CN108445216A

CN108445216A - A kind of anti-Miao Le Shi pipes hormone determination kit of people and the preparation method and application thereof

Info

Publication number: CN108445216A
Application number: CN201810143236.9A
Authority: CN
Inventors: 徐静心; 史舟芳; 强中华; 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: 2018-02-11
Filing date: 2018-02-11
Publication date: 2018-08-24
Anticipated expiration: 2038-02-11
Also published as: CN108445216B

Abstract

The invention discloses a kind of preparation methods of the anti-homogeneous detection kits of Miao Le Shi pipes hormone AMH of people, including：The component comprising receptor and the first combining unit in combination is prepared, is reacted with singlet oxygen by physical efficiency and generates detectable signal, the first combining unit can be in conjunction with the first epitope of AMH；It includes the first marker and second the second component of combining unit in combination to prepare, and the second combining unit can be the epitope of the epitope of the different binding characteristics of AMH or the identical combination characteristic of different location in conjunction with the second epitope of AMH, the second epitope and the first epitope；The third component of the specific junction mixture comprising donor and first marker in combination is prepared, donor can generate singlet oxygen in excited state.The kit prepared through this method being capable of efficient detection AMH.

Description

A kind of anti-Miao Le Shi pipes hormone determination kit of people and the preparation method and application thereof

Technical field

The invention belongs to biomolecule detection technical fields, are related to a kind of homogeneous immunological detection reagent of no matrix effect Box, and in particular to a kind of anti-human Miao Le Shi pipes hormone homogeneous luminescent immunity detection reagent and its preparation and application.

Background technology

Inhibiting substances of the anti-Miao Le Shi pipes hormone (anti-mullerian hormone, AMH) as Miao's Le Shi pipes belong to In the dimer glycoprotein of transforming growth actor βfamily.In sexual glands growth course, AMH plays an important role, and is true Protect man, woman organ is developed and one of the important substance of gonad function.In males, AMH is mainly produced by interstitial glands It is raw, it starts from embry ogenesis and runs through life always；In male fetus growth course, AMH causes Miao's Le Shi pipes to degenerate and formed Male genetic pipeline.The AMH contents of male neonate phase are higher, after birth the several months still rise, slowly decline after 2 years old, puberty Decline is most apparent, until adult minimizes.In women body, embryo's early stage causes Miao's Le Shi pipes to develop into because that cannot synthesize AMH The woman organs such as uterus, bilateral salpingo.When being developed to 36 weeks, gonad granulocyte generates AMH, by ovarian follicle before sinus after birth It is generated with little Dou granular cells.Internal AMH levels are related in forward direction to the age from baby, are gradually increasing with age, Puberty (about 15.8 years old) AMH peaks, until keeping stablizing before 25 years old.But AMH levels and age are in negative from after 25 years old It closes, is gradually reduced with age, until menopause drops to very low level and can not detect.

Currently, serum AMH quantitative analysis methods include enzyme-linked immunosorbent assay, enzymatic lighting immunity analysis and electrochemistry Luminescence immunoassay.Enzyme-linked immunosorbent assay is using alkaline phosphatase as marker, and 96 microwell plates are as solid phase carrier, packet Captured antibody.Enzymatic electrochemiluminescent immunoassay method analytical model is similar to enzyme-linked immunosorbent assay, and difference is to use luminous substrate, By the quantitative analysis for measuring optical signal.Electrochemical luminescence using tris (bipyridine) ruthenium be used as marker, with the use of tripropyl amine (TPA) with Electrode induced luminescence.

Above-mentioned three kinds of AMH immunoassay methods, it is that they belong to heterogeneous immunoassay to have common feature (Heterogeneous immunoassay).Heterogeneous immunoassay refers to being needed after antibody-antigene reacts, before detection signal Reaction, free state labelled antibody or other components are not participated in removal to be separated.Detach binding label and free label The common method of object is solid phase adsorption isolation technics.For AMH quantitative analyses, capture antibody is connect with solid phase carrier, Antigen to be checked is combined with labelled antibody and capture antibody respectively, in solid phase material surface formation double-antibody sandwich compound (in conjunction with Marker is located at solid phase surface), and free label antibody that is excessive, having neither part nor lot in antigen-antibody reaction is distributed in liquid phase.It goes Wash except liquid solution and repeatedly solid phase (particle).

There are two important links for solid phase adsorption separation method：Coating and washing.Such heterogeneous reaction pattern because coating and The presence for washing link, causes very big defect to labelling immunoassay, is mainly manifested in following two aspects：

1. in coating process, no matter physical absorption or be connected chemically, be coated in solid phase material surface antibody molecule its Conformation is different from the antibody molecule in liquid phase, with antigen molecule binding ability because space steric effect will be by a fixed limit System；No matter use microballoon or microwell plate as solid phase material, due to being coated with limited area, capture antibody molecule cannot be maximum Limit meets the needs of a large amount of determined antigens, thus will influence detection range.

2. " board-washing " or " washing ball " is the important link in non-homogeneous immunoassay and repeatedly occurs.Washing process increases The complexity of program is detected, detection time is increased and brings obstacle to realizing to automate.Further, since washing process is especially EIA enzyme immunoassay, it is difficult to realize and standardize, washing effect is different between each instrument connection, influences detection to a certain extent Precision occurs between criticizing, batch interior difference.

Solid phase adsorption separation method is also inevitable by washing separation institute band since each washing process needs elapsed time Come " error ".Washing error is the important sources of non-homogeneous immunoassay error, the precision of impact analysis method, to shadow Ring accuracy and the analysis susceptibility of analysis method.

For this reason, the present inventor is studied, be intended to provide it is a kind of have higher precision, Accuracy and sensitivity, operating method is easy, and can quantify the detection reagent for detecting the anti-Miao Le Shi pipes hormone AMH levels of people Box and detection method.

Invention content

In view of the deficiencies of the prior art, the present invention is based on homogeneous immunization method, the anti-Miao Le Shi pipes of quantitatively detection serum are prepared Swash AMH kits, establishes the anti-Miao Le Shi of double-antibody sandwich serum and manage and swash AMH quantitative analysis methods, optimized reagent preparation box, It is mainly manifested in：The preferably a pair of antibody that AMH is swashed for anti-Miao Le Shi pipes marks biotin and receptor respectively；Analysis system passes through Optimization (antibody dosage and buffer solution etc.) is crossed, analytical performance meets professional standard and clinical and R＆D units requirement.This production Product are mainly characterized by using homogeneous light immunoassay, and entire detection process not will produce additional useless without separating, washing process Liquid exempts washing error, has higher precision, accuracy and sensitivity.In addition, kit and the detection of the present invention Side has preferable enlarge-effect and analytical precision is higher, has very high sensitivity for analysis to assign the present invention.

It is sharp (anti-mullerian hormone) that the problem to be solved in the present invention is to provide a kind of anti-Miao Le Shi pipes of serum Homogeneous luminescent immunoassay kit and its preparation and application, to solve the existing cumbersome washing of heterogeneous immune reagent kit Step；Meanwhile overcoming radiommunoassay environmental pollution, the short defect of shelf life overcomes EIA enzyme immunoassay poor repeatability, easily " hook effect " defect occurs.The present invention uses luminescence immunoassay, has higher susceptibility and precision.

More specifically, first aspect present invention provides a kind of system of the anti-sharp homogeneous detection kit of Miao Le Shi pipes of people Preparation Method, the method includes：

The first component is prepared, first component includes receptor and the first combining unit in combination, the receptor Can be reacted with singlet oxygen and generate detectable signal, first combining unit can swash in conjunction with people's anti-Miao Le Shi pipe the One epitope；

The second component is prepared, second component includes the first marker and the second combining unit in combination, institute Stating the second combining unit can be with first epitope in conjunction with the second sharp epitope of the anti-Miao Le Shi pipes of people, second epitope The epitope of the epitope of the sharp different binding characteristics of the anti-Miao Le Shi pipes of people or the identical combination characteristic of different location；

Third component is prepared, the third component includes the special of donor and first marker in combination Conjugate, the donor can generate singlet oxygen in excited state.

In some embodiments of the present invention, first combining unit and second combining unit be independently Selected from the monoclonal antibody swashed with the anti-Miao Le Shi pipes of the people with binding specificity, antibody binding fragment, artificial antibody, repair The antibody of decorations, two or more Monoclonal Antibody Mixtures and polyclonal antibody, are preferably selected from polyclonal antibody and/or list Clonal antibody.

In some embodiments of the present invention, the marker is biotin, the specific bond of the marker Object is Streptavidin.

In some embodiments of the present invention, the receptor includes olefin(e) compound and metallo-chelate, is non-grain Sub- form, and it is solvable in water-bearing media；Or, the receptor is that the macromolecule filled with luminophor and lanthanide series is micro- Grain；And/or

The donor is photoactivation or chemical activation sensitizer, is non-particulate forms, and can in water-bearing media It is molten；And/or the donor is the high molecular particle filled with Photoactive compounds, and singlet oxygen can be generated under light excitation.

In some embodiments of the present invention, the preparation method further includes：

First chamber is prepared, the first chamber includes first component and the first buffer solution；

Second chamber is prepared, the second chamber includes second component and the second buffer solution；

Third composition is prepared, the third composition includes the third component and third buffer solution；

Preferably, first buffer solution, second buffer solution and the third buffer solution are separately selected from pH 8.0,0.1M Tris-HCl solution.

In some embodiments of the present invention, the preparation method of the first chamber includes：

The receptor is diluted to 4~6mg/mL using carbonate buffer solution, obtains receptor solution by step S1；

First anti-human anti-Miao Le Shi pipes are added in the receptor solution and swash antibody or its binding fragment by step S2, static The BSA solution for being diluted to 8~12mg/mL with carbonate buffer solution is added afterwards, is obtained and is managed with described first anti-human anti-Miao Le Shi Swash the receptor solution that antibody or its binding fragment combine；

Step S3 is detached and is swashed the receptor solution that antibody or its binding fragment are combined with described first anti-human anti-Miao Le Shi pipes In swash the receptor that antibody or its binding fragment are combined with described first anti-human anti-Miao Le Shi pipe, and the first buffering is added Solution obtains the first chamber.

In some embodiments of the present invention, the preparation method of the second chamber includes：

Second anti-human anti-Miao Le Shi pipes are swashed antibody or its binding fragment are placed in bag filter and are delayed using label by step T1 Fliud flushing is dialysed, and biotin solution is added after dialysis, and fill into elution buffer, static, and acquisition is combined with biotin Second anti-human anti-Miao Le Shi pipe swash antibody or its binding fragment；

Combined with biotin second anti-human anti-Miao Le Shi pipes are swashed antibody or its binding fragment are placed in dialysis by step T2 It is dialysed using elution buffer in bag, the second buffer solution is added after dialysis, obtains the second chamber.

Second aspect of the present invention provides a kind of kit sharp for homogeneously detecting the anti-Miao Le Shi pipes of people, is basis What the preparation method that the anti-Miao Le Shi pipes of people of first aspect present invention swash homogeneous detection kit was prepared.

In some embodiments of the present invention, the concentration of first component in the first chamber is selected from：10 ~300 μ g/mL, preferably 20~200 μ g/mL, more preferable 30~80 μ g/mL, and/or

The concentration of second component in the second chamber is selected from：0.5~15 μ g/mL, preferably 1~8 μ g/mL, More preferable 2~6 μ g/mL；And/or

The concentration of the third component in the third composition is selected from：10~300 μ g/mL, preferably 20~200 μ g/ ML, more preferable 30~80 μ g/mL.

In some embodiments of the present invention, the kit includes that the anti-Miao Le Shi pipes of people swash calibration object solution, described Calibration object solution is anti-Miao of people of concentration 0ng/mL, 0.16ng/mL, 0.6ng/mL, 4ng/mL, 10ng/mL, 24ng/mL series Le Shi pipes swash solution.

Third aspect present invention provides a kind of anti-sharp method homogeneously detected of Miao Le Shi pipes of people comprising uses basis Described in second aspect of the present invention chemiluminescence detection is carried out for homogeneously detecting the sharp kit of the anti-Miao Le Shi pipes of people.

In some embodiments of the present invention, step R1 mixes sample to be tested and first chamber and second chamber It closes, obtains the first mixture；

First mixture is mixed with third composition, obtains the second mixture by step R2；

Step R3 makes energy or reactive compound be contacted with second 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；

The presence of the chemiluminescence signal obtained in step R4, detecting step R3 and/or intensity, it is to be measured to judge to survey Swash and/or determine the sharp content of the anti-Miao Le Shi pipes of people with the presence or absence of the anti-Miao Le Shi pipes of people in sample.

In some embodiments of the present invention, the method further includes：It is managed using the anti-Miao Le Shi of the people and swashs calibration object Solution makes the step of anti-Miao Le Shi pipes of chemiluminescence signal-people swash the standard curve of concentration；The standard curve is for determining The sharp content of the anti-Miao Le Shi pipes of people in sample to be tested.

In some embodiments of the present invention, using described in the exciting light irradiation that wavelength is 600-700nm in step R3 Second mixture excites the donor to generate singlet oxygen, and the receptor is reacted with the singlet oxygen touched generates wave The transmitting light of a length of 520-620nm；In step R4, the signal for detecting the transmitting light exists and/or intensity, to judge to survey Swash and/or determine the sharp content of the anti-Miao Le Shi pipes of people with the presence or absence of the anti-Miao Le Shi pipes of people in sample to be tested.

In some embodiments of the present invention, described method includes following steps：

A. 10-30 μ l samples to be checked are added in reacting hole；

B. second chamber described in first chamber and 10-30 μ l described in 10-30 μ l is sequentially added in reacting hole；

C.35-45 DEG C incubation 10-30 minutes；

D. the third composition 100-250 μ l are added into reacting hole；

E.35-45 DEG C incubation 5-20 minutes；

F. utilize wavelength be 680nm laser irradiation reacting hole, excited donor generate singlet oxygen, receptor with touch Singlet oxygen reaction produce 612nm transmitting light；

G. the photon amount for detecting the transmitting light per hole calculates the sharp concentration of the anti-Miao Le Shi pipes of people according to standard curve.

Fourth aspect present invention provides a kind of method according to a third aspect of the present invention in quantitative detection sample to be tested Application in the anti-Miao Le Shi pipes of people sharp presence or absence and/or content.

Fifth aspect present invention provide according to method of the first aspect of the present invention prepare for assess ovarian reserve, Diagnose children development disease, assessment infertility, diagnosis of polycystic ovary syndrome or predict the reagent of menopause, kit, Application in detection device or detecting system or purposes.

Sixth aspect present invention provides according to method of the first aspect of the present invention, the kit of second aspect of the present invention Or application or purposes of the method for third aspect present invention in predicting menopause.

The technique effect that kit has in the present invention：Good linearity.Sensitivity is good.Accuracy is high.

Description of the drawings

Below in conjunction with attached drawing, the present invention will be described in detail.

Fig. 1 is kit testing principle schematic diagram of the present invention,

Wherein：

1 indicates the first anti-coated receptor of AMH antibody (FG-Ab1)；

2 indicate the second anti-AMH antibody (Bio-Ab2) of biotin labeling；

3 indicate the AMH in the AMH calibration objects of the AMH or known concentration in sample to be checked；

4 indicate the donor (SA-GG) for being coated with streptavidin.

Fig. 2 is the schematic diagram of kit of the present invention and Roche kit measurement results contrast.

Specific implementation mode

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

In the case where providing numberical range, it should be understood that the upper and lower bound of the range and the prescribed limit In any other regulation or between two parties each of between numerical value numerical value is encompassed by the present invention between two parties.These are small range of Upper and lower bound can independently be included in smaller range, and be also covered by within the present invention, obey any in prescribed limit The limit clearly excluded.In the case where defined range includes one or two limit, appointing for the limit that those include is excluded One or both range is also included in the present invention.

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

I. term

English corresponding to term " homogeneous " of the present invention is defined as " homogeneous ", and referring to need not be to special Property combine a member between pairing member in the compound and remaining free specific binding pair member that are bound to each other to form Or another member detaches and can be detected, for example, its can refer to need not antigen antibody complex to combination and remaining trip Carrying out separation from antigen or antibody can be detected.

Term " sample to be tested " of the present invention refers to may be containing a kind of mixture of analyte, analyte packet Include but be not limited to protein, hormone, antibody or antigen.The typical case that can be used in method disclosed by the invention or product waits for Test sample originally includes body fluid, as blood, blood derivatives, serum, blood plasma, urine, celiolymph, saliva, synovia and pulmonary emphysema are accumulated Liquid etc..Sample to be tested can be using it is preceding it is as needed using dilution or buffer solution to may be containing analyte Sample be diluted after solution.For example, in order to avoid HOOK effects, it can use Sample dilution will before upper machine testing Analyte is detected on detecting instrument again after being diluted, solution that at this time may be after the dilution containing analyte Collectively termed as sample to be tested.

Term " antibody " of the present invention and " immunoglobulin " are used with most wide meaning, including any isotype is anti- Body or immunoglobulin retain the antibody fragment of the specific binding to antigen, including but not limited to Fab, Fv, scFv and Fd Segment, chimeric antibody, humanized antibody, camelised antibodies, single-chain antibody, bispecific antibody and the antigen knot comprising antibody Close the fusion protein of part and non-antibody protein.In the case of any need, antibody can be all further with other parts As the specific junction mixture of marker and marker, such as biotin or Streptavidin etc. are conjugated.

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, or through the aforementioned of protokaryon or eukaryotic expression Immunoglobulin or through it is artificial reconstructed but retain or enhancing Proantigen binding characteristic.

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

Term " in conjunction with " of the present invention refers between different molecular that spatially duration adjoins by identification, including But the molecule, molecular complex, molecular complex etc. for being not limited to be formed bigger, specific recognition may include electrostatic, it is hydrophobic, from The interactions such as son and/or hydrogen bond, including but not limited to such as salt bridge and caused two of water bridge interaction are intermolecular straight Joint is connect, for example, antigen and antibody combine and forms compound, also can refer to will be different by the specific reaction of chemical group Molecular link collectively forms the molecule or compound of bigger, such as by active group by biotin and antibody covalent cross-linking.

Term " combining unit " of the present invention refers to the molecule that can be specifically bound with " analyte " or compound Object, in some cases, after the entirety or part of it of combining unit are combined with tested material, another part still keeps original Biological or chemical activity.

Term " component " of the present invention refers to the behaviour such as individually be purified, react, test, characterizing, mixing, preparing The minimum chemical unit or cell complex of work, in some cases, component refer to molecule, big point of polymer, chemical labeling Son.

Term " specific binding " of the present invention refers to that mutual discrimination between different molecular and selective binding are anti- Answer, said from stereochemical structure angle be exactly conformation between corresponding reactant correspondence, chemically react and say it is to have in angle There is the reaction of the combination between the different groups of specific reactivity.

Term " marker " of the present invention and " specific junction mixture of marker " refer to so a pair of of molecule, their energy Enough mutually specific bindings, for example, enzyme-substrate, Ag-Ab, ligand-receptor.One specific specific binding pair at The example of member couple is biotin-Streptavidin system, wherein " biotin " is widely present in animal vegetable tissue, molecule It is upper there are two cyclic structure, respectively imidazolone ring and thiphene ring, wherein imidazolone ring be combined with Streptavidin it is main Position.The biotin of activation can be coupled under the mediation of protein cross agent with known almost all creatures macromolecular, Including protein, nucleic acid, polysaccharide and lipid etc.；And " Streptavidin " is a kind of protein secreted by streptomycete, molecular weight For 65kD." Streptavidin " molecule is made of 4 identical peptide chains, wherein every peptide chain can combine a biotin.Cause This each antigen or antibody can be coupled multiple biotin molecules simultaneously, and sensitivity for analysis is improved to generate " tentacle effect ".

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 aromatics Object) or chemical activation (such as enzyme, metal salt).

In particular embodiments of the invention, the donor is photosensitizer, and the photosensitizer can be known in the art Photosensitizer, it is preferably stable with respect to light and the compound with singlet oxygen effecting reaction, non-limiting example do not include example Methylene blue, rose-red, porphyrin, phthalein as disclosed in United States Patent (USP) US5709994 (patent document is hereby incorporated by reference) The derivative with 1-50 replacing group of the compounds such as cyanines and chlorophyll and these compounds, the substituent group are used In making these compounds with more lipophilicity or with more hydrophily, and/or as being connected to specific binding pair member Linking group.The example of other photosensitizers well known by persons skilled in the art can also be used in the present invention, such as the U.S. Content described in patent US6406913, the patent document are hereby expressly incorporated by reference.

In other specific embodiments of the invention, the donor is other sensitizers of chemical activation, non-limiting Example be certain compounds, their catalyzing hydrogen peroxides are converted into singlet oxygen and water.The example packet of some other donor It includes：Isosorbide-5-Nitrae-dicarboxyethyl-Isosorbide-5-Nitrae-naphthalene endoperoxides object, 9,10- diphenylanthrancene -9,10- endoperoxides objects etc., heat these Compound or these compounds, which directly absorb light, can discharge singlet oxygen.

Term " receptor " of the present invention is to refer to react the substance that can generate detectable signal with singlet oxygen. Donor is by energy or reactive compound induced activation and the singlet oxygen for discharging upper state, the singlet oxygen quilt of the upper state The receptor of short distance is captured, to transmit energy to activate the receptor.

In some specific embodiments of the present invention, the receptor is such substance：It undergoes the change with singlet oxygen Reaction is learned to form unstable metastable state intermediate, the metastable state intermediate can be decomposed, subsequently or simultaneously be shone.This The exemplary of substances includes but not limited to a bit：Enol ether, enamine, 9- alkylidenes xanthans, 9- alkylidene-N- alkyl Acridane, virtue Vinethene, bicyclic ethylene oxide, thioxene, armaticity imidazoles or lucigenin.

In other specific embodiments of the present invention, the receptor can be reacted with singlet oxygen so that formed can be with Resolve into the hydroperoxides of ketone or carboxylic acid derivates or the olefines of dioxy cyclobutane；It can be decomposed by the effect of light Stabilization dioxy cyclobutane；It can be reacted with singlet oxygen to form the acetylene class of diones；Can be formed azo-compound or The hydrazone class or hydrazides of azo carbonyls, such as luminol；With the aromatic compounds that can form endoperoxides species. Specific, the non-limiting examples for the receptor that can be utilized according to the disclosure and claimed invention are recorded in United States Patent (USP) Number US5340716 (patent document is hereby incorporated by reference).

In other specific embodiments of the invention, the receptor includes olefin(e) compound and metallo-chelate, right and wrong Particlized and dissolve in water-bearing media, the preparation method of this receptor can be found in patent PCT/US2010/025433 (should Patent document is hereby incorporated by reference).

In the present invention, the donor can be coated on matrix to be formed filled with sensitized by functional group The high molecular particle for closing object can generate singlet oxygen under light excitation, and donor is referred to as photosensitive microballoon or photosensitive at this time Particle, including the solution of this photosensitive microballoon or photosensitive particulate is properly termed as photosensitive liquid or general liquid；And/or the receptor can To be to be coated on matrix to form the high molecular particle filled with luminophor and lanthanide series by functional group, this When be properly termed as shine microballoon or luminous particle.In this application, system is based on being coated on the luminescent substance of matrix surface through light Excitation and energy transmission induced luminescence signal, energy transmission is combined dependent on Ag-Ab leads to photosensitive microballoon and luminous microballoon It is close to each other and realize.There is no need to separation processes.The diameter smaller of nanoparticle, suspendability is stronger, uses simultaneously Three-level amplifies luminescent system, thus has higher sensitivity for analysis；Entire detection process is not necessarily to detach knot without cleaning Label and binding label are closed, therefore the reaction time is shorter；Probe material (emulsion and luminous agent) marks on matrix, and It is not to mark in biomolecule, the activity of biomolecule is not influenced, meanwhile, because there are larger specific surfaces for matrix Product, therefore can be coated with more probe materials and biomolecule on its surface, cause it in the effective concentration of reagent and sensitivity and The performance for detecting background etc. can be more excellent.

" 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-multi Hole, with any density, but preferably have and the close density of water, can preferably float in water, and by transparent, part Transparent or opaque material is constituted.Described matrix can be with or without charge, when with charge, preferably negative electrical charge.Institute State matrix can be solid (such as polymer, metal, glass, organic and inorganic matter such as mineral, salt and diatom), small oil droplet (such as Hydrocarbon, fluorocarbon, silicic fluid), vesica (such as such as phosphatide of synthesis or natural such as cell and thin Born of the same parents' organ).Matrix can be latex particle or other particles containing organic or inorganic polymer, lipid bilayer such as liposome, Phospholipid capsule bubble, small oil droplet, silicon particle, metal-sol, cell and crystallite dyestuff.Matrix usually has multifunctionality, Huo Zheneng Enough it is attached on donor or receptor by special or non-specific covalently or non-covalently interaction.It is there are many functional group It is available or be merged in.Typical functional group includes carboxylic acid, acetaldehyde, amino, cyano, vinyl, hydroxyl, sulfydryl Deng.A unrestricted example for being suitable for the invention matrix is carboxy-modified latex particle.This matrix it is detailed Situation 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 refer to but be not limited to can to specifically bind immunoglobulin or T cell by Any protein determinant of body.In some specific embodiments of the present invention, epitope is that antigenic surface can be by antibody specificity In conjunction with region.Epitopic determinants usually may include the chemically active surface group of molecule, such as, but not limited to：Amino acid, Carbohydrate side chain, phosphoryl and/or sulfonyl.In some other specific embodiment of the present invention, epitope can be specific three specific Structure feature and specific charge feature.In some cases, epitope can indicate there is specific binding properties on molecular surface Space structure, such as, but not limited to：Wholly or partly complementary nucleic acid sequence.

II. technical solution

First aspect present invention provides a kind of preparation method of the anti-sharp homogeneous detection kit of Miao Le Shi pipes of people, described Method includes：

Kit prepared by this method can quantitatively detect the anti-Miao Le Shi pipe of the people in sample and the presence or absence of swash or dense well Degree.

First marker and its specific junction mixture can be not reacting for liquid phase specific binding, not influence system Spectral characteristic molecular pairs, such as digoxin molecule and its antibody can also be that complementary both ends DNA is single-stranded or complementary Two sections of peptide nucleic acids.

The kit may also include Packaging box body.Insulating interlayer, cold storage agent packet, kit can be equipped in Packaging box body The attachmentes such as operation instructions.

Any component or composition of kit can be contained in independent container.Vessel surface can have mark label. Mark label may be selected from but not limited to the label that can write, bar code, Quick Response Code, magnetic labels, wireless signal receiver, wireless Signal projector.

This method is for non-diagnostic purpose.

A. 10-30 μ l samples to be checked are added in reacting hole；

C.35-45 DEG C incubation 10-30 minutes；

D. the third composition 100-250 μ l are added into reacting hole；

E.35-45 DEG C incubation 5-20 minutes；

III. embodiment

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.

Material and equipment

It is doubtful to include or the serum comprising AMH.

AMH sterlings.

Two plants of anti-AMH monoclonal antibodies, code name Ab1, Ab2, wherein Ab1 are purchased from Amy victory Science and Technology Ltd., Ab2 purchases From the Shanghai bio tech ltd Yu Bo.

Light-induced chemiluminescent detector：LICA-500 automatical analysis instruments, Bo Yang biotechnologies (Shanghai) Co., Ltd. Production.

(1) preparation method of antibody coating luminous particle (claiming R1 after being diluted by buffer solution)

Luminous particle：Microparticle surfaces contain aldehyde radical (- CHO), are connect with antibody molecule by aldehyde radical.It is interior to contain luminousization Close the chelate of object (derivative of thioxene) and lanthanide series (Eu) compound.

Receptor microballoon is purchased from platinum Elmer Co., Ltd, article No. 6762001.

Biological raw material：Anti- AMH antibody As b1.

Preparation process：It takes 2mg luminous particles solution and is diluted to 5mg/ with 9.6 carbonate buffer solutions of 0.05M pH (CB) Ml takes antibody 0.02mg to be transferred in particle pipe, mixes well 4 DEG C of coatings overnight；It adds and is diluted to CB buffer solutions The 20 μ l of BSA solution of 10mg/ml, room temperature rotate 2h；Fully washing particle, then use pH 8.0,0.1M Tris-HCl solution general Particle is diluted to 100 μ g/ml, that is, is used as working stock liquid (R1)

(2) preparation method of biotin labelled antibodies (claiming R2 after being diluted by buffer solution)

Activated biotin.For NHS biotins.

Biological raw material：Anti- AMH antibody As b2.

Preparation process：Antibody 0.5mg is taken to be transferred in 14KD bag filters, with label buffer solution (0.1M NaHCO₃) dialysis, 2h/ times, change liquid 1 time；The 10 μ l of activated biotin solution of addition 5mg/ml, rapid mixing, supplemental markers buffer solution to 500 μ l, 2-8 DEG C of mixing overnight, label ratio are 1:30 (antibody:Biotin-molar ratio)；Will label complete Bio-Ab reagents take to 14KD bag filters are dialysed with elution buffer (0.1M Tris-HCl), 2h/ times, change liquid 1 time；With pH 8.0,0.1M Tris- HCl solution is diluted to 5 μ g/ml.

(3) it is coated with the preparation method of the photosensitive particulate (claiming R3 after being diluted by buffer solution) of Streptavidin

Donor microballoon has included Photoactive compounds phthalein mountain valley with clumps of trees and bamboo dyestuff (luminol class chemiluminescent substance), while donor microballoon Inside contain active aldehyde, and is coated with Streptavidin in advance.

Donor microballoon is purchased from platinum Elmer Co., Ltd, article No. 6760002S.

Mentioned reagent can determine concentration or use pH 8.0,0.1M Tris-HCl solution dilute when in use according to actual needs It releases to required concentration.

(4) preparation process of AMH calibration objects

Aforementioned AMH sterlings are taken, are matched with 7.4 phosphate buffered saline solutions of 0.1M pH containing 20% inactivation calf serum Concentration 0, the AMH works of 0.16,0.6,4,10,24ng/mL series of calibration product solution each 0.5ml and concentration 200ng/mL is made Make storing solution, uses the diluted AMH working stock liquids of 7.4 phosphate buffered saline (PBS)s of 0.1M pH as needed.

Kit testing principle

The anti-Miao Le Shi pipes of people of the present invention swash the core of (AMH) assay kit (light-induced chemiluminescent method) technical solution Testing principle is as shown in Fig. 1, and the present invention is based on light-induced chemiluminescent analytical technologies, using dual antibody sandwich assay pattern, inspection It includes the first component, the second component, third component and sample to be detected to survey reaction system.

First group be divided into people's anti-Miao Le Shi pipe swash the coated luminous microballoon of first antibody of (AMH) binding specificity/ Particle (code name FG-Ab1), including the composition of the component is referred to as reagent 1 (R1).

Second group be divided into AMH binding specificities secondary antibody label biotin (code name Bio-Ab2, Biotin-Ab2), including the composition of the component is referred to as reagent 2 (R2).

Third component is to be coated with photosensitive microballoon/particle (code name SA-GG) of Streptavidin, includes the group of the component It closes object and is referred to as reagent 3 (R3).

Sample to be detected is AMH, AMH solution, the composition of (possibility) containing AMH, the serum of (possibility) containing AMH, group Knit liquid or tissue homogenate, the including but not limited to AMH of known concentration, it is known that the series of calibration product of AMH concentration, the low value of AMH, High level quality-control product.

Further include chemiluminescence detector and relevant conventional reagent and consumptive material.

When detection, said components are in liquid phase state, sample to be detected, R1 and R2 are mixed warm bath, in sample to be tested AMH is combined to form double-antibody sandwich compound with FG-Ab1 and Bio-Ab2 respectively, and R3, SA-GG are added then to reaction system Streptavidin part and double-antibody sandwich compound in biotin (Bio) combine, luminous particle and photosensitive particulate are mutual Close, after energy or reactive compound are contacted with photosensitive particulate, photosensitive particulate releases singlet oxygen, which expands Induced luminescence particle generates optical signal after dissipating and being attached to luminous particle.Distance of the singlet oxygen diffusion more than 200nm or so will Inactivation, therefore free luminous particle can hardly obtain energy, the generation of no optical signal.Therefore, light signal strength with react The proportional example functional relation of double-antibody sandwich complex content in system, when AMH molar content be no more than FG-Ab1 and When the minimum value of Bio-Ab2, light signal strength and the proportional example functional relation of AMH contents in sample to be tested pass through known content AMH calibration objects are formed by mathematical function, can calculate AMH concentration levels in non-key sample.

That is, clinical samples or calibration object are mixed with R1 solution and R2 solution, AMH to be checked respectively with shine it is micro- The specific antibody of ball surface and the specific antibody of biotin labeling combine, and form double-antibody sandwich compound；Two kinds at this time Antibody excess, there are the antibody molecules of unbound state.Be added the coated photosensitive microballoon of Streptavidin, Streptavidin with Biotin molecule combines (including compound or free biotin antibody), but only combines compound (FG-Ab1-AMH-Bio- Ab2 the biotin in) could will shine and photosensitive particulate is combined together, at this time in the effect of energy or reactive compound Under, for example in the case of laser irradiation, there are active oxygen species transfers, and luminous particle is caused to generate optical signal.Free coating is anti- Body luminous particle (FG) cannot generate optical signal far from photosensitive particulate (GG).Therefore, light signal strength and AMH contents in sample Proportional example functional relation.Mathematical function relationship is established using known AMH concentration calibrations product and corresponding light signal strength, not Know that sample AMH concentration is obtained by secondary function.

Compared with existing serum AMH quantitative in vitro detection reagents, this technology invents provided AMH quantitative detecting reagents The remarkable result of box is：This product is subordinate to homogeneous immunoassay, and whole process does not have separating, washing process, not only saves detection time, And avoid error caused by washing, have higher preci-sion and accuracy.

Embodiment 1- basic agent boxes

The core group of kit is divided into：

The AMH quantitative determination detection kits (light-induced chemiluminescent method) of the present embodiment are by including the first anti-AMH monoclonals Composition (reagent 1, R1), the comprising biotin labeling the second anti-AMH monoclonals of the coated luminous microballoon (FG-Ab1) of antibody The composition (reagent 2, R2) of antibody (Bio-Ab2) forms；It further include sample to be checked.In addition, including close comprising strepto- is coated with With the composition (reagent 3, R3) of the photosensitive microballoon (SA-GG) of element, mentioned reagent is the method system according to previous materials and equipment Standby, including light-induced chemiluminescent analyzer etc..

The application method of the kit of the present embodiment, including：

Detection process is automatically finished by automatic light-induced chemiluminescent analysis system and exports testing result.Based on aforementioned Testing principle the specific steps are：

A. sample to be checked is added in reacting hole；

B. R1 and R2 are sequentially added in reacting hole；

C. it incubates；

D. R3 is added in reacting hole；

E. it incubates；

F. the luminous photon amount of laser irradiation reacting hole and the every hole of calculating；

Optionally, g. calculates sample concentration.

When, there are when AMH to be measured, AMH is micro- with shining for the first anti-AMH monoclonal antibodies of coating simultaneously in the system of detection Ball and the second anti-AMH monoclonal antibody specificities combination for combining biotin, and form double antibody folder in luminous microsphere surface Heart compound；At this point, as being added the coated photosensitive microballoon of Streptavidin, biotin and Streptavidin in conjunction with and so that two Kind microballoon is close to each other, and under the excitation of excitation light source, photosensitive microballoon discharges singlet oxygen, encounters luminous microballoon in the solution After generate chemiluminescence, to further excite fluorophor on the same microballoon generate Cascaded amplification reaction generate it is glimmering Light.At this point, AMH to be measured is more, fluorescence intensity is stronger, and the content of AMH in serum is quantitatively detected according to luminous power.

Embodiment 2- core reagent boxes

The experiment of the present invention：

The preparation of kit material：

The preparation method of specific material and its corresponding reagent is prepared according to material and environment division.

The application method of the kit of the present embodiment, including：

The parallel addition reagent in different reacting holes, Parallel testing use the excitation photo-irradiation reaction of 680nm wavelength The transmitting light of 612nm wavelength is detected in hole.

Specifically detecting step is：

A. 25 μ l samples are separately added into differential responses hole；In calibration object sample AMH concentration difference 0,0.16,0.6, 4,10,24ng/mL, AMH concentration is referring to lower half portion in table 1 in sample to be tested.

B. 25 μ l R1 and 25 μ l R2 are sequentially added in each reacting hole；

C.37 DEG C incubation 15 minutes；

D. 175 μ l of R3 are added in each reacting hole；

E.37 DEG C incubation 10 minutes；

F. laser independently irradiates micropore and calculates the photon amount that shines per hole.

Wherein, R1 is 50 μ g/mL FG-Ab1；

R2 is 5 μ g/mL Biotin-Ab2；

R3 is 50 μ g/mL SA-GG.

The specific luminous value of calibration object (calibration) is not shown referring to 1 top half of table, the luminous value of sample to be tested in table 1.

Check experiment：

Kit：Roche AMH detection kits.

Testing principle is Electrogenerated chemiluminescent immunoassay.

Using double antibody sandwich method, total detection time 18min, detecting step is as follows：

It is incubated for the first time：50uL sample solutions, the first anti-AMH monoclonal antibody specifics, the ruthenium of biotin labeling are compound Second anti-AMH monoclonal antibody specifics of substance markers, reaction form antigen-antibody sandwich complex.

Second of incubation：After the bead particulates of coating Streptavidin are added, which passes through biotin and strepto- parent Interaction with element and solid phase binding.

Reaction solution is sucked in measuring cell, magnetic bead is adsorbed on by electrode surface by electromagnetic action.It is not combined with magnetic bead Substance is removed by ProCell/ProCell M.It gives electrode certain voltage, makes compound chemiluminescence, and pass through photoelectricity times Increase device and measures luminous intensity.

Testing result to the end is obtained by the calibration curve of detector, calibration curve is by 2 points of calibrations and reagent strip What the level-one calibration curve obtained on code obtained.

Detect sample：The diluted AMH working stock liquids of 7.4 phosphate buffered saline (PBS)s of 0.1M pH.The sample of detection it is dense Degree is referring to 1 lower half portion of table.

Detailed test method is carried out with reference to the step of Roche AMH detection kit specifications.

Detect instrument：LICA-500 automatical analysis instruments, the production of Bo Yang biotechnologies (Shanghai) Co., Ltd.. Software, which is carried, according to the instrument calculates concentration value.

1 kit of table calibrates and compares test 1

Principle, method, material and operating procedure are same as above, separately detect 2 batches of different samples, specifically indicate concentration, luminous value, The information such as concentration value are referring to table 2 and table 3.

2 kit of table calibrates and compares test 2

3 kit of table calibrates and compares test 3

In the detection of three batches, this inspection of the relationship digital simulation of AMH concentration and luminous value in each personal standard items The functional relation of AMH concentration and luminous value in the sample of survey is calculated by the functional relation using the luminous value of sample to be detected Go out the concentration of AMH in sample to be detected, the luminous value of sample to be tested and the AMH concentration being calculated by luminous value in table 1,2,3 It is not shown.

Hardware and software are carried using it, by programming, makes LICA-500 automatical analysis instrument not only and can show and is luminous Value, moreover it is possible to the concentration value of AMH in the sample that directly functional relation of the displaying through AMH concentration and luminous value is calculated.

For Roche kit, by the software systems of Roche, according to standard items and luminous value, (calibration object luminous value is in table 1, it is not shown in 2,3) relationship that both calculates, then directly product AMH in sample to be tested is worth to using the luminous of sample to be tested Concentration value, the concentration values for waiting for product AMH in test sample being specifically calculated referring to table 1,2,3 lower half portion.

The comparison of kit of the present invention and Roche kit, for three batches totally 27 samples to be tested, this in the present invention The AMH concentration that the method for embodiment is obtained is as y values (ordinate y-axis), the AMH concentration obtained using Roche kit As x values (abscissa x-axis), the functional relation of the two is calculated, obtains formula：Y=1.0408x+0.0337.Intuitively The two linear relationship is referring to Fig. 2.

Good, the 3 sample the method for the present invention measured values that both can be seen that linear relationship by above-mentioned functional relation With the correlation R of Roche definite value²=0.9915, correlation is good.As seen in Figure 2, the overall linear correspondence of the two Well.The effect of the present invention is close with the detection result of Roche kit.

But the present invention, using homogeneous detection, there is no magnetic beads etc. to detach purification process, reduces the behaviour in liquid phase Make step, reduce detection time, reduces the possibility of accumulated error.Decrease measuring instrument score complexity.

Embodiment 3- precision is tested

Precision meaning：Precision is to weigh the important indicator of vitro detection reagent within-run and between-run analysis, is evaluation production The important evidence of product validity generally includes analysis withinrun precision and betweenrun precision.

The basic ideas of the materials and methods of the present embodiment are same as Example 2, the two the difference is that detected sample Middle AMH concentration is different, and specific detecting step is：

A. 25 μ l samples are separately added into differential responses hole；In calibration object sample AMH concentration difference 0,0.16,0.6, 4、10、24ng/mL；Detected sample be low (L), in (M), high (H) value sample；

B. 25 μ l R1 and 25 μ l R2 are sequentially added in each reacting hole；

C.37 DEG C incubation 15 minutes；

D. 175 μ l of R3 are added in each reacting hole；

E.37 DEG C incubation 10 minutes；

F. laser independently irradiates micropore and calculates the photon amount that shines per hole；

G. sample concentration is calculated.

Wherein, R1 is 50 μ g/mL FG-Ab1；

R2 is 5 μ g/mL Biotin-Ab2；

R3 is 50 μ g/mL SA-GG.

It is fitted the functional relation of AMH concentration and luminous value according to AMH concentration in calibration object and luminous value, is closed with the function System calculates the concentration of AMH in sample to be tested.

Withinrun precision appraisal procedure：With low (L), in (M), high (H) value 3 batches of sample pair product carries out independence Analysis, each batch replication 10 times calculate the average value of 10 measurement resultsWith standard deviation (SD), according to formulaCalculate the coefficient of variation (CV).

Betweenrun precision appraisal procedure：With low (L), in (M), high (H) value sample, the product of 3 batches is carried out independent Analysis, each batch replication 10 times calculate the average value of 30 measurement resultsWith standard deviation (SD), according to formulaCalculate the coefficient of variation (CV).

Specific calibration object concentration and its luminous value, batch sample to be tested luminous value and dense according to AMH in this calibration object The AMH concentration that degree is calculated with the functional relation of luminous value respectively 10 average irradiances referring to table 4.

The AMH calculated according to AMH concentration in this calibration object and the functional relation of luminous value per three samples of a batch The average value of concentration, standard deviation and the coefficient of variation are referring to table 5.

Generally three batches of each three samples are calculated according to AMH concentration in this calibration object and the functional relation of luminous value The average value for the AMH concentration come, standard deviation and the coefficient of variation are referring to table 6.

4 precision of table tests initial data

5 serum AMH quantitative determination reagents (light-induced chemiluminescent method) of table analyze withinrun precision

6 serum AMH of table quantitative determines reagent (light-induced chemiluminescent method) betweenrun precision

From table 4 and table 5, table 6 it is found that kit testing result prepared by the method for the invention variation within batch coefficient It is equal with interassay coefficient of variation<5%, generally 3% or so, illustrate to carry out using kit prepared by the method for the invention Favorable repeatability, random error when detection is small.

Embodiment 4- accuracy is tested

Accuracy meaning：The degree that is consistent of measured value and actual value, the size of reaction system error.

Evaluation of accuracy：By 2 samples of the level containing different AMH, multipoint dilution is carried out with calibration object matrix liquid, The rate of recovery is calculated according to dilution ratio.

The basic ideas of the materials and methods of the present embodiment are same as Example 2, the two the difference is that reactant concentration Difference, specific detecting step are：

A. 25 μ l samples are separately added into differential responses hole；

B. 25 μ l R1 and 25 μ l R2 are sequentially added in each reacting hole；In calibration object sample AMH concentration difference 0, 0.16、0.6、4、10、24ng/mL；Detected sample is used according to concentration gradient as described below；

C.37 DEG C incubation 15 minutes；

D. 175 μ l of R3 are added in each reacting hole；

E.37 DEG C incubation 10 minutes；

G. sample concentration is calculated.

Wherein, R1 is 50 μ g/mL FG-Ab1；

R2 is 5 μ g/mL Biotin-Ab2；

R3 is 50 μ g/mL SA-GG.

The kit of the present embodiment in use, take two parts of high level AMH serum, respectively according to inspection determined by embodiment 2 Survey method uses 7.4 phosphate buffered saline (PBS)s of 0.1M pH and 1/2, under 1/4,1/8,1/10 times of dilution with original concentration Different reacting holes is added in the sample of concentration, is detected.

Using the detected value of original concentration as standard, compare the sample of the concentration under 1/2,1/4,1/8,1/10 times of dilution It is expected that concentration and measured concentration, to assess the accuracy of this kit assay.

Gradient dilution can ensure the relative concentration proportionate relationship of AMH in serial sample, this than independently preparing or The systematic error of the test of collecting sample is small, can be used to evaluate the performance of kit of the present invention well.

Specific calibration object luminous value and sample to be tested luminous value and its calculated concentration are referring to table 7.

7 accuracy initial data of table

8 first parts of serum AMH quantitative detecting reagents (light-induced chemiluminescent method) accuracy of table

9 second parts of serum AMH quantitative detecting reagents (light-induced chemiluminescent method) accuracy of table

It is measured, recycles after from table 7, table 8 and table 9 it is found that carrying out multipoint dilution with the AMH samples of 2 different levels Rate is in 97%~105% range, particularly, for first part of serum sample, under first three dilution, measured value with it is pre- The difference of measured value illustrates that the detection for the reagent sum that measured value is close with actual value, prepared by the method for the invention misses less than 1% Difference is small, and accuracy is good, favorable repeatability.

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.

Claims

1. a kind of anti-Miao Le Shi pipes of people swash the preparation method of homogeneous detection kit, which is characterized in that the method includes：

The first component is prepared, first component includes receptor and the first combining unit in combination, and the receptor can It is reacted with singlet oxygen and generates detectable signal, first combining unit can be in conjunction with the first sharp table of the anti-Miao Le Shi pipes of people Position；

Preparing the second component, second component includes the first marker and the second combining unit in combination, and described the Two combining units can be in conjunction with the second sharp epitope of the anti-Miao Le Shi pipes of people, and second epitope and first epitope are anti-Miao of people The epitope of the epitope of the sharp different binding characteristics of Le Shi pipes or the identical combination characteristic of different location；

Third component is prepared, the third component includes the specific bond of donor and first marker in combination Object, the donor can generate singlet oxygen in excited state.

2. preparation method according to claim 1, it is characterised in that：

First combining unit and second combining unit, which are respectively and independently selected to swash with the anti-Miao Le Shi pipes of the people, has knot Close monoclonal antibody, antibody binding fragment, artificial antibody, the antibody of modification, two or more monoclonal antibodies of specificity Mixture and polyclonal antibody are preferably selected from polyclonal antibody and/or monoclonal antibody.

3. preparation method according to claim 1, it is characterised in that：

The marker is biotin, and the specific junction mixture of the marker is Streptavidin.

4. preparation method according to claim 1, it is characterised in that：

The receptor includes olefin(e) compound and metallo-chelate, is non-particulate forms, and solvable in water-bearing media；Or, The receptor is the high molecular particle filled with luminophor and lanthanide series；And/or

The donor is photoactivation or chemical activation sensitizer, is non-particulate forms, and solvable in water-bearing media； And/or the donor is the high molecular particle filled with Photoactive compounds, and singlet oxygen can be generated under light excitation.

5. according to the preparation method described in any one of claim 1-4, which is characterized in that the preparation method further includes：

Preferably, first buffer solution, second buffer solution and the third buffer solution are separately selected from pH 8.0, 0.1M Tris-HCl solution.

6. preparation method according to claim 5, it is characterised in that：The preparation method of the first chamber includes：

First anti-human anti-Miao Le Shi pipe is added in the receptor solution and swashs antibody or its binding fragment by step S2, it is static after again Addition is diluted to the BSA solution of 8~12mg/mL with carbonate buffer solution, obtains sharp anti-with described first anti-human anti-Miao Le Shi pipes The receptor solution that body or its binding fragment combine；

Step S3, in the receptor solution that separation is combined with the described first sharp antibody of anti-human anti-Miao Le Shi pipe or its binding fragment with First anti-human anti-Miao Le Shi pipe swash antibody or its binding fragment in conjunction with the receptor, and the first buffer solution is added, obtains Obtain the first chamber.

7. preparation method according to claim 5, it is characterised in that：The preparation method of the second chamber includes：

Step T1, by second anti-human anti-Miao Le Shi pipe swash antibody or its binding fragment be placed in bag filter using label buffer solution into Row dialysis, is added biotin solution, and fill into elution buffer after dialysis, static, obtains second combined with biotin Anti-human anti-Miao Le Shi pipes swash antibody or its binding fragment；

Combined with biotin second anti-human anti-Miao Le Shi pipes are swashed antibody or its binding fragment are placed in profit in bag filter by step T2 It is dialysed with elution buffer, the second buffer solution is added after dialysis, obtain the second chamber.

8. a kind of kit sharp for homogeneously detecting the anti-Miao Le Shi pipes of people, is wanted described in any one of 1-7 according to right What the preparation method that the anti-Miao Le Shi pipes of people swash homogeneous detection kit was prepared.

9. kit according to claim 8, it is characterised in that：

The concentration of first component in the first chamber is selected from：10~300 μ g/mL, preferably 20~200 μ g/mL, more It is preferred that 30~80 μ g/mL, and/or

The concentration of second component in the second chamber is selected from：0.5~15 μ g/mL, preferably 1~8 μ g/mL, it is more excellent Select 2~6 μ g/mL；And/or

The concentration of the third component in the third composition is selected from：10~300 μ g/mL, preferably 20~200 μ g/mL, more It is preferred that 30~80 μ g/mL.

10. kit according to claim 8 or claim 9, which is characterized in that the kit includes that the anti-Miao Le Shi pipes of people swash school Quasi- product solution, the calibration object solution are concentration 0ng/mL, 0.16ng/mL, 0.6ng/mL, 4ng/mL, 10ng/mL, 24ng/mL The anti-Miao Le Shi pipes of people of series swash solution.

11. a kind of anti-Miao Le Shi pipes of people swash the method homogeneously detected comprising wanted described in any one of 8-10 using according to right Carry out chemiluminescence detection for homogeneously detecting the sharp kit of people's anti-Miao Le Shi pipe.

12. according to the method for claim 11, which is characterized in that described method includes following steps：

Sample to be tested is mixed with first chamber and second chamber, obtains the first mixture by step R1；

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

The presence of the chemiluminescence signal obtained in step R4, detecting step R3 and/or intensity, to judge to survey in sample to be tested Swash and/or determine the sharp content of the anti-Miao Le Shi pipes of people with the presence or absence of the anti-Miao Le Shi pipes of people.

13. according to the method for claim 11, which is characterized in that the method further includes：Utilize the anti-Miao Le Shi of the people Pipe swashs calibration object solution and makes the step of anti-Miao Le Shi pipes of chemiluminescence signal-people swash the standard curve of concentration；The standard is bent Line is used to determine the content that the anti-Miao Le Shi pipes of people are sharp in sample to be tested.

14. according to the method for claim 12, which is characterized in that utilize the excitation that wavelength is 600-700nm in step R3 Light irradiates second mixture, and the donor is excited to generate singlet oxygen, the receptor and the singlet oxygen touched Reaction generates the transmitting light that wavelength is 520-620nm；In step R4, the signal for detecting the transmitting light exists and/or intensity, from And judges to survey in sample to be tested and swash and/or determine the sharp content of the anti-Miao Le Shi pipes of people with the presence or absence of the anti-Miao Le Shi pipes of people.

15. according to the method for claim 13, which is characterized in that described method includes following steps：

A. 10-30 μ l samples to be checked are added in reacting hole；

C.35-45 DEG C incubation 10-30 minutes；

D. the third composition 100-250 μ l are added into reacting hole；

E.35-45 DEG C incubation 5-20 minutes；

F. it is the laser irradiation reacting hole of 680nm to utilize wavelength, and excited donor generates singlet oxygen, receptor and the single line touched The reaction of state oxygen produces the transmitting light of 612nm；

16. a kind of method according to any one of claim 11~15 anti-Miao Le of people in quantitative detection sample to be tested The presence or absence and/or the application in content of family name Guan Ji.

17. wanting the method described in any one of 1-7 preparing for assessing ovarian reserve, diagnosis children development disease according to right Disease, assessment infertility, diagnosis of polycystic ovary syndrome or reagent, kit, detection device or the detection system of predicting menopause Application in system or purposes.

18. want the method described in any one of 1-7 according to right, right wants kit described in any one of 8-10 or right to want Application or purposes of the method in predicting menopause described in any one of 11-15.