CN107402308A - Immue quantitative detection reagent boxes of IL 6 and preparation method thereof - Google Patents
Immue quantitative detection reagent boxes of IL 6 and preparation method thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/54326—Magnetic particles
- G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
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Abstract
The invention discloses a kind of immue quantitative detection reagent boxes of IL 6 and preparation method thereof.Wherein, the immue quantitative detection reagent boxes of IL 6 include：Coupling has the magnetic microsphere reagent of the antibody of IL 6, reacts diluent reagent and the calibration objects of IL 6.Using the detection kit of the present invention, serum sample detection can be carried out on existing a variety of Biochemical Analyzers, easy to operate quick, providing testing result only needs about 10~15 minutes, it is easy to inflammation, pyemia early diagnosis, is more suitable for popularizing and applying in basic medical units at different levels.
Description
Technical field
The present invention relates to quantitative measurement technology field, in particular to a kind of IL-6 immue quantitative detection reagent boxes and its system
Preparation Method.
Background technology
The glycoprotein that interleukin-6 (interleukin-6, IL-6) is made up of 212 amino acid, body is by inflammation
Secreted after stimulation by T cell, B cell, mononuclear macrophage and endothelial cell etc..IL-6 is a kind of manifold effect cell factor, it
There are various biological activity, including infection reaction and cytoprotection function before mediation.IL-6 participates in generation and the hair of many diseases
Exhibition, its blood plasma level and inflammation, virus infection, autoimmune disease are closely related, and its change is than CRP earlier.It is now recognized that
It is the quantization mark of inflammation, pyemic early stage sensitiveness " warning " mark and chronic inflammation.
IL-6 detection method mainly has chemiluminescence immunoassay and Electrochemiluminescence assay at present.Two kinds
Method uses double antibody sandwich method, and difference is that label is different：Chemoluminescence method is alkaline phosphatase；Electrochemical luminescence
Method is ruthenium.Chemical luminous immune detection method, which represents reagent, to be had：The interleukin 6 measure kit (chemistry of U.S. Bake Man
Luminescence method), the kit chief component is the paramagnetic particles for being coated with goat anti rat IgG, and Goat anti human IL-6 is alkaline
Phosphatase (ox) conjugate；The interleukin-6 measure kit (chemoluminescence method) of England Siemens AG, the kit are main
Part has the coating pearl for being coated with monoclonal mouse anti-IL-6 antibodies, the polyclonal goat-anti of alkaline phosphatase (small Roll) mark
IL-6 antibody.Electrochemical luminescence immunologic detection method represents interleukin 6 detection kit (electrochemistry hair of the reagent as German Roche
Light method), the kit chief component is is coated with the bead particulates of streptavidin, biotinylated anti-interleukin-6 resists
The anti-interleukin-6 antibody of body, ruthenium mark.
Chemoluminescence method and Electrochemiluminescince can accurately, quick detection go out result, but this method instrument cost is relative
It is higher that and the requirement to laboratory operation personnel is of a relatively high, it is necessary to specific chemiluminescence instrument, thus clinically without
Method really popularization and use, only some relatively large laboratories just possess corresponding condition.
Therefore, clinic needs to research and develop a kind of sensitiveness height, high specificity, suitable clinic simple and efficient to handle, inexpensive
The IL-6 of popularization detection method.
The content of the invention
The present invention is intended to provide a kind of IL-6 immue quantitative detection reagent boxes and preparation method thereof, to solve IL-6 in the prior art
Quantitative testing cost is high, the technical problem of complex operation.
To achieve these goals, according to an aspect of the invention, there is provided a kind of IL-6 immue quantitative detection reagent boxes.Should
IL-6 immue quantitative detection reagent boxes include：Coupling has the magnetic microsphere reagent of IL-6 antibody, reacts diluent reagent and IL-6 calibration objects.
Further, the magnetic microsphere reagent that coupling has IL-6 antibody includes：Coupling has the magnetic microsphere and matrix of IL-6 antibody
Solution, wherein, matrix solution includes：0.5~20g/L of stabilizer, 2~200g/L of coagulant, 0.1~10mL/ of surfactant
L, preservative 0.5~5mL/L and 10~50mmol/L phosphate buffer, matrix solution pH value are 7.0~9.0.
Further, it be paramagnetism microballoon that coupling, which has the magnetic microsphere used in the magnetic microsphere of IL-6 antibody, particle diameter for 100~
200nm, it is 1~4mg/mL that coupling, which has mass concentration of the magnetic microsphere of IL-6 antibody in matrix solution,.
Further, stabilizer is bovine serum albumin(BSA) and/or casein；Coagulant is sucrose and/or ficoll -400；
Surfactant is Tween-20 and/or triton x-100；Preservative is Proclin300.
Further, the magnetic microsphere reagent that coupling has IL-6 antibody includes：Stabilizer be 5g/L bovine serum albumin(BSA) and
5g/L casein, the sucrose and 50g/L ficoll -400 that coagulant is 100g/L, the tween that surfactant is 1mL/L -
20 and 1mL/L triton x-100, preservative are 1mL/L Proclin300, and 25mmol/L phosphate buffers, matrix is molten
Liquid pH value is 7.4；It is 2mg/mL that coupling, which has mass concentration of the magnetic microsphere of IL-6 antibody in matrix solution,.
Further, it is using the coupling of EDC-NHS methods to be coupled IL-6 antibody and magnetic microsphere in the magnetic microsphere for having IL-6 antibody
's.
Further, reaction diluent reagent includes：0.5~20g/L of stabilizer, 2~50mL/L of coagulant, surface-active
0~50mmol/L of 0.1~3mL/L of agent, 0.5~5mL/L of preservative and phosphate buffer 1, react the pH value of diluent reagent
For 7.0~9.0.
Further, stabilizer is bovine serum albumin(BSA), and coagulant is PEG 8000, surfactant be tween-
20, preservative Proclin300.
Further, reaction diluent reagent includes：Stabilizer be 5g/L bovine serum albumin(BSA), coagulant 20mL/L
PEG 8000, surfactant are 1mL/L Tween-20, and preservative is 1mL/L Proclin300, and cushioning liquid is
25mmol/L phosphate buffer, the pH value for reacting diluent reagent are 7.4.
Further, IL-6 calibration objects include：PH7.4 50mmol/L phosphate buffer, recombination human source IL-6,
10g/L bovine serum albumin(BSA) and 1mL/L preservative.
Further, the concentration range of IL-6 calibration objects is 0~1000pg/mL.
According to another aspect of the present invention, there is provided a kind of preparation method of IL-6 immue quantitative detection reagent boxes.The preparation side
Method includes preparing the magnetic microsphere reagent for being coupled and having IL-6 antibody, prepares reaction diluent reagent and prepares the step of IL-6 calibration objects
Suddenly；Wherein, the magnetic microsphere reagent that preparing coupling has IL-6 antibody includes：Preparing coupling has the matrix of the magnetic microsphere of IL-6 antibody molten
Liquid, prepare IL-6 antibody and magnetic microsphere conjugate and the matrix solution and IL-6 antibody of the magnetic microsphere for having IL-6 antibody will be coupled
It is mixed to prepare to prepare with magnetic microsphere conjugate and is coupled the magnetic microsphere reagent for having IL-6 antibody；Preparing reaction diluent reagent includes：
PH7.4 phosphate buffers are taken to add after stabilizer, coagulant, surfactant, preservative are fully mixed and dissolved with 0.22 μ
M filtering with microporous membrane, finally with corresponding buffer solution constant volume, it is sealed standby；Preparing IL-6 calibration objects includes：Prepare calibration
The calibration object of product dilution and various concentrations gradient.
Further, the matrix solution that preparing coupling has the magnetic microsphere of IL-6 antibody specifically includes：PH7.4 phosphate is taken to delay
With 0.22 μm of filtering with microporous membrane after fliud flushing adds stabilizer, coagulant, surface-active, preservative are fully mixed and dissolved, most
Eventually with corresponding buffer solution constant volume, it is sealed standby；Wherein, 0.5~20g/L of stabilizer, 2~200g/L of coagulant, surface
0.1~10mL/L of activating agent, preservative 0.5~5mL/L and 10~50mmol/L phosphate buffer, matrix solution pH value are
7.0~9.0；Stabilizer is bovine serum albumin(BSA) and/or casein；Coagulant is sucrose and/or ficoll -400；Surface-active
Agent is Tween-20 and/or triton x-100；Preservative is Proclin300.
Further, IL-6 antibody is prepared to specifically include with magnetic microsphere conjugate：By magnetic microsphere, EDC and NHS in mass ratio
For 1：1：1, and pH 5.0 50mmol/L MES solution is added, it is 4mg/mL to make magnetic microsphere mass concentration, is placed on vertical rotation
Turn to activate on instrument, 25 DEG C of room temperature lucifuges react 30min, by the magnetic microsphere after activation and IL-6 monoclonal antibodies mass ratio 80：1 enters
Row mixing, is placed on vertical rotary instrument and marks, and 25 DEG C of room temperature lucifuges react 3h, by reacted magnetic microsphere with pH7.2's
25mmol/L Tris-HCl wash liquids 3 times, after magnetic separation removes supernatant, add containing glycine, 5g/L bovine serum albumins
In vain, 0.5mL/L triton x-100s, pH 7.4 phosphate buffer, it is 4mg/mL to make magnetic microsphere mass concentration, is placed on vertical
Terminated on straight gyroscope, 25 DEG C of room temperature lucifuges react 2h, the Tris- by the magnetic microsphere after termination with pH 7.2 25mmol/L
HCl wash liquids 3 times, matrix solution is added, the magnetic microsphere mass concentration for making to be coupled IL-6 antibody is 10mg/mL, 2~8 DEG C
Preserve.
Further, the matrix solution for being coupled the magnetic microsphere for having IL-6 antibody is mixed with IL-6 antibody and magnetic microsphere conjugate
Close, coupling is had the final concentration of 1~4mg/mL of the magnetic microsphere of IL-6 antibody.
Using the detection kit of the present invention, serum sample detection can be carried out on existing a variety of Biochemical Analyzers, is grasped
It is quick to make simplicity, providing testing result only needs about 10~15 minutes, is easy to inflammation, pyemia early diagnosis, is more suitable at different levels
Basic medical unit is popularized and application.
Brief description of the drawings
The Figure of description for forming the part of the application is used for providing a further understanding of the present invention, and of the invention shows
Meaning property embodiment and its illustrate be used for explain the present invention, do not form inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 is shown to be verified according to the range of linearity of the IL-6 immue quantitative detection reagent boxes of embodiment 1；And
Fig. 2 shows the IL-6 immue quantitative detection reagent boxes and commercially available IL-6 chemiluminescence detection kits according to embodiment 1
The correlation of testing result compares.
Embodiment
It should be noted that in the case where not conflicting, the feature in embodiment and embodiment in the application can phase
Mutually combination.Describe the present invention in detail below with reference to the accompanying drawings and in conjunction with the embodiments.
For the quantitative testing cost height of IL-6 in the prior art, the technical problem of complex operation, according to a kind of allusion quotation of the present invention
The embodiment of type, there is provided a kind of IL-6 magnetic microspheres immunoturbidimetry detection method and detection kit, its main purpose is to be used for
Detect the content of IL-6 in human serum sample.Its principle is using super paramagnetic microsphere as carrier, and IL-6 antibody and magnetic microsphere are carried out
Coupling, in the detection means of Biochemical Analyzer, the IL-6 albumen in sample contacts with the magnetic microsphere after coupling, produces magnetic microsphere
Raw aggregation, forms magnetic microsphere-antibody-antigen-antibody-magnetic microsphere compound, turbidity occurs in reaction solution, when antibody concentration is fixed
When, the amount of the immune complex of formation increases with the increase of IL-6 amounts in sample, and the turbidity of reaction solution is consequently increased.When
Can be by different degrees of absorption during the reaction solution of incident light irradiation difference turbidity, and the amplitude of variation of this absorbance is with treating sample
IL-6 content passes through the variable quantity for determining reaction solution absorbance and known IL-6 concentration within the specific limits into positive correlation in this
Calibration object, you can draw the content of IL-6 in sample to be checked.
According to a kind of typical embodiment of the present invention, there is provided a kind of IL-6 immue quantitative detection reagent boxes.The kit includes：
Coupling has the magnetic microsphere reagent of IL-6 antibody, reacts diluent reagent and IL-6 calibration objects.
Using the detection kit of the present invention, serum sample detection can be carried out on existing a variety of Biochemical Analyzers, is grasped
It is quick to make simplicity, providing testing result only needs about 10~15 minutes, is easy to inflammation, pyemia early diagnosis, is more suitable at different levels
Basic medical unit is popularized and application.
Preferably, the magnetic microsphere reagent that coupling has IL-6 antibody includes：Coupling has the magnetic microsphere of IL-6 antibody and matrix molten
Liquid, wherein, matrix solution includes：0.5~20g/L of stabilizer, 2~200g/L of coagulant, 0.1~10mL/L of surfactant,
Preservative 0.5~5mL/L and 10~50mmol/L phosphate buffer, matrix solution pH value are 7.0~9.0.
Preferably, it be paramagnetism microballoon that coupling, which has the magnetic microsphere used in the magnetic microsphere of IL-6 antibody, particle diameter for 100~
200nm, it is 1~4mg/mL that coupling, which has mass concentration of the magnetic microsphere of IL-6 antibody in matrix solution,.
The particle size for being coupled the magnetic microsphere of IL-6 antibody has very for the sensitivity of detection and range of linearity etc.
Significant impact.The magnetic microsphere of small particle makes reagent have wide detection range, can detect thing to be detected in high concentration range
Content, but particle diameter is unsuitable too small, and reaction speed otherwise can be caused slack-off, and prepares complex, and difficulty is higher；And big particle diameter
Magnetic particle make reagent that there is higher sensitivity, the content of thing to be detected in low strength range, but particle diameter can be detected
It is unsuitable excessive, conjugate aggregation sedimentation can be caused further to influence measurement result.Thus, it is the inspection prepared by ensureing in the present invention
Test agent box causes antigen-antibody reaction experiment to be carried out in a wider scope, and can ensure there is high sensitivity
And high accuracy, the particle diameter of used super-paramagnetism nano microballoon is preferably 100~200nm.
Preferably, the magnetic microsphere reagent that coupling has IL-6 antibody includes：Stabilizer is 5g/L bovine serum albumin(BSA) and 5g/
L casein, the sucrose and 50g/L ficoll -400 that coagulant is 100g/L, surfactant is 1mL/L Tween-20
With 1mL/L triton x-100, preservative be 1mL/L Proclin300,25mmol/L phosphate buffers, matrix solution
PH value is 7.4；It is 2mg/mL that coupling, which has mass concentration of the magnetic microsphere of IL-6 antibody in matrix solution,.
According to a kind of typical embodiment of the present invention, IL-6 antibody and magnetic microsphere in the magnetic microsphere for having IL-6 antibody are coupled
It is to be coupled using EDC-NHS methods.
According to a kind of typical embodiment of the present invention, reaction diluent reagent includes：0.5~20g/L of stabilizer, promote solidifying
0~50mmol/L of 2~50mL/L of agent, 0.1~3mL/L of surfactant, 0.5~5mL/L of preservative and phosphate buffer 1,
The pH value for reacting diluent reagent is 7.0~9.0.Preferably, stabilizer is bovine serum albumin(BSA), and coagulant is polyethylene glycol
8000, surfactant is Tween-20, preservative Proclin300.It is furthermore preferred that reaction diluent reagent includes：It is stable
Agent is 5g/L bovine serum albumin(BSA), and coagulant is 20mL/L PEG 8000s, and surfactant is 1mL/L polysorbas20,
Preservative is 1mL/L Proclin300, and cushioning liquid is 25mmol/L phosphate buffer, reacts the pH of diluent reagent
It is worth for 7.4.
According to a kind of typical embodiment of the present invention, IL-6 calibration objects include：PH7.4 50mmol/L phosphate delays
The bovine serum albumin(BSA) of fliud flushing, recombination human source IL-6,10g/L, preservative are 1mL/L Proclin300.
Preferably, the concentration range of IL-6 calibration objects is 0~1000pg/mL.
According to a kind of typical embodiment of the present invention, there is provided a kind of preparation method of IL-6 immue quantitative detection reagent boxes.Should
Preparation method includes preparing the magnetic microsphere reagent for being coupled and having IL-6 antibody, prepares reaction diluent reagent and prepares IL-6 calibration objects
The step of；Wherein, the magnetic microsphere reagent that preparing coupling has IL-6 antibody includes：Preparing coupling has the base of magnetic microsphere of IL-6 antibody
Matter solution, prepare IL-6 antibody and magnetic microsphere conjugate and the matrix solution and IL-6 of the magnetic microsphere for having IL-6 antibody will be coupled
Antibody is mixed to prepare to prepare with magnetic microsphere conjugate is coupled the magnetic microsphere reagent for having IL-6 antibody；Prepare reaction diluent reagent bag
Include：Take pH7.4 phosphate buffers to add stabilizer, coagulant, surfactant, preservative to use after fully mixing and dissolve
0.22 μm of filtering with microporous membrane, finally with corresponding buffer solution constant volume, it is sealed standby；Preparing IL-6 calibration objects includes：Match somebody with somebody
The calibration object of calibration object dilution processed and various concentrations gradient.
Preferably, the matrix solution that preparing coupling has the magnetic microsphere of IL-6 antibody specifically includes：Take pH7.4 phosphate-buffereds
With 0.22 μm of filtering with microporous membrane after liquid adds stabilizer, coagulant, surface-active, preservative are fully mixed and dissolved, finally
With corresponding buffer solution constant volume, it is sealed standby；Wherein, 0.5~20g/L of stabilizer, 2~200g/L of coagulant, surface are lived
Property 0.1~10mL/L of agent, preservative 0.5~5mL/L and 10~50mmol/L phosphate buffer, matrix solution pH value be 7.0
~9.0；Stabilizer is bovine serum albumin(BSA) and/or casein；Coagulant is sucrose and/or ficoll -400；Surfactant
For Tween-20 and/or triton x-100；Preservative is Proclin300.Wherein, in stabilizer, coagulant, surfactant
Limited between every two kinds of components without proportioning, be synergy；Proclin300 main actives are the different thiophenes of 2- methyl -4-
Oxazoline -3- ketone (MCI) and CMIT (CMCI), Chinese：Proclin300 preservatives.
Preferably, the magnetic microsphere reagent that preparing coupling has IL-6 antibody specifically includes：Preparing coupling has the magnetic of IL-6 antibody
The matrix solution of microballoon：800mL 25mmol/L pH7.4 phosphate buffers are taken to add bovine serum albumin(BSA) 5g, casein 5g,
Sucrose 100g, the 50g of ficoll -400, Tween-20 1mL, triton x-100 1mL, Proclin300 1mL are fully mixed simultaneously
With 0.22 μm of filtering with microporous membrane after dissolving, 1000mL finally is settled to buffer solution, that is, matrix solution is made, is sealed standby
With；Prepare IL-6 antibody and magnetic microsphere conjugate：It is 1 by magnetic microsphere, EDC and NHS mass ratio：1：1, and add pH's 5.0
50mmol/L MES solution, it is 4mg/mL to make magnetic microsphere mass concentration, is placed on vertical rotary instrument and activates, and 25 DEG C of room temperatures are kept away
Light reaction 30min, by the magnetic microsphere after activation and IL-6 monoclonal antibodies mass ratio 80：1 is mixed, and is placed on vertical rotary
Marked on instrument, 25 DEG C of room temperature lucifuges react 3h, by reacted magnetic microsphere pH 7.2 25mmol/L Tris-HCl washing lotions
Washing 3 times, after magnetic separation removes supernatant, add containing glycine, 5g/L bovine serum albumin(BSA)s, 0.5mL/L triton x-100s,
PH 7.4 phosphate buffer, it is 4mg/mL to make magnetic microsphere mass concentration, is placed on vertical rotary instrument and terminates, 25 DEG C of room temperatures
Lucifuge reacts 2h, the Tris-HCl wash liquids 3 times by the magnetic microsphere after termination with pH 7.2 25mmol/L, it is molten to add matrix
Liquid, the magnetic microsphere mass concentration for making to be coupled IL-6 antibody is 10mg/mL, and 2~8 DEG C preserve；Coupling has the magnetic of IL-6 antibody micro-
It is prepared by the reagent of ball：The magnetic microsphere for being coupled IL-6 antibody is proportionally mixed with matrix solution, detection kit institute is made
What is needed has been coupled the reagent of the magnetic microsphere of IL-6 antibody.
Preferably, the matrix solution for being coupled the magnetic microsphere for having IL-6 antibody is mixed with IL-6 antibody and magnetic microsphere conjugate
Close, coupling is had the final concentration of 1~4mg/mL of the magnetic microsphere of IL-6 antibody (matrix solution and IL-6 antibody and magnetic microsphere conjugate
Mixed proportion：2.5~10：1).
Preferably, IL-6 calibration objects are prepared to specifically include：1) preparation of calibration object dilution：1. measure purified water 200mL
In liquid dispensing container；2. weigh NaH2PO4.2H2O 1.25g are dissolved in liquid dispensing container, are mixed；3. weigh Na2HPO4.12H2O
15.04g is dissolved in liquid dispensing container, is mixed；4. with 0.45 μm of membrane filtration；5. plus purified water mixes to the 3/4 of required volume；
6. weighing bovine serum albumin(BSA) 10g to be dissolved in liquid dispensing container, mix；7. measuring biological preservative 1mL to be dissolved in liquid dispensing container, mix
It is even；8. it is settled to 1000mL with purified water；9. it is 7.4 to determine pH value；2) by recombination human source IL-6 albumen calibration object dilution
Be configured to the calibration object of various concentrations gradient, the concentration of calibration object be respectively 0pg/mL, 5pg/mL, 20pg/mL, 100pg/mL,
500pg/mL and 1000pg/mL.
Beneficial effects of the present invention are further illustrated below in conjunction with embodiment.
Embodiment 1
Prepare IL-6 immue quantitative detection reagent boxes
Comprise the following steps that：
(1) reaction diluent reagent is prepared：800mL 25mmol/L pH7.4 phosphate buffers are taken to add bovine serum albumin
White 5g, PEG 8000 20mL, Tween-20 1mL, Proclin300 1mL fully mix and dissolve after with 0.22 μm of micropore
Membrane filtration, 1000mL finally is settled to 25mmol/L pH7.4 phosphate buffers, is sealed standby.
(2) the matrix solution for the magnetic microsphere for being coupled IL-6 antibody is prepared：800mL 25mmol/L pH7.4 phosphate is taken to delay
Fliud flushing adds bovine serum albumin(BSA) 5g, casein 5g, sucrose 100g, the 50g of ficoll -400, Tween-20 1mL, and Qula leads to X-
100 1mL, Proclin300 1mL fully mix and dissolve after with 0.22 μm of filtering with microporous membrane, finally use 25mmol/L
PH7.4 phosphate buffers are settled to 1000mL, i.e., manufactured matrix solution, are sealed standby.
(3) IL-6 antibody and magnetic microsphere conjugate are prepared：It is 1 by magnetic microsphere, EDC and NHS mass ratio：1：1, and add
PH 5.0 50mmol/L MES solution, it is 4mg/mL to make magnetic microsphere mass concentration, is placed on vertical rotary instrument and activates, 25
DEG C room temperature lucifuge reaction 30min.By the magnetic microsphere after activation and IL-6 monoclonal antibodies mass ratio 80：1 is mixed, and is placed on
Marked on vertical rotary instrument, 25 DEG C of room temperature lucifuges react 3h.Tris- by reacted magnetic microsphere with pH 7.2 25mmol/L
HCl wash liquids 3 times, after magnetic separation removes supernatant, add and lead to containing glycine, 5g/L bovine serum albumin(BSA)s, 0.5mL/L Qulas
X-100, pH 7.4 phosphate buffer, it is 4mg/mL to make magnetic microsphere mass concentration, is placed on vertical rotary instrument and terminates, 25
DEG C room temperature lucifuge reaction 2h.Tris-HCl wash liquids 3 times by the magnetic microsphere after termination with pH 7.2 25mmol/L, are added
Matrix solution, the magnetic microsphere mass concentration for making to be coupled IL-6 antibody is 10mg/mL, and 2~8 DEG C preserve.
(4) the preparation of the magnetic microsphere reagent of IL-6 antibody has been coupled：The magnetic microsphere and matrix solution of IL-6 antibody will be coupled
(making the final concentration of 2mg/mL of magnetic microsphere for being coupled IL-6 antibody) is mixed according to a certain percentage, is made required for detection kit
The magnetic microsphere for being coupled IL-6 antibody reagent；
(5) the preparation of IL-6 calibration objects：
1. the preparation of calibration object dilution：1. purified water 200mL is measured in liquid dispensing container；2. weigh sodium dihydrogen phosphate
(NaH2PO4.2H2O) 1.25g is dissolved in liquid dispensing container, is mixed；3. weigh disodium hydrogen phosphate (Na2HPO4.12H2O) 15.04g is molten
In liquid dispensing container, mix；4. with 0.45 μm of membrane filtration；5. plus purified water mixes to the 3/4 of required volume；6. weigh ox
Seralbumin (BSA) 10g is dissolved in liquid dispensing container, is mixed；Match somebody with somebody 7. measuring biological preservative (Proclin300) 1.0mL and being dissolved in
In liquid container, mix；8. it is settled to 1000mL with purified water；9. it is 7.4 to determine pH value.
2. the calibration object by commercially available recombination human source IL-6 albumen with calibration object diluent preparing into various concentrations gradient, is adopted
Demarcated with the commercial kit of food and medicine Surveillance Authority approval listing, each concentration gradient detects 3 times, takes detection
As a result average value, definite value is 0pg/mL (calibration object dilution, being not added with IL-6 albumen), 5pg/mL, 20pg/mL respectively,
100pg/mL, 500pg/mL, 1000pg/mL.
React diluent reagent, be coupled IL-6 antibody magnetic microsphere reagent and IL-6 calibration objects packing：Will be upper
State that step obtained component is dispensed according to dimension with suitable container, labeling is semi-finished product.
(7) finished product assembles：Extracting three boxes out can just be assembled into by specificity, accuracy, sensitivity and stability assay approval
IL-6 immue quantitative detection reagent boxes.
Embodiment 2.
Prepare interleukin-6 immue quantitative detection reagent box
(1) reaction diluent reagent is prepared：800mL 50mmol/L pH7.4 phosphate buffers are taken to add bovine serum albumin
White 20g, PEG 8000 50mL, Tween-20 3mL, Proclin300 5mL fully mix and dissolve after with 0.22 μm it is micro-
Hole membrane filtration, 1000mL finally is settled to corresponding buffer solution, be sealed standby.
(2) the matrix solution for the magnetic microsphere for being coupled IL-6 antibody is prepared：800mL 50mmol/L pH7.4 phosphate is taken to delay
Fliud flushing adds bovine serum albumin(BSA) 20g, casein 20g, sucrose 200g, the 200g of ficoll -400, Tween-20 10mL, Qula
Lead to 0.45 μm of filtering with microporous membrane after X-100 10mL, Proclin300 5mL are fully mixed and dissolved, it is final with corresponding
Buffer solution is settled to 1000mL, i.e., manufactured matrix solution, is sealed standby.
Remaining step is same as Example 1.
Embodiment 3
Prepare interleukin-6 immue quantitative detection reagent box
(1) reaction diluent reagent is prepared：800mL 10mmol/L pH7.4 phosphate buffers are taken to add bovine serum albumin
White 0.5g, PEG 8000 2mL, Tween-20 0.1mL, Proclin300 0.5mL fully mix and dissolve after with 0.22 μ
M filtering with microporous membrane, 1000mL finally is settled to corresponding buffer solution, be sealed standby.
(2) the matrix solution for the magnetic microsphere for being coupled IL-6 antibody is prepared：800mL 10mmol/L pH7.4 phosphate is taken to delay
Fliud flushing adds bovine serum albumin(BSA) 0.5g, casein 0.5g, sucrose 2g, the 2g of ficoll -400, Tween-20 0.1mL, and Qula is led to
X-1000.1mL, Proclin300 1mL are delayed accordingly after fully mixing and dissolve with 0.22 μm of filtering with microporous membrane, final use
Fliud flushing is settled to 1000mL, i.e., manufactured matrix solution, is sealed standby.
Remaining step is same as Example 1.
Embodiment 4
Prepare interleukin-6 immue quantitative detection reagent box
(1) reaction diluent reagent is prepared：800mL 50mmol/L pH7.4 phosphate buffers are taken to add bovine serum albumin
White 10g, PEG 8000 20mL, Tween-20 2mL, Proclin300 1mL fully mix and dissolve after with 0.22 μm it is micro-
Hole membrane filtration, 1000mL finally is settled to corresponding buffer solution, be sealed standby.
(2) the matrix solution for the magnetic microsphere for being coupled IL-6 antibody is prepared：800mL 50mmol/L pH7.4 phosphate is taken to delay
Fliud flushing adds bovine serum albumin(BSA) 10g, casein 10g, sucrose 100g, the 50g of ficoll -400, Tween-20 5mL, and Qula is led to
X-1005mL, Proclin300 1mL are buffered accordingly after fully mixing and dissolve with 0.22 μm of filtering with microporous membrane, final use
Liquid is settled to 1000mL, i.e., manufactured matrix solution, is sealed standby.
Remaining step is same as Example 1.
Embodiment 5
Prepare interleukin-6 immue quantitative detection reagent box
(1) reaction diluent reagent is prepared：800mL 20mmol/L pH7.4 phosphate buffers are taken to add bovine serum albumin
White 2g, PEG 8000 20mL, Tween-20 0.5mL, Proclin300 1mL fully mix and dissolve after with 0.22 μm it is micro-
Hole membrane filtration, 1000mL finally is settled to corresponding buffer solution, be sealed standby.
(2) the matrix solution for the magnetic microsphere for being coupled IL-6 antibody is prepared：800mL 20mmol/L pH7.4 phosphate is taken to delay
Fliud flushing adds bovine serum albumin(BSA) 2g, casein 2g, sucrose 100g, the 50g of ficoll -400, Tween-20 0.5mL, and Qula is led to
X-1000.5mL, Proclin300 1mL are delayed accordingly after fully mixing and dissolve with 0.22 μm of filtering with microporous membrane, final use
Fliud flushing is settled to 1000mL, i.e., manufactured matrix solution, is sealed standby.
Remaining step is same as Example 1.
Comparative example 1
Prepare interleukin-6 immue quantitative detection reagent box
(1) reaction diluent reagent is prepared：800mL 25mmol/L pH7.4 phosphate buffers are taken to add polyethylene glycol
800020mL, Tween-20 1mL, Proclin300 1mL fully mix and dissolve after with 0.22 μm of filtering with microporous membrane, finally
1000mL is settled to corresponding buffer solution, is sealed standby.
(2) the matrix solution for the magnetic microsphere for being coupled IL-6 antibody is prepared：800mL 25mmol/L pH7.4 phosphate is taken to delay
Fliud flushing adds sucrose 100g, the 50g of ficoll -400, Tween-20 1mL, and triton x-100 1mL, Proclin300 1mL fill
Divide after mixing and dissolving with 0.22 μm of filtering with microporous membrane, be finally settled to 1000mL with corresponding buffer solution, i.e., manufactured base
Matter solution, it is sealed standby.
Remaining step is same as Example 1.
Comparative example 2
Prepare interleukin-6 immue quantitative detection reagent box
(1) reaction diluent reagent is prepared：800mL 25mmol/L pH7.4 phosphate buffers are taken to add bovine serum albumin
White 25g, PEG 8000 20mL, Tween-20 4mL, Proclin300 5mL fully mix and dissolve after with 0.22 μm it is micro-
Hole membrane filtration, 1000mL finally is settled to corresponding buffer solution, be sealed standby.
(2) the matrix solution for the magnetic microsphere for being coupled IL-6 antibody is prepared：800mL 25mmol/L pH7.4 phosphate is taken to delay
Fliud flushing adds bovine serum albumin(BSA) 25g, casein 25g, sucrose 100g, the 50g of ficoll -400, Tween-20 12mL, and Qula is led to
X-100 12mL, Proclin300 5mL are delayed accordingly after fully mixing and dissolve with 0.45 μm of filtering with microporous membrane, final use
Fliud flushing is settled to 1000mL, i.e., manufactured matrix solution, is sealed standby.
Remaining step is same as Example 1.
The application method of the IL-6 immue quantitative detection reagent boxes of experimental example 1
The detection method concrete operations of IL-6 contents are as follows in IL-6 immue quantitative detection reagent boxes detection sample：
1. kit is taken out from 4 DEG C of refrigerators, equilibrium at room temperature 15 minutes；
2. instrument parameter is set：Detection wavelength 500nm, and reagent and sample are placed on the position specified；
3. add 240 μ L's into 5 μ L samples to be checked (calibration pipe makees sample with calibration object, and blank is using distilled water as sample)
Diluent reagent is reacted, is fully mixed, is incubated 5 minutes in 37 DEG C；
4. adding the reagent of the 60 μ L magnetic microsphere for being coupled IL-6 antibody into mixed system again, mix；
After 5.37 DEG C incubate 1 minute, each pipe absorbance A 2 is determined after determining each pipe absorbance A Isosorbide-5-Nitrae minute, calculates absorbance
Difference DELTA A=A2-A1；
6. calibrate QC replication 2 times, it is right using the average value Δ A of every 2 absorbance differences measured of pipe as ordinate
The calibration object concentration answered is abscissa, " concentration-absorbance " standard curve is drawn using multiple spot gamma correction pattern, respectively to treat
Survey the concentration that sera absorbance changing value calculates IL-6 in the serum sample on standard curve.
Experimental example 2
The performance evaluation of the IL-6 detection kits of embodiment 1
The kit detection range of embodiment 1 is 0~1000pg/mL, dose-response curve coefficient correlation (r) it is absolute
Value is not less than 0.9900.Such as want the content of IL-6 in accurate determination sample, in sample IL-6 concentration value should be no more than 0~
1000p g/mL concentration range, the measurement result beyond this scope are the result of calculation drawn by calibration object curve extension.
(1) the detection of sensitivity for analysis
By the use of the calibration object dilution (S0) in the detection kit prepared by embodiment 1 as dummy, according to described
Detection method repeat detection 20 times, calculate the average and standard deviation of dummy, two added with dummy detection average
Standard deviation is counter to bring standard curve into, and obtained concentration value is the sensitivity for analysis of this kit, and its sensitivity for analysis is 2pg/
mL。
(2) the range of linearity is verified
A clinical high level pooled serum is taken, concentration is close to 1000pg/mL (956pg/mL), with the calibration in embodiment 1
High level pooled serum sample is carried out multiple proportions by product dilution according to 1/2,1/4,1/8,1/16,1/32,1/64,1/128,1/256
Dilution, collectively constitutes the sample group of 9 concentration levels, is detected with the kit in embodiment 1, each pattern detection three
It is secondary, once completed in experiment, calculate the average value of measurement result three times respectively.With diluted concentration (X) for abscissa, to determine knot
The average value (Y) of fruit is ordinate, calculates the coefficient correlation (r) of equation of linear regression and equation of linear regression, is computed drawing
Equation of linear regression is Y=1.004X-0.465, correlation coefficient r=0.9999, the results showed that kit is in 5~1000pg/mL
Good relationship in the range of linearity, as a result as shown in table 1 and Fig. 1.
Table 1
(3) the detection of accuracy
Each replication of serum sample 10 times with the kit of embodiment 1 to two parts of various concentrations, calculates two parts respectively
The average and standard deviation of sample, the coefficient of variation CV of two parts of samples is calculated with average/standard deviation, as a result show point of two parts of samples
The coefficient of variation is respectively 3.11% and 2.93% (being shown in Table 2) in analysis
The accuracy testing result of table 2.
(4) methodology compares
With chemoluminescence method principle detection IL-6 results accurately and reliably, it is widely recognized as, due to not having in the market
IL-6 commercial reagent is detected with magnetic microsphere immunoturbidimetry, so selection is using the commercially available of chemoluminescence method principle detection IL-6
Import reagent reagent as a comparison.
The serum sample 100 that import IL-6 chemical luminescence reagent kits detected, wherein 61, positive reaction sample are collected,
39, negative reaction sample.Sample is measured with the kit of embodiment 1, linear regression point is carried out to all sample results
Analysis, calculate kit and the coefficient correlation (r) of commercially available IL-6 chemiluminescence detection kits testing result.As a result show, two kinds
Correlation coefficient r=0.9981 of kit testing result, linear regression mode are y=0.992x-0.264 (see Fig. 2).
(5) stability
By the detection kit point prepared by the detection kit prepared by the embodiment of the present invention 1~5 and comparative example 1~2
Taken out after not being placed in 37 DEG C of heat treatment 15 days, commercially available import IL-6 chemiluminescences are detected according to detection method on Biochemical Analyzer
Kit definite value is 15.43pg/mL and 165.18pg/mL two parts of blood samples, every part of blood sample replication four times, calculates detection knot
Fruit and the relative deviation and measured value rate of change, result of the test of commercially available import reagent definite value result are shown in Table 3.Can from result of the test
See, the thermostabilization of detection kit of the present invention is preferable, and is better than comparative example detection kit.
Each detection kit stability control experiment result of table 3.
As can be seen from the above description, the above embodiments of the present invention realize following technique effect：Using this skill
Art can accurately and reliably, simply and rapidly detect IL-6 contents in human serum, be particularly suitable for general in basic medical units at different levels
And and application.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.Within the spirit and principles of the invention, that is made any repaiies
Change, equivalent substitution, improvement etc., should be included in the scope of the protection.
Claims (15)
- A kind of 1. IL-6 immue quantitative detection reagent boxes, it is characterised in that including：Coupling has the magnetic microsphere reagent of IL-6 antibody, reaction Diluent reagent and IL-6 calibration objects.
- 2. IL-6 immue quantitative detection reagent boxes according to claim 1, it is characterised in that described to be coupled the magnetic for having IL-6 antibody Microballoon reagent includes：Coupling has the magnetic microsphere and matrix solution of IL-6 antibody, wherein, the matrix solution includes：Stabilizer 0.5 ~20g/L, 2~200g/L of coagulant, 0.1~10mL/L of surfactant, preservative 0.5~5mL/L and 10~50mmol/L Phosphate buffer, the matrix solution pH value be 7.0~9.0.
- 3. IL-6 immue quantitative detection reagent boxes according to claim 2, it is characterised in that described to be coupled the magnetic for having IL-6 antibody Magnetic microsphere used in microballoon is paramagnetism microballoon, and particle diameter is 100~200nm, and the coupling has the magnetic microsphere of IL-6 antibody in institute It is 1~4mg/mL to state the mass concentration in matrix solution.
- 4. IL-6 immue quantitative detection reagent boxes according to claim 2, it is characterised in that the stabilizer is bovine serum albumin White and/or casein；The coagulant is sucrose and/or ficoll -400；The surfactant is Tween-20 and/or song Draw logical X-100；Preservative is Proclin300.
- 5. IL-6 immue quantitative detection reagent boxes according to claim 2, it is characterised in that described to be coupled the magnetic for having IL-6 antibody Microballoon reagent includes：The stabilizer is 5g/L bovine serum albumin(BSA) and 5g/L casein, and the coagulant is 100g/L Sucrose and 50g/L ficoll -400, the surfactant be 1mL/L Tween-20 and 1mL/L triton x-100, The preservative is 1mL/L Proclin300, and 25mmol/L phosphate buffers, the matrix solution pH value is 7.4, described It is 2mg/mL that coupling, which has mass concentration of the magnetic microsphere of IL-6 antibody in the matrix solution,.
- 6. IL-6 immue quantitative detection reagent boxes according to claim 2, it is characterised in that described to be coupled the magnetic for having IL-6 antibody IL-6 antibody is coupled with magnetic microsphere using EDC-NHS methods in microballoon.
- 7. IL-6 immue quantitative detection reagent boxes according to claim 1, it is characterised in that the reaction diluent reagent bag Contain：0.5~20g/L of stabilizer, 2~50mL/L of coagulant, 0.1~3mL/L of surfactant, 0.5~5mL/L of preservative and phosphorus 10~50mmol/L of phthalate buffer, the pH value of the reaction diluent reagent is 7.0~9.0.
- 8. IL-6 immue quantitative detection reagent boxes according to claim 7, it is characterised in that the stabilizer is bovine serum albumin In vain, the coagulant is PEG 8000, and the surfactant is Tween-20, preservative Proclin300.
- 9. IL-6 immue quantitative detection reagent boxes according to claim 8, it is characterised in that the reaction diluent reagent bag Contain：Stabilizer is 5g/L bovine serum albumin(BSA), and coagulant is 20mL/L PEG 8000s, and surfactant is 1mL/L's Tween-20, preservative are 1mL/L Proclin300, and cushioning liquid is 25mmol/L phosphate buffer, and the reaction is dilute The pH value for releasing liquid reagent is 7.4.
- 10. IL-6 immue quantitative detection reagent boxes according to claim 1, it is characterised in that the IL-6 calibration objects include： PH7.4 50mmol/L phosphate buffer, recombination human source IL-6,10g/L bovine serum albumin(BSA) and 1mL/L anti-corrosion Agent.
- 11. IL-6 immue quantitative detection reagent boxes according to claim 10, it is characterised in that the concentration of the IL-6 calibration objects Scope is 0~1000pg/mL.
- 12. a kind of preparation method of IL-6 immue quantitative detection reagent boxes as any one of claim 1 to 11, its feature exist In, including the magnetic microsphere reagent for being coupled and having IL-6 antibody is prepared, prepare reaction diluent reagent and prepare the step of IL-6 calibration objects Suddenly；Wherein, preparing the magnetic microsphere reagent for having IL-6 antibody that is coupled includes：Preparation coupling has the magnetic microsphere of IL-6 antibody Matrix solution, prepare matrix solution of the IL-6 antibody with magnetic microsphere conjugate and by the magnetic microsphere for being coupled and having IL-6 antibody Described prepare, which is mixed to prepare, with the IL-6 antibody and magnetic microsphere conjugate is coupled the magnetic microsphere reagent for having IL-6 antibody；Preparing the reaction diluent reagent includes：Take pH7.4 phosphate buffers add stabilizer, coagulant, surface-active, Preservative fully mix and dissolve after with 0.22 μm of filtering with microporous membrane, finally with corresponding buffer solution constant volume, be sealed standby With；Preparing the IL-6 calibration objects includes：Prepare calibration object dilution and the calibration object of various concentrations gradient.
- 13. preparation method according to claim 12, it is characterised in that prepare the magnetic microsphere for being coupled and having IL-6 antibody Matrix solution specifically include：Take pH7.4 phosphate buffers to add stabilizer, coagulant, surface-active, preservative to use after fully mixing and dissolve 0.22 μm of filtering with microporous membrane, finally with corresponding buffer solution constant volume, it is sealed standby；Wherein, 0.5~20g/L of stabilizer, 2~200g/L of coagulant, 0.1~10mL/L of surfactant, preservative 0.5~ 5g/L and 10~50mmol/L phosphate buffers, the matrix solution pH value are 7.0~9.0；The stabilizer is bovine serum albumin(BSA) and/or casein；The coagulant is sucrose and/or ficoll -400；It is described Surfactant is Tween-20 and/or triton x-100；Preservative is Proclin300.
- 14. preparation method according to claim 12, it is characterised in that prepare IL-6 antibody and magnetic microsphere conjugate is specific Including：It is 1 in mass ratio by magnetic microsphere, EDC and NHS：1：1, and pH 5.0 50mmol/L MES solution is added, make magnetic micro- Ball mass concentration is 4mg/mL, is placed on vertical rotary instrument and activates, and 25 DEG C of room temperature lucifuges react 30min, by the magnetic after activation Microballoon and IL-6 monoclonal antibodies mass ratio 80：1 is mixed, and is placed on vertical rotary instrument and is marked, and 25 DEG C of room temperature lucifuges are anti- 3h is answered, the Tris-HCl wash liquids 3 times by reacted magnetic microsphere with pH 7.2 25mmol/L, magnetic separation removes supernatant Afterwards, add containing glycine, 5g/L bovine serum albumin(BSA)s, 0.5mL/L triton x-100s, pH 7.4 phosphate buffer, make Magnetic microsphere mass concentration is 4mg/mL, is placed on vertical rotary instrument and terminates, and 25 DEG C of room temperature lucifuges react 2h, by the magnetic after termination Microballoon pH 7.2 25mmol/L Tris-HCl wash liquids 3 times, add the matrix solution, make to be coupled IL-6 antibody Magnetic microsphere mass concentration be 10mg/mL, 2~8 DEG C of preservations.
- 15. preparation method according to claim 12, it is characterised in that the coupling is had to the magnetic microsphere of IL-6 antibody Matrix solution mixes with the IL-6 antibody with magnetic microsphere conjugate, make coupling have the magnetic microsphere of IL-6 antibody final concentration of 1~ 4mg/mL。
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