CN108445216B

CN108445216B - Human anti-mullerian hormone determination kit and preparation method and application thereof

Info

Publication number: CN108445216B
Application number: CN201810143236.9A
Authority: CN
Inventors: 徐静心; 史舟芳; 强中华; 刘宇卉; 李临
Original assignee: Chemclin Diagnostics Corp
Current assignee: Chemclin Diagnostics Corp
Priority date: 2018-02-11
Filing date: 2018-02-11
Publication date: 2021-10-15
Anticipated expiration: 2038-02-11
Also published as: CN108445216A

Abstract

The invention discloses a preparation method of a human anti-mullerian hormone (AMH) homogeneous detection kit, which comprises the following steps: preparing a composition comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a first binding moiety capable of binding to a first epitope of AMH, bound thereto; preparing a second component comprising a first label and a second binding member capable of binding to a second epitope of AMH, which second epitope is an epitope of different binding properties of AMH or an epitope of the same binding properties at a different location from the first epitope, bound thereto; preparing a third component comprising a specific binder of a donor capable of generating singlet oxygen in an excited state and said first label bound thereto. The kit prepared by the method can efficiently detect AMH.

Description

Human anti-mullerian hormone determination kit and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomolecule detection, relates to a homogeneous immunoassay kit without a matrix effect, and particularly relates to a homogeneous luminescent immunoassay kit for antihuman mullerian hormone, and a preparation method and a use method thereof.

Background

Anti-mullerian hormone (AMH) is a dimeric glycoprotein belonging to the transforming growth factor- β family, as an inhibitory substance of mullerian tubes. In the process of gonadal organ development, AMH plays an important role, and is one of important substances for ensuring the development of male and female organs and gonadal function. In males, AMH is produced primarily by leydig cells, starting from embryogenesis and throughout life; during male fetal development, AMH causes the mullerian duct to degenerate and form the male reproductive tract. The male has high content of AMH in neonatal period, and still rises in months after birth, slowly falls after 2 years of age, and falls in adolescence most obviously until adults are at the lowest. In female body, mullerian tube develops into female organs such as uterus and bilateral fallopian tube due to the inability of the embryo to synthesize AMH in early stage. By 36 weeks of development, ovarian granulosa cells produce AMH, postnatally from pre-antral follicles and small antral granulosa cells. AMH levels are positively correlated with age from the beginning of the infant, rising gradually with age, with peak AMH at puberty (about 15.8 years) and remaining stable by 25 years. However, after age 25, AMH levels are negatively correlated with age and gradually decline with age until menopause falls to a low level and is undetectable.

Currently, methods for quantitative analysis of serum AMH include enzyme-linked immunoassay, enzymatic luminescence immunoassay, and electrochemical luminescence immunoassay. The enzyme-linked immunoassay method adopts alkaline phosphatase as a marker, a 96 micro-porous plate as a solid phase carrier and coats a capture antibody. The enzymatic luminescence immunoassay is similar to enzyme-linked immunoassay, and is different from enzyme-linked immunoassay in that a luminescent substrate is used for quantitative analysis by measuring an optical signal. The electrochemical luminescence adopts terpyridyl ruthenium as a marker, and tripropylamine and an electrode are used in combination to induce luminescence.

The three AMH immunoassay methods have the common characteristic that the three AMH immunoassay methods belong to Heterogeneous immunoassay (Heterogeneous immunoassay). Heterogeneous immunoassay refers to the need to separate and remove unreacted free labeled antibody or other components after antibody-antigen reaction and before detecting signals. A common method for separating bound and free labels is a solid phase adsorptive separation technique. For AMH quantitative analysis, a capture antibody is connected with a solid phase carrier, an antigen to be detected is respectively combined with a labeled antibody and the capture antibody, a double-antibody sandwich complex is formed on the surface of a solid phase material (the combined label is positioned on the surface of the solid phase), and excessive free labeled antibody which is not involved in antigen-antibody reaction is distributed in a liquid phase. The liquid phase solution was removed and the solid phase (microparticles) was washed repeatedly.

The solid phase adsorption separation method has two important links: coating and washing. The heterogeneous reaction mode causes great defects to the labeled immunoassay due to the existence of coating and washing links, and is mainly shown in the following two aspects:

1. in the coating process, no matter physical adsorption or chemical connection, the conformation of the antibody molecules coated on the surface of the solid phase material is different from that of the antibody molecules in the liquid phase, and the binding capacity with the antigen molecules is limited to a certain extent due to the steric hindrance effect; whether microspheres or micro-porous plates are adopted as solid phase materials, the captured antibody molecules cannot meet the requirements of a large number of antigens to be detected to the maximum extent due to the limited coating area, so that the detection range is influenced.

"plate washing" or "ball washing" is an important step in heterogeneous immunoassays and occurs multiple times. The washing process adds complexity to the detection procedure, increases detection time and provides obstacles to automation. In addition, because the washing process, especially the enzyme immunoassay, is difficult to realize standardization, the washing effect is different among each test hole, the precision of detection is influenced in a certain degree, and the batch-to-batch and batch-to-batch differences occur.

The solid phase adsorption separation method is time-consuming in each washing process, and is also bound to cause errors caused by washing separation. The washing error is an important source of heterogeneous immunoassay error, and affects the precision of the analysis method, thereby affecting the accuracy and the analysis sensitivity of the analysis method.

In view of the above, the present inventors have conducted studies and have desired to provide a detection kit and a detection method which have high precision, accuracy and sensitivity, are easy to handle, and can quantitatively detect the AMH level of human anti-mullerian hormone.

Disclosure of Invention

Aiming at the defects of the prior art, the invention prepares a kit for quantitatively detecting serum anti-Mullerian duct laser AMH based on a homogeneous immunization method, establishes a double-antibody sandwich serum anti-Mullerian duct laser AMH quantitative analysis method, and is mainly represented by the following steps of: preferably a pair of antibodies directed against mullerian kinase AMH label biotin and the receptor, respectively; the analysis system is optimized (antibody dosage, buffer solution and the like), and the analysis performance of the system meets the industrial standard and the requirements of clinical and scientific research laboratories. The product is mainly characterized in that homogeneous phase photoimmunoassay is adopted, the whole detection process has no separation and washing process, no extra waste liquid is generated, washing errors are avoided, and the product has higher precision, accuracy and sensitivity. In addition, the kit and the detection method have good amplification effect and high analysis precision, so that the kit and the detection method have very high analysis sensitivity.

The invention aims to solve the problem of providing a serum anti-mullerian hormone (anti-mullerian hormone) homogeneous luminescent immunoassay kit and a preparation and use method thereof, so as to solve the problem of complex washing steps of the existing heterogeneous immunoassay kit; meanwhile, the defects of environmental pollution and short shelf life of radioimmunoassay are overcome, and the defects of poor repeatability and easy occurrence of hook effect of enzyme immunoassay are overcome. The invention adopts luminescence immunoassay, and has higher sensitivity and precision.

More specifically, the invention provides a preparation method of a human anti-mullerian tube laser homogeneous detection kit, which comprises the following steps:

preparing a first component comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a first binding unit capable of binding to a first epitope of human anti-muller's tube kinase bound thereto;

preparing a second component comprising a first label and a second binding unit bound thereto, said second binding unit being capable of binding to a second epitope of human anti-mullerian kinase which is an epitope of different binding properties or of the same binding properties at different positions than said first epitope;

preparing a third component comprising a donor capable of producing singlet oxygen in an excited state and a specific conjugate of the first label bound thereto.

In some embodiments of the invention, the first binding unit and the second binding unit are each independently selected from a monoclonal antibody, an antibody binding fragment, an artificial antibody, a modified antibody, a mixture of two or more monoclonal antibodies, and a polyclonal antibody, preferably from a polyclonal antibody and/or a monoclonal antibody, with binding specificity to the human anti-mullerian hormone.

In some embodiments of the invention, the label is biotin and the specific binder for the label is streptavidin.

In some embodiments of the invention, the acceptor comprises an olefinic compound and a metal chelate, which is in non-particulate form and soluble in an aqueous medium; or, the receptor is a polymer particle filled with a luminescent compound and a lanthanide element; and/or the presence of a gas in the gas,

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

In some embodiments of the invention, the method of making further comprises:

preparing a first composition comprising the first component and a first buffer;

preparing a second composition comprising the second component and a second buffer;

preparing a third composition comprising the third component and a third buffer;

preferably, the first buffer, the second buffer and the third buffer are each independently selected from a 0.1M Tris-HCl solution at pH 8.0.

In some embodiments of the invention, the method of preparing the first composition comprises:

step S1, diluting the receptor to 4-6 mg/mL by using a carbonate buffer solution to obtain a receptor solution;

step S2, adding a first anti-human anti-Mullerian antibody or a binding fragment thereof into the receptor solution, standing, and then adding a BSA solution diluted to 8-12 mg/mL by using a carbonate buffer solution to obtain a receptor solution bound with the first anti-human anti-Mullerian antibody or the binding fragment thereof;

step S3, isolating the receptor bound to the first anti-human anti-mullerian antibody or binding fragment thereof in a receptor solution bound to the first anti-human anti-mullerian antibody or binding fragment thereof and adding a first buffer solution to obtain the first composition.

In some embodiments of the invention, the method of preparing the second composition comprises:

step T1, placing the second anti-human anti-Mullerian antibody or binding fragment thereof in a dialysis bag, dialyzing with a labeled buffer solution, adding a biotin solution after dialysis, supplementing a dialysis buffer solution, and standing to obtain a second anti-human anti-Mullerian antibody or binding fragment thereof bound with biotin;

and a step T2 of placing the second anti-human anti-mullerian antibody or binding fragment thereof bound to biotin in a dialysis bag and dialyzing the resulting solution with a dialysis buffer, and adding the second buffer after the completion of dialysis to obtain the second composition.

In a second aspect, the present invention provides a kit for the homogeneous detection of human anti-mullerian kinase, which is prepared according to the preparation method of the human anti-mullerian kinase homogeneous detection kit of the first aspect of the present invention.

In some embodiments of the invention, the concentration of the first component in the first composition is selected from: 10 to 300. mu.g/mL, preferably 20 to 200. mu.g/mL, more preferably 30 to 80. mu.g/mL, and/or

The concentration of the second component in the second composition is selected from: 0.5-15 μ g/mL, preferably 1-8 μ g/mL, more preferably 2-6 μ g/mL; and/or

The concentration of the third component in the third composition is selected from: 10 to 300. mu.g/mL, preferably 20 to 200. mu.g/mL, and more preferably 30 to 80. mu.g/mL.

In some embodiments of the invention, the kit comprises a human anti-mullerian kinase calibrator solution in a concentration of 0ng/mL, 0.16ng/mL, 0.6ng/mL, 4ng/mL, 10ng/mL, 24ng/mL series of human anti-mullerian kinase solutions.

A third aspect of the invention provides a method for the homogeneous detection of human anti-mullerian kinase comprising performing a chemiluminescent detection using a kit for the homogeneous detection of human anti-mullerian kinase according to the second aspect of the invention.

In some embodiments of the invention, step R1, mixing the sample to be tested with the first composition and the second composition to obtain a first mixture;

step R2, mixing the first mixture with a third composition to obtain a second mixture;

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

and step R4, detecting the existence and/or intensity of the chemiluminescence signal obtained in the step R3, so as to judge whether human anti-Mullerian kinase exists in the sample to be detected and/or determine the content of the human anti-Mullerian kinase.

In some embodiments of the invention, the method further comprises: a step of preparing a standard curve of a chemiluminescence signal-human anti-mullerian excimer concentration by using the human anti-mullerian excimer calibration solution; the standard curve is used for determining the content of human anti-mullerian shock in the sample to be detected.

In some embodiments of the present invention, the second mixture is irradiated with excitation light with a wavelength of 600-700nm in step R3 to excite the donor to generate singlet oxygen, and the acceptor reacts with the contacted singlet oxygen to generate emission light with a wavelength of 520-620 nm; in step R4, the presence and/or intensity of the emitted light signal is detected, so as to determine whether human anti-mullerian laser is present in the sample to be tested and/or determine the content of human anti-mullerian laser.

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

a. adding 10-30 mul of sample to be detected into the reaction hole;

b. sequentially adding 10-30 μ l of the first composition and 10-30 μ l of the second composition into the reaction well;

c, incubating at 35-45 ℃ for 10-30 minutes;

d. adding 250 μ l of the third composition 100-;

e.35-45 ℃ for 5-20 minutes;

f. irradiating the reaction hole by using laser with the wavelength of 680nm to excite the donor to generate singlet oxygen, and reacting the acceptor with the contacted singlet oxygen to generate emitted light with the wavelength of 612 nm;

g. the amount of photons emitted per well was measured and the concentration of human anti-mullerian excitation was calculated from the standard curve.

A fourth aspect of the invention provides the use of a method according to the third aspect of the invention for quantitatively determining the presence, absence and/or amount of human anti-mullerian stimulation in a sample to be tested.

A fifth aspect of the invention provides the use or use of a method according to the first aspect of the invention in the manufacture of a reagent, kit, test device or test system for the assessment of ovarian reserve, diagnosis of sexual developmental disorders in children, assessment of infertility, diagnosis of polycystic ovarian syndrome or prediction of menopause time.

A sixth aspect of the invention provides a use or use of a method according to the first aspect of the invention, a kit according to the second aspect of the invention or a method according to the third aspect of the invention in predicting downtime.

The kit has the following technical effects: the linearity is good. The sensitivity is good. The accuracy is high.

Drawings

The present invention will be described in detail below with reference to the accompanying drawings.

FIG. 1 is a schematic diagram of the detection principle of the kit of the invention,

wherein:

1 represents the receptor coated with a primary anti-AMH antibody (FG-Ab 1);

2 represents a biotin-labeled secondary anti-AMH antibody (Bio-Ab 2);

3 represents AMH in the sample to be detected or AMH in an AMH calibrator with known concentration;

4 represents a streptavidin-coated donor (SA-GG).

FIG. 2 is a schematic diagram showing the comparison of the measurement results of the kit of the present invention and the Roche kit.

Detailed Description

In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the present invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

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

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

I. Term(s) for

The term "homogeneous" as used herein in the context of the present invention is defined as "homogeneous" and means that the detection is performed without the need to separate the complex formed by the specific binding partner from the remaining free specific binding partner, e.g.the bound antigen-antibody complex from the remaining free antigen or antibody.

The term "test sample" as used herein refers to a mixture that may contain an analyte, including but not limited to a protein, hormone, antibody or antigen. Typical test samples that may be used in the methods or products disclosed herein include body fluids such as blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid, emphysema, and the like. The sample to be tested may be a solution obtained by diluting a sample that may contain an analyte with a diluent or a buffer solution as needed before use. For example, to avoid the HOOK effect, the analyte may be diluted with a sample diluent before the on-line detection, and then the detection may be performed on the detection apparatus, in which case the diluted solution that may contain the analyte is collectively referred to as the sample to be detected.

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

The term "monoclonal antibody" as used herein refers to an immunoglobulin secreted by a monoclonal B lymphocyte, which may be prepared by methods well known to those skilled in the art, or which is expressed by prokaryotic or eukaryotic cells or which has been engineered to retain or enhance the original antigen binding properties.

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

The term "binding" as used herein refers to the continuous spatial juxtaposition of different molecules by recognition, including but not limited to the formation of larger molecules, molecular complexes, etc., and specific recognition may include electrostatic, hydrophobic, ionic, and/or hydrogen bonding interactions, including but not limited to direct association between two molecules such as salt bridges and water bridges, for example, where an antigen and an antibody bind to form a complex, and may also refer to the bonding of different molecules together to form a larger molecule or complex by a specific reaction of chemical groups, for example, where biotin is covalently cross-linked to an antibody via a reactive group.

The term "binding member" as used herein refers to a molecule or complex that specifically binds to an "analyte" and, in some cases, retains biological or chemical activity after the binding member, in whole or in part, binds to a test substance.

The term "component" as used herein refers to the smallest chemical unit or complex of units that can be individually subjected to purification, reaction, testing, characterization, mixing, formulation, etc., and in some cases, refers to a molecule, polymer, chemically-tagged macromolecule.

The term "specific binding" as used herein refers to a mutual discrimination and selective binding reaction between different molecules, i.e., a conformation correspondence between corresponding reactants in terms of a steric structure, and a binding reaction between different groups having a specific reactivity in terms of a chemical reaction.

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

The term "donor" as used herein refers to a sensitizer capable of generating a reactive intermediate such as singlet oxygen that reacts with a receptor upon activation by energy or an active compound. The donor may be photoactivated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.).

In some embodiments of the invention, the donor is a photosensitizer which may be a photosensitizer known in the art, preferably a compound that is relatively light stable and does not react efficiently with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrins, phthalocyanines, and chlorophylls disclosed in, for example, U.S. patent No. 5709994, which is incorporated herein by reference in its entirety, and derivatives of these compounds having 1-50 atom substituents that serve to render these compounds more lipophilic or more hydrophilic, and/or as a linker group to a member of a specific binding pair. Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, such as those described in US patent No. US6406913, which is incorporated herein by reference.

In other embodiments of the invention, the donor is a chemically activated other sensitizer, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Other examples of donors include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, etc., which are heated or directly absorb light to release singlet oxygen.

The term "acceptor" as used herein refers to a substance capable of reacting with singlet oxygen to produce a detectable signal. The donor is induced by energy or an active compound to activate and release singlet oxygen in a high energy state that is trapped by a close proximity acceptor, thereby transferring energy to activate the acceptor.

In some embodiments of the invention, the receptor is a substance that: it undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate that can decompose with or following luminescence. Typical examples of such substances include, but are not limited to: enol ether, enamine, 9-alkylidene xanthan gum, 9-alkylidene-N-alkyl acridin, aromatic vinyl ether, diepoxy ethylene, dimethyl thiophene, aromatic imidazole or lucigenin.

In other embodiments of the invention, the acceptor is an olefin capable of reacting with singlet oxygen to form a hydroperoxide or dioxetane that can be decomposed into ketones or carboxylic acid derivatives; a stable dioxetane which can be decomposed by the action of light; acetylenes which can react with singlet oxygen to form diketones; hydrazones or hydrazides which can form azo compounds or azocarbonyl compounds, such as luminol; and aromatic compounds that can form endoperoxides. Specific, non-limiting examples of receptors that can be utilized in accordance with the disclosed and claimed invention are described in U.S. patent No. US5340716, which is incorporated herein by reference in its entirety.

In other embodiments of the invention, the receptor comprises an olefinic compound and a metal chelate, which is non-particulated and soluble in an aqueous medium, and the method of making such receptor can be found in patent PCT/US2010/025433 (which is incorporated herein by reference in its entirety).

In the invention, the donor can be polymer particles filled with photosensitive compounds formed by coating functional groups on a substrate, and can generate singlet oxygen under the excitation of light, at the moment, the donor can also be called photosensitive microspheres or photosensitive particles, and the solution containing the photosensitive microspheres or photosensitive particles can be called photosensitive solution or general solution; and/or the receptor can be a polymer particle which is coated on the substrate through a functional group to form a luminescent compound and lanthanide elements filled in the polymer particle, and the polymer particle is called a luminescent microsphere or a luminescent particle. In the application, the system induces a luminescent signal through optical excitation and energy transfer based on a luminescent substance coated on the surface of a substrate, and the energy transfer is realized by leading a photosensitive microsphere and a luminescent microsphere to be close to each other depending on antigen-antibody combination. Therefore, no separation process is required. The diameter of the nano microsphere is smaller, the suspension performance of the nano microsphere is stronger, and meanwhile, a three-level amplification luminescent system is adopted, so that the nano microsphere has higher analysis sensitivity; the whole detection process does not need cleaning, namely, the binding label and the binding label do not need to be separated, so the reaction time is shorter; the tracer substances (photosensitizer and luminous agent) are marked on the substrate instead of the biomolecule, so that the activity of the biomolecule is not influenced, and the substrate has a large specific surface area, so that more tracer substances and biomolecules can be coated on the surface of the substrate, and the performance of the substrate on the aspects of effective concentration and sensitivity of the reagent, detection background and the like is better.

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

The term "epitope" as used herein refers to, but is not limited to, any protein determinant capable of specifically binding to an immunoglobulin or T cell receptor. In some embodiments of the invention, an epitope is a region of the antigen surface that is capable of being specifically bound by an antibody. Epitope determinants may generally include chemically active surface groups of the molecule, such as, but not limited to: amino acids, sugar side chains, phosphoryl groups and/or sulfonyl groups. In other embodiments of the invention, epitopes may have specific three-dimensional structural characteristics as well as specific charge characteristics. In some cases, an epitope may represent a spatial structure on the surface of a molecule with specific binding properties, such as, but not limited to: a nucleic acid sequence that is fully or partially complementary.

II, technical scheme

The invention provides a preparation method of a human anti-mullerian tube laser homogeneous detection kit, which comprises the following steps:

The kit prepared by the method can be used for well and quantitatively detecting the existence or concentration of human anti-mullerian tube laser in a sample.

The first marker and the specific binding substance thereof can be liquid phase specific binding pair molecules which do not react and influence the spectral characteristics of the system, such as digoxin molecules and antibodies thereof, and can also be complementary double-end DNA single chains or complementary two-segment peptide nucleic acids.

In some embodiments of the invention, the method of making further comprises:

The kit may further comprise a packaging box. The packing box body can be internally provided with a heat-insulating interlayer, a refrigerant storage bag, a reagent box use instruction and other accessories.

Any component or composition of the kit can be contained in a separate container. The container surface may have identifying indicia. The identification mark may be selected from, but not limited to, a writeable label, a bar code, a two-dimensional code, a magnetic label, a wireless signal receiver, a wireless signal transmitter.

The method is for non-diagnostic purposes.

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

a. adding 10-30 mul of sample to be detected into the reaction hole;

c, incubating at 35-45 ℃ for 10-30 minutes;

d. adding 250 μ l of the third composition 100-;

e.35-45 ℃ for 5-20 minutes;

Example III

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

Materials and apparatus

A serum suspected of containing or comprising AMH.

Pure AMH product.

Two anti-AMH monoclonal antibodies, code numbers Ab1 and Ab2, wherein Ab1 is purchased from Aimeijie science and technology Limited company, and Ab2 is purchased from Shanghai Yubo Biotechnology Limited company.

Light-activated chemiluminescence detector: LICA-500 full-automatic analyzer, manufactured by Boyang Biotechnology (Shanghai) Limited.

(1) Preparation method of antibody-coated luminescent particles (R1 after being diluted by buffer solution)

Luminescent particles: the surface of the particle contains aldehyde group (-CHO), and is connected with antibody molecules through the aldehyde group. Chelate compounds containing a luminescent compound (derivative of dimethylthiophene) and a lanthanide (Eu) compound.

The receptor microspheres are available from platinum elmer limited under the designation 6762001.

Biological raw materials: anti-AMH antibody Ab 1.

The preparation process comprises the following steps: diluting 2mg of luminescent microparticle solution with 0.05M pH 9.6 Carbonate Buffer (CB) to 5mg/ml, transferring 0.02mg of antibody into microparticle tube, mixing well, and coating overnight at 4 deg.C; then adding 20 mu l of BSA solution diluted to 10mg/ml by using CB buffer solution, and rotating for 2h at room temperature; the microparticles were washed thoroughly and diluted to 100. mu.g/ml with 0.1M Tris-HCl solution, pH 8.0, as a working stock solution (R1)

(2) Preparation method of biotin labeled antibody (R2 after being diluted by buffer solution)

Activating biotin. Is NHS biotin.

Biological raw materials: anti-AMH antibody Ab 2.

The preparation process comprises the following steps: transfer 0.5mg of antibody into a 14KD dialysis bag and use with labeling buffer (0.1M NaHCO)₃) Dialyzing for 2 h/time, and changing the solution for 1 time; adding 10 μ l of 5mg/ml activated biotin solution, rapidly mixing, supplementing labeling buffer solution to 500 μ l, mixing at 2-8 deg.C overnight, and labeling at a ratio of 1:30 (antibody: biotin-molar ratio); taking the marked Bio-Ab reagent to a 14KD dialysis bag, dialyzing with a dialysis buffer solution (0.1M Tris-HCl) for 2 h/time, and changing the solution for 1 time; diluted to 5. mu.g/ml with 0.1M Tris-HCl solution, pH 8.0.

(3) Preparation method of photosensitive particles (R3 after being diluted by buffer solution) coated with streptavidin

The donor microsphere contains a photosensitive compound phthalocyanine dye (luminol-type chemiluminescent substance), and contains an active aldehyde group which is coated with streptavidin in advance.

Donor microspheres are available from platinum elmer ltd under the designation 6760002S.

The concentration of the reagent can be determined according to actual needs or diluted to the required concentration by a 0.1M Tris-HCl solution with the pH value of 8.0 when in use.

(4) Process for preparing an AMH calibrator

Taking the pure AMH product, preparing 0.5mL of each of 0, 0.16, 0.6, 4, 10 and 24ng/mL series of calibrator solutions by using 0.1M phosphate buffered saline with pH 7.4 and containing 20 percent inactivated calf serum, and preparing AMH working stock solution with the concentration of 200ng/mL, and diluting the AMH working stock solution by using 0.1M phosphate buffered saline with pH 7.4 according to requirements.

Principle of detection of kit

The core detection principle of the technical scheme of the human anti-mullerian tube laser (AMH) determination kit (light-excited chemiluminescence method) is shown in the attached figure 1, the detection reaction system is based on a light-excited chemiluminescence analysis technology and adopts a double-antibody sandwich analysis mode, and the detection reaction system comprises a first component, a second component, a third component and a sample to be detected.

The first component was a first antibody coated luminescent microsphere/microparticle (code no FG-Ab1) with human anti-mullerian kinase (AMH) binding specificity, and the composition containing this component was designated reagent 1 (R1).

The second component is Biotin labeled with a second antibody having binding specificity for AMH (code No. Bio-Ab2, Biotin-Ab2), and the composition comprising this component is referred to as reagent 2 (R2).

The third component is streptavidin-coated photosensitive microspheres/microparticles (code SA-GG), and the composition containing this component is referred to as reagent 3 (R3).

The sample to be tested is AMH, an AMH solution, a composition (possibly) containing AMH, serum, tissue fluid or tissue homogenate (possibly) containing AMH, and comprises but is not limited to AMH with known concentration, a series of calibrators with known AMH concentration, and a low-value and high-value quality control product of AMH.

Also comprises a chemiluminescence detector and related conventional reagents and consumables.

During detection, the components are in a liquid phase state, a sample to be detected, R1 and R2 are mixed in a warm bath, AMH in the sample to be detected is combined with FG-Ab1 and Bio-Ab2 respectively to form a double-antibody sandwich complex, then R3 is added into a reaction system, a streptavidin part of SA-GG is combined with biotin (Bio) in the double-antibody sandwich complex, luminescent particles and photosensitive particles are close to each other, energy or active compounds are contacted with the photosensitive particles, the photosensitive particles release singlet oxygen, and the singlet oxygen diffuses and is combined with the luminescent particles to induce the luminescent particles to generate optical signals. The singlet oxygen will be deactivated by diffusion beyond a distance of about 200nm, and thus the free luminescent particles will hardly gain energy and no light signal will be generated. Therefore, the intensity of the light signal is in a direct proportional function relation with the content of the double-antibody sandwich complex in the reaction system, when the molar content of AMH does not exceed the minimum value of FG-Ab1 and Bio-Ab2, the intensity of the light signal is in a direct proportional function relation with the content of AMH in the sample to be detected, and the AMH concentration level in the unknown sample can be calculated through a mathematical function formed by calibrating products with known content of AMH.

That is, mixing the clinical specimen or the calibrator with the R1 solution and the R2 solution, and respectively binding the AMH to be detected with the specific antibody on the surface of the luminescent microsphere and the specific antibody marked by biotin to form a double-antibody sandwich compound; at this point, there is an excess of both antibodies and there are antibody molecules in an unbound state. Adding a photosensitive microsphere coated by streptavidin, wherein the streptavidin is combined with a biotin molecule (comprising a compound or a free biotin antibody), but only the biotin in the combined compound (FG-Ab1-AMH-Bio-Ab2) can combine the luminescent particle and the photosensitive particle, and the active oxygen molecule is transferred under the action of energy or an active compound, such as laser irradiation, so that the luminescent particle generates a light signal. The free coated antibody luminescent particles (FG) are far away from the photosensitive particles (GG) and cannot generate optical signals. Therefore, the intensity of the optical signal is proportional to the AMH content of the specimen. And establishing a mathematical function relation by adopting a known AMH concentration calibrator and corresponding optical signal intensity, and obtaining the AMH concentration of an unknown sample through a quadratic function.

Compared with the existing serum AMH quantitative in-vitro detection reagent, the AMH quantitative detection kit provided by the invention has the following remarkable effects: the product belongs to homogeneous immunoassay, has no separation and washing process in the whole process, saves detection time, avoids errors caused by washing, and has higher precision and accuracy.

Example 1 basic kit

The core components of the kit are as follows:

the AMH quantitative determination detection kit (light-activated chemiluminescence method) of this example was composed of a composition (reagent 1, R1) comprising a luminescent microsphere (FG-Ab1) coated with a first anti-AMH monoclonal antibody, a composition (reagent 2, R2) comprising a biotin-labeled second anti-AMH monoclonal antibody (Bio-Ab 2); also comprises a sample to be detected. Further, a composition (reagent 3, R3) comprising streptavidin-coated photosensitive microspheres (SA-GG) prepared by the method of the aforementioned materials and devices, including a light-activated chemiluminescence analyzer, and the like, was included.

The method of using the kit of the present embodiment comprises:

the detection process is completed by an automatic light-activated chemiluminescence analysis system in a full-automatic manner, and a detection result is output. The detection principle comprises the following specific steps:

a. adding a sample to be detected into the reaction hole;

b. sequentially adding R1 and R2 into the reaction hole;

c. incubation;

d. adding R3 into the reaction hole;

e. incubation;

f. irradiating the reaction holes by laser and calculating the quantity of light-emitting photons of each hole;

optionally, calculating the sample concentration.

When AMH to be detected exists in a detection system, the AMH is specifically combined with a luminescent microsphere coated with a first anti-AMH monoclonal antibody and a second anti-AMH monoclonal antibody combined with biotin at the same time, and a double-antibody sandwich compound is formed on the surface of the luminescent microsphere; at this time, if the streptavidin-coated photosensitive microspheres are added, biotin and streptavidin are combined to enable the two microspheres to approach each other, and under the excitation of an excitation light source, the photosensitive microspheres release singlet oxygen to generate chemiluminescence after contacting with the luminescent microspheres in a solution, so that the fluorescent groups on the same microsphere are further excited to generate cascade amplification reaction to generate fluorescence. At this time, the more AMH to be detected, the stronger the fluorescence intensity, and the content of AMH in serum is quantitatively detected according to the intensity of luminescence.

Example 2 core kit

The experiment of the invention:

preparation of kit materials:

the preparation method of the specific material and the corresponding reagent thereof is prepared according to the material and equipment.

The method of using the kit of the present embodiment comprises:

reagents are added into different reaction wells in parallel, detection is carried out in parallel, excitation light with the wavelength of 680nm is used for irradiating the reaction wells, and emission light with the wavelength of 612nm is detected.

The specific detection steps are as follows:

a. adding 25 mul of samples into different reaction holes respectively; the AMH concentration in the calibrator sample is respectively 0, 0.16, 0.6, 4, 10 and 24ng/mL, and the AMH concentration in the sample to be tested is shown in the lower half part of the table 1.

b. 25 mu l R1 and 25 mu l R2 were added to each reaction well in sequence;

c.37 ℃ temperature 15 minutes;

d. adding R3175 μ l into each reaction hole;

e.37 ℃ incubation for 10 min;

f. the laser light irradiates the micropores independently and calculates the quantity of light photons emitted from each hole.

Wherein R1 is FG-Ab1 of 50 mu g/mL;

r2 is Biotin-Ab2 at 5. mu.g/mL;

r3 is 50. mu.g/mL SA-GG.

The specific luminescence values (calibration) of the calibrator are shown in the upper part of table 1, and the luminescence values of the samples to be tested are not shown in table 1.

Control test:

the kit comprises: roche AMH detection kit.

The detection principle is electrochemical luminescence immunoassay.

The total detection time is 18min by using a double antibody sandwich method, and the detection steps are as follows:

first incubation: the 50uL sample solution, a biotin-labeled first anti-AMH specific monoclonal antibody and a ruthenium complex-labeled second anti-AMH specific monoclonal antibody react to form an antigen-antibody sandwich complex.

And (3) incubation for the second time: after adding streptavidin-coated magnetic bead particles, the complex is bound to the solid phase through the interaction of biotin and streptavidin.

And (3) sucking the reaction liquid into a measuring cell, and adsorbing magnetic beads on the surface of the electrode through electromagnetic action. The material not bound to the magnetic beads was removed by ProCell/ProCell M. The electrodes are given a voltage to cause the compound to chemiluminesce and the intensity of the luminescence is measured by a photomultiplier.

The final detection result is obtained by the calibration curve of the detector, which is obtained by 2-point calibration and the primary calibration curve obtained on the reagent bar code.

Detecting a sample: 0.1M AMH working stock diluted in phosphate buffered saline pH 7.4. The concentrations of the samples tested are seen in the lower part of table 1.

The detailed test method was performed with reference to the procedures of the Roche AMH assay kit instructions.

The detection instrument is as follows: LICA-500 full-automatic analyzer, manufactured by Boyang Biotechnology (Shanghai) Ltd. The concentration values were calculated according to the instrument's own software.

TABLE 1 kit calibration and control test 1

The principle, method, materials and operation steps are the same as above, and 2 different batches of samples are detected, and the specific information of labeled concentration, luminous value, concentration value and the like is shown in tables 2 and 3.

TABLE 2 kit calibration and control tests 2

TABLE 3 kit calibration and control test 3

In the three batches of detection, the relationship between the AMH concentration in the standard substance and the luminescence value is used for calculating and fitting the functional relationship between the AMH concentration in the sample to be detected and the luminescence value, the luminescence value of the sample to be detected is used for calculating the AMH concentration in the sample to be detected according to the functional relationship, and the luminescence value of the sample to be detected and the AMH concentration calculated by the luminescence value in tables 1, 2 and 3 are not shown.

By using the self-contained hardware and software and programming, the LICA-500 full-automatic analyzer can not only display the luminous value, but also directly display the concentration value of AMH in the sample calculated by the functional relation between the concentration of AMH and the luminous value.

For the Roche kit, the Roche software system calculates the relationship between the standard substance and the luminescence value (the luminescence value of the calibrator is not shown in tables 1, 2 and 3), then the luminescence value of the sample to be detected is directly used to obtain the concentration value of the AMH in the sample to be detected, and the concentration value of the AMH in the sample to be detected obtained through specific calculation is referred to the lower half parts of tables 1, 2 and 3.

Compared with the roche kit, aiming at 27 samples to be detected in three batches, the AMH concentration obtained by the method of the embodiment of the invention is used as a y value (ordinate y axis), the AMH concentration obtained by the roche kit is used as an x value (abscissa x axis), and a functional relation between the AMH concentration and the x value is calculated to obtain a formula: y 1.0408x + 0.0337. The two linear relationships are visualized as shown in fig. 2.

As can be seen from the functional relation, the linear relation between the two is good, and the correlation R between the measured value of the sample for 3 times and the Roche definite value is obtained by using the method of the invention²The correlation was good when 0.9915. As can be seen from fig. 2, the overall linear correspondence between the two is good. The effect of the invention is close to the detection effect of the Roche kit.

However, the invention adopts homogeneous detection, and has no separation and purification processes of magnetic beads and the like, thereby reducing the operation steps in a liquid phase, reducing the detection time and reducing the possibility of accumulated errors. The complexity of the measurement instrument score is also reduced.

Example 3 precision testing

The significance of precision is as follows: precision is an important index for measuring the variation of in-vitro detection reagents in batches and among batches, is an important basis for evaluating the effectiveness of products, and generally comprises the analysis of the in-batch precision and the inter-batch precision.

The basic idea of the material and method of this embodiment is the same as that of embodiment 2, and the difference between the two is that the AMH concentration in the sample to be detected is different, and the specific detection steps are as follows:

a. adding 25 mul of samples into different reaction holes respectively; the AMH concentration in the calibrator sample is respectively 0, 0.16, 0.6, 4, 10 and 24 ng/mL; samples to be detected are samples with low (L), medium (M) and high (H) values;

b. 25 mu l R1 and 25 mu l R2 were added to each reaction well in sequence;

c.37 ℃ temperature 15 minutes;

d. adding R3175 μ l into each reaction hole;

e.37 ℃ incubation for 10 min;

f. the laser respectively and independently irradiates the micropores and calculates the quantity of light photons emitted by each hole;

g. the sample concentration was calculated.

Wherein R1 is FG-Ab1 of 50 mu g/mL;

r2 is Biotin-Ab2 at 5. mu.g/mL;

r3 is 50. mu.g/mL SA-GG.

And fitting a functional relation between the AMH concentration and the luminous value according to the AMH concentration and the luminous value in the calibrator, and calculating the AMH concentration in the sample to be tested by using the functional relation.

The evaluation method of the precision in the batch comprises the following steps: independent analysis of 3 batches of product was performed using low (L), medium (M), high (H) value samples, the assay was repeated 10 times for each batch, and the average of the 10 measurements was calculated

And Standard Deviation (SD), according to the formula

The Coefficient of Variation (CV) was calculated.

The method for evaluating the batch precision comprises the following steps: independent analysis of 3 batches of product was performed using low (L), medium (M) and high (H) value samples, the assay was repeated 10 times for each batch, and the average of 30 measurements was calculated

And Standard Deviation (SD), according to the formula

The Coefficient of Variation (CV) was calculated.

The specific concentration of the calibrator, the luminescence value thereof, the luminescence value of the samples to be measured in the batch, and the 10-time average luminescence value of each of the AMH concentrations calculated according to the functional relationship between the AMH concentration and the luminescence value in the calibrator are shown in Table 4.

The mean, standard deviation and coefficient of variation of the AMH concentration calculated for each of the three samples as a function of the AMH concentration and luminescence value in the calibrator are shown in table 5.

The mean, standard deviation and coefficient of variation of the AMH concentration calculated from the AMH concentration as a function of the luminescence value in the calibrator for the present time for three samples are shown in table 6.

TABLE 4 precision measurement raw data

TABLE 5 serum AMH quantitation reagent (light activated chemiluminescence) assay precision within batch

TABLE 6 serum AMH quantitation reagent (light activated chemiluminescence) batch-to-batch precision

As can be seen from tables 4, 5 and 6, the kit prepared by the method of the present invention has an intra-batch variation coefficient and an inter-batch variation coefficient of less than 5%, which are both about 3% in total, indicating that the kit prepared by the method of the present invention has good repeatability and small random error when used for detection.

Example 4 accuracy testing

The accuracy significance is as follows: the coincidence degree of the measured value and the actual value reflects the magnitude of the system error.

The accuracy evaluation method comprises the following steps: the 2 samples containing different levels of AMH were diluted in calibrator matrix solution at multiple points and the recovery was calculated from the dilution ratio.

The basic idea of the materials and methods of this example is the same as that of example 2, the difference between them is that the concentrations of the reactants are different, and the specific detection steps are as follows:

a. adding 25 mul of samples into different reaction holes respectively;

b. 25 mu l R1 and 25 mu l R2 were added to each reaction well in sequence; the AMH concentration in the calibrator sample is respectively 0, 0.16, 0.6, 4, 10 and 24 ng/mL; the sample to be tested is used according to the concentration gradient as follows;

c.37 ℃ temperature 15 minutes;

d. adding R3175 μ l into each reaction hole;

e.37 ℃ incubation for 10 min;

g. the sample concentration was calculated.

Wherein R1 is FG-Ab1 of 50 mu g/mL;

r2 is Biotin-Ab2 at 5. mu.g/mL;

r3 is 50. mu.g/mL SA-GG.

In the use of the kit of this example, two high-value AMH sera were taken, and the original concentration thereof was measured by adding 0.1M pH 7.4 phosphate buffered saline and 1/2, 1/4, 1/8, 1/10 times diluted samples to different wells according to the measurement method determined in example 2.

The expected concentration and the measured concentration of the samples at concentrations 1/2, 1/4, 1/8, 1/10-fold diluted were compared using the measured values of the original concentrations as standards to evaluate the accuracy of the assay of the kit.

The gradient dilution can ensure the relative concentration proportional relation of AMH in the series of samples, has smaller system error than the test of independently preparing or collecting samples respectively, and can be well used for evaluating the service performance of the kit.

The specific luminescence values of the calibrator, the sample to be tested and the calculated concentration are shown in Table 7.

TABLE 7 accuracy raw data

TABLE 8 accuracy of AMH quantitative determination reagent (light-activated chemiluminescence method) for first serum

TABLE 9 accuracy of second serum AMH quantitative determination reagent (light activated chemiluminescence method)

From table 7, table 8 and table 9, it can be seen that the recovery rates of the measurement after the multi-point dilution with 2 AMH samples of different levels were all within the range of 97% to 105%, and particularly, for the first serum sample, the difference between the measured value and the predicted value was less than 1% at the first three dilutions, which indicates that the measured value and the true value are close to each other.

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

Claims

1. A preparation method of a homogeneous detection kit for human anti-mullerian hormone by a light-activated chemiluminescence method is characterized by comprising the following steps:

preparing a first composition by taking 2mg of a luminescent particle solution containing aldehyde groups and diluting the solution to 5mg/ml with 0.05M of a pH 9.6 carbonate buffer, and adding 0.02mg of a first monoclonal antibody capable of binding to a first epitope of human anti-Mullerian hormone; adding BSA solution diluted to 10mg/ml with carbonate buffer solution to obtain a first component; fully washing the particles, and diluting the particles to 100 mu g/ml by using a first buffer solution to prepare a first composition;

preparing a second composition, taking 0.5mg of a second monoclonal antibody capable of binding a second epitope of human anti-mullerian hormone, transferring the second monoclonal antibody into a 14KD dialysis bag, dialyzing the second monoclonal antibody by using a labeling buffer solution, adding 10 mu l of 5mg/ml activated biotin solution, supplementing the labeling buffer solution to 500 mu l for labeling, and continuously dialyzing the labeled biotin-second monoclonal antibody by using the 14KD dialysis bag to obtain a second component; diluting the second component to 5 μ g/ml with a second buffer to form a second composition;

preparing a third composition, and diluting a photosensitive microsphere coated with streptavidin by using a third buffer solution, wherein the photosensitive microsphere contains active aldehyde groups;

preparing human anti-mullerian hormone calibrator solution, and preparing 0ng/mL, 0.16ng/mL, 0.6ng/mL, 4ng/mL, 10ng/mL and 24ng/mL series of standard solutions by using 0.1M phosphate buffered saline solution with pH 7.4 and containing 20% inactivated calf serum;

wherein the first buffer, the second buffer and the third buffer are independently selected from a 0.1M Tris-HCl solution at pH 8.0.

2. A kit for homogeneously detecting human anti-mullerian hormone, which is prepared according to the preparation method of claim 1.

3. A method for homogeneous detection of human anti-mullerian hormone comprising performing a chemiluminescent detection using the kit of claim 2, which is a method for non-disease diagnostic purposes.

4. A method according to claim 3, characterized in that the method comprises the steps of:

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

step R3 of contacting an energy or active compound with said second mixture to excite the donor to produce singlet oxygen and the acceptor to react with said singlet oxygen received to generate a detectable chemiluminescent signal;

and step R4, detecting the existence and/or intensity of the chemiluminescence signal obtained in the step R3, so as to judge whether the human anti-Mullerian hormone exists in the sample to be detected and/or determine the content of the human anti-Mullerian hormone.

5. The method of claim 3, further comprising: preparing a standard curve of a chemiluminescence signal-human anti-mullerian hormone concentration by using a human anti-mullerian hormone calibrator solution; the standard curve is used for determining the content of human anti-mullerian hormone in the sample to be detected.

6. The method as claimed in claim 4, wherein in step R3, the second mixture is irradiated with excitation light having a wavelength of 600-700nm to excite the donor to generate singlet oxygen, and the acceptor reacts with the singlet oxygen to generate emission light having a wavelength of 520-620 nm; in step R4, the presence and/or intensity of the emitted light signal is detected, so as to determine whether human anti-mullerian hormone is present in the sample to be tested and/or to determine the content of human anti-mullerian hormone.

7. The method according to claim 5, characterized in that it comprises the steps of:

a. adding 10-30 mul of sample to be detected into the reaction hole;

b. sequentially adding 10-30 mu l of the first composition and 10-30 mu l of the second composition into reaction holes;

c. incubating at 35-45 deg.C for 10-30 min;

d. adding 100-250 mu l of the third composition into a reaction hole;

e. incubating at 35-45 deg.C for 5-20 min;

g. the amount of photons emitted per well was measured and the concentration of human anti-mullerian hormone was calculated from the standard curve.

8. Use of a method according to any one of claims 3 to 7 for detecting the presence, absence and/or amount of human anti-mullerian hormone in a test sample.

9. Use or use of the method according to claim 1 for the preparation of a reagent, kit, test device or test system for assessing ovarian reserve, diagnosing a childhood sexual development disorder, assessing infertility, diagnosing polycystic ovarian syndrome, or predicting menopause time.

10. Use or use of the method of claim 1, the kit of claim 2 or the method of any one of claims 3-7 in predicting downtime.