CN107817354A - A kind of chemiluminescence detection kit of interleukin 6 and preparation method thereof - Google Patents
A kind of chemiluminescence detection kit of interleukin 6 and preparation method thereof Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
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- 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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Abstract
The invention discloses a kind of chemiluminescence detection kit of interleukin 6 and preparation method thereof.The kit includes：Sample dilution, the magnetic particle for being coated with interleukin 6 monoclonal antibody, the interleukin 6 monoclonal antibody for being marked with acridinium ester, interleukin 6 serial standards solution, chemiluminescence exciting liquid A, chemiluminescence exciting liquid B, cleaning fluid.Chemiluminescence is combined by kit of the present invention with immune magnetic particle, there is provided a kind of close to homogeneous reaction system.Compared with prior art, kit of the present invention has the advantages that high sensitivity, high specificity, reaction time are short.
Description
Technical field
The invention belongs to in-vitro diagnosis field, specifically with the kit of interleukin 6 in chemoluminescence method detection human serum
And preparation method thereof.
Background technology
Interleukin 6, full name interleukin-6（IL-6）, it is a kind of cell factor of manifold effect, belongs to interleukins
One kind.It is by fibroblast, Monocytes/Macrophages, T lymphocytes, bone-marrow-derived lymphocyte, epithelial cell, horn cell with
And a variety of oncocytes produce.Interleukin 6 can be by the induction such as IL-1, TNF-a, PDGF, virus infection, double-stranded RNA and c AMP
Normal cell produces.Interleukin 6, which can stimulate, to be participated in the cell propagation of immune response, breaks up and improve its function.Interleukin 6
Main function is：Promote the differentiation of B cell hyperplasia and secretory antibody, also have to liver cell, T cell, nerve fiber, the hematopoiesis decorum
Extensive effect；With Antineoplastic effect, also can directly or indirectly strengthen NK cells and CTL kills tumor activity.Thus, interleukin 6 is again
There are many synonyms, such as β2Interferon, hepatocyte-stimulating factor, hybridoma cell growth factor, B cell differential factor etc..
Interleukin 6 plays an important role in the defense mechanism of human body.It also plays the part of in the generation of a variety of diseases simultaneously
Important role.Detection interleukin has very important meaning for understanding the state of an illness, judging prognosis, research pathogenesis.
At present, the interleukin 6 detection kit of domestic-developed uses ELISA more.Examined using ELISA
The shortcomings that survey is that HRPO or alkaline phosphatase label easy in inactivation, chromogenic substrate are shown in that light easily decomposes, and sensitivity is low, to knot
The similar compound of structure has certain cross reaction, causes test result inaccurate.The present invention uses acridinium ester label, has
Following advantage：1. reaction does not need catalyst, background luminescence is low, and signal to noise ratio is high, and luminescence-producing reaction disturbing factor is few；2. flash type is sent out
Light, the quick concentration of light release, luminous efficiency is high, luminous intensity is big；3. acridinium ester molecular weight is small, avoid covering antibody binding position
Point, improve system overall sensitivity；4. it is easy to protein bind and photon yield is not reduced after being coupled；5. label is stable,
Do not influenceed by ambient oxidant, the several months long can be preserved at 2-8 DEG C.Therefore acridine substituent is a kind of very effectiveization
Learn luminous marker.
The content of the invention
The invention provides a kind of detection kit with higher sensitivity and specific interleukin 6.
To achieve the above object, the present invention provides following technical scheme：
The chemiluminescence detection kit and preparation method of a kind of interleukin 6, the kit include Sample dilution, be coated with it is white
The magnetic particle of the monoclonal antibody of interleukin 6, the interleukin 6 monoclonal antibody for being marked with chemiluminescent labels, exciting liquid A, swash
Lotion B, interleukin 6 calibration object solution and cleaning fluid.
The chemiluminescent labels are acridinium ester, such as：NSP-DMAE-NHS, NSP-SA-NHS etc..
The Sample dilution is the phosphate buffer that pH is 7.4, includes 1% bovine serum albumin(BSA), 0.1%TritonX-
100 and 0.3% Proclin.
The surface modification group of the magnetic bead can be carboxyl, amino, Streptavidin-biotin or tolysulfonyl
Base, magnetic bead particle diameter can be 1.5~3 μm.
The interleukin 6 monoclonal antibody is the monoclonal antibody specific of interleukin 6 to be checked.
Described acridinium ester and the mol ratio of antibody are 5~10:1, preferred molar ratio 7.4:1.
Magnetic particle and Streptavidin can be also coupled, simultaneously by described magnetic particle directly with interleukin 6 antibody coupling
Using biotin labeling interleukin 6 antibody.
The preparation method of the chemiluminescence detection kit of described interleukin 6, comprises the following steps：
1）Prepare sample dilution buffer：The Sample dilution is the phosphate buffer that pH is 7.4, and it is pure to include 1% ox blood
Albumen, 0.1%TritonX-100 and 0.3% Proclin.
2）Prepare the magnetic particle for being coated with interleukin 6 monoclonal antibody：After the magnetic particle solution Magneto separate of carboxylated
Supernatant is removed, is resuspended with MES buffer solutions, EDC solution is added, is activated on vertical rotary instrument, the magnetic particle after activation is situated between with white
Plain 6 antibody, which are placed on vertical rotary instrument, to be coupled, and Magneto separate removes supernatant, washs 3 times, is added the buffer solution containing 1%BSA and is carried out
Closing, is finally placed in 2-8 DEG C of preservation by magnetic particle suspension.
3）Prepare the interleukin 6 monoclonal antibody of acridinium ester label：The μ g of interleukin 6 monoclonal antibody 10 are taken, addition contains
NaCl phosphate buffer to volume is 60 μ L, adds 1 μ L acridinium ester mother liquors, and concussion mixes, the reaction of room temperature lucifuge
30min, phosphate buffers of the 40 μ L containing NaCl and glycine is added after reaction, mixed, room temperature lucifuge reaction 30min.Reaction
Product is moved into bag filter afterwards, dialyzate is the 20mM PBSs that pH is 7.4,2-8 DEG C of lucifuge dialysis, one is changed at interval of 2h
Secondary PBS, at least change 3 times, last time dialysed overnight, to remove unlabelled acridinium ester.Label is taken out, by actual body
Product add 10% bovine serum albumin(BSA) to its final concentration of 0.1%, add isometric glycerine, mix, -20 DEG C are kept in dark place.
4）Prepare interleukin 6 antigen standard solution：With containing 1%BSA, 0.1%TritonX-100,0.3% Proclin,
Phosphate buffer that pH is 7.4 dilution interleukin 6 sterling to concentration is 0pg/mL, 10pg/mL, 50pg/mL, 150pg/mL,
600pg/mL, 1000pg/mL interleukin 6 antigen standard solution.
5）Prepare exciting liquid：Exciting liquid includes exciting liquid A and exciting liquid B.Wherein exciting liquid A is H2O2And HNO3Mixing
Liquid, wherein H2O2Mass fraction is 0.05-5%, HNO3Concentration is 0.05-2.5 mol/L；Exciting liquid B be Triton X-100 and
NaOH mixed liquor, wherein Triton X-100 concentration are that 0.05-2.0 mol/L, NaOH concentration are 0.05-1.0 mol/
L。
6）Prepare cleaning fluid：Cleaning fluid is containing surfactant TritionX-100 and biological preservative Proclin
Phosphate buffer.
Described acridinium ester is dissolved in DMSO or DMF, and the ultimate density of prepared solution is 1-10mmol/L, preferably dense
Spend for 6.5 mmol/L.
The principle that interleukin 6 detection kit of the present invention uses is double antibody sandwich method, particular by magnetic particle
Conjugated monoclonal antibodies are combined with the antigentic specificity in sample, then with the monoclonal of acridinium ester label to see that topic combines to form compound
Thing, finally utilize photon detection measured object concentration caused by acridinium ester.
The present invention has advantages below：
Using Magneto separate chemiluminescence as detection means, while acridinium ester label technology is used to be detected to establish a kind of inspection
Survey the direct chemical luminescence reagent kit of interleukin 6.Acridinium ester have molecular weight it is small, to the effect of the steric hindrance of association reaction less,
Background luminescence is low, signal to noise ratio is high, the participation without enzyme, luminous efficiency is high, mark is stable, can improve the bright of system overall sensitivity
Aobvious advantage.
The present invention requires low to Sample pretreatment, and sample can be used to detect after the processing such as extraction, dilution, and can realize
High-volume automatic detection.
To sum up, kit of the present invention is simple to operate, has luminous value stabilization, high sensitivity, detection quick and convenient, accurate
The advantages that true property is high, reproducible, high specificity.
Embodiment
Technical scheme is further detailed and described below by way of embodiment.
Embodiment 1：The establishment and preparation of kit
1）Kit is set up
A kind of chemiluminescence detection kit of interleukin 6 is set up, it contains following component：
Sample dilution；
It is coated with the magnetic particle of interleukin 6 monoclonal antibody；
It is marked with the interleukin 6 monoclonal antibody of chemiluminescent labels；
Exciting liquid A, exciting liquid B；
Interleukin 6 calibration object solution, concentration be respectively 0pg/mL, 10pg/mL, 50pg/mL, 150pg/mL, 600pg/mL,
1000pg/mL；
Cleaning fluid.
2）The preparation of Sample dilution
Specially pH is 7.4 phosphate buffer, includes 1% bovine serum albumin(BSA), 0.1%TritonX-100 and 0.3% anti-corrosion
Agent Proclin.
3）Prepare the magnetic particle suspension for being coated with interleukin 6 monoclonal antibody
Supernatant will be removed after the magnetic particle solution Magneto separate of carboxylated, be resuspended with MES buffer solutions, added EDC solution, vertically revolving
Turning to activate on instrument, the magnetic particle after activation is placed on vertical rotary instrument with interleukin 6 antibody and is coupled, Magneto separate removes supernatant,
Washing 3 times, adds the buffer solution containing 1%BSA and is closed, magnetic particle suspension finally is placed in into 2-8 DEG C of preservation.
4）Prepare the interleukin 6 monoclonal antibody of acridinium ester label
The μ g of interleukin 6 monoclonal antibody 10 are taken, it is 60 μ L to add the phosphate buffer containing NaCl to volume, adds 1 μ L a word used for translations
Pyridine ester mother liquor, concussion mix, and room temperature lucifuge reaction 30min, phosphate-buffereds of the 40 μ L containing NaCl and glycine are added after reaction
Liquid, mix, room temperature lucifuge reaction 30min.Product is moved into bag filter after reaction, dialyzate is that the 20mM PBS that pH is 7.4 delay
Fliud flushing, 2-8 DEG C of lucifuge dialysis, changes a PBS at interval of 2h, at least changes 3 times, last time dialysed overnight, do not marked with removing
The acridinium ester of note.Label is taken out, by actual volume add 10% bovine serum albumin(BSA) to its final concentration of 0.1%, add etc.
Volume glycerine, mix, -20 DEG C are kept in dark place.
5）Exciting liquid A and exciting liquid B preparation
Exciting liquid A is H2O2And HNO3Mixed liquor, wherein H2O2Mass fraction is 0.05-5%, HNO3Concentration is 0.05-2.5
mol/L；
Exciting liquid B is Triton X-100 and NaOH mixed liquor, and wherein Triton X-100 concentration is 0.05-2.0 mol/
L, NaOH concentration are 0.05-1.0 mol/L.
6）The preparation of interleukin 6 calibration object solution
It is white with being diluted containing the phosphate buffer that 1%BSA, 0.1%TritonX-100,0.3% preservative Proclin, pH are 7.4
The sterling of interleukin 6 to concentration is 0pg/mL, 10pg/mL, 50pg/mL, 150pg/mL, 600pg/mL, 1000pg/mL；
Interleukin 6 antigen standard solution.
7）The preparation of cleaning fluid
Cleaning fluid is the phosphoric acid of the pH7.4 containing surfactant 1%TritionX-100 and biological preservative 0.3%Proclin
Salt buffer.
Embodiment 2：The detection of interleukin 6 in actual sample
The use operation sequence of interleukin 6 immue quantitative detection reagent box of the present invention is as follows：
The detection of kit
1）By μ L of sample to be tested 50, μ L of magnetic particle suspension 150, the μ L of acridinium ester label 150, reaction tube is sequentially added, is vibrated
Mix, 37 DEG C of 15 min of incubation.
2）Separation, clean 5 times.
3）Reaction vessel after washing, which is fully vibrated, makes magnetic particle scatter.
4）100 μ L chemiluminescence exciting liquid B are added after adding 100 μ L chemiluminescence exciting liquids A, 1 s, it is relative to measure its
Luminous intensity, the content of Procalcitonin luminous intensity proportion relation corresponding thereto in sample.
Embodiment 3：The performance indications of kit
1）The sensitivity of kit
By the use of 0 ng/mL calibration objects in kit as sample to be tested, replication 20 times, the RLU values of 20 measurement results are drawn
（Relative light unit）, calculate its average value（M）And standard deviation（SD）, RLU values corresponding to M+2SD are drawn, according to zero-dose school
Concentration-RLU values result between quasi- product and adjacent calibration object carries out 2 regression fits and draws linear function, by M+2SD's
RLU values are brought into above-mentioned equation, obtain corresponding concentration value.By minimum detection limit detection method in detection scheme, it is repeated 3 times
Experiment, it is 3.8 pg/mL to the sensitivity for analysis of interleukin 6 finally to obtain this kit.
2) precision determines
With interleukin 6 kit test concentrations it is 50pg/mL and 600pg/mL sample, each concentration retest 10 times, meter
Calculate kit precision, the results showed that kit CV is respectively less than 5%.
3）Stability
Interleukin 6 detection kit is taken to carry out normal storage stability test, 2-8 DEG C is placed respectively temporally 1,3,5,7,9,
Detected within 11,12,13,14,15 months；Uncap stability test respectively by 2-8 DEG C place 0 day, 7 days, 14 days, 16 days, 18
My god, be measured within 20 days, 22 days, 24 days, 26 days, 28 days, 30 days, 32 days.As a result show that interleukin 6 detection kit is stored in
2-8 DEG C, in light protected environment, the term of validity is 12 months.Uncap and be stored in 2-8 DEG C, in light protected environment, the term of validity is 30 days.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires rather than described above limits, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.
Moreover, it will be appreciated that although the present specification is described in terms of embodiments, not each embodiment is only wrapped
Containing an independent technical scheme, this narrating mode of specification is only that those skilled in the art should for clarity
Using specification as an entirety, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
It is appreciated that other embodiment.
Claims (7)
- A kind of 1. chemiluminescence detection kit of interleukin 6, it is characterised in that：The kit includes Sample dilution, coating The magnetic particle that has interleukin 6 monoclonal antibody, the interleukin 6 monoclonal antibody for being marked with chemiluminescent labels, exciting liquid A, exciting liquid B, interleukin 6 calibration object solution and cleaning fluid.
- 2. interleukin 6 chemiluminescence detection kit according to claim 1, it is characterised in that：The chemiluminescence mark Note thing is acridinium ester, such as NSP-DMAE-NHS, NSP-SA-NHS.
- A kind of 3. chemiluminescence detection kit of interleukin 6 according to claim 1, it is characterised in that：Described a word used for translation Pyridine ester mark is interleukin 6 monoclonal antibody.
- A kind of 4. chemiluminescence detection kit of interleukin 6 according to claim 1, it is characterised in that：Described magnetic Property particulate or magnetic particle and Streptavidin can be coupled directly with interleukin 6 antibody coupling, it is while white using biotin labeling The antibody of interleukin 6.
- A kind of 5. chemiluminescence detection kit of interleukin 6 according to claim 1, it is characterised in that：Described school Quasi- product are using the phosphate buffer that the pH containing 1% BSA, 0.1%TritonX-100 and 0.3% Proclin is 7.4 as base Matter, add the calibration object solution for the series concentration gradient that the configuration of interleukin 6 sterling forms.
- A kind of 6. chemiluminescence detection kit of interleukin 6 according to claim 1, it is characterised in that：Described change Luminous exciting liquid A is learned by H2O2 And HNO3Mixed liquor composition, chemiluminescence exciting liquid B is by Triton X-100 and NaOH Mixed liquor forms.
- 7. the chemiluminescence detection kit of a kind of interleukin 6 according to claim 1, it is characterised in that including following Step：A certain amount of sample to be tested is added in reaction cup, adds magnetic particle suspension liquid and acridinium ester label, is mixed, 37 DEG C of incubations, add cleaning fluid and remove supernatant, add chemiluminescence exciting liquid A and exciting liquid B, determine corresponding luminous intensity RLU/s。
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