CN108445238A - Detect the homogeneous immunological detection reagent suit and its preparation method and application of interstitialcellstimulating hormone (ICSH) - Google Patents
Detect the homogeneous immunological detection reagent suit and its preparation method and application of interstitialcellstimulating hormone (ICSH) Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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Abstract
The present invention relates to the homogeneous immunological detection reagent of a kind of detection interstitialcellstimulating hormone (ICSH) of biotechnology suits and its preparation method and application.The homogeneous immunological detection reagent suit of detection interstitialcellstimulating hormone (ICSH) prepared by the method for the invention, when being analyzed using light-induced chemiluminescent immunoassay platform, high sensitivity, precision are good, and by optimizing analysis system (antibody dosage and buffer solution etc.), analytical performance meets professional standard, and reference is provided for the clinical monitoring onset of ovulation.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of homogeneous immunological detection reagent of detection interstitialcellstimulating hormone (ICSH)
Suit and its preparation method and application.
Background technology
Interstitialcellstimulating hormone (ICSH) is also known as luteotropin (luteinizing hormone, LH), is thin by anterior pituitary basophil
The glycoprotein of intracrine.Molecular weight about 30000.Chemical constitution：For glycoprotein, by two subunit peptide chains of a and b with Covalent bonding together
It forms.In the presence of follicular stimulating hormone, conjunction with which effect stimulates ovarian estrogen secretion, makes follicle maturity and ovulation, make
Ruptured follicle forms corpus luteum and secretes estrogen and progestational hormone.Stimulation interstitial glands develop and promote its Testosterone Secretion.Therefore
Also known as interstitial cell augmentor.
For the detection method of interstitialcellstimulating hormone (ICSH), no matter radiommunoassay, EIA enzyme immunoassay or chemiluminescence immunoassay
Analysis is required to the labelled antibody that reaction is not participated in removal since labelled antibody is excessive before detection.Existing heterogeneous immune point
Analysis method is required for cumbersome washing step, and still, repeatedly washing will necessarily be that immunoassay brings washing " error ", due to washing
Process especially EIA enzyme immunoassay, it is difficult to realize and standardize, washing effect is different between each instrument connection, to a certain extent shadow
The precision of sound detection between appearance batch, criticizes interior difference, to accuracy and the analysis susceptibility of impact analysis method.Meanwhile it needing
Overcome radiommunoassay environmental pollution, the short defect of shelf life to overcome EIA enzyme immunoassay poor repeatability, easily occurs " hook-shaped
Effect " defect.
Therefore, it is necessary to research and develop a kind of rush corpus luteum generation having compared with high sensitive and precision, without washing step
Plain detection method.
Invention content
The problem to be solved in the present invention is to provide a kind of homogeneous immune detection examination preparing detection interstitialcellstimulating hormone (ICSH) (LH)
Agent is set with, and the interstitialcellstimulating hormone (ICSH) in homogeneous immunization method detection sample to be tested can be used in the reagent set obtained by this method,
Entire detection process not only saves detection time, and not will produce additional waste liquid without separating, washing process, exempts washing error,
With higher precision, accuracy and sensitivity, analytical performance meets professional standard and clinical labororatory requires.
The present invention provides a kind of homogeneous immunological detection reagent suit preparing detection interstitialcellstimulating hormone (ICSH) in first aspect
Method comprising：
First chamber is prepared, the first chamber includes component a and the first buffer solution, and wherein component a is by energy
It is enough that the receptor for generating detectable signal and first antibody in combination or its binding fragment composition are reacted with singlet oxygen；Institute
Stating first antibody or its binding fragment can be combined with the first epitope specificity of interstitialcellstimulating hormone (ICSH)；
Second chamber is prepared, the second chamber includes component b and the second buffer solution, and wherein component b is and the
The secondary antibody that can be combined with the second epitope specificity of interstitialcellstimulating hormone (ICSH) or its binding fragment structure that one marker combines
At, and second epitope and the first epitope non-overlapping；
Third composition is prepared, the third composition includes component c and third buffer solution, wherein the component c is
The donor that singlet oxygen can be generated in excited state that first marker specific junction mixture combines.
First marker of the present invention and the first label combination are not limited to biotin and Streptavidin.
In certain embodiments of the present invention, the first antibody and secondary antibody be selected from monoclonal antibody and/or
Polyclonal antibody.
In other embodiments of the present invention, the receptor includes olefin(e) compound and metallo-chelate, is non-
Particulate forms, and it is solvable in water-bearing media；And/or the receptor is the high score filled with luminophor and lanthanide series
Sub- particle；
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.
In certain embodiments of the present invention, the method further includes preparing interstitialcellstimulating hormone (ICSH) calibration object to be serially diluted
Liquid.
In certain specific embodiments of the invention, the interstitialcellstimulating hormone (ICSH) calibration object serial dilutions it is a concentration of
0~200mIU/mL.
In certain embodiments of the present invention, in the first chamber component a a concentration of 0.1~1.0mg/mL；
Preferably, in the first chamber component a a concentration of 0.35~0.65mg/mL.
In other embodiments of the present invention, a concentration of 1~10 μ g/mL of component b in the second chamber；It is excellent
Selection of land, a concentration of 3.5~6.5 μ g/mL of component b in the second chamber.
In certain embodiments of the present invention, in the third composition component c a concentration of 50~150 μ g/mL；It is excellent
Selection of land, a concentration of 80~120 μ g/mL of component c in the third composition.
In certain embodiments of the present invention, the preparation method of the first chamber includes：
Receptor is diluted to 1~10mg/mL using carbonate buffer solution, obtains receptor solution by step S1；
First antibody or its binding fragment are added in receptor solution, is added after static and uses carbonate buffer by step S2
Liquid is diluted to the BSA solution of 5~15mg/mL, obtains the receptor solution combined with first antibody or its binding fragment；
Step S3, detach in the receptor solution that is combined with first antibody or its binding fragment with first antibody or its combine
The receptor that segment combines, and the first buffer solution is added, obtain first chamber.
In other embodiments of the present invention, the preparation method of the second chamber includes：
Secondary antibody or its binding fragment are placed in bag filter and are dialysed using label buffer solution, dialysed by step T1
After the first marker solution is added, and fill into elution buffer, it is static, obtain the secondary antibody combined with the first marker
Or its binding fragment；
The secondary antibody combined with the first marker or its binding fragment are placed in bag filter slow using dialysis by step T2
Fliud flushing is dialysed, and the second buffer solution is added after dialysis, obtains second chamber solution.
In certain embodiments of the present invention, the NaHCO that the label buffer solution is 0.05~0.15M3Solution；It is described
Elution buffer is the Tris-HCl solution of 0.05~0.15M.
In other embodiments of the present invention, mole of the secondary antibody or its binding fragment and the first marker
Than being 1:(28~32).
In certain embodiments of the present invention, it is 7.5 that first buffer solution and the second buffer solution, which are pH value,
The Tris-HCl solution of~8.5 0.05~0.15M.
The second aspect of the present invention provides a kind of interstitialcellstimulating hormone (ICSH) prepared by method as described in the first aspect of the invention
Homogeneous immunological detection reagent suit.
Third aspect present invention provides a kind of using reagent set detection rush corpus luteum as described in respect of the second aspect of the invention
The homogeneous immunization method of element is generated, the method detects for light-induced chemiluminescent.
In certain embodiments of the present invention, the method includes：
Sample to be tested is mixed with first chamber and second chamber, obtains the first mixture by step R1；
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 wait for test sample to judge to survey
It whether there is interstitialcellstimulating hormone (ICSH) in product.
In other embodiments of the present invention, the method further includes：Utilize interstitialcellstimulating hormone (ICSH) calibration object series
Dilution makes the step of standard curve of chemiluminescence signal-interstitialcellstimulating hormone (ICSH) concentration；The standard curve is for determining
The content of interstitialcellstimulating hormone (ICSH) in sample to be tested.
In the present invention, after all reagent combinations, mixing can be carried out as needed and/or incubates (incubation).Specifically,
The temperature of the incubation can be 35-45 DEG C, and the time can be 5-30min；Preferably, the temperature of the incubation can be selected from 36
DEG C, 37 DEG C, 38 DEG C, 39 DEG C, 40 DEG C, 41 DEG C or 42 DEG C；The time of incubation can be selected from 10min, 12min, 15min, 16min,
18min, 20min, 25min are stayed overnight.
In certain specific embodiments of the invention, it the described method comprises the following steps：
A. the sample to be tested of 10~30 μ L is 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,
A concentration of 0.2~0.8mg/mL of component a in middle first chamber, a concentration of 2~8 μ g/ of the component b in second chamber
mL；
C.35~45 DEG C incubation 10~30 minutes；
D. 100~250 μ L of the third composition are added into reacting hole；Component c's wherein in third composition is dense
Degree is 50~150 μ g/mL；
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 is touched
Singlet oxygen reaction produces the transmitting light of 612nm；
G. the photon amount for detecting transmitting light in each reacting hole, the concentration of interstitialcellstimulating hormone (ICSH) is calculated according to standard curve.
Here, it should be strongly noted that the above method is the method for non-disease diagnostic purpose.
Fourth aspect present invention provides a kind of reagent set as described in respect of the second aspect of the invention and is preparing for monitoring
Application in the kit of the onset of ovulation.
Beneficial effects of the present invention are：The homogeneous immune detection of detection interstitialcellstimulating hormone (ICSH) prepared by the method for the invention
Reagent set, when being analyzed using light-induced chemiluminescent immunoassay platform, high sensitivity, precision are good, and pass through optimization
Analysis system (antibody dosage and buffer solution etc.), analytical performance meets professional standard, and reference is provided for the clinical monitoring onset of ovulation.
Description of the drawings
To make the present invention be readily appreciated that, illustrate the present invention below in conjunction with the accompanying drawings.
Fig. 1 is the reaction mechanism figure in the homogeneous immunological detection reagent suit for detect interstitialcellstimulating hormone (ICSH)；Wherein：1 anti-LH
The luminous microballoon that antibody combines；The 2 anti-LH antibody combined with biotin；3 LH to be measured；4 combined with Streptavidin it is photosensitive micro-
Ball.
Fig. 2 is LH calibration graphs.
Specific implementation mode
To make the present invention be readily appreciated that, the present invention is described more detail below.But before describing the present invention in detail, it should be understood that
The present invention is not limited to the specific implementation modes of description.It is also understood that term used herein is embodied only for description
Mode, and be not offered as restrictive.
In the case where providing numberical range, it should be understood that in the upper and lower bound of the range and the prescribed limit
Any other regulation or between two parties each of between numerical value numerical value is encompassed by the present invention between two parties.These small range of upper limits
It can independently be included in smaller range with lower limit, and be also covered by within the present invention, obey any clear in prescribed limit
The limit of exclusion.Defined range include one or two limit in the case of, exclude any of the limit that those include or
The range of the two is also included in the present invention.
Unless otherwise defined, the usual reason of all terms used herein and those skilled in the art
Solve meaning having the same.Although similar or equivalent any method and material also can be with method described herein and material
It is used in the implementation or test of the present invention, but preferred method and material will now be described.
I, terms
English corresponding to term " homogeneous " of the present invention is defined as " homogeneous ", and referring to need not be to combining
Antigen antibody complex and remaining free antigen or antibody detached and can be both detected.
Term " sample to be tested " of the present invention refers to that may include containing a kind of mixture of analyte, analyte
But it is not limited to protein, hormone, antibody or antigen.The typical sample to be tested that can be used in method disclosed by the invention includes
Body fluid, such as serum, urine.Sample to be tested can be using it is preceding it is as needed using dilution or buffer solution to that may contain
The sample of analyte be diluted after solution.For example, in order to avoid HOOK effects, sample can be used before upper machine testing
Dilution is detected on detecting instrument again after being diluted analyte, at this time may be after the dilution containing analyte
Solution be collectively termed as sample to be tested.
Term " antibody " of the present invention and " immunoglobulin " are used with most wide meaning, include the antibody of any isotype
Or immunoglobulin, retain the antibody fragment of the specific binding to antigen, including but not limited to Fab, Fv, scFv and Fd piece
Section, chimeric antibody, humanized antibody, single-chain antibody, bispecific antibody and the antigen-binding portion thereof comprising antibody and non-antibody
The fusion protein of albumen.In the case of any need, antibody can further with other parts, such as specific binding pair
Member, such as biotin or Streptavidin (a member in biotin-Streptavidin specific binding pair member) etc. are sewed
It closes.
Term " monoclonal antibody " of the present invention refers to the immunoglobulin secreted by the bone-marrow-derived lymphocyte of monoclonal,
It can be prepared by method known in those skilled in the art.
Term " polyclonal antibody " of the present invention refers to the immune globulin generated by more than one bone-marrow-derived lymphocyte clone
White set, can be prepared by method known in those skilled in the art.
Term " antigen " of the present invention is to refer to stimulation body to generate immune response, and with immune response product can resist
Body and sensitized lymphocyte combine in vivo and in vitro, and the substance of immunological effect occurs.
Term " in conjunction with " of the present invention refers to due to interactions such as example covalent, electrostatic, hydrophobic, ion and/or hydrogen bonds,
Including but not limited to two intermolecular direct joints caused by such as salt bridge and the interaction of water bridge.
Term " specific binding " of the present invention refers to that mutual discrimination between two kinds of substances and selective binding are anti-
Answer, said from stereochemical structure angle be exactly conformation between corresponding reactant correspondence.
Term " biotin " of the present invention is widely present in animal vegetable tissue, there are two cyclic structure on molecule,
Respectively imidazolone ring and thiphene ring, wherein imidazolone ring are the main portions combined with Streptavidin.The biotin of activation
It can be coupled with known almost all creatures macromolecular under the mediation of protein cross agent, including protein, nucleic acid, more
Sugar and lipid etc.；And " Streptavidin " is a kind of protein secreted by streptomycete, molecular weight 65kD." Streptavidin "
Molecule is made of 4 identical peptide chains, wherein every peptide chain can combine a biotin.Therefore each antigen or antibody can be same
When be coupled multiple biotin molecules, to generate " tentacle effect " improve sensitivity for analysis.In the case of any need, this hair
Any reagent used in bright, including antigen, antibody, receptor or donor, can according to actual needs biotin-conjugated-strepto- it is affine
Any member in plain specific binding pair member.
Term " donor " of the present invention refers to that can generate after the activation by energy or reactive compound and receptor
The sensitizer of the reactive intermediate of such as singlet oxygen of reaction.Donor can be photoactivation (such as dyestuff and aromatic compound)
Or (such as enzyme, metal salt) of chemical activation.In particular embodiments of the invention, the donor is photosensitizer, described
Photosensitizer can be photosensitizer known in the art, preferably stable with respect to the light and not compound with singlet oxygen effecting reaction,
Its non-limiting example includes sub- disclosed in such as United States Patent (USP) US5709994 (patent document is hereby incorporated by reference)
The compounds such as methyl blue, rose-red, porphyrin, phthalocyanine and chlorophyll and these compounds have 1-50 replacing group
Derivative, the substituent group for so that these compounds with more lipophilicity or with more hydrophily, and/or as connection
To the linking group of specific binding pair member.The example of other photosensitizers well known by persons skilled in the art can also be at this
It is used in invention, such as the content described in United States Patent (USP) US6406913, which is hereby expressly incorporated by reference.At this
It invents in other specific embodiments, the donor is other sensitizers of chemical activation, and non-limiting example is certain
Compound, their catalyzing hydrogen peroxides are converted into singlet oxygen and water.The example of some other donor includes：1,4- dicarboxyl second
Base-Isosorbide-5-Nitrae-naphthalene endoperoxides object, 9,10- diphenylanthrancene -9,10- endoperoxides objects etc. heat these compounds or these changes
Conjunction object, which directly absorbs light, can discharge singlet oxygen.
Term " receptor " of the present invention is to refer to react the substance that can generate detectable signal with singlet oxygen.For
Body is by energy or reactive compound induced activation and the singlet oxygen for discharging upper state, and the singlet oxygen of the upper state is by low coverage
From receptor capture, to transmit energy to activate the receptor.In some specific embodiments of the present invention, the receptor is
Such substance, experience and the chemical reaction of singlet oxygen are to form unstable metastable state intermediate, in the metastable state
Mesosome can decompose, and subsequently or simultaneously shine.The exemplary of these substances includes but not limited to：Enol ether, enamine, 9- alkylidenes
Xanthans, 9- alkylidene-N- alkyl Acridane, aromatic ethylene ether, bicyclic ethylene oxide, thioxene, armaticity imidazoles or lucigenin.
In other specific embodiments of the present invention, the receptor is can to react that be formed ketone can be resolved into singlet oxygen
Or the hydroperoxides of carboxylic acid derivates or the olefines of dioxy cyclobutane；The stabilization dioxy ring that can be decomposed by the effect of light
Butane；It can be reacted with singlet oxygen to form the acetylene class of diones；Azo-compound or azo carbonyl compound can be formed
The hydrazone class or hydrazides of object, such as luminol；With the aromatic compounds that can form endoperoxides species.It can be according to the disclosure
Specific, the non-limiting examples of the receptor utilized with claimed invention are recorded in U.S. Patent number US5340716, and (this is special
Sharp document is hereby incorporated by reference).In other specific embodiments of the invention, the receptor includes olefin(e) compound and gold
The case where category chelate is non-particlized and solvable in water-bearing media, this receptor can be found in patent PCT/
US2010/025433 (patent document is hereby incorporated by reference).
In the present invention, the donor can be coated on to be formed on matrix by functional group to be filled with photosensitive chemical combination
The high molecular particle of object can generate singlet oxygen under light excitation, and donor is referred to as photosensitive microballoon or photosensitive micro- at this time
Grain, including the solution of this photosensitive microballoon or photosensitive particulate is properly termed as photosensitive liquid or general liquid；And/or the receptor can be with
It is to be coated on matrix to form the high molecular particle filled with luminophor and lanthanide series by functional group, at this time may be used
To be known as shine microballoon or luminous particle.In this application, system is excited based on the luminescent substance for being coated on matrix surface through light
With energy transmission induced luminescence signal, energy transmission is combined dependent on Ag-Ab causes photosensitive microballoon and luminous microballoon mutual
It is close and realize.There is no need to separation processes.The diameter smaller of nanoparticle, suspendability is stronger, while using three-level
Amplify luminescent system, thus there is higher sensitivity for analysis；Entire detection process is not necessarily to detach binding marker without cleaning
And binding label, therefore the reaction time is shorter；Probe material (emulsion and luminous agent) marks on matrix, rather than marks
In biomolecule, the activity of biomolecule is not influenced, meanwhile, because there are larger specific surface areas for matrix, therefore its surface
On can be coated with more probe materials and biomolecule, cause it in the effective concentration of reagent and sensitivity and detection background etc. side
The performance in face 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-porous
, with any density, but preferably have and the close density of water, can preferably float in water, and by transparent, partially transparent
Or opaque material is constituted.Described matrix can be with or without charge, when with charge, preferably negative electrical charge.The base
Body can be that solid (such as polymer, metal, glass, organic and inorganic matter such as mineral, salt and diatom), small oil droplet are (such as hydrocarbon
Compound, fluorocarbon, silicic fluid), vesica (such as phosphatide such as synthesis or natural such as cell and organelle
Official).Matrix can be latex particle or other particles, lipid bilayer such as liposome, phosphatide containing organic or inorganic polymer
Vesica, small oil droplet, silicon particle, metal-sol, cell and crystallite dyestuff.Matrix usually has multifunctionality, or can pass through
Special or non-specific covalently or non-covalently interaction and be attached on donor or receptor.Be available there are many functional group or
Person is merged in.Typical functional group includes carboxylic acid, acetaldehyde, amino, cyano, vinyl, hydroxyl, sulfydryl etc..It is suitable for
One unrestricted example of the matrix of the present invention is carboxy-modified latex particle.The details of this matrix can be found in
United States Patent (USP) US5709994 and US5780646 (this two patents document is hereby incorporated by reference).
Term " epitope " of the present invention is any egg for referring to specific binding immunoglobulin or T cell receptor
White determinant.In some specific embodiments of the present invention, epitope is that antigenic surface can be by the region of antibody specificity set.
Epitopic determinants usually may include the chemically active surface group of molecule, such as, but not limited to：Amino acid, carbohydrate side chain, phosphinylidyne
Base and/or sulfonyl.In some other specific embodiment of the present invention, epitope can specific specific three-valued structures feature and
Specific charge feature.
Term " homogeneous immunological detection reagent suit " of the present invention refers to the whole that homogeneous immune detection must use
The combination of reagent or medicament.
II, specific embodiments
The testing principle of the present invention is as shown in Figure 1：It is anti-in receptor surface coating mouse using homogeneous luminescent method of immunity
People's LH monoclonal antibodies, obtain the receptor combined with antibody；By the anti-human LH labeling of monoclonal antibodies biotin of another plant of mouse, prepare
The antibody combined with biotin；Meanwhile in donor pan coating Streptavidin, obtaining the donor combined with Streptavidin.
Its specific works mechanism is as follows：When detecting in system there are LH to be measured, the LH specificity with receptor surface simultaneously
Antibody and the specific antibody combined with biotin combine, and form double-antibody sandwich compound；Two kinds of antibody excess at this time, exist
The antibody molecule of unbound state.The donor combined with Streptavidin is added, Streptavidin is combined (packet with biotin molecule
Include compound or free biotin antibody), but only combine compound (FG-Ab-LH-Ab-Bio) in biotin, could incite somebody to action
The distance of receptor and donor furthers, and under the excitation of energy or reactive compound, donor discharges singlet oxygen, contacts in the solution
Chemiluminescence is generated after to receptor, it is glimmering to further excite the fluorophor on receptor to generate Cascaded amplification reaction generation
Light.The free receptor combined with antibody cannot obtain energy far from donor, generate optical signal.Therefore, light signal strength with
The proportional example functional relation of LH contents in sample.Mathematics is established using known LH concentration calibrations product and corresponding light signal strength
Functional relation can calculate the concentration level of unknown LH samples to be measured.
In certain specific embodiments of the invention, prepared by the method for the invention detects the equal of interstitialcellstimulating hormone (ICSH)
Phase immunologic function test reagent is set with, including：
(1) LH calibration objects serial dilutions：6 concentration are 200mIU/ml, 100mIU/ml, 50mIU/ml, 10m respectively
IU/ml、1mIU/ml、0mIU/ml；
(2) the anti-human LH monoclonal antibody solutions of mouse combined with biotin, a concentration of 1~10 μ g/mL；
(3) the receptor microspheres solution combined with the anti-human LH monoclonal antibodies of mouse, a concentration of 0.1~1.0mg/mL；
(4) the donor microspheres solution combined with Streptavidin.
The anti-human LH monoclonal antibodies of mouse are commercial reagents, are available.
The anti-human LH monoclonal antibodies of mouse should meet following leading indicator：
Specificity：Immunogene uses people source LH or recLH, has a good specificity with people LH, but with people TSH, FSH, hCG
Cross reacting rate is less than 0.01%；
Affinity (0~200mIU/ml) in detection range shows good combination, and it is bent can to obtain good dose-response
Line；
Potency enzyme-linked immunosorbent assay (ELISA) is more than 1/15000；
Purity protein A affinity chromatography is pure, and purity is more than 99%.
The luminous microballoon (receptor), photosensitive microballoon (donor) are commercial reagents.
Microballoon shine for being combined with the anti-human LH monoclonal antibodies of mouse.The microballoon that shines is modified with aldehyde radical, passes through aldehyde radical
It is connect with antibody molecule, forms the receptor microballoon combined with antibody.The microballoon that shines is interior to contain luminophor and lanthanide series
The chelate of object is closed, luminophor is the derivative of thioxene, and lanthanide compound can be Eu (TTA) 3/TOPO
Or Eu (TTA) 3/Phne.Receptor microballoon is purchased from platinum Elmer Co., Ltd, article No. 6762001.
Photosensitive microballoon (donor) has included Photoactive compounds phthalein mountain valley with clumps of trees and bamboo dyestuff (luminol class chemiluminescent substance), while photosensitive
Contain active aldehyde in microballoon, and is combined with Streptavidin.Photosensitive microballoon is purchased from platinum Elmer Co., Ltd, article No.
6760002S。
Further include opaque microwell plate, such as 96 hole reaction plate of homogeneous luminescent in the reagent set.
III, embodiments
To keep the present invention easier to understand, below in conjunction with embodiment, present invention be described in more detail, these realities
Apply example only serve it is illustrative, it is not limited to application range of the invention.If the raw material used in the present invention or component nothing
Specified otherwise can be made by commercial sources or conventional method.
Embodiment 1：Prepare the homogeneous immunological detection reagent suit of detection interstitialcellstimulating hormone (ICSH)
(1) preparation of the luminous microballoon combined with antibody：
Receptor as the present embodiment is to be coated on to be formed on latex particle containing aldehyde radical (- CHO) to be filled with dimethyl thiophene
The particle (luminous microballoon) of the derivative of pheno and the chelate of lanthanide series Eu.Receptor is connect by aldehyde radical with antibody molecule.
Biological raw material：LH monoclonal antibodies
Preparation process：It takes 2mg to shine microspheres solution, the carbonate buffer solution (CB) of 0.05M pH 9.6 is used in combination to be diluted
At 5mg/ml；Take LH monoclonal antibodies 0.02mg be transferred to dilution after luminous microspheres solution in, mix well, 4 DEG C overnight；Again
The BSA solution 20uL that 10mg/ml is diluted to the CB buffer solutions of 0.05M pH 9.6 are added, room temperature rotates 2h；It fully washs micro-
Microballoon is finally diluted to 0.5mg/ml with the Tris-HCl solution of 8.0 0.1M of pH, obtains reagent R1 by ball.
(2) preparation process of the antibody combined with biotin labeling
Biological raw material：Activated biotin and FSH monoclonal antibodies
Preparation process：LH monoclonal antibodies 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 biotin solution 10ul of 5mg/ml, rapid mixing, supplemental markers buffer solution is added
To 500 μ l, 2-8 DEG C of mixing overnight, label ratio is 1:30 (antibody:Biotin-molar ratio)；The Bio-Ab that label is completed turns
14KD bag filters are moved to, is dialysed with elution buffer (0.1M Tris-HCl), 2h/ times, changes liquid 1 time；With 8.0 0.1M of pH
Tris-HCl solution is diluted to 5 μ g/ml, obtains reagent R2.
(3) preparation of the donor combined with Streptavidin
A, photosensitive microballoon (donor) suspension processing：Draw a certain amount of photosensitive microballoon in high speed freezing centrifuge from
The heart discards supernatant, and a certain amount of MES buffer solutions are added, and ultrasound suspends again to particle on ultrasonic cell disintegration instrument, and it is slow that MES is added
Fliud flushing adjusts photosensitive microballoon concentration to 100mg/ml.
B, solution of streptavidin is prepared：A certain amount of Streptavidin is weighed, adds MES buffer solutions to 8mg/ml.
C, it mixes：Photosensitive microballoon (donor) suspension handled well, the solution of streptavidin of 8mg/ml and MES is slow
Fliud flushing, with 2:5:1 volume ratio is mixed, and rapid mixing obtains reaction solution.
D, it reacts：The NaBH of MES buffers 25mg/ml3CN solution, according to reaction solution 1:25 volume ratio adds
Enter, rapid mixing, 37 DEG C of revolving reactions 48 hours.
E, it closes：The Gly solution of MES buffers 75mg/ml and the NaBH of 25mg/ml3CN solution, according to it is anti-
Answer liquid 2:1:10 volume ratio is added in above-mentioned solution, mixing, 37 DEG C of revolving reactions 2 hours.The BSA for adding 200mg/ml is molten
Liquid (MES buffer solutions) is 5 with reaction solution volume ratio:8, rapid mixing, 37 DEG C of revolving reactions 16 hours.
F, it cleans：MES buffer solutions are added into completely reacted solution, supernatant is abandoned in high speed freezing centrifuge centrifugation, is added new
Fresh MES buffer solutions ultrasonic method suspends again, centrifuges again, so cleaning 3 times, is finally suspended with photo-sensitire reagent buffer solution,
Obtain third composition.Wherein, in third composition component c a concentration of 100 μ g/mL, used as general liquid, obtain reagent
R3。
(4) preparation process of LH calibration objects serial dilutions
LH sterlings are taken, are configured to respectively with 7.4 phosphate buffered saline solutions of 0.1M pH containing 20% inactivation calf serum
0.5ml 0,1,10,50,100,200mIU/mL calibration object serial dilutions.
Embodiment 2：Interstitialcellstimulating hormone (ICSH) in the reagent set detection sample prepared using the method for the invention.
Used reagent set is the homogeneous immunological detection reagent set for the interstitialcellstimulating hormone (ICSH) that embodiment 1 is prepared.
Detection process is automatically finished and exports testing result, specific steps by automatic light-induced chemiluminescent analysis system：
1) 10 μ l samples to be tested or calibration object, quality-control product are separately added into reacting hole；
2) 25 μ LR1 and 25 μ l R1 are sequentially added in reacting hole；
3) it incubates 15 minutes for 37 DEG C；
4) 175 μ l R3 are added；
5) it incubates 15 minutes for 37 DEG C；
6) signal value of laser irradiation micropore and calculating per hole；Standard curve is drawn according to the signal value (table 1) of calibration object,
As shown in Figure 2.Then it by the signal value of the standard curve of drafting and sample to be tested, calculates and promotees corpus luteum generation in sample to be tested
The concentration of element.
Table 1：The testing result of calibration object
| Calibration object is numbered | Sign value (IU/L) | Hole 1 | Hole 2 | Mean value |
| 1 | 0 | 403 | 399 | 401 |
| 2 | 2 | 7785 | 8247 | 8016 |
| 3 | 10 | 38532 | 37357 | 37944.5 |
| 4 | 25 | 98158 | 90733 | 94445.5 |
| 5 | 100 | 362174 | 387963 | 375068.5 |
| 6 | 200 | 713977 | 712917 | 713447 |
Embodiment 3：The detection precision of reagent set prepared by the method for the invention
Precision is to weigh the important indicator of reagent within-run and between-run analysis, is that the important of commercialized product validity is intended in evaluation
Foundation generally includes withinrun precision and betweenrun precision.
Withinrun precision appraisal procedure：With low (L), high (H) value sample, independent analysis is carried out to the product of 3 batches, often
A batch replication 10 times (being measured using method described in embodiment 2) calculates the average value of 10 measurement results
(x) and standard deviation (SD) calculates the coefficient of variation (CV), the results are shown in Table 2 according to formula CV=SD/x × 100%.
Betweenrun precision appraisal procedure：With low (L), high (H) value sample, independent analysis is carried out to the product of 3 batches, often
A batch replication 10 times (being measured using the method that embodiment 2 describes), calculates the average value (x) of 30 measurement results
The coefficient of variation (CV) is calculated, the results are shown in Table 3 according to formula CV=SD/x × 100% with standard deviation (SD).
Table 2：The withinrun precision of reagent set prepared by the method for the invention
Table 3：The betweenrun precision of reagent set prepared by the method for the invention
| Quality-control product | X (n=30) | SD | CV (%) |
| L | 10.38 | 0.3870 | 3.73 |
| H | 46.95 | 2.9785 | 6.34 |
From table 2 and table 3 it is found that the withinrun precision of reagent set prepared by the method for the invention is equal<5%, essence between batch
Density is equal<6.5%, illustrate using the method for the invention prepare reagent set be detected when it is reproducible, with chance error
Difference is small.
Embodiment 4：The accuracy in detection of reagent set prepared by the method for the invention
Accuracy is the degree that is consistent of measured value and actual value, and reaction reagent is set with the size of detection error.
Evaluation of accuracy：Be separately added into the solution of LH a concentration of zero high (H), in (M), low (L) concentration LH
Antigen, the ratio for calculating each sample measured value and theoretical value are recycled rate, and the results are shown in Table 4.
Table 4：The accuracy in detection of reagent set prepared by the method for the invention
As known from Table 4, the accuracy in detection deviation for the reagent set that prepared by the method for the invention is small, and rate of recovery deviation exists
Within 2%, illustrate that the detection error for the reagent set that measured value is close with theoretical value, prepared by the method for the invention is small.
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 (16)
1. a kind of method for the homogeneous immunological detection reagent suit preparing detection interstitialcellstimulating hormone (ICSH) comprising：
Prepare first chamber, the first chamber includes component a and the first buffer solution, and wherein component a is by can be with
The receptor and first antibody in combination of singlet oxygen reaction generation detectable signal or its binding fragment are constituted；Described
One antibody or its binding fragment can be combined with the first epitope specificity of interstitialcellstimulating hormone (ICSH)；
Second chamber is prepared, the second chamber includes component b and the second buffer solution, and wherein component b is and the first mark
Remember the secondary antibody that can be combined with the second epitope specificity of interstitialcellstimulating hormone (ICSH) or its binding fragment composition that object combines, and
Second epitope and the first epitope non-overlapping；
Third composition is prepared, the third composition includes component c and third buffer solution, wherein the component c is first
The donor that singlet oxygen can be generated in excited state that marker specific junction mixture combines.
2. according to the method described in claim 1, it is characterized in that, the first antibody and secondary antibody are anti-selected from monoclonal
Body and/or polyclonal antibody.
3. method according to claim 1 or 2, which is characterized in that the receptor includes olefin(e) compound and metal-chelating
Object is non-particulate forms, and solvable in water-bearing media；And/or the receptor is filled with luminophor and group of the lanthanides member
The high molecular particle of element；
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.
4. according to the method described in any one of claim 1-3, which is characterized in that the method further includes preparing to promote corpus luteum
Generate plain calibration object serial dilutions.
5. according to the method described in claim 4, it is characterized in that, the interstitialcellstimulating hormone (ICSH) calibration object serial dilutions it is dense
Degree is 0~200mIU/mL.
6. according to the method described in any one of claim 1-5, which is characterized in that component a in the first chamber
A concentration of 0.1~1.0mg/mL；Preferably, in the first chamber component a a concentration of 0.35~0.65mg/mL；With/
Or,
A concentration of 1~10 μ g/mL of component b in the second chamber；Preferably, in the second chamber component b it is dense
Degree is 3.5~6.5 μ g/mL；And/or
A concentration of 50~150 μ g/mL of component c in the third composition；Preferably, component c in the third composition
A concentration of 80~120 μ g/mL.
7. according to the method described in any one of claim 1-6, which is characterized in that the preparation method of the first chamber
Including：
Receptor is diluted to 1~10mg/mL using carbonate buffer solution, obtains receptor solution by step S1；
First antibody or its binding fragment are added in receptor solution, is added after static dilute with carbonate buffer solution by step S2
It releases to the BSA solution of 5~15mg/mL, obtains the receptor solution combined with first antibody or its binding fragment；
Step S3, detach in the receptor solution that is combined with first antibody or its binding fragment with first antibody or its binding fragment
In conjunction with receptor, and be added the first buffer solution, obtain first chamber.
8. method according to any one of claims 1-7, which is characterized in that the preparation method of the second chamber
Including：
Secondary antibody or its binding fragment are placed in bag filter and are dialysed using label buffer solution by step T1, and dialysis terminates
After the first marker solution is added, and fill into elution buffer, it is static, obtain the secondary antibody combined with the first marker or its
Binding fragment；
The secondary antibody combined with the first marker or its binding fragment are placed in bag filter and utilize elution buffer by step T2
It dialyses, the second buffer solution is added after dialysis, obtain second chamber solution.
9. according to the method described in claim 8, it is characterized in that, the NaHCO that the label buffer solution is 0.05~0.15M3It is molten
Liquid；The elution buffer is the Tris-HCl solution of 0.05~0.15M；And/or
The secondary antibody or the molar ratio of its binding fragment and the first marker are 1:(28~32).
10. according to the method described in any one of claim 1-10, which is characterized in that first buffer solution and second
Buffer solution is the Tris-HCl solution for 0.05~0.15M that pH value is 7.5~8.5.
11. a kind of homogeneous immunological detection reagent for the interstitialcellstimulating hormone (ICSH) for wanting any one of 1-10 the methods to prepare such as right
Suit.
12. a kind of homogeneous immunization method detecting interstitialcellstimulating hormone (ICSH) using reagent set as claimed in claim 11, described
Method detects for light-induced chemiluminescent.
13. according to the method for claim 12, which is characterized in that the method includes：
Sample to be tested is mixed with first chamber and second chamber, obtains the first mixture by step R1；
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 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
With the presence or absence of interstitialcellstimulating hormone (ICSH).
14. according to the method for claim 13, which is characterized in that the method further includes：Utilize interstitialcellstimulating hormone (ICSH) school
Quasi- product serial dilutions make the step of standard curve of chemiluminescence signal-interstitialcellstimulating hormone (ICSH) concentration；The standard curve
Content for determining interstitialcellstimulating hormone (ICSH) in sample to be tested.
15. according to the method for claim 13, which is characterized in that the described method comprises the following steps：
A. the sample to be tested of 10~30 μ L is 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, wherein the
A concentration of 0.2~0.8mg/mL of component a in one composition, a concentration of 2~8 μ g/mL of the component b in second chamber；
C.35~45 DEG C incubation 10~30 minutes；
D. 100~250 μ L of the third composition are added into reacting hole；Component c's wherein in third composition is a concentration of
50~150 μ g/mL；
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；
G. the photon amount for detecting transmitting light in each reacting hole, the concentration of interstitialcellstimulating hormone (ICSH) is calculated according to standard curve.
16. a kind of application of reagent set as claimed in claim 11 in preparing the kit for monitoring the onset of ovulation.
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| CN118209739A (en) * | 2024-03-19 | 2024-06-18 | 厦门宝太和瑞生物技术有限公司 | Luteinizing hormone detection kit |
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Application publication date: 20180824 |