CN109633171A - The quickly fluorescence immune chromatography test paper bar and its preparation and application of detection GFAP - Google Patents
The quickly fluorescence immune chromatography test paper bar and its preparation and application of detection GFAP Download PDFInfo
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Abstract
The present invention relates to the fluorescence immune chromatography test paper bar of quickly detection GFAP a kind of and its preparations and reference, quickly the fluorescence immune chromatography test paper bar of detection GFAP includes bottom plate, the sample pad set gradually along floor length, conjugate release pad, chromatographic film and water absorption pad, the chromatographic film is equipped with detection line and nature controlling line, the detection line is separated from each other with nature controlling line, the fluorescence probe of fluorescent microsphere label GFAP monoclonal antibody is fixed in the conjugate release pad, the GFAP antigen with the pairing of microballoon labelled antibody is coated at the detection line, sheep anti-mouse igg antibody is coated at the nature controlling line.Compared with prior art, the present invention passes through preparation GFAP recombinant antigen and monoclonal antibody, the lateral immunochromatography technique of binding time resolved fluorometric, it is prepared for quickly detecting the fluorescence immune chromatography test paper bar of GFAP, and a kind of method for establishing quickly detection serum midbrain injury marker GFAP, it being capable of quick, GFAP in quantitative detection human serum concentration.
Description
Technical field
The invention belongs to fluorescence immunoassay detection technique fields, more particularly, to the fluorescence immunoassay layer of quickly detection GFAP a kind of
Analyse test strips and its preparation and application.
Background technique
Traumatic brain injury (traumatic brain injury, TBI) is the most common disease in neurosurgery, is generation
One of main dead and disability-causing factor within the scope of boundary；Its incidence and case fatality rate occupy third position, are only second to cancer and painstaking effort
Pipe disease；Top priority is accounted in the traumatic cause of death.TBI can be divided into primary brain and secondary according to the sequence of generation
Property cerebral injury.Primary brain refers to the damage occurred immediately when violence directly or indirectly acts on head, and pathological change includes
Contusion, lacerated wound, bleeding etc..Secondary brain injury refers to the cerebral injury lesion occurred after injury, is related to changing for cell, molecule etc.
Become, it may occur however that the pathological changes such as ischemic, anoxic, oedema, inflammation.Secondary brain injury is cytokine profiles and biochemical substances
The pathophysiological process of the damage and antibody Monoclonal that participate in jointly.
Although modern imaging technology greatly improves the treatment level and ability to TBI in recent years, it is still difficult
With brain cell injury situation during comprehensive, dynamic evaluation TBI.Recent studies have shown that, selection wherein have high degree of specificity and
Biomarker of the cell factor and biochemical substances of sensibility as monitoring craniocerebral injury, can reflect cranium to a certain extent
By the time after the severity of brain injury and damage.Biomarker generally refers to for a generic physiological of objective determination and evaluation
Or certain characteristic biochemical indicator in pathology or therapeutic process, it can know what body was presently in by the measurement to it
Process in biological process.The biomarker for checking a kind of disease specific, for the identification of disease, early diagnosis and pre-
Monitoring anti-, in therapeutic process may play help.Usually along with intracranial vessel inner film injury and blood brain after TBI generation
The destruction of barrier causes the irreversible injury of neuron, Deiter's cells, capilary.Glial fibrillary acid protein (glial
Fibrillary acidic protein, GFAP) one of significant albumen as astroglia, it is expressed in and develops into
In ripe spongiocyte, there are the sensibility and specificity of height, its table after spongiocyte damages to spongiocyte damage
Up to can change rapidly.
Glial fibrillary acid protein (glial fibrillary acidic protein, GFAP) is in 1971 by Eng etc.
It is successfully separated for the first time, is a kind of acidic protein that molecular weight is about 50~53kDa, be the distinctive skeleton egg of astroglia
It is white.A large amount of GFAP monomer polymerization forms the cytoskeleton of astroglia, and with soluble protein and intermediate filament protein two
Kind form is present in the cytoplasm of Deiter's cells.GFAP is made of 8 different subunits, and gene is by 9 exons and 8
Introne composition, along with 4 selective exons and 2 selective introne DNAs, so that forming a size is about 10kb
DNA fragmentation.GFAP comes across the 8th week of embryonic development earliest in human brain, is gradually expressed in spongiocyte, main to express
In astroglia, and it is also distributed in spongiocytes such as ependymocyte, oligodendroglias.GFAP is astrocyte
In main monomer intermediate filament protein, be the exclusive specific cell skelemin of central nervous system, be astrocyte
Significant albumen.
When central nervous system is damaged, along with the generation of c-fos protein, GFAP is in astroglia
In largely synthesis and accumulation.Then, the destruction of a large amount of necrosis and blood-brain barrier of nerve cell and spongiocyte, GFAP can be released
It is put into cerebrospinal fluid, peripheral blood is then entered by impaired blood-brain barrier, cause GFAP concentration in peripheral blood significantly raised.Have
Studies have shown that about 1h can detect GFAP in patients serum, and serum GFAP is dense after slight TBI generation after TBI occurs
Degree will increase.Craniocerebral injury degree is more serious, and downright bad Deiter's cells is more, and the extent of the destruction of blood-brain barrier is tighter
Weight, the GFAP for being released into peripheral blood will be more.Speculate that the concentration of GFAP in serum is positively correlated with cerebral injury severity, it is early
Phase just has scholar to confirm this point.GFAP clinically commonly used as specific biomarkers evaluate the serious journey of cerebral injury
Degree and prognosis situation.In addition to this, GFAP shows timing variation with the passage of cerebral injury time.Scholar is different by simulation
Animal injury model finds that GFAP is changed over time and expresses changing rule.It to sum up finds, GFAP peak concentration after damage
Time of occurrence is different, it may be possible to due to groups of animals model, degree of injury, experimental method difference caused by.But it shows big
Identical rule is caused, reflects that the expression of GFAP is time to time change, this is damaged to be inferred in Forensic Identification using GFAP
Hurting the time provides theoretical foundation.
U.S. FDA in 2017 has approved diagnostic reagent of the GFAP of Banyan Biomarker company as TBI, still,
Its detection method is the mode of ELISA, this technology be in principle it is effective, but complicated for operation and time cost compared with
It is high.
Patent CN 107102146A discloses a kind of double-antibody sandwich quantitative detection blood based on carbon dots for fluorescent marker
The method of GFAP in clear, this technology are effective in principle, but since the stability of carbon dots is not strong, so practicability
It is insufficient.
Patent CN 106568750A discloses a kind of detection side GFAP for being based on fluorescence resonance energy transfer method (FRET)
Method, but preparation process complexity and high process cost, do not have operability.
In the phase at the end of the 20th century, the technologies such as monoclonal antibody technique, colloidal gold immunochromatographimethod technology and new material technology are rapid
Development, the novel in vitro diagnostic techniques that grown up on this basis therewith is lateral chromatography technology, it have it is convenient,
Single part detection, economical and practical advantage.
Lateral chromatography immunity test strip (Lateral Flow Immunochromatographic Strips, LFIS) by
In the high degree of specificity for the separating capacity and routine immunization detection method for being provided simultaneously with chromatography brilliance, reported for the first time after 1980
After human chorionic gonadotropin (β-HCG) detection, medicine detection, food quality prison have been widely used in it
In the live fast qualitative quantitative detection and real-time test in fields such as survey, environmental monitoring, agricultural and animal husbandry and legal medical expert's verdict.Side
Testing principle to chromatography immunoreagent item is to be fixed on layer by that can add lustre to or generate the probe of electrochemical signals to mark
The antibody or antigen in test strips conjugate release pad are analysed, luminous intensity, electrification are measured through visual color reaction or instrument
Signal etc. is learned, to realize the measurement to target analyte.With the development with rapid changepl. never-ending changes and improvements of emerging technology and new material, constantly
Emerge the lateral chromatography immunity test strip of many new models.Test strips can substantially be divided according to label probe type
Class, such as colloid gold nanoparticle (Colloidal Gold Nanoparticles, AuNPs) immunity test strip, fluorescence immunoassay
Test strips (lanthanide series, fluorchrome etc.), up-converting phosphor (Upconverting Phosphor, UCP) immunity test strip,
Quantum dot (Quantum Dots, QDs) immunity test strip and colloidal carbon particle (Carbon Nanotubes
Nanoparticles, CNPs) immunity test strip etc..Be most commonly used to quantitative detection is that the time resolution that lanthanide series marks is exempted from
Epidemic disease fluorescence probe.
Timed-resolved fluoroimmunoassay (Time-resolved Fluoroimmunoassay, TRFIA) is ultramicron detection
An emerging detection technique in field is after radiating immuning analysis technology, Enzyme Immunoassay, luminescence immunoassay technology
Later, on the basis of conventional fluorescent immunoassay, immune point of a kind of nonradioactive labeling to come out at the beginning of the eighties of last century age
Analysis technology.It has many advantages, such as high sensitivity, specificity, high stability and on-radiation, by preclinical medicine, clinical detection,
The concern and application in the fields such as molecular biology.TRFIA lanthanide series labelled antigen or antibody, according to lanthanide chelate
Fluorescence lifetime is long, stoke (stokes) displacement is big, quantum yield is high, emission peak is narrow, excitation and launch wavelength ideal etc. shine
Feature measures fluorescence with TIME RESOLVED TECHNIQUE, while two parameters of Detection wavelength and time carry out signal resolution, can effectively arrange
Unless the interference of specific fluorescence, greatly improves sensitivity for analysis.
Summary of the invention
Based on to occur cerebral injury reality situation, the diagnosis key of TBI be quickly, execute-in-place it is significant, because
This develops a kind of live or bedside use, and it is convenient to operate, and as a result quick diagnostic reagent has great value.Based on this, originally
Application provides the fluorescence immune chromatography test paper bar and its preparation and application of a kind of quickly detection GFAP.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of fluorescence immune chromatography test paper bar of quick detection GFAP, including bottom plate, by chromatography direction, along floor length according to
Sample pad, conjugate release pad, chromatographic film and the water absorption pad of secondary setting, the chromatographic film are equipped with detection line (T line) and matter
It controls line (C line), the detection line is separated from each other with nature controlling line, and fluorescent microsphere label is fixed in the conjugate release pad
The fluorescence probe of GFAP monoclonal antibody is coated with the GFAP antigen with the pairing of microballoon labelled antibody at the detection line, described
Nature controlling line at be coated with sheep anti-mouse igg antibody.
Fluorescent microsphere marks GFAP monoclonal antibody as indicator markers.
The conjugate release pad is glass fibre material, and the chromatographic film uses nitrocellulose filter (NC film), described
Sample pad is equipped with well.
The preparation method of the fluorescence immune chromatography test paper bar of the quick detection GFAP, comprising the following steps:
(1) fluorescence probe of fluorescent microsphere label GFAP monoclonal antibody is prepared；
(2) fluorescence probe of preparation is fixed in conjugate release pad with the mode for impregnating conjugate release pad, is freezed
After drying, it is sealed spare；
(3) it is coated with GFAP antigen and sheep anti-mouse igg antibody lines in chromatographic film, respectively detection line and nature controlling line；
(4) by the paste of sample pad, conjugate release pad, chromatographic film and water absorption pad on bottom plate, quickly detection GFAP is formed
Fluorescence immune chromatography test paper bar.
The GFAP antigen the preparation method comprises the following steps:
1.1, the building of expression plasmid:
1.1.1 double digestion: the restriction endonuclease of GFAP gene two sides is determined；
1.1.2 it connects: transfecting suitable purpose bacterial strain after target fragment is connect with carrier segments；
1.1.3 turn bacterium: turning bacterium and apply plate, the presence of segment is identified by bacterium colony PCR；
1.1.4 fungi preservation: picking positive bacteria fall in suitable resistance LB culture medium culture and is stored in sterile glycerol-
80℃；
1.2, the expression and purifying of recombinant protein:
1.2.1 cultivate: picking contains GFAP expression plasmid, is inoculated in LB culture medium, shake culture；
1.2.2 inducing expression: being induced with IPTG, and E.coli expression GFAP simultaneously collects thallus；
1.2.3 protein purification: nickel column is crossed after bacterial cell disruption, is purified by flash albumen with imidazoles；
1.3, the identification of recombinant protein:
1.3.1 purifying protein identifies purity using the method for SDS-PAGE；
1.3.2 whether correctly collected using GFAP kit identification destination protein.
The GFAP monoclonal antibody the preparation method comprises the following steps:
2.1, animal immune: Balb/C mouse is immunized in GFAP albumen；
2.2 cell fusions: it is merged using the splenocyte and SP2/0 cell of immune mouse, obtains hybridoma；
2.3 cell screenings: it screens to obtain positive monoclonal hybridoma using limiting dilution assay combination indirect ELISA method
Cell；
2.4 Antibody preparations: monoclonal antibody is enriched with come amplification in vitro using mouse ascites；
2.5 antibody purifications: ascites is purified using caprylic acid-ammonium and affinity chromatography, is measured after dialysis
A280 determines that protein concentration is stand-by.
In the fluorescence probe of the fluorescent microsphere label GFAP monoclonal antibody, the fluorescent microsphere is time-resolved fluorescence
Microballoon；
Time-resolved fluorescence microballoon is a kind of to have the lanthanide series and its chelating agent (such as EuCl of unique fluorescent characteristic3)
As the specific function microballoon of tracer, micro-sphere material is usually polystyrene, surface modification have proper density carboxyl or its
Its functional group improves the stability of marker for the covalent coupling with albumen or antibody.
The method of the fluorescence probe of the fluorescent microsphere label GFAP monoclonal antibody are as follows:
(1), with NHS and EDC solution activation microballoon (dosage: 1mg microballoon uses 0.2mg NHS and 0.2mgEDC)；
(2), after mixing well, in 37 DEG C of standing 30min；
(3), GFAP monoclonal antibody is added in activated microballoon, makes the final concentration of 0.5mg/ml of microballoon, first
37 DEG C of coupling 30min are stood, then stand 4 DEG C overnight (not less than 8H)；
(4), confining liquid and 10%Tween-20 are added in the microballoon being coupled, mixes, stands 37 DEG C of 30min；
(5), 13000rpm, centrifugation 15min cleaning microballoon is twice；
(6), after sucking supernatant, microballoon is added and saves liquid, is uniformly dispersed, 4 DEG C of preservations, as GFAP monoclonal antibody is glimmering
Light probe.
The application of the fluorescence immune chromatography test paper bar of the quick detection GFAP, sample are added dropwise to the well in sample pad
It chromatographs forward under capillary action afterwards, by the conjugate release pad containing marker, makes marker aquation again, and micro- with fluorescence
Ball label GFAP monoclonal antibody reacts to each other, then swimming forward together, if containing GFAP in sample, meeting and fluorescent microsphere
It marks GFAP monoclonal antibody to combine, moves forward to detection zone along chromatographic film under chromatography effect, cross detection line by matter
Line capture is controlled, and unbonded fluorescent microsphere marks GFAP monoclonal antibody and by GFAP antigen capture shape coated in chromatographic film
It is adsorbed on detection line region at compound, under the action of laser source, fluorescent material emits the fluorescence signal of certain wave, and fluorescence is exempted from
Epidemic disease analyzer captures fluorescence signal.
Amount of the sample containing GFAP is more in a certain range, and the compound gathered in detection line is fewer, and fluorescence signal is weaker,
Since fluorescent microsphere label GFAP monoclonal antibody is present in excess, so no matter whether containing GFAP, fluorescent microsphere in sample
Label GFAP monoclonal antibody can chromatograph compound to nature controlling line formation fluorescent microsphere label GFAP monoclonal antibody-sheep anti-mouse igg
Object shows fluorescence signal.
The fluorescence signal that nature controlling line is shown be determine sample-adding amount whether enough and the whether normal standard of chromatography process.
According to the ratio T/C of detection line fluorescence intensity and nature controlling line fluorescence intensity, according to fluorescence immunoassay quantitative analysis instrument
The concentration of GFAP in built-in curve interpretation sample；
The built-in curve of fluorescence immunoassay quantitative analysis instrument is by a series of standard items of known GFAP concentration, using described fast
The fluorescence immune chromatography test paper bar of speed detection GFAP, it is glimmering with the standard items detection line of standard items GFAP concentration and known GFAP concentration
The ratio T/C of luminous intensity and nature controlling line fluorescence intensity is the curve of two factors fitting.
Compared with prior art, the present invention differentiates glimmering by preparation GFAP recombinant antigen and monoclonal antibody, binding time
The lateral immunochromatography technique of light is prepared for quickly detecting the fluorescence immune chromatography test paper bar of GFAP, and establishes a kind of quickly inspection
The method for surveying serum midbrain injury marker GFAP, being capable of quick, GFAP in quantitative detection human serum concentration.
Detailed description of the invention
Fig. 1 is the fluorescence immune chromatography test paper bar structural schematic diagram of quickly detection GFAP.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment
A kind of fluorescence immune chromatography test paper bar of quick detection GFAP, as described in Figure 1, including bottom plate 1, chromatography direction is pressed,
Sample pad 2, conjugate release pad 3, chromatographic film 4 and the water absorption pad 5 set gradually along 1 length of bottom plate, chromatographic film 4 are equipped with detection
Line 6 (T line) and nature controlling line 7 (C line), detection line 6 is separated from each other with nature controlling line 7, is fixed with fluorescent microsphere in conjugate release pad 3
The fluorescence probe of GFAP monoclonal antibody is marked, the GFAP antigen with the pairing of microballoon labelled antibody, Quality Control are coated at detection line 6
Sheep anti-mouse igg antibody is coated at line 7.
Fluorescent microsphere marks GFAP monoclonal antibody as indicator markers.The conjugate release pad is glass fibre material
Matter, the chromatographic film use nitrocellulose filter (NC film), and the sample pad is equipped with well.
The preparation method of the fluorescence immune chromatography test paper bar of the quick detection GFAP, comprising the following steps:
(1) fluorescence probe of fluorescent microsphere label GFAP monoclonal antibody is prepared；
(2) fluorescence probe of preparation is fixed in conjugate release pad with the mode for impregnating conjugate release pad, is freezed
After drying, it is sealed spare；
(3) it is coated with GFAP antigen and sheep anti-mouse igg antibody lines in chromatographic film, respectively detection line and nature controlling line；
(4) by the paste of sample pad, conjugate release pad, chromatographic film and water absorption pad on bottom plate, quickly detection GFAP is formed
Fluorescence immune chromatography test paper bar.
The GFAP antigen the preparation method comprises the following steps:
1.1, the building of expression plasmid:
1.1.1 double digestion: the restriction endonuclease of GFAP gene two sides is determined；
1.1.2 it connects: transfecting suitable purpose bacterial strain after target fragment is connect with carrier segments；
1.1.3 turn bacterium: turning bacterium and apply plate, the presence of segment is identified by bacterium colony PCR；
1.1.4 fungi preservation: picking positive bacteria fall in suitable resistance LB culture medium culture and is stored in sterile glycerol-
80℃；
1.2, the expression and purifying of recombinant protein:
1.2.1 cultivate: picking contains GFAP expression plasmid, is inoculated in LB culture medium, shake culture；
1.2.2 inducing expression: being induced with IPTG, and E.coli expression GFAP simultaneously collects thallus；
1.2.3 protein purification: nickel column is crossed after bacterial cell disruption, is purified by flash albumen with imidazoles；
1.3, the identification of recombinant protein:
1.3.1 purifying protein identifies purity using the method for SDS-PAGE；
1.3.2 whether correctly collected using GFAP kit identification destination protein.
A kind of method for specifically obtaining GFAP antigen protein is given in the present embodiment, as follows:
1, the expression of GFAP recombinant protein
The building of 1.1 expression plasmids
1.1.1 double digestion: opening bearer documents with Vector NTI, click digestion point search, add all restriction enzyme sites,
Common restriction enzyme site is selected, and clicks Remove all in search restriction enzyme site and adds again, it is ensured that selected digestion position
Point is unique in the carrier, and in target gene two sides, this time that selection is HindIII and NotI restriction enzyme
1.1.2 connect: by target fragment: carrier segments=3:1 mode connects, and carrier is PET-28a (+), the company of addition
Connect enzyme and buffer.Suitable purpose bacterial strain (DH5 α) is transfected after 16 DEG C of connection 1h
1.1.3 turn bacterium: turning bacterium and apply plate, the presence of segment is identified by bacterium colony PCR
1.1.4 fungi preservation: picking positive bacteria falls in suitable resistance LB culture medium 37 DEG C, and 200rpm is incubated overnight pumping
The overnight culture and 50% isometric sterile glycerol for taking 0.5mL mix, and are stored in -80 DEG C and mark.
The expression and purifying of 1.2 recombinant proteins
1.2.1 picking contains the BL21 bacterial strain of GFAP expression plasmid pET-28a, is inoculated with into containing 50 μ g/mL kanamycins
LB culture medium 100mL in, shake culture is overnight.
1.2.2 2mL blank LB culture medium is taken out as background, as blank zeroing setting light splitting light under 600nm wavelength
Degree meter.
1.2.3 overnight culture is poured into the LB culture medium 100mL of four bottles of 500mL, each bottle of addition 50mg/mL card that
Two bottles of 500mL culture medium that kanamycins and overnight culture is added is merged into one bottle, divided after mixing is rocked when pouring by mycin
Reinstall the state of two bottles of 500mL, it is ensured that the homogeneity between two bottles.To other two bottles same operations.
1.2.4 shake culture 2-3h is measured OD value (600nm), and 12.5mL filtration sterilization is added according to every 500mL
IPTG after every two bottles are merged into one bottle, after pouring into the IPTG mixing of the 100mM of filtration sterilization, dispenses back the shape of two bottles of 500mL
State, it is ensured that the homogeneity between two bottles.To other two bottles same operations.
1.2.5 thalline were collected by centrifugation.
1.2.6 take thallus that 200ml disruption buffer (the 50mM phosphate buffer of pH7.4) is added to mix on ice, ice bath is super
Sound is crushed 2h.Supernatant is collected in centrifugation.
1.2.7 nickel column is crossed, is purified by flash albumen with imidazoles, collects eluent to get GFAP antigen protein is arrived.
The GFAP monoclonal antibody the preparation method comprises the following steps:
2.1, animal immune: Balb/C mouse is immunized in GFAP albumen；
2.2 cell fusions: it is merged using the splenocyte and SP2/0 cell of immune mouse, obtains hybridoma；
2.3 cell screenings: it screens to obtain positive monoclonal hybridoma using limiting dilution assay combination indirect ELISA method
Cell；
2.4 Antibody preparations: monoclonal antibody is enriched with come amplification in vitro using mouse ascites；
2.5 antibody purifications: ascites is purified using caprylic acid-ammonium and affinity chromatography, is measured after dialysis
A280 determines that protein concentration is stand-by.
A kind of preparation method of specific GFAP monoclonal antibody is given in the present embodiment, as follows:
1, it is immunized: taking the female balb/c mouse of 8-12 week old, GFAP antigen is immunized using the hypodermic mode of multiple spot.
It is as follows that this implements immune programme:
2, it merges:
2.1, in first 2 days of fusion, expand culture myeloma cell SP2/0 and myeloma cell's suspension is prepared.
2.2, in first 2 days of fusion, non-immunized balb/c mouse is taken, neck dislocation is lethal.Take macrophage about 3-5*106
It is a, it is resuspended with HAT to 2*105/ ml spreads 96 orifice plates, in 37 DEG C, 6%CO2It is cultivated in incubator spare.
2.3, the balb/c mouse that GFAP has been immunized is taken, eyeball blood sampling is extractd and is used as positive control serum.
2.4, the lethal mouse of neck dislocation, solution take spleen cell, obtain about 6*108Splenocyte, and cell is resuspended in
In 10ml incomplete culture medium.
2.5, by 1-108Splenocyte and 2*107 myeloma cell SP2/0 are mixed in a 50ml fusion pipe, are added endless
Full culture medium 30ml, after centrifugation plus 50%PEG1ml is merged.
2.6, add incomplete culture medium 20-30ml in 90S, in 37 DEG C of standing 10min.
2.7, supernatant is removed in centrifugation, is resuspended, and add the HAT culture medium containing macrophage to 80ml.
2.8,96 orifice plates are dispensed, every hole 0.1ml, in 37 DEG C, 6%CO2Culture in incubator.
2.9, it is swapped out 1/2 culture medium after 5 days with HAT culture medium；It is swapped out HAT culture medium after 7-10 days with HT culture medium；14
Ordinary culture medium is used after it.
2.10, supernatant is sucked out when its length to 1/10 or more hole floor space in the production for observing hybridoma
3, antibody capture Elisa screens hybridoma:
3.1, sheep anti-mouse igg coated elisa plate, every hole add 100ul, 37 DEG C of 2h.
3.2, it washs, 2.10 supernatants, 37 DEG C of 2h is added after patting dry.
3.3, it washs, GFAP antigen, 37 DEG C of 2h is added after patting dry.
3.4, it washs, HRP-IgG, 37 DEG C of 2h is added after patting dry.
3.5, it washs, after patting dry plus tmb substrate colour developing, interpretation result filter out positive hybridoma cell hole.
4, the cloning of hybridoma:
4.1, positive hybridoma cell is filtered out to 3.5 using limiting dilution assay to be serially diluted to 10 cell/ml,
Every hole is inoculated with 100ul.
4.2,37 DEG C, 7.5%CO2 is cultivated 7-10 days, until there is macroscopic clone.
4.3, supernatant is drawn, antibody is detected, cloning is continued in positive hole.
4.4, this implementation obtains full positive colony after three-wheel cloning.
4.5, obtained monoclonal cell strain is expanded culture and is frozen.
5, ascites is beaten:
5.1, adult balb/c mouse, intraperitoneal inoculation norphytane, every mouse 0.5ml are taken.
5.2, the intraperitoneal inoculation diluted hybridoma of PBS, every mouse 5*10 after 7 days5/0.2ml。
5.3, find that mouse web portion obviously expands and hair is blown after 10 days, be slow in action, can CO2 asphyxia put to death mouse,
The ascites in abdominal cavity is drawn with pipettor.Every mouse about 2-3ml.
6, antibody purification:
6.1,5.3 ascites are purified using octanoic acid-ammonium sulfate precipitation method, obtains GFAP monoclonal antibody crude extract.
6.2, column purification GFAP monoclonal antibody crude extract is crossed using ProteinG affinity column, obtains purified GFAP monoclonal antibody
Solution.
It 6.3, is about 2mg/ml with A280 detection GFAP antibody concentration with 1*PBS dialysis GFAP monoclonal antibody solution.
In the fluorescence probe of the fluorescent microsphere label GFAP monoclonal antibody, the fluorescent microsphere is time-resolved fluorescence
Microballoon；
Time-resolved fluorescence microballoon is a kind of to have the lanthanide series and its chelating agent (such as EuCl of unique fluorescent characteristic3)
As the specific function microballoon of tracer, micro-sphere material is usually polystyrene, surface modification have proper density carboxyl or its
Its functional group improves the stability of marker for the covalent coupling with albumen or antibody.
The method of the fluorescence probe of the fluorescent microsphere label GFAP monoclonal antibody are as follows:
(1), with NHS and EDC solution activation microballoon (dosage: 1mg microballoon uses 0.2mg NHS and 0.2mgEDC)；
(2), after mixing well, in 37 DEG C of standing 30min；
(3), GFAP monoclonal antibody is added in activated microballoon, makes the final concentration of 0.5mg/ml of microballoon, first
37 DEG C of coupling 30min are stood, then stand 4 DEG C overnight (not less than 8H)；
(4), confining liquid and 10%Tween-20 are added in the microballoon being coupled, mixes, stands 37 DEG C of 30min；
(5), 13000rpm, centrifugation 15min cleaning microballoon is twice；
(6), after sucking supernatant, microballoon is added and saves liquid, is uniformly dispersed, 4 DEG C of preservations, as GFAP monoclonal antibody is glimmering
Light probe.
The application of the fluorescence immune chromatography test paper bar of the quick detection GFAP, sample are added dropwise to the well in sample pad
It chromatographs forward under capillary action afterwards, by the conjugate release pad containing marker, makes marker aquation again, and micro- with fluorescence
Ball label GFAP monoclonal antibody reacts to each other, then swimming forward together, if containing GFAP in sample, meeting and fluorescent microsphere
It marks GFAP monoclonal antibody to combine, moves forward to detection zone along chromatographic film under chromatography effect, cross detection line by matter
Line capture is controlled, and unbonded fluorescent microsphere marks GFAP monoclonal antibody and by GFAP antigen capture shape coated in chromatographic film
It is adsorbed on detection line region at compound, under the action of laser source, fluorescent material emits the fluorescence signal of certain wave, and fluorescence is exempted from
Epidemic disease analyzer captures fluorescence signal.
Amount of the sample containing GFAP is more in a certain range, and the compound gathered in detection line is fewer, and fluorescence signal is weaker,
Since fluorescent microsphere label GFAP monoclonal antibody is present in excess, so no matter whether containing GFAP, fluorescent microsphere in sample
Label GFAP monoclonal antibody can chromatograph compound to nature controlling line formation fluorescent microsphere label GFAP monoclonal antibody-sheep anti-mouse igg
Object shows fluorescence signal.The fluorescence signal that nature controlling line is shown is that whether enough and chromatography process is normal for judgement sample-adding amount
Standard.According to the ratio T/C of detection line fluorescence intensity and nature controlling line fluorescence intensity, according to built in fluorescence immunoassay quantitative analysis instrument
The concentration of GFAP in curve interpretation sample；The built-in curve of fluorescence immunoassay quantitative analysis instrument is by a series of known GFAP concentration
Standard items are dense with standard items GFAP concentration and known GFAP using the fluorescence immune chromatography test paper bar of the quick detection GFAP
The standard items detection line fluorescence intensity of degree and the ratio T/C of nature controlling line fluorescence intensity are the curve of two factors fitting.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
Claims (10)
1. a kind of fluorescence immune chromatography test paper bar of quickly detection GFAP, including bottom plate, the sample set gradually along floor length
Pad, conjugate release pad, chromatographic film and water absorption pad, the chromatographic film are equipped with detection line and nature controlling line, the detection line
It is separated from each other with nature controlling line, which is characterized in that fluorescent microsphere label GFAP monoclonal is fixed in the conjugate release pad
The fluorescence probe of antibody, is coated with the GFAP antigen with the pairing of microballoon labelled antibody at the detection line, at the nature controlling line
It is coated with sheep anti-mouse igg antibody.
2. the fluorescence immune chromatography test paper bar of quickly detection GFAP according to claim 1 a kind of, which is characterized in that described
Conjugate release pad is glass fibre material, and the chromatographic film uses nitrocellulose filter, and the sample pad is equipped with well.
3. quickly detecting the preparation method of the fluorescence immune chromatography test paper bar of GFAP as described in claim 1, which is characterized in that packet
Include following steps:
(1) fluorescence probe of fluorescent microsphere label GFAP monoclonal antibody is prepared；
(2) fluorescence probe of preparation is fixed in conjugate release pad with the mode for impregnating conjugate release pad, is freeze-dried
Afterwards, it is sealed spare；
(3) it is coated with GFAP antigen and sheep anti-mouse igg antibody lines in chromatographic film, respectively detection line and nature controlling line；
(4) paste of sample pad, conjugate release pad, chromatographic film and water absorption pad is formed on bottom plate and quickly detects the glimmering of GFAP
Light immuno-chromatographic test paper strip.
4. quickly detecting the preparation method of the fluorescence immune chromatography test paper bar of GFAP according to claim 3, which is characterized in that
The GFAP antigen the preparation method comprises the following steps:
1.1, the building of expression plasmid:
1.1.1 double digestion: the restriction endonuclease of GFAP gene two sides is determined；
1.1.2 it connects: transfecting purpose bacterial strain after target fragment is connect with carrier segments；
1.1.3 turn bacterium: turning bacterium and apply plate, the presence of segment is identified by bacterium colony PCR；
1.1.4 fungi preservation: picking positive bacteria falls in suitable resistance LB culture medium culture and is saved with sterile glycerol；
1.2, the expression and purifying of recombinant protein:
1.2.1 cultivate: picking contains GFAP expression plasmid, is inoculated in LB culture medium, shake culture；
1.2.2 inducing expression: being induced with IPTG, and E.coli expression GFAP simultaneously collects thallus；
1.2.3 protein purification: nickel column is crossed after bacterial cell disruption, is purified by flash albumen with imidazoles；
1.3, the identification of recombinant protein:
1.3.1 purifying protein identifies purity using the method for SDS-PAGE；
1.3.2 whether correctly collected using GFAP kit identification destination protein.
5. quickly detecting the preparation method of the fluorescence immune chromatography test paper bar of GFAP according to claim 3, which is characterized in that
The GFAP monoclonal antibody the preparation method comprises the following steps:
2.1, animal immune: Balb/C mouse is immunized in GFAP albumen；
2.2 cell fusions: it is merged using the splenocyte and SP2/0 cell of immune mouse, obtains hybridoma；
2.3 cell screenings: it screens to obtain positive monoclonal hybridoma using limiting dilution assay combination indirect ELISA method thin
Born of the same parents；
2.4 Antibody preparations: monoclonal antibody is enriched with come amplification in vitro using mouse ascites；
2.5 antibody purifications: purifying ascites using caprylic acid-ammonium and affinity chromatography, and it is true that A280 is measured after dialysis
It is stand-by to determine protein concentration.
6. quickly detecting the preparation method of the fluorescence immune chromatography test paper bar of GFAP according to claim 3, which is characterized in that
In the fluorescence probe of the fluorescent microsphere label GFAP monoclonal antibody, the fluorescent microsphere is time-resolved fluorescence microballoon；
The method of the fluorescence probe of the fluorescent microsphere label GFAP monoclonal antibody are as follows:
(1), microballoon is activated with NHS and EDC solution；
(2), it after mixing well, stands；
(3), GFAP monoclonal antibody is added in activated microballoon, makes the final concentration of 0.5mg/ml of microballoon, first stands
Coupling, then stand overnight；
(4), confining liquid and 10%Tween-20 are added in the microballoon being coupled, mixes, stands；
(5), eccentric cleaning microballoon；
(6), after sucking supernatant, microballoon is added and saves liquid, is uniformly dispersed, as fluorescent microsphere label GFAP monoclonal antibody is glimmering
Light probe.
7. the quickly application of the fluorescence immune chromatography test paper bar of detection GFAP as described in claim 1, which is characterized in that sample drop
It is chromatographed forward under capillary action after sample pad is added, by the conjugate release pad containing marker, makes marker aquation again,
And react to each other with fluorescent microsphere label GFAP monoclonal antibody, then swimming forward together, if containing GFAP in sample, meeting
In conjunction with fluorescent microsphere label GFAP monoclonal antibody, detection zone is moved forward to along chromatographic film under chromatography effect, is crossed
Detection line is captured by nature controlling line, and unbonded fluorescent microsphere marks GFAP monoclonal antibody and by GFAP coated in chromatographic film
Antigen capture forms compound and is adsorbed on detection line region, and under the action of laser source, fluorescent material emits the fluorescence of certain wave
Signal, fluorescence immunity analyzer capture fluorescence signal.
8. the quickly application of the fluorescence immune chromatography test paper bar of detection GFAP according to claim 7, which is characterized in that one
Determine in range that amount of the sample containing GFAP is more, the compound gathered in detection line is fewer, and fluorescence signal is weaker, due to fluorescent microsphere
Label GFAP monoclonal antibody is present in excess, so no matter whether containing GFAP in sample, fluorescent microsphere marks GFAP Dan Ke
Grand antibody can chromatograph to nature controlling line and form fluorescent microsphere label GFAP monoclonal antibody-sheep anti-mouse igg compound, display fluorescence letter
Number.
9. the quickly application of the fluorescence immune chromatography test paper bar of detection GFAP according to claim 7, which is characterized in that Quality Control
The fluorescence signal that line is shown be determine sample-adding amount whether enough and the whether normal standard of chromatography process.
10. the quickly application of the fluorescence immune chromatography test paper bar of detection GFAP according to claim 7, which is characterized in that root
According to the ratio T/C of detection line fluorescence intensity and nature controlling line fluorescence intensity, sentenced according to the built-in curve of fluorescence immunoassay quantitative analysis instrument
Read the concentration of GFAP in sample；
The built-in curve of fluorescence immunoassay quantitative analysis instrument is by a series of standard items of known GFAP concentration, using the quick inspection
The fluorescence immune chromatography test paper bar of GFAP is surveyed, it is strong with the standard items detection line fluorescence of standard items GFAP concentration and known GFAP concentration
Degree and the ratio T/C of nature controlling line fluorescence intensity are the curve of two factors fitting.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110488007A (en) * | 2019-09-26 | 2019-11-22 | 天津华科泰生物技术有限公司 | A kind of immunochromatographydetection detection card and preparation method thereof of quick detection glial fibrillary acidic albumen |
| CN110567926A (en) * | 2019-09-11 | 2019-12-13 | 江苏宜偌维盛生物技术有限公司 | Novel time-resolved fluorescent microsphere coupling process and application of fluorescent microspheres |
| CN111323579A (en) * | 2020-03-24 | 2020-06-23 | 苏州星烁纳米科技有限公司 | Immunochromatographic test strip, kit and immunoassay method |
| CN113588959A (en) * | 2021-09-10 | 2021-11-02 | 河南农业大学 | Turn-on test strip for detecting T-2 toxin and preparation method thereof |
| CN116124753A (en) * | 2023-04-14 | 2023-05-16 | 北京芯迈微生物技术有限公司 | Microfluidic quantitative detection kit and method based on fluorescence conversion capability |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060240480A1 (en) * | 2003-09-24 | 2006-10-26 | Roche Diagnostics Operations, Inc. | Use of GFAP for identification of intracerebral hemorrhage |
| CN104142404A (en) * | 2013-05-10 | 2014-11-12 | 深圳市安群生物工程有限公司 | Fluorescent immunochromatographic test paper for detecting human GFAP protein, and preparation method thereof |
| US20170234867A1 (en) * | 2012-10-31 | 2017-08-17 | Astute Medical, Inc. | Quantitative lateral flow assay |
| CN108107202A (en) * | 2017-12-27 | 2018-06-01 | 石家庄市畜产品质量监测中心 | A kind of time-resolved fluoroimmunoassay chromatograph test strip and preparation method thereof |
| CN108997496A (en) * | 2018-10-16 | 2018-12-14 | 无锡傲锐东源生物科技有限公司 | Anti- GFAP protein monoclonal antibody and application thereof |
-
2018
- 2018-12-29 CN CN201811639111.1A patent/CN109633171A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060240480A1 (en) * | 2003-09-24 | 2006-10-26 | Roche Diagnostics Operations, Inc. | Use of GFAP for identification of intracerebral hemorrhage |
| US20170234867A1 (en) * | 2012-10-31 | 2017-08-17 | Astute Medical, Inc. | Quantitative lateral flow assay |
| CN104142404A (en) * | 2013-05-10 | 2014-11-12 | 深圳市安群生物工程有限公司 | Fluorescent immunochromatographic test paper for detecting human GFAP protein, and preparation method thereof |
| CN108107202A (en) * | 2017-12-27 | 2018-06-01 | 石家庄市畜产品质量监测中心 | A kind of time-resolved fluoroimmunoassay chromatograph test strip and preparation method thereof |
| CN108997496A (en) * | 2018-10-16 | 2018-12-14 | 无锡傲锐东源生物科技有限公司 | Anti- GFAP protein monoclonal antibody and application thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110567926A (en) * | 2019-09-11 | 2019-12-13 | 江苏宜偌维盛生物技术有限公司 | Novel time-resolved fluorescent microsphere coupling process and application of fluorescent microspheres |
| CN110488007A (en) * | 2019-09-26 | 2019-11-22 | 天津华科泰生物技术有限公司 | A kind of immunochromatographydetection detection card and preparation method thereof of quick detection glial fibrillary acidic albumen |
| CN111323579A (en) * | 2020-03-24 | 2020-06-23 | 苏州星烁纳米科技有限公司 | Immunochromatographic test strip, kit and immunoassay method |
| CN113588959A (en) * | 2021-09-10 | 2021-11-02 | 河南农业大学 | Turn-on test strip for detecting T-2 toxin and preparation method thereof |
| CN116124753A (en) * | 2023-04-14 | 2023-05-16 | 北京芯迈微生物技术有限公司 | Microfluidic quantitative detection kit and method based on fluorescence conversion capability |
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Application publication date: 20190416 |