CN108089010A - A kind of chemiluminescence detection kit of E-Selectin and preparation method thereof - Google Patents
A kind of chemiluminescence detection kit of E-Selectin and preparation method thereof Download PDFInfo
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- 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
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Abstract
The invention discloses a kind of chemiluminescence detection kits of E selectins and preparation method thereof.The kit includes：The E selectins of magnetic particle coupling（Es）Capture antibody, the E selectins of acridinium ester label（Es）Detect antibody, E selectins calibration object, chemiluminescence preexciting liquid A, chemiluminescence exciting liquid B, cleaning solution.Chemiluminescence is combined by kit of the present invention with immune magnetic particle, is provided a kind of close to homogeneous reaction system.Compared with prior art, the present invention establishes direct chemoluminescence method high sensitivity, high specificity, accurate quick, detection time is short, and testing result has higher accuracy and repeatability, which is applicable to various luminometer devices.
Description
Technical field
The invention belongs to the immunochemiluminescence detection kits of immune detection analysis field, specifically a kind of E-Selectin
And preparation method thereof.
Background technology
E-Selectin（Es）It is referred to as " endothelial leukocyte adhesion molecule "（EL-AM-1）, it is by Uniform Name
Es or CD62E.Es is identical with other members of selectin family, is a type single chain glycoprotein, relative molecular mass 115x103,
It is made of a series of protein domain segments, including amino-terminal C-type phytolectin（leotin）Factor print section, one
Epidermal growth factor（EGF）Print section, 6 shorter common repetitive sequence（SCR）（Or complement adjusting/binding protein repetitive sequence,
Containing about 60 amino acid of each sequence）, single trans-membrane region and a cytoplasmic domain.Compared with other selectins, the main knot of Es
Structure feature is：Multiple repetitive sequences of complement adjustment type are combined with agglutinin and EGF areas city, and each complement adjusts repeated fragment and contains
6 rather than 4 cysteines.Es agglutinins region and mannose-binding protein close-packed arrays, c-type aggegation region and EGF areas
Domain has the conservative of height, makes it with playing an important role in terms of ligand binding.
Inflammation is increasingly valued by people in generation, the developing effect of coronary atherosclerosis, chronic inflammation
Property reaction promote the progress of atherosclerosis.The cell adhesion molecule of substantial connection is formed with coronary atherosclerosis
Mainly there are selectin family, integrin family, immunoglobulin superfamily.Es is a member of selectin family, is in inflammation mistake
The adhesion molecule initially occurred in journey.The effect of Es is the white blood cell flow slowed down in blood flow, it is made to be rolled to intravascular epidermis
Then face makes integrin family and its ligand intercellular adhesion molecule-1（ICAM-1）Or Es is combined, and makes leucocyte close adhesion
In inner skin surface, and mediate the exudation carefully run in vain, and migration of the leucocyte to blood vessel endothelium stick be atherosclerosis weight
Want mechanism.Patients with coronary heart disease serum soluble E s expression increases, and Es may take part in the pathogenic process and coronary artery of coronary heart disease
The formation of atherosis.So we contribute to the detection of Es to judge the stabilization of patch in patient with angina pectoris and coronary artery
Property.
The method of E-Selectin detection at present has：Enzyme-linked immunization etc..The enzyme that enzyme-linked immunization uses is horseradish peroxidating
Object enzyme, use horseradish peroxidase major defect for：Luminol exists in no horseradish peroxidase, also can be by
H2O2It aoxidizes itself to shine, background is relatively high, influences signal-to-noise ratio, and kinetics is complicated, and influence factor is more, as a result not steady enough
Fixed, the substrate that obtain high sensitivity and plateau length is not easy.The present invention is had the following advantages that using acridinium ester label：①
Background luminescence is low, and signal-to-noise ratio is high, and luminescence-producing reaction disturbing factor is few；2. flash type shines, the quick concentration of light release, luminous efficiency
Height, luminous intensity are big；3. acridinium ester molecular weight is small, masking antibody combining site is avoided, improves system overall sensitivity；4. it is easy to
With protein bind and after being coupled, photon yield is not reduced；5. label stablize, from ambient oxidant influence (at 2-8 DEG C
Under can preserve the several months long).Therefore acridine substituent is a kind of very effective chemiluminescent labels.
For chemoluminescence method, advantage is the method that the present invention uses：High sensitivity, high specificity, accurate quick, detection
Time is short, the range of linearity is wide, and testing result has higher accuracy and repeatability.
The content of the invention
The present invention provides a kind of kits of the detection E-Selectin with higher sensitivity and specificity.
To achieve the above object, the present invention provides following technical solution：
The chemiluminescence detection kit and preparation method of a kind of E-Selectin, chemoluminescence method provided by the present invention detect E-
The kit of selectin takes magnetic particle to be coupled E-Selectin（Es）Capture antibody, acridinium ester label E-Selectin（Es）Detection
Antibody.Kit further includes E-Selectin calibration object, chemiluminescence preexciting liquid A, chemiluminescence exciting liquid B and cleaning solution.
As further embodiment of the present invention, the magnetic particle can be directly with E-Selectin antibody coupling, also can be by magnetic
Particle is coupled with Streptavidin, while uses biotin labeling E-Selectin antibody.
As further embodiment of the present invention, the surface modification group of the magnetic bead is carboxyl, amino, Streptavidin-
Biotin or p-toluenesulfonyl.Magnetic bead can influence accuracy using the endless bulk deposition of preceding solid phase, therefore while selecting should be selected point
It is good to dissipate property, the slow magnetic bead of sinking speed.
As further embodiment of the present invention, the chemiluminescent labels are acridinium ester, such as：NSP-DMAE-NHS、
NSP-SA-NHS etc..
As further embodiment of the present invention, the acridinium ester label is E-Selectin（Es）Detect antibody.
As further embodiment of the present invention, the acridinium ester label E-Selectin（Es）Detecting the buffer solution of antibody is
PH 8.0-11.0, the Na that concentration is 0.01-0.20mol/L2CO3-NaHCO3Buffer solution.
As further embodiment of the present invention, the molar ratio of the acridinium ester and antibody is 5-100:1.
As further embodiment of the present invention, the acridinium ester is dissolved in DMSO or DMF, and prepared solution is final
Concentration is 0.1-100 mmol/L.
As further embodiment of the present invention, the detection antibody and capture antibody are E-Selectins（Es）Monoclonal
Antibody.
As further embodiment of the present invention, the calibration object be with the Tris containing 0.5-5.0% BSA or PBS or
HEPES buffer solution is matrix, adds in E-Selectin sterling and configures.
As further embodiment of the present invention, the E-Selectin calibration object solution concentration is respectively 0 ng/mL, 5
ng/mL、20 ng/mL、80 ng/mL、320 ng/mL、1280 ng/mL。
As further embodiment of the present invention, the chemiluminescence preexciting liquid A is H2O2And HNO3Mixed liquor,
Middle H2O2Mass fraction is 0.01-5.0%, HNO3Concentration is 0.01-1.0 mol/L.
As further embodiment of the present invention, the chemiluminescence exciting liquid B is the mixed of Triton X-100 and NaOH
Liquid is closed, the wherein mass fraction of Triton X-100 is 0.01-2.0%, and the concentration of NaOH is 0.05-1.0 mol/L.
As further embodiment of the present invention, the cleaning solution includes following components：PH value 7.0-9.0, concentration are
Tris or HEPES or the other solution of 5.0-50.0 mmol/L, wherein containing the Tween- that mass fraction is 0.01-0.25%
20。
The principle of the present invention is to be combined the high degree of specificity of antibody-antigene reaction with the high sensitivity that acridinium ester shines
Get up, the photon detection production concentration generated using acridinium ester catching reaction.
The advantage of the invention is that sandwich method joint magnetic microparticle chemiluminescence technology is used to measure the content of E-Selectin.
Acridinium ester has a clear superiority as the direct chemiluminescence of label, is mainly manifested in：Catalyst is not required in reaction, as long as alkali
Property environment can carry out, be swift in response, can catching reaction generates completely photon, background luminescence is low, and signal-to-noise ratio is high, interference because
Element is few, and reagent stability is good, and system is simple, and exciting liquid is at low cost, acridinium ester easily and protein bind, and photon yield after being coupled
It does not reduce.
Specific embodiment
The present invention provides a kind of chemiluminescence detection kit of E-Selectin, to make the object of the invention, technical solution
And effect is clearer, clear and definite, the present invention is described in detail below.
The present invention provides a kind of chemiluminescence detection kit and preparation method of E-Selectin, the E- choosings of magnetic particle coupling
Select element（Es）Capture antibody, the E-Selectin of acridinium ester label（Es）Detect antibody.Kit further include E-Selectin calibration object,
Chemiluminescence preexciting liquid A, chemiluminescence exciting liquid B and cleaning solution.
Specifically, during magnetic particle coupling capture antibody is prepared, the buffer solution of the coupled antibody is the present invention
PH value 5.0-6.0, the buffer solution that concentration is 20-200 mmol/L MES.
Specifically, for the present invention during magnetic particle coupling capture antibody is prepared, the Block buffer is containing 1%
The buffer solution of BSA.
Specifically, the present invention is during acridinium ester label detection antibody is prepared, acridinium ester label E- selections
Element（Es）The Na that the buffer solution of detection antibody is pH 8.0-11.0, concentration is 0.01-0.20mol/L2CO3-NaHCO3Buffer solution.
Specifically, calibration object of the present invention is to be by the Tris containing 0.5-5.0% BSA or PBS or HEPES buffer solution
Matrix adds in E-Selectin sterling and configures.
Specifically, the chemiluminescence preexciting liquid A is H2O2And HNO3Mixed liquor, wherein H2O2Mass fraction is
0.01-5.0%, HNO3Concentration is 0.01-1.0 mol/L.
Specifically, chemiluminescence exciting liquid B of the present invention is the mixed liquor of Triton X-100 and NaOH, wherein
The mass fraction of Triton X-100 is 0.01-2.0%, and the concentration of NaOH is 0.05-1.0 mol/L.
Specifically, cleaning solution of the present invention is pH value 7.0-9.0, concentration is 5.0-50.0 mmol/L Tris or
HEPES or other solution, wherein containing the Tween-20 that mass fraction is 0.01-0.25%.
Below by embodiment, the present invention is described in detail.
Embodiment 1：A kind of establishment of chemiluminescence detection kit of E-Selectin and preparation method
1. the establishment of kit
The E-Selectin monoclonal antibody of magnetic particle coupling；
The E-Selectin of acridinium ester label（Es）Monoclonal antibody；
E-Selectin series of calibration product solution；
Chemiluminescence preexciting liquid A and chemiluminescence exciting liquid B；
Cleaning solution.
2. it is coupled the preparation of the magnetic particle suspension of E-Selectin monoclonal antibody
（1）1 mg carboxyls magnetic particle is taken in 0.5 mL centrifuge tubes, adds in the MES buffer solutions that 200 μ L concentration are 0.1 mol/L,
Vortex mixing, is placed on magnetic frame, and standing 5 min makes magnetic particle be separated with liquid, and supernatant discarding washs 3 times, adds 200
The MES of μ L（PH is 6.0）Buffer solution is vortexed.
（2）15 μ g E-Selectin monoclonal antibodies are added in, it is 150 to make the molar ratio of carboxyl and antibody:1, it is vortexed, rotation
Turn reaction tube, be incubated at room temperature 30 min.
（3）The coupling reagent EDC that 10 μ L concentration are 10 mg/mL is added in, in being vortexed on rotatable reactor, is incubated at room temperature 2
h。
（4）The glycine buffer that 200 μ L is taken to contain 1% BSA（Concentration is 50mmol/L）It is closed, the time is 1 h.
（5）Supernatant is removed, adds in the cleaning buffer solution of 200 μ L（TBS+0.05% Tween-20）, wash 3 times.
（6）The above-mentioned magnetic particle suspension prepared is placed in the preservation liquid of 500 μ L（The glycine of 300 mmol/L, 2%
Glycerine, 5% sucrose）In, 2-8 DEG C of preservation.
3. acridinium ester label E-Selectin（Es）The preparation of monoclonal antibody
（1）Purify E-Selectin（Es）Antibody：By the E-Selectin of 100 μ g（Es）Antibody is placed in bag filter, and by bag filter
It is placed in the mark buffer solution not less than 1L and dialyses, during which buffer fluid exchange 5 times, last time dialysed overnight marks buffer solution
It is pH 9.8, the Na that concentration is 50 mmol/L2CO3-NaHCO3Buffer solution.
（2）The acridinium ester NSP-DMAE-NHS of 1.7 mg is weighed, is dissolved in 447 μ L anhydrous dimethyl formamides（DMF）In, match somebody with somebody
Into the NSP-DMAE-NHS DMF solutions of 6.5 mmol/L.
（3）By the E-Selectin after dialysis（Es）Monoclonal antibody is placed in 500 μ L centrifuge tubes, adds in 200 μ L pH
9.8th, concentration is the Na of 50 mmol/L2CO3-NaHCO3Buffer solution（It is protected from light）, 759 μ L are added in, 6.5 mmol/L's
NSP-DMAE-NHS DMF solutions, make acridinium ester and E-Selectin（Es）The molar ratio of monoclonal antibody is 7.4:1, vibration is mixed
It is even, 1 h is reacted at room temperature, and 100 μ L concentration of addition are 10 g/L lysines, stand 15 min, make mark reaction terminating.
（4）Label NSP-DMAE-NHS-Ab and free NSP-DMAE-NHS passes through Sephadex G-50 columns（1×
25cm）Separation is balanced with purification buffer pH 6.3, the PBS that concentration is 0.1 mol/L and elutes chromatographic column.
（5）Protein peak is detected with chromatograph in separation process, measures the chemiluminescence intensity and 280 nm of efflux respectively
Absorbance.
（6）The eluent that shading value is high and absorbance is big is collected, stored frozen is dispensed after adding in 1% BSA.
4. the preparation of E-Selectin series of calibration product solution
It is matrix with the Tris-HCl buffer solutions containing 1% BSA, addition E-Selectin sterling is made into mark concentration and is respectively：0
Ng/mL, 5 ng/mL, 20 ng/mL, 80 ng/mL, 320 ng/mL, the solution of 1280 ng/mL.
5. the preparation of shine exciting liquid A, B
（1）Chemiluminescence preexciting liquid A is by H2O2And HNO3Composition.Wherein, wherein H2O2Mass fraction be 1.5%, HNO3It is dense
It spends for 0.1 mol/L, is distributed into 20 mL/ branch with brown bottle, 2-8 DEG C saves backup.
（2）Chemiluminescence exciting liquid B is made of the mixed liquor of Triton X-100 and NaOH.Wherein, Triton X-100
Mass fraction be 1.0%, NaOH concentration be 0.4 mol/L, be distributed into 20 mL/ branch with brown bottle, 2-8 DEG C saves backup.
6. the preparation of cleaning solution
Weigh 3.05 g Tris, in the beaker of 8.775 g NaCl to 1000 mL, it is 0.05% to add in 1mL mass fractions
Tween-20 is stirred and evenly mixed, constant volume, is saved backup after pH is adjusted to 7.6.
Embodiment 2：With kit detection and interpretation of result
（1）In reaction cup add in 50 μ L of sample to be tested, add magnetic particle coupling 150 μ L of suspension, vibrate mixing, 37 DEG C
It is incubated 8 min.
（2）Separation is cleaned 3 times.Reaction vessel after washing, which is fully vibrated, makes magnetic particle scatter..
（3）150 μ L of acridinium ester label are added in into reaction cup, vibrate mixing, 37 DEG C of 7 min of incubation.
（4）Separation is cleaned 3 times.Reaction vessel after washing, which is fully vibrated, makes magnetic particle scatter.
（5）100 μ L chemiluminescence exciting liquid B are added in after adding in 100 μ L chemiluminescence preexciting liquid A, 1s, measure its phase
To luminous intensity, the content of E-Selectin luminous intensity proportion relation corresponding thereto in sample.
Embodiment 3：The performance indicator 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 draws the RLU values of 20 measurement results
（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, and corresponding concentration value is obtained.By minimum detection limit detection method in detection scheme, it is repeated 3 times
Experiment, it is 4 ng/mL to the sensitivity for analysis of E-Selectin that this kit, which is finally obtained,.
2. precision measures
It is the sample of (12 ± 1.2) ng/mL and (45 ± 4.5) ng/mL with Es kits test concentrations, each concentration repeats to survey
Examination 10 times calculates kit precision, the results showed that kit CV is respectively less than 5%.
3. stability
E-Selectin detection kit is taken to carry out normal storage stability test, 2-8 DEG C is placed respectively temporally 1,3,5,7,9,
It is 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.The results show E-Selectin detection kit is stored
In 2-8 DEG C, light protected environment, the term of validity is 12 months.It uncaps and is 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 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, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Profit requirement rather than above description limit, it is intended that all by what is fallen within the meaning and scope of the equivalent requirements of the claims
Variation is included within the present invention.
Moreover, it will be appreciated that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
Using specification as an entirety, the technical solutions in each embodiment can also be properly combined, forms those skilled in the art
It is appreciated that other embodiment.
Claims (9)
1. the chemiluminescence detection kit and its composition of a kind of E-Selectin, it is characterised in that：The E- selections of magnetic particle coupling
Element（Es）Capture antibody, the E-Selectin of acridinium ester label（Es）Detect antibody, E-Selectin calibration object, chemiluminescence preexciting
Liquid A, chemiluminescence exciting liquid B, cleaning solution.
2. the chemiluminescence detection kit and its composition of a kind of E-Selectin according to claim 1, it is characterised in that：
The kernel of the magnetic bead is ferroso-ferric oxide or di-iron trioxide, and the surface modification group of magnetic particle is one or more activity
Functional group includes but not limited to carboxyl, amino, p-toluenesulfonyl or Streptavidin-biotin.
3. a kind of chemiluminescence detection kit of E-Selectin according to claim 1, it is characterised in that：The magnetic
Particle directly can capture antibody coupling with E-Selectin or be coupled magnetic particle and Streptavidin, while use biotin mark
Remember E-Selectin capture antibody.
4. the chemiluminescence detection kit and its composition of a kind of E-Selectin according to claim 1, it is characterised in that：
The chemiluminescent labels are acridinium ester, such as NSP-DMAE-NHS, NSP-SA-NHS.
5. the chemiluminescence detection kit and its composition of a kind of E-Selectin according to claim 1, it is characterised in that：
The acridinium ester label is E-Selectin（Es）Detect antibody.
6. a kind of chemiluminescence detection kit of E-Selectin according to claim 1, it is characterised in that：The inspection
It is E-Selectin to survey antibody and capture antibody（Es）Monoclonal antibody.
7. a kind of chemiluminescence detection kit of E-Selectin according to claim 1, it is characterised in that：The school
Quasi- product are the calibration objects that the series concentration gradient that the configuration of E-Selectin sterling forms is added in using the buffer solution containing BSA as matrix
Solution.
8. a kind of chemiluminescence detection kit of E-Selectin according to claim 1, it is characterised in that：The change
The preexciting liquid A that shines is learned by H2O2And HNO3Mixed liquor composition, chemiluminescence exciting liquid B is by Triton X-100 and NaOH
Mixed liquor forms.
9. the chemiluminescence detection kit and its composition of a kind of E-Selectin according to claim 1, including following step
Suddenly：A certain amount of sample to be tested is added in reaction cup, adds magnetic particle coupling suspension, mixing, 37 DEG C of incubations add in clear
Washing lotion removes supernatant；Adding in acridinium ester label according to a certain percentage afterwards, 37 DEG C of incubations add in cleaning solution and remove supernatant,
Chemiluminescence preexciting liquid A and exciting liquid B are added in, measures corresponding luminous intensity RLU/s.
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| 王利平: "布地奈德雾化对AECOPD患者治疗前后血清ICAM-1、E-选择素的影响", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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