first antibody in combination or Its binding fragment, the first antibody or its binding fragment can be combined with the first epitope specificity of 14-3-3eta albumen；Group Point b includes the secondary antibody that can be combined with the second epitope specificity of 14-3-3eta albumen or its binding fragment, and described second Epitope and the first epitope non-overlapping.

In certain specific embodiments of the invention, the step of reagent preparation I (component a, the coated luminous particle of antibody) Suddenly include：

Step S1, by certain density receptor microspheres solution and first antibody or its binding fragment in being coated with buffer solution It is mixed to get I mixture；

Step S2 is added terminate liquid and terminates and react, then adds confining liquid and closed, through clear into I mixture Buffer solution is added after washing, obtains component a.

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

In some preferred embodiments of the present invention, the confining liquid is glycine solution, the glycine solution A concentration of 10-200mg/ml, preferably 20-150mg/ml, more preferable 50-100mg/ml.

In certain specific embodiments of the invention, reagent preparation II (component b, antibody label biotin) the step of Including：

Step T1 mixes certain density biotin solution and secondary antibody or its binding fragment in marking buffer solution Conjunction obtains Section II mixture；

Step T2 dialyses to Section II mixture, after buffered solution dilution, obtains the component b.

In some preferred embodiments of the present invention, the coating buffer solution and label buffer solution are bicarbonate Sodium buffer solution.

The homogeneous immunological detection reagent box for detecting 14-3-3eta albumen involved by third aspect present invention, packet Containing the homogeneous immunological detection reagent suit for detecting 14-3-3eta albumen described in second aspect of the present invention.It can pass through The homogeneous immunological detection reagent for detecting 14-3-3eta albumen is fitted into kit and is made.

14-3-3eta albumen is homogeneous immune in fourth aspect present invention, detection sample to be tested according to the present invention Detection method, including homogeneous immunological detection reagent as described in respect of the second aspect of the invention is used to be set with to judge to survey in sample to be tested With the presence or absence of the content of 14-3-3eta albumen and/or determining 14-3-3eta albumen.

Similarly, the homogeneous immunologic detection method according to the present invention for detecting 14-3-3eta albumen in sample to be tested, also It whether there is 14- in sample to be tested including using the homogeneous immunological detection reagent box as described in third aspect present invention to judge to survey 3-3eta albumen and/or the content for determining 14-3-3eta albumen.

In certain embodiments of the present invention, inspection is immunized in the homogeneous of 14-3-3eta albumen in the detection sample to be tested Survey method includes：

Third mixture is mixed with component c, obtains the 4th mixture by step R2；

In some embodiments of the invention, the method further includes the making 14-3-3eta albumen before step R1 The step of standard working curve.In some specific embodiments of the present invention, the making 14-3-3eta protein standards work is bent The step of line includes：First according to step R1-R4, the working calibration product of the 14-3-3eta albumen containing various concentration are detected The chemiluminescence signal value of solution fits 14-3-3eta protein standard works then according to the correspondence of concentration and signal value Make curve, obtains the functional relation between the concentration of 14-3-3eta albumen and chemiluminescence signal value.

In some further embodiments of the present invention, in step R4, the letter of chemiluminescence described in detecting step R3 Number intensity, and the content of 14-3-3eta albumen in sample to be tested is determined based on 14-3-3eta protein standards working curve.

In the present invention, measuring samples react under homogeneous phase condition, process without washing, that is, between step R1 and R2 and The step of not detaching and/or wash between step R2 and R3.

In some specific embodiments of the present invention, judge to survey with the presence or absence of 14-3-3eta albumen in sample to be tested, it is described The homogeneous immunologic detection method of 14-3-3eta albumen includes in detection sample to be tested：

(1) it by sample to be tested and component a and combines b and mixes, obtain third mixture；

(2) third mixture is mixed with component c, obtains the 4th mixture；

(3) exciting light of 600-700nm wavelength is used to irradiate the 4th mixture, it being capable of excited donor generation singlet Oxygen, receptor react the transmitting light for generating 520-620nm with the singlet oxygen touched as detectable chemiluminescence signal；

(4) chemiluminescence signal in detecting step (4) whether there is.

In other specific embodiments of the present invention, the content of 14-3-3eta albumen, the detection sample to be tested are determined The homogeneous immunologic detection method of middle 14-3-3eta albumen includes：

Step 1: making 14-3-3eta protein standard working curves.

(1) by as the 14-3-3eta albumen sterling of calibration object with calibration object dilution proportionally gradient dilution at not With the working calibration product solution of concentration；

(2) working calibration product solution and component a are taken and combines b mixing, obtains third mixture；

(3) third mixture is mixed with component c, obtains the 4th mixture；

(4) exciting light of 600-700nm wavelength is used to irradiate the 4th mixture, excited donor generates singlet oxygen, receptor The transmitting light for generating 520-620nm is reacted with the singlet oxygen touched as detectable chemiluminescence signal；

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

(6) repetition step (2)-(5) detect the working calibration product solution of the 14-3-3eta albumen containing various concentration Chemiluminescence signal value (intensity) fits 14-3-3eta protein standard works then according to the correspondence of concentration and signal value Make curve, obtains the functional relation between the concentration of 14-3-3eta albumen and chemiluminescence signal value.

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

(2) third mixture is mixed with component c, obtains the 4th mixture；

(3) exciting light of 600-700nm wavelength is used to irradiate the 4th mixture, excited donor generates singlet oxygen, receptor The transmitting light for generating 520-620nm is reacted with the singlet oxygen touched as detectable chemiluminescence signal；

(4) intensity of the chemiluminescence signal generated in detecting step (4), and worked based on 14-3-3eta protein standards Curve determines the content of 14-3-3eta albumen in sample to be tested.

In fifth aspect present invention, the homogeneous immune detection examination provided by the present invention provided such as second aspect of the present invention Agent is sleeved on the presence and/or the application in content of 14-3-3eta albumen in detection sample to be tested, it can be understood as utilizes this hair The homogeneous immunological detection reagent that bright second aspect is provided is set with whether there is 14-3-3eta albumen to judge to survey in sample to be tested And/or the method for determining the content of 14-3-3eta albumen, wherein the sample to be tested is selected from blood, blood plasma, serum, synovial fluid And tissue, the preferably described sample to be tested are selected from blood, blood plasma and serum, the further preferred sample to be tested is serum.

Similarly, the homogeneous immunological detection reagent box provided by the present invention provided such as third aspect present invention is detecting The presence and/or the application in content of 14-3-3eta albumen in sample to be tested, it can be understood as utilize third aspect present invention institute The homogeneous immunological detection reagent box of offer whether there is 14-3-3eta albumen and/or determining 14-3- to judge to survey in sample to be tested The method of the content of 3eta albumen, wherein the sample to be tested is selected from blood, blood plasma, serum, synovial fluid and tissue, preferably institute It states sample to be tested and is selected from blood, blood plasma and serum, the further preferred sample to be tested is serum.

Similarly, the homogeneous immunologic detection method provided by the present invention provided such as fourth aspect present invention is waited in detection The presence and/or the application in content of 14-3-3eta albumen in sample, it can be understood as carried using second aspect of the present invention The homogeneous immunological detection reagent of confession is set with, and judges to survey using the homogeneous immunologic detection method as described in fourth aspect present invention With the presence or absence of 14-3-3eta albumen and/or the method for the content for determining 14-3-3eta albumen in sample to be tested, wherein described to wait for Sample is selected from blood, blood plasma, serum, synovial fluid and tissue, and the preferably described sample to be tested is selected from blood, blood plasma and serum, into The preferably described sample to be tested of one step is serum.

Similarly, the homogeneous immunologic detection method provided by the present invention provided such as fourth aspect present invention is waited in detection The presence and/or the application in content of 14-3-3eta albumen in sample, it can be understood as carried using third aspect present invention The homogeneous immunological detection reagent box of confession, and waited for using the homogeneous immunologic detection method as described in fourth aspect present invention to judge to survey With the presence or absence of 14-3-3eta albumen and/or the method for the content for determining 14-3-3eta albumen in sample, wherein described to be measured Sample is selected from blood, blood plasma, serum, synovial fluid and tissue, and the preferably described sample to be tested is selected from blood, blood plasma and serum, into one The step preferably sample to be tested is serum.

Reagent set as described in respect of the second aspect of the invention involved by sixth aspect present invention is being prepared for detecting class Application in the kit of rheumatic arthritis, it can be understood as made using reagent set as described in respect of the second aspect of the invention The method being ready for use in the kit of detection rheumatoid arthritis comprising：

Step M1 provides the sample to be tested from main body to be measured；

Step M2, using the homogeneous immunologic detection method described in second aspect of the present invention come judge survey sample to be tested in whether There are the contents of 14-3-3eta albumen and/or determining 14-3-3eta albumen；

Wherein, the sample to be tested is selected from blood, blood plasma, serum, synovial fluid and tissue, and the preferably described sample to be tested is selected from Blood, blood plasma and serum, the further preferred sample to be tested are serum.

In the present invention, and all condition ratios of normal control, the presence of 14-3-3eta albumen is described in the sample to be tested The diagnostic indicant of rheumatoid arthritis in main body to be measured.

In the present invention, and all condition ratios of normal control, the increase of the content of 14-3-3eta albumen in the sample to be tested It is the diagnostic indicant of rheumatoid arthritis in the main body to be measured.

In some preferred embodiments, and all condition ratios of normal control, 14-3-3eta albumen in the sample to be tested Content increase 0.2ng/ml be rheumatoid arthritis in the main body to be measured diagnostic indicant.

In the present invention, compared with rheumatoid arthritis control sample, the phase of 14-3-3eta albumen in the sample to be tested It is the prognostic indicant of rheumatoid arthritis in the main body to be measured to content.

In the present invention, compared with the sample before the treatment from same main body to be measured, 14-3-3eta in the sample to be tested The effect of the relative amount instruction therapeutic scheme of albumen.

III, embodiments

To keep the present invention easier to understand, below in conjunction with embodiment, present invention be described in more detail, these realities Apply example only serve it is illustrative, it is not limited to application range of the invention.If the raw material used in the present invention or component nothing Specified otherwise can be made by commercial sources or conventional method.

In the method for the invention, all reagents after combination or mixing, can carry out according to actual needs mixing and/ Or it incubates.Specifically, the temperature of the incubation can be the arbitrary temp in 25-45 DEG C of temperature range, and incubative time can be Overnight or 10-20min.

Embodiment 1：

1, the preparation of reagent I (the coated receptor of antibody)：

(1) preparation of receptor and donor

Preparation method, composed structure and its content of receptor and donor as the present invention may refer to Chinese patent Embodiment 1 in CN100429197C (patent document is hereby incorporated by reference).

(2) it pre-processes

Pending 0.2mg antibody starting materials are fitted into bag filter (molecular cut off 14KD), bag filter is put into beaker, 100 times of volume 0.05M pH9.6CB elution buffers are added in beaker, are placed on magnetic stirring apparatus, 2-8 DEG C is dialysed. It at least replaces dialyzate 1 time, at least dialyses 4-5 hours every time.The albumen dialysed suction is transferred in clean centrifuge tube, is taken Sample measures albumen concentration.

(3) it is coated with process

3.1 take 2mg receptors that centrifuge tube is added, and 0.05M pH9.6CB cross-linking buffers are added, and centrifuge 7500rpm, 15min, Supernatant is abandoned, 400ul cross-linking buffers are added into centrifuge tube, carries out ultrasonic cleaning particle, centrifuges again.

Particle is resuspended 3.2 addition 200ul cross-linking buffers, and it is 10mg/ml to make particle concentration, adds 0.1mg antibody Centrifuge tube is placed in 37 DEG C after mixing by raw material, 25-40rpm mixing overnights on vertical rotary mixer.

Centrifuge tube is put into 2-8 DEG C of cooling 10min by 3.3, takes the 8mg/ml NaBH of 4 μ L₄Centrifuge tube is added in solution immediately Interior and mixing, room temperature, 25-40rpm reacts 2 hours on vertical rotary mixer.

3.4 are added the 75mg/mL Gly solution mixings of 32 μ L in centrifuge tube, and 25-40rpm is anti-on vertical rotary mixer It answers 1 hour.

(4) it cleans

Centrifuge tube is weighed after trim, and 7500rpm, 15min are centrifuged, and abandons supernatant, and 0.1M pH7.4PBST cleaning bufferings are added Liquid carries out ultrasonic cleaning particle.It is repeated twice, then primary with particle preservation buffer solution for cleaning.

(5) it prepares

4ml Working dilutions are added, it is 50 μ g/mL to make its working concentration, completes the preparation of reagent I, spare.

2, the preparation of reagent II (biotin of antibody label)：

(1) it pre-processes

It takes pending 0.2mg antibody starting materials to be fitted into bag filter (molecular cut off 14KD), bag filter is put into beaker, 100 times of volume 0.1M pH8.0NaHCO are added in beaker₃Elution buffer is placed on magnetic stirring apparatus, and 2-8 DEG C carries out thoroughly Analysis.It at least replaces dialyzate 1 time, at least dialyses 4-5 hours every time.The albumen dialysed suction is transferred to clean centrifuge tube In, albumen concentration is measured by sampling.

(2) labeling process

Take 200 μ L0.1M pH8.0NaHCO₃It marks buffer solution that centrifuge tube is added, 0.1mg antibody starting materials, mixing is added.Again 8 μ L of prepared 5mg/mL biotin solutions, rapid mixing is added.25-40rpm reacted on 2-8 DEG C of vertical rotary mixer Night.

(3) it dialyses

It takes pending biotin labeling solution to be fitted into bag filter (molecular cut off 14KD), bag filter is put into burning 100 times of volume 0.1M pH7.4PBS elution buffers are added in beaker, are placed on magnetic stirring apparatus for cup, and 2-8 DEG C carries out thoroughly Analysis.It at least replaces dialyzate 1 time, at least dialyses 4-5 hours every time.

(4) it prepares

20ml Working dilutions are added, it is 1ug/ml to make its working concentration, completes the preparation of reagent 2, spare.

3, the preparation of 14-3-3eta albumen calibration object：

The preparation of 1.1 calibration object dilutions：HEPES 4.77g, NaCl 1.7g are weighed, purified water 160g mixings are added 30min, using the concentrated hydrochloric acid of 1M and the NaOH solution tune pH value of 1M to 7.4 ± 0.2, continue to add Proclin300 0.1g, BSA 30g、1M MgCl₂ 0.5ml、0.1M MgCl₂0.1ml, addition purified water is weighed surely to 200g, repetition measurement pH after stirring 30min After value, 2-8 DEG C spare.

The preparation of 1.2 calibration objects：With calibration object dilution, proportionally gradient dilution is at working calibration product, then by by working Calibration object calibrates product calibration object antibody concentration, completes the preparation of calibration object.

4, experimental implementation：

After above-mentioned component is assembled into 14-3-3eta protein determination boxes, it is loaded in full-automatic photo-induced chemiluminescence immunoassay point In analyzer, detecting step is set：

1) Tip are loaded to draw in 20 μ L calibration objects to reaction microwell plate；

2) Tip are loaded to draw in 25 μ L reagent Is to reaction microwell plate；

3) Tip are loaded to draw in 25 μ L reagent IIs to reaction microwell plate；

4) horizontal oscillations mixing is incubated 17min for 37 DEG C after 20 seconds；

5) mixed liquor (instrument is mating) that Tip are drawn the donor that 175 μ L include Streptavidin modification is loaded extremely to react In microwell plate；

6) horizontal oscillations mixing is incubated 15min for 37 DEG C after 20 seconds；

7) under the 680nm exciting lights irradiation that instrument generates, donor is induced to activate, and discharge the active oxygen of upper state from Son.The active oxygen ion of the upper state is captured in short distance by luminous particle, to transmit energy to activate in luminous particle Luminophor.After number microsecond, the luminophor in receptor will release the high level feux rouges of 612nm, use single photon counting Device measures these high level photons；

8) according to the signal value of calibration object, standard curve is fitted according to five parameter fitness methods, obtains signal value and 14- Equation between 3-3eta albumen concentration；

9) equally according still further to step 1) -7) detection sample to be tested, 14- in sample to be tested is calculated by the equation in 8) 3-3eta albumen concentration.

5, experimental result

5.1 examine the range of linearity of work calibration object

The range of linearity for examining work calibration object, the results are shown in Table 1.

Table 1

As it can be seen from table 1 standard curve degree fit equation R2 ＞ 0.99.Have in the measurement range of 0.2-20ng/ml Have extraordinary linear.

5.2 sensitivity

It is repeated 20 times measured value with low concentration sample, the results are shown in Table 2.From table 2 it can be seen that its testing result precision is 7.42%, so Functional Sensitivity is up to 0.2ng/ml.When containing 0.2ng/ml detection objects in sample, area just can be generated Not in the signal value of normal population (zero-dose sample), has high sensitivity.

Table 2

5.3 evaluation result

46 rheumatoid case group samples and 50 Normal group samples are evaluated using prepared kit, are as a result seen Table 3, Fig. 1, meanwhile, other arthritis samples are detected, 4 and Fig. 1 are the results are shown in Table.

Table 3

Table 4

Conclusion：14-3-3 η albumen concentration is below 0.2ng/ml in health examination group sample, has in rheumatoid arthritis group 19 sample detection 14-3-3 η albumen concentration are higher than 0.2ng/ml, show 14-3-3 η subtype proteins Gao Shui in arthritic Flat expression, is detected significantly to increase in RA patients serums.Meanwhile when detecting other arthritis samples, 14-3-3 η albumen is dense Degree is below 0.2ng/ml, therefore, it is considered that 14-3-3 η Protein Detections results are related to clinical diagnosis rheumatoid, prompts to detect this egg The white diagnosis that can be improved to rheumatoid arthritis, helps clinic to make a definite diagnosis RA.

It should be noted that embodiment described above is only used for explaining the present invention, do not constitute to any of the present invention Limitation.By referring to exemplary embodiments, invention has been described, it should be appreciated that word used in it is descriptive With explanatory vocabulary, rather than limited vocabulary.The present invention can be made within the scope of the claims by regulation Modification, and the present invention is revised in without departing substantially from scope and spirit of the present invention.Although the present invention described in it relates to And specific method, material and embodiment, it is not intended that the present invention is limited to particular case disclosed in it, on the contrary, this hair It is bright to can be extended to other all methods and applications with the same function.

Sequence table

<110>Beijing Kemei Biological Technology Co., Ltd.

<120>Detect homogeneous immunological detection reagent box and its application of 14-3-3 eta albumen

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 246

<212> PRT

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

<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. detection 14-3-3eta albumen or its segment or the 14-3-3eta albumen or its segment are formed at least one antibody The existing of immune complex preparing for evaluating subject's through the following steps using homogeneous immunologic detection method Purposes in the reagent of rheumatoid arthritis：A) it provides from the to be measured of subject of the suspection with rheumatoid arthritis Sample；B) detect in the sample to be tested 14-3-3eta albumen or its segment or the 14-3-3eta albumen or its segment with extremely The immune complex that a kind of few antibody is formed；The presence of the wherein described 14-3-3eta albumen or its segment or immune complex refers to Show the rheumatoid arthritis of subject；Wherein, the 14-3-3eta albumen or its segment include at least one 14-3- 3eta epitopes, the sample to be tested are selected from blood, blood plasma, serum, synovial fluid and tissue.

2. purposes according to claim 1, which is characterized in that the step further include measure 14-3-3eta albumen or its The content for the immune complex that segment or the 14-3-3eta albumen or its segment are formed at least one antibody.

3. purposes according to claim 2, which is characterized in that determined based on 14-3-3eta protein standards working curve The content of 14-3-3eta albumen in sample to be tested.

4. purposes according to claim 2, which is characterized in that the step further includes by measured 14-3-3eta eggs The content for the immune complex that white or its segment or the 14-3-3eta albumen or its segment are formed at least one antibody, with 14-3- described in sample before normal reference sample, rheumatoid arthritis control sample or treatment from same subject The immune complex that 3eta albumen or its segment or the 14-3-3eta albumen or its segment are formed at least one antibody contains Amount is compared.

5. purposes according to claim 1, which is characterized in that the step include by the sample with comprising can be with At least one specific epitopes of 14-3-3eta albumen or its segment specifically bind the antibody to form immune complex.

6. according to the purposes described in any one of claim 1-5, which is characterized in that the antibody includes can be with 14-3- First antibody that first epitope specificity of 3eta albumen combines and can be with the second epitope specificity of 14-3-3eta albumen In conjunction with secondary antibody, wherein second epitope and the first epitope non-overlapping.

7. purposes according to claim 6, which is characterized in that the first antibody is combined with receptor, and the receptor can It is reacted with singlet oxygen and generates detectable chemiluminescence signal.

8. purposes according to claim 7, which is characterized in that the receptor includes olefin(e) compound and metallo-chelate, It is non-particulate forms, and solvable in water-bearing media；And/or the receptor is filled with luminophor and lanthanide series High molecular particle.

9. purposes according to claim 6, which is characterized in that the first antibody and secondary antibody are separately selected from Monoclonal antibody and/or polyclonal antibody, preferably monoclonal antibody.

10. purposes according to claim 1, which is characterized in that the amino acid sequence of the 14-3-3eta albumen or its segment Row are as shown in SEQUENCE NO.1.

11. purposes according to claim 10, which is characterized in that it is 14-3-3eta that the epitope, which is selected from amino acid fragment, The relative specificity segment of the sequence of albumen：1-6aa、27-38aa、71-83_aa、112-119_aaAnd 141-154aa.

12. a kind of homogeneous immunological detection reagent suit for detecting 14-3-3eta albumen comprising：

Component a, it includes the receptor for generating detectable signal and first antibody in combination can be reacted with singlet oxygen Or its binding fragment, the first antibody or its binding fragment can be combined with the first epitope specificity of 14-3-3eta albumen；

Component b, it includes the secondary antibody that can be combined with the second epitope specificity of 14-3-3eta albumen or its bonding pads Section, second epitope and the first epitope non-overlapping；

13. reagent set according to claim 12, which is characterized in that the amino acid sequence of the 14-3-3eta albumen As shown in SEQUENCE NO.1.

14. reagent set according to claim 13, which is characterized in that second epitope and first epitope difference Independently selected from the relative specificity segment for the sequence that amino acid fragment is 14-3-3eta albumen：1-6aa、27-38aa、71- 83aa, 112-119aa and 141-154aa.

15. reagent set according to claim 12, which is characterized in that the first antibody and secondary antibody difference are only On the spot it is selected from monoclonal antibody and/or polyclonal antibody, preferably monoclonal antibody.

16. according to the reagent set described in any one of claim 12-15, which is characterized in that the reagent set also wraps Include the 14-3-3eta albumen sterlings as calibration object, the calibration object be calibrated product dilution proportionally gradient dilution at not With the working calibration product solution of concentration.

17. according to the reagent set described in any one of claim 12-16, which is characterized in that the secondary antibody or its Binding fragment is combined with a member in specific binding pair member, and the donor with it is another in specific binding pair member A member combines.

18. reagent set according to claim 17, which is characterized in that the secondary antibody or its binding fragment and biology Element combines, and the donor is combined with Streptavidin.

19. according to the reagent set described in any one of claim 12-18, which is characterized in that in the component a receptor with And a concentration of 10-200 μ g/mL of first antibody or its binding fragment in combination, preferably 20-150 μ g/mL, more preferable 30- 100 μ g/mL, most preferably 40-80 μ g/mL；And/or a concentration of 0.1-8 μ of secondary antibody or its binding fragment in the component b G/mL, preferably 0.2-6 μ g/mL, more preferable 0.4-4 μ g/mL, most preferably 0.6-2 μ g/mL；And/or donor described in component c A concentration of 5-20 μ g/mL, preferably 8-15 μ g/mL, more preferable 10-12 μ g/mL.

20. according to the reagent set described in any one of claim 12-19, which is characterized in that the receptor includes alkene Compound and metallo-chelate are non-particulate forms, and solvable in water-bearing media；And/or the receptor is filled with hair The high molecular particle of optical compounds and lanthanide series.

21. according to the reagent set described in any one of claim 12-19, which is characterized in that the donor is photoactivation Or chemical activation sensitizer, be non-particulate forms, and it is solvable in water-bearing media；And/or the donor be filled with The high molecular particle of Photoactive compounds can generate singlet oxygen under light excitation.

22. a kind of homogeneous immunological detection reagent box for detecting 14-3-3eta albumen, it includes appoint in claim 12-21 Homogeneous immunological detection reagent suit described in meaning one.

23. the homogeneous immunologic detection method of 14-3-3eta albumen in a kind of detection sample to be tested comprising using such as claim Homogeneous immunological detection reagent described in any one of 12-21 is set with or using homogeneous immune detection as claimed in claim 20 Kit judges to survey in sample to be tested with the presence or absence of 14-3-3eta albumen and/or determines the content of 14-3-3eta albumen.

24. according to the method for claim 23, which is characterized in that this method includes：

Third mixture is mixed with component c, obtains the 4th mixture by step R2；

Step R3 makes energy or reactive compound be contacted with the 4th mixture, and the donor is excited to generate singlet oxygen, The receptor can be reacted with the singlet oxygen received generates detectable chemiluminescence signal；

Step R4, the presence of chemiluminescence signal and/or intensity described in detecting step R3 are to judge to survey in sample to be tested It is no that there are the contents of 14-3-3eta albumen and/or determining 14-3-3eta albumen.

25. according to the method for claim 24, which is characterized in that the method further includes the making before step R1 The step of 14-3-3eta protein standard working curves.

26. according to the method for claim 25, which is characterized in that in step R4, chemistry hair described in detecting step R3 The intensity of optical signal, and containing for 14-3-3eta albumen in sample to be tested is determined based on 14-3-3eta protein standards working curve Amount.

27. according to the method described in any one of claim 24-26, which is characterized in that between step R1 and R2 and walk Rapid the step of not detaching and/or wash between R2 and R3.

28. according to the method for claim 24, which is characterized in that in step R3, use the excitation of 600-700nm wavelength Light irradiates the 4th mixture, and excited donor generates singlet oxygen, and receptor is reacted with the singlet oxygen touched generates 520-620nm Transmitting light.

29. a kind of homogeneous immunological detection reagent suit or such as claim 22 as described in any one of claim 12-21 The homogeneous immunological detection reagent box or the method as described in any one of claim 23-28 are in detecting sample to be tested The presence and/or the application in content of 14-3-3eta albumen, wherein the sample to be tested is selected from blood, blood plasma, serum, synovial membrane Liquid and tissue.

30. a kind of reagent set as described in any one of claim 12-21 is being prepared for detecting rheumatoid joint Application in scorching kit comprising：

Step M1 provides the sample to be tested from main body to be measured；

Step M2 judges to survey in sample to be tested with the presence or absence of the content of 14-3-3eta albumen and/or determining 14-3-3eta albumen；

Step M3, by it with normal reference sample, rheumatoid arthritis control sample or the treatment from same main body to be measured The comparision contents of 14-3-3eta albumen described in preceding sample；

31. application according to claim 30, which is characterized in that with all condition ratios of normal control, the sample to be tested The presence of middle 14-3-3eta albumen is the diagnostic indicant of rheumatoid arthritis in the main body to be measured.

32. application according to claim 30, which is characterized in that with all condition ratios of normal control, the sample to be tested The increase of the content of middle 14-3-3eta albumen is the diagnostic indicant of rheumatoid arthritis in the main body to be measured.

33. application according to claim 32, which is characterized in that with all condition ratios of normal control, the sample to be tested The content of middle 14-3-3eta albumen increases the diagnostic instruction that 0.2ng/ml is rheumatoid arthritis in the main body to be measured Object.

34. application according to claim 30, which is characterized in that described compared with rheumatoid arthritis control sample The relative amount of 14-3-3eta albumen is the prognostic instruction of rheumatoid arthritis in the main body to be measured in sample to be tested Object.

35. application according to claim 30, which is characterized in that with the sample phase before the treatment from same main body to be measured Than the effect of the relative amount instruction therapeutic scheme of 14-3-3eta albumen in the sample to be tested.

36. according to the application described in any one of claim 30-35, which is characterized in that in step M2, using such as right It is required that the method described in any one of 22-26 whether there is 14-3-3eta albumen and/or determination to judge to survey in sample to be tested The content of 14-3-3eta albumen.