CN109633162A - S-100 β protein detection kit - Google Patents
S-100 β protein detection kit Download PDFInfo
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- CN109633162A CN109633162A CN201811434397.XA CN201811434397A CN109633162A CN 109633162 A CN109633162 A CN 109633162A CN 201811434397 A CN201811434397 A CN 201811434397A CN 109633162 A CN109633162 A CN 109633162A
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- 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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The present invention provides S-100 β protein detection kit, belongs to fluorescence immune chromatography technical field in Medical Immunology, including detection card, IC card and dilution, and detection card includes the bonding pad for being adsorbed with the S-100 β monoclonal antibody of rare-earth fluorescent microballoon label；Nitrocellulose filter, the detection line of coating C reaction S-100 β monoclonal antibody and the nature controlling line of coating T reaction sheep anti-mouse igg antibody；Sample pad；Liner plate and blotting paper.The test sample of kit of the present invention is serum, blood plasma or the whole blood sample of people, have good detection specificity, higher sensitivity, stability, the simplicity of operation, its purposes is the content of S-100 β albumen in quantitative detection human serum, blood plasma or whole blood sample in vitro, for the early stage auxiliary diagnosis of clinically cerebral injury, assessment the severity of brain injury, guiding treatment and judging prognosis.
Description
Technical field
The invention belongs to fluorescence immune chromatography technical fields in Medical Immunology, and in particular to S-100 β protein assay reagent
Box.
Background technique
S100 albumen is the small acidic protein that molecular weight is 10-12kDa size, and forms homodimer and different dimerization
Body.S100 albumen usually participates in a large amount of cellular activity event, such as signal transduction, cell differentiation, cell mobility adjust, turn
Record and cell cycle progression etc..S100 albumen is combined by Liang Ge subunit α, β and forms S100 α α, S100 α β, S100 β β.Wherein,
S100- β (S100 α β and S100 β β) albumen is a kind of acid calbindin of low molecular weight, is called the special egg of nervous centralis
It is white, largely it is present in central nervous system.Content is less than 0.2ng/mL, the S100 β under physiological concentration in normal adult's serum
Albumen has neurotrophic effect.When people is in cerebral infarction, brain trauma or cardiac operation, S100- β albumen is seeped from cytosol
Enter cerebrospinal fluid out, then enter blood through impaired blood-brain barrier, is increased so as to cause the concentration of S100- β albumen in blood, it can
Stimulation spongiocyte and neuron generate proinflammatory cytokine and are relied on to further activate spongiocyte by nitric oxide
Mechanism kills neuron.The overexpression of S100 β albumen will increase brain to ischemic, and the neurological susceptibility of anoxic leads to withering for neuron
It dies.After central nervous system damage, the concentration in blood and cerebrospinal fluid all increases, and S100 β albumen is seeped from Deiter's cells
Out into cerebrospinal fluid, blood is entered by impaired blood-brain barrier, so that serum detection S100 β protein concentration increases.
S100- β albumen has regular hour changing rule as the biochemical marker of cerebral injury after brain injury, and stability is preferable,
It is pre- that the detection of concentration value facilitates the early stage auxiliary diagnosis of clinically cerebral injury, assessment the severity of brain injury, guiding treatment and judgement
Afterwards etc..Therefore serum S100- β determination of protein concentration is to assess cerebral apoplexy, the reliable markers object of development.
Fluorescent quantitation immunochromatography technique principle is similar to ELISA method, and this method is reacted based on antigen and antibody specific
Principle, pass through labelled antibody or antigen and form immune complex to achieving the purpose that detection.It is different from ELISA method
It is that the immunoreaction process of fluorescent quantitation immunochromatography technique is to occur in chromatographic film and pass through capillarity to front layer
Analysis.In recent years, the chromatographic technique including fluorescent mark immunity chromatographic technique is widely used in the detection of low concentration sample, by
There is certain advantage in terms of sensibility, operability and accuracy in detection method, therefore this method is including clinical chemistry, life
Every subjects field including object analysis and environmental analysis all has wide practical use.Based on fluorescent quantitation immunochromatographic method
Kit can be used for the detection of high-volume sample, but can not substitute current instrument analysis technology, it can be used as one kind
Important complementary technology, it should and other analytical technologies are used in conjunction or corroborate each other, the sensitivity for increasing detection can reduce intersection again
Reaction can also carry out more accurately quantitative.Therefore, fluorescent quantitation immunochromatographic method monitors at the scene, simple Basic Laboratory
Have wide practical use in the preliminary screening of detection or even large-scale experiment room.Therefore, anti-by synthesis haptens and completely
Former, fluorescent quantitation immunochromatography platform pairing antigen-antibody, fluorescent quantitation immunochromatography pad optimization, kit Performance Evaluation and
The conceptual phases such as clinical application are high performance to establish effective detection S100- β protein fluorescence quantitative immunochromatographic method and research and development
S100- β protein detection kit has great importance.
Summary of the invention
The purpose of the present invention is to provide serum, blood plasma or whole blood samples that a kind of test sample is people, have good
The S-100 β protein detection kit of specificity, higher sensitivity, stability, the simplicity of operation is detected, purposes includes
Clinically the early stage auxiliary diagnosis of cerebral injury, assessment the severity of brain injury, guiding treatment and judging prognosis.
The technical solution that the present invention is taken to achieve the above object are as follows:
The present invention discloses a kind of S-100 β protein detection kit, including detection card, IC card and dilution, wherein detection card
Bonding pad including being adsorbed with the S-100 β monoclonal antibody of rare-earth fluorescent microballoon label.
Preferably, bonding pad is prepared using following steps: glass fibre cotton is placed in containing 1.2-1.5%BSA, 0.5-
In the 1.5-1.6M Tris-HCL treatment fluid of 0.8% fucose, 15-25min is impregnated under the Ultrasonic Conditions of 28-34KHz,
Then the dry 2-3h at 60-70 DEG C, takes out spare.The bonding pad is the S-100 β monoclonal antibody of rare-earth fluorescent microballoon label
That is S-100 β monoclonal antibody-microsphere compound provides a drying, preservations, the place for moistening and discharging again, fucose with
Ultrasonic wave can play gain effect, and the effect of ultrasonic wave can promote to occur or accelerate certain chemical reactions, so that bonding pad table
Face quickly forms one layer of smooth surface being made of sugar, uniform, reduces later period S-100 β monoclonal antibody-microsphere compound
With the suction-operated of bonding pad；It can also be in S-100 β monoclonal antibody-microsphere compound surface shape in addition combined with the sugar on pad surface
S-100 β monoclonal antibody-microsphere compound is remained in the drying process using its significant water retention at a glace
Good dispersibility and uniformity are kept, while being also avoided that the activity decline of antibody, after sample is added dropwise, it is fast that carbohydrate meets liquid
Instant solution, S-100 β monoclonal antibody-microsphere compound also can quickly thoroughly be discharged from bonding pad, so above-mentioned preparation method
The sensitivity and stability of kit can be improved in obtained bonding pad.In addition, the D- fucose in fucose containing 2.8-3.2%
When, the surface tension of chromatography samples can be reduced, can better ensure that S-100 β monoclonal antibody-microballoon is multiple in again wet process
Uniformity and stationarity of the object on bonding pad are closed, S-100 β monoclonal antibody-microsphere compound on bonding pad can be improved and released
Put, optimize S-100 β monoclonal antibody-microsphere compound releasing effect, while can also accelerating liquid Tomography Velocity, improve
The detection rates of kit.
Preferably, detection card further includes nitrocellulose filter, the detection line of coating C reaction S-100 β monoclonal antibody
With the nature controlling line of coating T reaction sheep anti-mouse igg antibody.S-100 β monoclonal antibody is recombination human source S-100 β albumen, by complete
Gene chemical synthesis source of people S-100 β gene constructs S-100 β recombinant plasmid；Using S-100 β recombinant plasmid as expression vector, pass through yeast
Eukaryotic expression prepares S-100 β albumen；S-100 β monoclonal antibody is prepared using the S-100 β albumen of expression.
Further preferably, nitrocellulose filter is handled using following steps:
1) S-100 β monoclonal antibody and goat anti-mouse igg antibody coating dilution adjustment concentration to 1-3mg/ml, film
Liquid measure is 1-2 μ l/cm；
2) the S-100 β monoclonal antibody that obtains step 1) and goat anti-mouse igg antibody solution as detection line and
Nature controlling line is sprayed on nitrocellulose filter in parallel and is coated with, and is divided into 3-4mm between the detection line and nature controlling line, is subsequently placed in
In baking oven, 35-40 DEG C is dried 2 hours.
Still more preferably, coating dilution is the 4-6mM BS buffering containing 0.5-0.8% fucose, pH 7-7.5
Liquid；D- fucose in fucose containing 4-5%.Under normal circumstances, the expansion with coating buffer BS on nitrocellulose filter film
It dissipating, some surface reactive materials on film can cause film hydrophobicity to enhance by with to the movement of tunica fibrosa both sides, after point sample, when
When S-100 β monoclonal antibody-microsphere compound forward position chromatography is reached near detection line, since hydrophobicity is strong in detection line, solution
Bottom plate of the meeting under detection line is swum underwater until detection line another side hydrophily strength, just normal chromatography, and above-mentioned coating dilutes
Liquid can be improved hydrophily of the coated antibody on nitrocellulose filter, in point sample, can lower the expansion of S-100 β monoclonal antibody
Scattered speed reduces the surfactant at detection line and is largely lost, even if surfactant is all lost, fucose it is fast
Fast dissolution properties also can make the film at detection line position infiltrate rapidly, and the presence of D- fucose can reduce chromatography in fucose
The surface tension of sample, it is ensured that the disappearance for phenomenon of diving under water；Simultaneously during film is dry, carbohydrate hydroxyl is mono- in combination with S-100 β
The polar group on clonal antibody surface forms ammonia key, thus instead of the hydrone around S-100 β monoclonal antibody polar group,
The hydration shell of one layer of hypothesis is formed on the β monoclonal antibody surface S-100, protects the ammonia of antibody to be keyed position and is not directly exposed to
In external environment, stablize the higher structure of S-100 β monoclonal antibody, even if being reduced as far as in the case where dry dehydration
The inactivation of S-100 β monoclonal antibody improves the sensitivity and stability of kit.
Further preferably, the anti-S-100 β monoclonal antibody of the fluorescent material label in bonding pad and nitrocellulose filter are examined
Correspondence monoclonal antibody on survey line sandwiches S-100 β albumen in detection sample.
Preferably, detection card includes sample pad, liner plate and blotting paper；Sample pad is made by following steps: by glass
Tunica fibrosa be soaked in containing 1.0%TritonX-100,2.5%BSA, pH 7-7.5 4-6mM BS buffer in, in 0-5 DEG C
3-5h is impregnated, is subsequently placed in baking oven, 37 DEG C dry 2 hours.
Preferably, the test sample of kit is serum, blood plasma or the whole blood of people.
Kit is contained using S-100 β albumen in immunofluorescence double-antibody method quantitative detection human serum, blood plasma or whole blood
Amount.After sample is added drop-wise to detection card well, the anti-S- of the fluorescent material label in sample in S-100 β albumen and bonding pad
100 β monoclonal antibodies, which combine, forms reaction compound, and reaction compound is acted on chromatography and being moved forward along nitrocellulose filter, quilt
Correspondence monoclonal antibody in nitrocellulose filter detection line captures, S-100 β albumen quantity of the catch and detection zone signal in sample
Intensity is positively correlated, by fluorescence analyser, the content of S-100 β albumen in quantitative detection sample.
Invention additionally discloses above-mentioned S-100 β protein detection kit quantitative detection human serum, blood plasma or whole bloods in vitro
The purposes of the content of S-100 β albumen in this.
Preferably, purposes include clinically cerebral injury early stage auxiliary diagnosis, assessment the severity of brain injury, guiding treatment and
Judging prognosis.
Compared with prior art, the invention has the benefit that 1) test sample of kit of the present invention be people serum,
Blood plasma or whole blood sample have good detection specificity, higher sensitivity, stability, the simplicity of operation；2) of the invention
The content of S-100 β albumen in kit energy quantitative detection sample can be used for the early stage auxiliary diagnosis of clinically cerebral injury, comment
Estimate the severity of brain injury, guiding treatment and judging prognosis, have many advantages, such as more rapidly, more effectively, it is more acurrate；3) kit of the present invention
The sensitivity and stability of kit can be improved with bonding pad, moreover it is possible to which the Tomography Velocity of accelerating liquid improves the detection of kit
Rate；4) kit of the present invention can ensure that the disappearance of diving phenomenon with nitrocellulose filter, improve the sensitivity of kit and steady
It is qualitative.
Present invention employs above-mentioned technical proposals to provide S-100 β protein detection kit, compensates for the prior art not
Foot, reasonable design, easy operation.
Specific embodiment
In the following, being described further in conjunction with specific embodiments to embodiment of the present invention.The embodiments described below is to show
Example property, for explaining only the invention, and it is not considered as limiting the invention.
Embodiment according to the present invention provides embodiment below and facilitates a better understanding of the present invention, but and unlimited
The fixed present invention.Experimental method in following embodiments is unless otherwise specified conventional method.In addition, the term in the present invention
" coating " is the term in immune field, includes the meaning adsorbed and fixed.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1:
A kind of S-100 β protein detection kit, including detection card, IC card and dilution, wherein detection card includes being adsorbed with
The bonding pad of the S-100 β monoclonal antibody of rare-earth fluorescent microballoon label；Nitrocellulose filter, coating C react S-100 β Dan Ke
The detection line of grand antibody and the nature controlling line of coating T reaction sheep anti-mouse igg antibody；Sample pad；Liner plate and blotting paper are (such as 1 institute of table
Show).S-100 β monoclonal antibody is recombination human source S-100 β albumen, synthesizes source of people S-100 β gene by full genome, constructs S-
100 β recombinant plasmids；Using S-100 β recombinant plasmid as expression vector, S-100 β albumen is prepared by yeast eukaryotic expression；Utilize table
The S-100 β albumen preparation S-100 β monoclonal antibody reached.Kit uses immunofluorescence double-antibody method quantitative detection people's blood
Clearly, in blood plasma or whole blood S-100 β albumen content.After sample is added drop-wise to detection card well, in sample S-100 β albumen and
The anti-S-100 β monoclonal antibody of fluorescent material label in bonding pad, which combines, forms reaction compound, reacts compound with chromatography
Effect moves forward along nitrocellulose filter, is captured by the correspondence monoclonal antibody in nitrocellulose filter detection line, in sample
S-100 β albumen quantity of the catch and detection zone signal strength are positively correlated, by fluorescence analyser, S-100 β egg in quantitative detection sample
White content.
Above-mentioned bonding pad is prepared using following steps: glass fibre cotton is placed in containing 1.3%BSA, 0.6% fucose
In 1.55M Tris-HCL treatment fluid, 20min is impregnated under the Ultrasonic Conditions of 30KHz, then the dry 2.5h at 65 DEG C, takes
It is spare out.The bonding pad is that S-100 β monoclonal antibody, that is, S-100 β monoclonal antibody-microballoon of rare-earth fluorescent microballoon label is multiple
It closes object and one drying, preservation, the place for moistening and discharging again is provided, fucose and ultrasonic wave can play gain effect, surpass
The effect of sound wave can promote to occur or accelerate certain chemical reactions so that bonding pad surface quickly formed one layer by sugar form,
Uniform smooth surface reduces the suction-operated of later period S-100 β monoclonal antibody-microsphere compound and bonding pad；In addition it ties
The sugar for closing pad surface can also form a glace on S-100 β monoclonal antibody-microsphere compound surface, significantly be protected using it
Water effect makes S-100 β monoclonal antibody-microsphere compound still be able to maintain good dispersibility and uniformity in the drying process,
It is also avoided that the activity decline of antibody simultaneously, after sample is added dropwise, carbohydrate is met liquid and quickly dissolved, S-100 β monoclonal antibody-
Microsphere compound also can quickly thoroughly be discharged from bonding pad, so kit can be improved in the bonding pad that above-mentioned preparation method obtains
Sensitivity and stability.
Nitrocellulose filter is handled using following steps:
1) S-100 β monoclonal antibody and goat anti-mouse igg antibody coating dilution adjustment concentration to 2mg/ml, film liquid
Amount is 1.5 μ l/cm；
2) the S-100 β monoclonal antibody that obtains step 1) and goat anti-mouse igg antibody solution as detection line and
Nature controlling line is sprayed on nitrocellulose filter in parallel and is coated with, and is divided into 3.5mm between the detection line and nature controlling line, is subsequently placed in
In baking oven, 37 DEG C are dried 2 hours.Wherein, coating dilution is the 5mM BS buffer containing 0.6% fucose, pH 7.4；Rock
Algae sugar in containing 4.5% D- fucose.Under normal circumstances, the diffusion with coating buffer BS on nitrocellulose filter film, film
On some surface reactive materials film hydrophobicity can be caused to enhance, after point sample, as S-100 β by with mobile to tunica fibrosa both sides
When monoclonal antibody-microsphere compound forward position chromatography is reached near detection line, since hydrophobicity is strong in detection line, solution can be along
Bottom plate under detection line is swum underwater until detection line another side hydrophily strength, just normal chromatography, and above-mentioned coating dilution can
Hydrophily of the coated antibody on nitrocellulose filter is improved, in point sample, the speed of S-100 β monoclonal antibody diffusion can be lowered
Degree reduces being largely lost for the surfactant at detection line, even if surfactant is all lost, the quick dissolution of fucose
Property also can make the film at detection line position infiltrate rapidly, and the presence of D- fucose can reduce chromatography samples in fucose
Surface tension, it is ensured that the disappearance for phenomenon of diving under water；Simultaneously during film is dry, carbohydrate hydroxyl is anti-in combination with S-100 β monoclonal
The polar group in body surface face forms ammonia key, thus instead of the hydrone around S-100 β monoclonal antibody polar group, in S-
100 β monoclonal antibody surfaces form the hydration shell of one layer of hypothesis, protect the ammonia of antibody to be keyed position and are not directly exposed to the external world
In environment, stablize the higher structure of S-100 β monoclonal antibody, even if being reduced as far as S- in the case where dry dehydration
The inactivation of 100 β monoclonal antibodies improves the sensitivity and stability of kit.
Sample pad is made by following steps: glass fibre membrane is soaked in containing 1.0%TritonX-100,2.5%
It in the 5mM BS buffer of BSA, pH 7.4, in 4 DEG C of immersion 4h, is subsequently placed in baking oven, 37 DEG C dry 2 hours.
Liner plate: selection PVC board.
Blotting paper is made by following steps: blotting paper is cut into suitable size and serves as water absorption pad, is in detection process
The chromatography of solution provides power.
The composition of 1 S-100 β protein detection kit of table
In the anti-S-100 β monoclonal antibody and nitrocellulose filter detection line of fluorescent material label in above-mentioned bonding pad
Correspondence monoclonal antibody sandwich detection sample in S-100 β albumen.
The condition of storage of mentioned reagent box and validity period: 4~30 DEG C of detection reagent preservations, validity period 24 months, reagent aluminium
Behind foil bag Kaifeng, in 2 hours effectively.
The use instrument of mentioned reagent box: the NP007 dry type fluorescence immunity analyzer of Weihai Niu Pu Bioisystech Co., Ltd,
The AFS1000 dry type fluorescence immunity analyzer of Guangzhou Lan Bo Biotechnology Co., Ltd.
The test sample of mentioned reagent box is serum, blood plasma or the whole blood of people.Serum, blood plasma, whole blood sample are routinely square
Sodium citrate, EDTA or Heparin plasma can be used in method acquisition, anti-coagulants；It is please small in 4 after separation for serum or blood plasma
When interior measurement, serum or 2~8 DEG C of blood plasma can be reserved for 5 days, need to be stored in -20 DEG C, before detection more than 5 days, sample is restored to room
Detection is completed after temperature in 1 hour.It please don't multigelation；Whole blood sample can be reserved for 3 days in 2~8 DEG C, must not freeze.
The application method of mentioned reagent box such as table 2:
The application method of 2 kit of table
Nominal reference section are as follows: S-100 β < 0.2ng/mL.
Note: due to differences such as geography, ethnic group, gender and ages, each laboratory should establish the reference interval of oneself.
The performance indicator of mentioned reagent box:
1. the range of linearity: within the scope of 0.1ng/mL~10.0ng/mL, linearly dependent coefficient r >=0.9900.
2. repeatability: the coefficient of variation (CV)≤15%.
3. difference between batch: relatively very poor (R)≤20%.
4. accuracy: being detection sample with internal reference, measurement result and sign value relative deviation should be in ± 15% models
Within enclosing.
5. minimum detection limit: being not higher than 0.1ng/mL.
The points for attention of mentioned reagent box:
1. this product is disposable external diagnosis reagent, it please don't reuse, please use before the deadline.
2. test card and component are only applicable to relevant fluorescence analyser.
3. product is asked before use not break a seal, apparent damage person is packed, please don't be used.
4. the reagent component of different lot numbers cannot be used with, IC card must not block with detection mixes lot number use.
5. safeguard measure appropriate should be taken in collection, disposition, storage, mixing sample and detection process.
6. there is desiccant in aluminium foil bag, must not eat.
7. should be avoided, experimental situation temperature is excessively high, and the test card of cryo-conservation needs to restore to room temperature to open again, in order to avoid
The moisture absorption.
8. trembling and electromagnetic environment should be avoided in detection card and fluorescence analyser when in use；During normal use instrument itself
It generates trembling and belongs to normal phenomenon；Detection please don't extract IC card when carrying out.
9. suggesting using fresh sample, disturbed test and mistake knot can be led to if having obvious haemolysis or blood clot in sample
Fruit, be sure not using.
10. the testing result of reagent should combine its symptom, sign, disease to the clinical diagnosis and treatment of patient only for clinical reference
Situations such as history, the inspection of other laboratories and therapeutic response, comprehensively considers.
11. all samples, waste liquid, pipe etc. are handled by infectious contaminants processing mode.It should will be finished after detection
Detection card is discarded in corresponding biohazard product container, is handled by biohazard object.
12. if you have questions or being suggested in the process using this reagent, please contacted with producer.
S-100 β protein detection kit S-100 β albumen in quantitative detection human serum, blood plasma or whole blood sample in vitro
The purposes of content.Purposes includes that the early stage auxiliary diagnosis of clinically cerebral injury, assessment the severity of brain injury, guiding treatment and judgement are pre-
Afterwards.
Embodiment 2:
A kind of S-100 β protein detection kit, including detection card, IC card and dilution, wherein detection card includes being adsorbed with
The bonding pad of the S-100 β monoclonal antibody of rare-earth fluorescent microballoon label；Nitrocellulose filter, coating C react S-100 β Dan Ke
The detection line of grand antibody and the nature controlling line of coating T reaction sheep anti-mouse igg antibody；Sample pad；Liner plate and blotting paper.
Above-mentioned bonding pad is prepared using following steps: glass fibre cotton is placed in containing 1.2%BSA, 0.5% fucose
In 1.5M Tris-HCL treatment fluid, 15min is impregnated under the Ultrasonic Conditions of 28KHz, then the dry 2h at 60 DEG C, takes out
It is spare.
Nitrocellulose filter is handled using following steps:
1) S-100 β monoclonal antibody and goat anti-mouse igg antibody coating dilution adjustment concentration to 1mg/ml, film liquid
Amount is 1 μ l/cm；
2) the S-100 β monoclonal antibody that obtains step 1) and goat anti-mouse igg antibody solution as detection line and
Nature controlling line is sprayed on nitrocellulose filter in parallel and is coated with, and is divided into 3mm between the detection line and nature controlling line, is subsequently placed in baking
In case, 35 DEG C are dried 2 hours.Wherein, coating dilution is the 4mM BS buffer containing 0.5% fucose, pH 7；Fucose
In containing 4% D- fucose.
Sample pad is made by following steps: glass fibre membrane is soaked in containing 1.0%TritonX-100,2.5%
BSA, pH 7-7.5 4-6mM BS buffer in, in 0-5 DEG C of immersion 3-5h, be subsequently placed in baking oven, 37 DEG C dry 2 hours.
Liner plate: selection PVC board.
Blotting paper is made by following steps: blotting paper is cut into suitable size and serves as water absorption pad, is in detection process
The chromatography of solution provides power.
Embodiment 3:
A kind of S-100 β protein detection kit, including detection card, IC card and dilution, wherein detection card includes being adsorbed with
The bonding pad of the S-100 β monoclonal antibody of rare-earth fluorescent microballoon label；Nitrocellulose filter, coating C react S-100 β Dan Ke
The detection line of grand antibody and the nature controlling line of coating T reaction sheep anti-mouse igg antibody；Sample pad；Liner plate and blotting paper.
Above-mentioned bonding pad is prepared using following steps: glass fibre cotton is placed in containing 1.3%BSA, 0.6% fucose
In 1.55M Tris-HCL treatment fluid, wherein in fucose containing 3.0% D- fucose, under the Ultrasonic Conditions of 30KHz
20min is impregnated, then the dry 2.5h at 65 DEG C, takes out spare.When containing D- fucose in fucose, chromatography samples can be reduced
Surface tension, can better ensure that S-100 β monoclonal antibody-microsphere compound on bonding pad in again wet process
Uniformity and stationarity can improve S-100 β monoclonal antibody-microsphere compound release on bonding pad, optimize S-100 β Dan Ke
The releasing effect of grand antibody-microspheres compound, at the same can also accelerating liquid Tomography Velocity, improve the detection rates of kit.
Nitrocellulose filter is handled using following steps:
1) S-100 β monoclonal antibody and goat anti-mouse igg antibody coating dilution adjustment concentration to 2mg/ml, film liquid
Amount is 1.5 μ l/cm；
2) the S-100 β monoclonal antibody that obtains step 1) and goat anti-mouse igg antibody solution as detection line and
Nature controlling line is sprayed on nitrocellulose filter in parallel and is coated with, and is divided into 3.5mm between the detection line and nature controlling line, is subsequently placed in
In baking oven, 37 DEG C are dried 2 hours.Wherein, coating dilution is the 5mM BS buffer containing 0.6% fucose, pH 7.4；Rock
Algae sugar in containing 4.5% D- fucose.
Sample pad is made by following steps: glass fibre membrane is soaked in containing 1.0%TritonX-100,2.5%
It in the 5mM BS buffer of BSA, pH 7.4, in 4 DEG C of immersion 4h, is subsequently placed in baking oven, 37 DEG C dry 2 hours.
Liner plate: selection PVC board.
Blotting paper is made by following steps: blotting paper is cut into suitable size and serves as water absorption pad, is in detection process
The chromatography of solution provides power.
The kit in use, draws 150 μ L sample of whole blood and adds a drop dilution or serum, 100 μ L sample of blood plasma
Originally it is added in the well of test card, will test card insertion after five minutes and enter to be applicable in the card slot of instrument, carry out quantitative interpretation knot
Fruit.When illustrating in fucose containing D- fucose, the Tomography Velocity of energy accelerating liquid improves the detection rates of kit.
Embodiment 4:
A kind of S-100 β protein detection kit, including detection card, IC card and dilution, wherein detection card includes being adsorbed with
The bonding pad of the S-100 β monoclonal antibody of rare-earth fluorescent microballoon label；Nitrocellulose filter, coating C react S-100 β Dan Ke
The detection line of grand antibody and the nature controlling line of coating T reaction sheep anti-mouse igg antibody；Sample pad；Liner plate and blotting paper.
Above-mentioned bonding pad is prepared using following steps: glass fibre cotton is placed in containing 1.2%BSA, 0.5% fucose
In 1.5M Tris-HCL treatment fluid, 15min is impregnated under the Ultrasonic Conditions of 28KHz, then the dry 2h at 60 DEG C, takes out
It is spare.
Nitrocellulose filter is handled using following steps:
1) S-100 β monoclonal antibody and goat anti-mouse igg antibody coating dilution adjustment concentration to 1mg/ml, film liquid
Amount is 1 μ l/cm；
2) the S-100 β monoclonal antibody that obtains step 1) and goat anti-mouse igg antibody solution as detection line and
Nature controlling line is sprayed on nitrocellulose filter in parallel and is coated with, and is divided into 3mm between the detection line and nature controlling line, is subsequently placed in baking
In case, 35 DEG C are dried 2 hours.Wherein, coating dilution is the 4mM BS buffer containing 0.5% fucose, pH 7；Fucose
In containing 4% D- fucose.
Sample pad is made by following steps: glass fibre membrane is soaked in containing 1.0%TritonX-100,2.5%
It in the 4mM BS buffer of BSA, pH 7, in 0 DEG C of immersion 3h, is subsequently placed in baking oven, 37 DEG C dry 2 hours.
Liner plate: selection PVC board.
Blotting paper is made by following steps: blotting paper is cut into suitable size and serves as water absorption pad, is in detection process
The chromatography of solution provides power.
Comparative example 1:
A kind of S-100 β protein detection kit, including detection card, IC card and dilution, wherein detection card includes being adsorbed with
The bonding pad of the S-100 β monoclonal antibody of rare-earth fluorescent microballoon label；Nitrocellulose filter, coating C react S-100 β Dan Ke
The detection line of grand antibody and the nature controlling line of coating T reaction sheep anti-mouse igg antibody；Sample pad；Liner plate and blotting paper.
Above-mentioned bonding pad is prepared using following steps: glass fibre cotton is placed in the 1.55M Tris- containing 1.3%BSA
In HCL treatment fluid, 20min is impregnated under the Ultrasonic Conditions of 30KHz, then the dry 2.5h at 65 DEG C, takes out spare.Remaining
It is consistent with embodiment 1.
Comparative example 2:
A kind of S-100 β protein detection kit, including detection card, IC card and dilution, wherein detection card includes being adsorbed with
The bonding pad of the S-100 β monoclonal antibody of rare-earth fluorescent microballoon label；Nitrocellulose filter, coating C react S-100 β Dan Ke
The detection line of grand antibody and the nature controlling line of coating T reaction sheep anti-mouse igg antibody；Sample pad；Liner plate and blotting paper.
Above-mentioned bonding pad is prepared using following steps: glass fibre cotton is placed in containing 1.3%BSA, 0.6% fucose
20min is impregnated in 1.55M Tris-HCL treatment fluid, then the dry 2.5h at 65 DEG C, takes out spare.Remaining and embodiment 1 one
It causes.
Comparative example 3:
A kind of S-100 β protein detection kit, including detection card, IC card and dilution, wherein detection card includes being adsorbed with
The bonding pad of the S-100 β monoclonal antibody of rare-earth fluorescent microballoon label；Nitrocellulose filter, coating C react S-100 β Dan Ke
The detection line of grand antibody and the nature controlling line of coating T reaction sheep anti-mouse igg antibody；Sample pad；Liner plate and blotting paper.
Nitrocellulose filter is handled using following steps:
1) S-100 β monoclonal antibody and goat anti-mouse igg antibody coating dilution adjustment concentration to 2mg/ml, film liquid
Amount is 1.5 μ l/cm；
2) the S-100 β monoclonal antibody that obtains step 1) and goat anti-mouse igg antibody solution as detection line and
Nature controlling line is sprayed on nitrocellulose filter in parallel and is coated with, and is divided into 3.5mm between the detection line and nature controlling line, is subsequently placed in
In baking oven, 37 DEG C are dried 2 hours.Wherein, the 5mM BS buffer that coating dilution is pH 7.4.Remaining is consistent with embodiment 1.
Comparative example 4:
A kind of S-100 β protein detection kit, including detection card, IC card and dilution, wherein detection card includes being adsorbed with
The bonding pad of the S-100 β monoclonal antibody of rare-earth fluorescent microballoon label；Nitrocellulose filter, coating C react S-100 β Dan Ke
The detection line of grand antibody and the nature controlling line of coating T reaction sheep anti-mouse igg antibody；Sample pad；Liner plate and blotting paper.
Nitrocellulose filter is handled using following steps:
1) S-100 β monoclonal antibody and goat anti-mouse igg antibody coating dilution adjustment concentration to 2mg/ml, film liquid
Amount is 1.5 μ l/cm；
2) the S-100 β monoclonal antibody that obtains step 1) and goat anti-mouse igg antibody solution as detection line and
Nature controlling line is sprayed on nitrocellulose filter in parallel and is coated with, and is divided into 3.5mm between the detection line and nature controlling line, is subsequently placed in
In baking oven, 37 DEG C are dried 2 hours.Wherein, coating dilution is the 5mM BS buffer containing 0.6% fucose, pH 7.4.Its
It is remaining consistent with embodiment 1.
Test example 1:
Sensitivity experiment
Sample dilution is detected as sample, replication 20 times, is obtained 20 measurement results, is calculated its average value
(M) and standard deviation (SD) it, obtains M+2SD, the result of M+2SD is substituted into the normal equation established, corresponding concentration value is found out.
The corresponding concentration value of embodiment 1 is 0.0384ng/mL, and the corresponding concentration value of embodiment 3 is 0.0352ng/mL, and comparative example 1 is corresponding
Concentration value be 0.0587ng/mL, the corresponding concentration value of comparative example 2 is 0.0601ng/mL, and the corresponding concentration value of comparative example 3 is
0.0556ng/mL, the corresponding concentration value of comparative example 4 are 0.0537ng/mL, illustrate that the zero degree of embodiment 3 is better than embodiment 1, real
The sensitivity for applying the detection card of example 1 is better than comparative example 1, comparative example 2, comparative example 3, comparative example 4.Comparative example 1 and embodiment 3
Known to: when containing D- fucose in fucose, optimize S-100 β monoclonal antibody-microsphere compound releasing effect, improves examination
The sensitivity of agent box；Known to comparative example 1 and comparative example 1, comparative example 2: fucose and ultrasonic wave can play gain effect,
The sensitivity of kit can be improved in obtained bonding pad；Known to comparative example 1 and comparative example 3, comparative example 4: coating dilution
In fucose containing D- fucose exist, it can be ensured that the advanced of S-100 β monoclonal antibody is stablized in the disappearance for phenomenon of diving under water
Structure improves the sensitivity of kit.
Test example 2:
Stability experiment
Detect the stability of card: the detection card in kit is generally held in the environment in drying at room temperature cool place.Detection card
It is placed one month in 50 DEG C of baking ovens and carries out the stability test that accelerates the failure, be equivalent under detection card room temperature and save 1 year effective
Property.Will test be stuck in 50 DEG C of baking ovens place respectively 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks after take out be put into drying
It in room, is compared with the reagent card of room temperature in drying shed, Sample dilution detects the stability of card using 4 DEG C of preservations.
The detection card of embodiment 1 be placed in 50 DEG C of baking ovens accelerate the failure 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks in standard items
Response curve in the case where test coincide substantially with the detection card being stored at room temperature, the fluorescence signal T/C value base of low middle and high concentration
This does not change, and after illustrating the accelerated destruction test of fluorescent quantitation immunochromatographydetection detection card, has good stability, can be with room temperature two
Year.And the detection card of comparative example 1, comparative example 2, comparative example 3, comparative example 4 is placed in 50 DEG C of baking ovens accelerate the failure 1 week, 2 weeks, 3
Week, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks response curve in the case where standard items test and detecting of being stored at room temperature block it is identical
Degree is worse than the detection card of embodiment 2, illustrate the stability of the detection card of embodiment 1 be better than comparative example 1, comparative example 2, comparative example 3,
Comparative example 4.Known to comparative example 1 and comparative example 1, comparative example 2: fucose and ultrasonic wave can play gain effect, obtain
Bonding pad the stability of kit can be improved；Known to comparative example 1 and comparative example 3, comparative example 4: containing in coating dilution
With the presence of the fucose of D- fucose, it can be ensured that the higher structure of S-100 β monoclonal antibody is stablized in the disappearance for phenomenon of diving under water,
Improve the stability of kit.
The prior art of routine techniques dawn known to those skilled in the art in above-described embodiment, therefore herein no longer in detail
It repeats.
The above embodiments are only used to illustrate the present invention, and not limitation of the present invention, the ordinary skill people of this field
Member can also make a variety of changes and modification without departing from the spirit and scope of the present invention.Therefore, all equivalent
Technical solution also belong to scope of the invention, scope of patent protection of the invention should be defined by the claims.
Claims (10)
1.S-100 β protein detection kit, including detection card, IC card and dilution, it is characterised in that: the detection, which blocks, includes
It is adsorbed with the bonding pad of the S-100 β monoclonal antibody of rare-earth fluorescent microballoon label.
2. S-100 β protein detection kit according to claim 1, it is characterised in that: the bonding pad is using following step
Rapid preparation: glass fibre cotton is placed at the 1.5-1.6M Tris-HCL containing 1.2-1.5%BSA, 0.5-0.8% fucose
It manages in liquid, 15-25min is impregnated under the Ultrasonic Conditions of 28-34KHz, then the dry 2-3h at 60-70 DEG C, takes out spare.
3. S-100 β protein detection kit according to claim 1, it is characterised in that: the detection card further includes nitric acid
Cellulose membrane, the detection line of coating C reaction S-100 β monoclonal antibody and the nature controlling line of coating T reaction sheep anti-mouse igg antibody.
4. S-100 β protein detection kit according to claim 3, it is characterised in that: the nitrocellulose filter uses
Following steps processing:
1) S-100 β monoclonal antibody and goat anti-mouse igg antibody coating dilution adjustment concentration to 1-3mg/ml, film liquid amount
For 1-2 μ l/cm；
2) the S-100 β monoclonal antibody that obtains step 1) and goat anti-mouse igg antibody solution are as detection line and Quality Control
Line is sprayed on nitrocellulose filter in parallel and is coated with, and drying is divided into 3-4mm between the detection line and nature controlling line.
5. S-100 β protein detection kit according to claim 4, it is characterised in that: the coating dilution be containing
The 4-6mM BS buffer of 0.5-0.8% fucose, pH 7-7.5；D- fucose in the fucose containing 4-5%.
6. S-100 β protein detection kit according to claim 3, it is characterised in that: the fluorescence in the bonding pad
The anti-S-100 β monoclonal antibody of matter label is sandwiched with the corresponding monoclonal antibody in the nitrocellulose filter detection line to be examined
S-100 β albumen in test sample sheet.
7. S-100 β protein detection kit according to claim 1, it is characterised in that: the detection card includes sample
Pad, liner plate and blotting paper；The sample pad is made by following steps: glass fibre membrane is soaked in containing 1.0%
TritonX-100,2.5%BSA, pH 7-7.5 4-6mM BS buffer in, in 0-5 DEG C of immersion 3-5h, drying.
8. S-100 β protein detection kit according to claim 1-7, it is characterised in that: the kit
Test sample is serum, blood plasma or the whole blood of people.
9. the described in any item S-100 β protein detection kits of claim 1-8 in vitro quantitative detection human serum, blood plasma or
The purposes of the content of S-100 β albumen in whole blood sample.
10. according to the purposes of the described in any item S-100 β protein detection kits of claim 9, it is characterised in that: described
Purposes includes the early stage auxiliary diagnosis of clinically cerebral injury, assessment the severity of brain injury, guiding treatment and judging prognosis.
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| CN201811434397.XA CN109633162A (en) | 2018-11-28 | 2018-11-28 | S-100 β protein detection kit |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811434397.XA CN109633162A (en) | 2018-11-28 | 2018-11-28 | S-100 β protein detection kit |
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| CN109856403A (en) * | 2018-12-21 | 2019-06-07 | 河北省医疗器械与药品包装材料检验研究院(河北省医疗器械技术审评中心) | A kind of S100- β albumen time-resolved fluoroimmunoassay chromatography detection kit |
| CN112798793A (en) * | 2020-12-30 | 2021-05-14 | 中国农业科学院油料作物研究所 | Test strip and test card for detecting PAT/bar protein, and preparation method and application thereof |
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