CN102955036A - Nanometer detection test paper strip of high-sensitivity neutrophils gelatinase associated lipocalin (NGAL) and preparation method thereof - Google Patents
Nanometer detection test paper strip of high-sensitivity neutrophils gelatinase associated lipocalin (NGAL) and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a nanometer detection test paper strip of high-sensitivity neutrophils gelatinase associated lipocalin (NGAL), which comprises a sample pad, a nanometer pad, a nitrocellulose membrane and an absorption pad, wherein the sample pad, the nanometer pad, the nitrocellulose membrane and the absorption pad are arranged on a fixing plate and connected in sequence, a detection line antibody and a quality control line antibody are arranged on the nitrocellulose membrane, and a silver wrapped gold nanometer shell compound is arranged on the nanometer pad. The invention further discloses a preparation method of the nanometer detection test paper strip of the high-sensitivity NGAL. When the nanometer detection test paper strip is applied to detect the NGAL in human blood and/or urine, the specificity of early diagnosing acute and chronic kidney injuries is high; compared with spherical gold nanoparticles, a better resolution optical signal can be generated in the white background, and the acceleration of immunochromatography is high, so as to be favorable for quick detection of the test paper; and a detection signal is enhanced by 1000 times. Results are easy to observe, the sensitivity reaches 10 pg/ml, the results can be read within 15 minutes, and the nanometer detection test paper strip has good stability and repeatability.
Description
Technical field
The present invention relates to biological technology application, specifically a kind of nano material test strip and preparation method thereof.
Background technology
NGAL(NeutrophiL GeLatinase Associated LipocaLin) molecular weight 22KD, also report 25KD is arranged, mainly be present in the neutral class cell, storage seldom in its hetero-organization.When a variety of causes (wound, poisoning, infection, disease) caused acute and Chronic Renal Impairment, renal tissue NGAL amount obviously raise and is released into blood.Detection blood and/or urine NGAL are judgements and assess acute and biomarker Chronic Renal Impairment, be the index of kidney substantial structure damage.Goldstandard cardiac muscle troponin I, the T of similar diagnosing acute myocardial infarction damage.Acute and the Chronic Renal Impairment (insufficiency) of at present clinical diagnosis mainly is to detect serum creatinine (creatine), urea nitrogen, urinates trace of albumin, is the indirect functional parameter of injury of kidney.Behind the acute injury of kidney, serum creatinine side occurred in 2-3 days.NGAL just obviously raise in blood and urine after acute injury of kidney 2-3 hour, detected the time that NGAL has improved the ability of diagnosis kidney essence damage in advance and greatly, and to reasonable diagnosis and treatment, reducing mortality ratio provides objective basis.
Acute injury of kidney renal dysfunction patient accounts for total 5-7% among the United States Hospital inpatient, and ratio is up to 25% in the patient of hospital severe ward (IC Μ), and mortality ratio is up to 50-80%.Acute and Chronic Renal Impairment relates to openheart surgery, chemotherapy and radiation, severe hypertension, diabetes, septicemia, heart failure and all kinds Cardiorenal syndrome.8,000,000,000 dollars of 23 in masschusetts, u.s.a hospital's acute injury of kidney of such disease (AKI) year cost conservative estimations.Therefore, use the application's patent detection NGAL and will bring into play huge society and economic benefit.
Summary of the invention
Deficiency for the prior art existence, first technical matters that the present invention will solve provides the nano material test strip of a kind of hypersensitivity neutrophil leucocyte gelatinase relative carrier lipoprotein NGAL, be specifically designed to the NGAL that detects in human urine or the blood, it has easy and simple to handle, highly sensitive, detects 0.01-0.1ng/mL than advantages such as 1000 times of classic method susceptibility raisings, result stablize.Another technical matters that the present invention solves provides the preparation method of the nano material test strip of a kind of hypersensitivity neutrophil leucocyte gelatinase relative carrier lipoprotein NGAL.
For achieving the above object, the technical solution used in the present invention is the nano material test strip of a kind of hypersensitivity neutrophil leucocyte gelatinase relative carrier lipoprotein NGAL, comprise and be located at sample pad, nano material pad, nitrocellulose filter and the absorption pad that links to each other successively on the fixed head, be provided with detection line antibody and nature controlling line antibody at nitrocellulose filter, be provided with golden contracted payment nm of gold shell compound at the nano material pad.
Preferably, wherein golden contracted payment nm of gold shell compound is the compound that 40-60nm, 10-20nm nm of gold contracted payment shell label is linked by specific single stranded DNA.
Preferably, the wherein nucleotide sequence of specific single stranded DNA such as SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4.
Preferably, wherein include colour developing antibody mouse-anti human neutrophil genatinase associated lipocalin antibody BOT05 in the golden contracted payment nm of gold shell compound, include anti-human NGAL monoclonal antibody in the detection line antibody and be also referred to as seizure antibody BOT06, described nature controlling line antibody is sheep anti-mouse igg.
Preferably, wherein specific single stranded DNA length is 30-80bp, and specific single stranded DNA 3 ' end indicates biotin, and specific single stranded DNA 5 ' end is connected with sulfydryl (SH).
Preferably, the antibody that wherein develops the color is that two strains can be matched mutually and formed sandwich anti-human NGAL monoclonal antibody with catching antibody.
Preferably, described nano material pad is the pad that the dacron film is made.
Preferably, wherein sample pad is the pad that glass fibre membrane is made.
Another technical scheme of the present invention is the preparation method of the nano material test strip of hypersensitivity neutrophil leucocyte gelatinase relative carrier lipoprotein NGAL, may further comprise the steps:
(1) preparation of nm of gold shell solution;
(2) golden shell mark preparation and confirm golden contracted payment shell nanometer material (40-60nm and 10-20nm) size and purity is determined;
(3) preparation of golden contracted payment nm of gold shell compound is about to the compound that signal antibody BOT05 and 10-20nm and 40-60nm gold contracted payment shell label links;
Regulate PH8.5-9.0 with 0.1M K2CO3; NGAL signal monoclonal antibody BOT05 and nanoshell are adjusted into 4:100(μ g/ml than row)+single stranded DNA 3' end was indicated the 30-80bpSSDNA of biotin and nanoshell signal antibody valency chain coupled reaction 6-8 hour, 25 ℃, its final concentration is 0.5-2.0 μ M;
It is 0.2-1.0% that SSDNA and nanoshell signal antibody compound are added the PEG8000 final concentration, and 2MNaCl/20mM phosphate regulates SSDNA nanoshell complex PH to 7.4;
Above-mentioned PH7.4 complex and streptavidin, horseradish peroxidase (final concentration 0.4 μ M) under 25 ℃ of conditions, are hatched 4 hours, and centrifugal, precipitation is cleaned 3 times, preserve 4 ℃ for subsequent use;
(4) golden shell nanometer material pad preparation;
(5) nitrocellulose filter pre-service and for subsequent use;
(6) prepare and assemble golden shell nanometer material pad;
(7) detection line preparation;
(8) nature controlling line preparation;
(9) test strips assembling;
(10) detect.
Wherein, single stranded DNA length is 43bp in the step (3), and described SSDNA and nanoshell signal antibody compound are added the PEG8000 final concentration is 0.5.
Beneficial effect: the present invention uses the gold nanoshell of sizes hollow to replace present colloidal gold strip spherical nanoparticle commonly used.Be applied to human blood and/or urine NGAL detects, specificity is high, based on producing better resolved light signal than spherical gold nano grain under the white background.And because the weight of hollow gold shell is lighter than onesize solid nanogold particle, the speed of its immunochromatography is accelerated, and is conducive to the fast detecting of test paper.Use nano particle and DNA and protein covalent bonds mechanism under the specified conditions, biotin labeled single stranded DNA connects the nano antibody of two kinds of sizes, strengthens detection signal and reaches 1000 times.The result easily observes, and sensitivity reaches 10pg/mL, with regard to the energy sentence read result, has good stability and repeated in 15 minutes.Easy and simple to handle, need not special instruments and equipment, can be applicable to the early diagnosis of the acute injury of kidney that a variety of causes causes, chronic renal insufficiency dialysis and result for the treatment of are passed judgment on.Especially suit in vast basic hospital promotion and application.
Description of drawings
Fig. 1 is the one-piece construction synoptic diagram of most preferred embodiment of the present invention;
1, sample pad 2, nano material pad: functionalization 10nm gold shell, signal antibody BOT05, functionalization 50nm signal antibody BOT05 3, nitrocellulose filter 4, absorption pad 5, detection line antibody 6, nature controlling line antibody
Embodiment
Below in conjunction with the drawings and specific embodiments, further illustrate the present invention, present embodiment is implemented under take technical solution of the present invention as prerequisite, should understand these embodiment and only be used for explanation the present invention and be not used in and limit the scope of the invention.
The material that this experiment is used:
Detection line antibody is that anti-human NGAL monoclonal antibody BOT06 is seizure antibody, and nature controlling line antibody is sheep anti-mouse igg, and it is mouse-anti human neutrophil genatinase associated lipocalin antibody BOT05 that golden shell labelled antibody includes colour developing antibody, buys in Genscript The Biology CRO Nanjing company.
The used specific single stranded DNA of gold contracted payment nm of gold shell compound is to make by our company's design is synthetic.
Other materials is all bought on market.
Embodiment 1
As shown in Figure 1, the nano material test strip of a kind of hypersensitivity neutrophil leucocyte gelatinase relative carrier lipoprotein NGAL, comprise and be located at sample pad 1, nano material pad 2, nitrocellulose filter 3 and the absorption pad 4 that links to each other successively on the fixed head, be provided with detection line antibody 5 and nature controlling line antibody 6 at nitrocellulose filter 3, be provided with golden contracted payment nm of gold shell compound at nano material pad 2.Wherein, golden contracted payment nm of gold shell compound is the compound that 40-60nm and 10-20nm nm of gold contracted payment shell label is linked by specific single stranded DNA.Wherein, the nucleotide sequence of specific single stranded DNA such as SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4.Wherein, golden contracted payment nm of gold shell compound is the compound that 50nm and 10nm nm of gold contracted payment shell label is linked by specific single stranded DNA.Wherein include colour developing antibody mouse-anti human neutrophil genatinase associated lipocalin antibody BT05 in the golden contracted payment nm of gold shell compound, include anti-human NGAL monoclonal antibody in the detection line antibody and be also referred to as seizure antibody BT06, described nature controlling line antibody is sheep anti-mouse igg.Described specific single stranded DNA length is 30-80bp, and specific single stranded DNA 3' end indicates biotin, and specific single stranded DNA 5 ' end is connected with sulfydryl (SH).
Colour developing antibody is that two strains can be matched mutually and formed sandwich anti-human NGAL monoclonal antibody with catching antibody.Described nano material pad is the pad that the dacron film is made.Described sample pad is the pad that glass fibre membrane is made.
Embodiment 2
(1) preparation of nm of gold shell solution:
Take sodium citrate and tannic acid as bistable agent and reductive agent, prepare monodispersed, the torispherical silver nano-grain solution that size is adjustable.Under 50-70 ℃ water-bath, with the 1%(AgNO of 1.5-2.0ml
3Solution joins in the 50-70ml deionized water, and is heated to constant temperature.This solution system is designated as A; Under 50-70 ℃ water-bath, 1% the sodium citrate solution of 0.5% the tan-ooze of 0.05-5.0ml and 1-3ml is joined in the deionized water of 20-40ml, and be heated to constant temperature, this solution is designated as B.Under vigorous stirring, B is imported among the A, and continue to be stirred to solution and present glassy yellow, after this reaction solution is heated to boiling, and keeps refluxing, continue 10-30 minute, constant volume is to 100ml after the room temperature cooling.
Take monodispersed silver nano-grain as sacrificing masterplate, prepare but the gold nanoshell solution of dispersion by displacement reaction: get 20-40ml silver nano-grain solution, at 12000-15000g, 4 ℃ were descended centrifugal 15-30 minute, the sucking-off supernatant disperses again with deionized water again, and constant volume is to 100ml.The silver nano-grain solution of this 100ml is heated to rapidly boiling, is keeping adding 2mM HAuCl under backflow and the vigorous stirring
4(or NaAuCl
4/ KAuCl
4) 1-4ml, the reactant liquor color is changed into blackish green gradually by yellow, continuing room temperature cooling in the situation about stirring afterwards.
(2) the golden mark of colour developing antibody:
Use 0.05-0.20MK
2CO
3Regulate nm of gold shell solution to pH be 7.4-9.0.In 80-120ml nm of gold shell solution, dropwise add colour developing antibody (mouse-anti human neutrophil genatinase associated lipocalin antibody BOT05) 2.0-4.0mg, stirred 3-10 minute), left standstill 20-60 minute.Add again 15-35ml, 2-10% BSA and 20mM Tris-HClpH7.5-9.0,2-8 ℃ of sealing spent the night, and gets reactant liquor; Reactant liquor is in 2-8 ℃, 3000-5000g, and centrifugal 20-40 minute, precipitation was used 100-150ml, and 0.5-2%BSA and 20mM Tris-HClpH8.0-9.0 are resuspended, and repeated centrifugation twice, centrifugal speed are followed successively by 4000-2000g and 3000-1000g must precipitate.(composition is the 20-40% trehalose, 0.5-2.0% BSA, 0.005-0.02%Tween-20,0.01-0.03% NaN with the resuspended liquid of 1000-1500 μ l
3, 10-30mMTris-HCl, pH8.0-9.0) and the precipitation that suspends, get golden labeling antibody;
(3) preparation of nano material pad and spray pad:
Film treating fluid preparation: 0.15-0.35%Triton X-100,0.10-0.20mMNaCl, 0.01-0.1mM) TrispH7.5; The dacron film soaks (1-2h), vacuum drying in the film treating fluid; With 1.5-3.0 μ l/cm, with golden labeling antibody spray printing on the nano material pad;
(4) detection line preparation:
Getting the anti-human NGAL monoclonal antibody of 1 strain BOT06(can form sandwich with mouse-anti human neutrophil genatinase associated lipocalin antibody BOT05 pairing) do detection line antibody, with damping fluid 5-20% trehalose, the dilution of 3-8% methyl alcohol is 0.5-2.0mg/mL; With drawing the film instrument antibody is drawn on nitrocellulose filter, concentration is that 1 μ l/cm forms detection line, vacuum drying;
(5) nature controlling line preparation:
Sheep anti-mouse igg is 0.2-1.0mg/ml with damping fluid 5-20% trehalose, 3-8% methyl alcohol dilution; With drawing the film instrument antibody is drawn on nitrocellulose filter, concentration is that 0.5 μ l/cm forms nature controlling line, vacuum drying;
(6) test strips assembling:
Sample pad 1, nano material pad 2, nitrocellulose filter 3 and thieving paper 4 are sticked on the plastic plate of single face pressure sensitive adhesive in order, be provided with detection line antibody 5 and nature controlling line antibody 6 at nitrocellulose filter 3; Plastic plate after cutting is pasted, the test card of packing into; Use with the heat-sealing Fresco Bag of drying agent and pack room temperature preservation;
(7) detect:
Sample drop is added well, observe in the time of 10-20 minute, interpretation ELISA test strip result's standard is: the positive, and all there is the colour developing band at detection line place and nature controlling line place; Feminine gender, the detection line place is without the colour developing band, and there is the colour developing band at the nature controlling line place; Test strips lost efficacy, and the nature controlling line place is without the colour developing band.
Embodiment 3
(1) preparation of nm of gold shell solution:
Take sodium citrate and tannic acid as bistable agent and reductive agent, prepare monodispersed, the torispherical silver nano-grain solution that size is adjustable.Under 50-70 ℃ water-bath, with the 1%(AgNO of 1.5-2.0ml
3Solution joins in the 50-70ml deionized water, and is heated to constant temperature.This solution system is designated as A; Under 50-70 ℃ water-bath, 1% the sodium citrate solution of 0.5% the tan-ooze of 0.05-5.0ml and 1-3ml is joined in the deionized water of 20-40ml, and be heated to constant temperature, this solution is designated as B.Under vigorous stirring, B is imported among the A, and continue to be stirred to solution and present glassy yellow, after this reaction solution is heated to boiling, and keeps refluxing, continue 10-30 minute, constant volume is to 100ml after the room temperature cooling.
Take monodispersed silver nano-grain as sacrificing masterplate, prepare but the gold nanoshell solution of dispersion by displacement reaction: get 20-40mL silver nano-grain solution, at 12000-15000g, 4 ℃ were descended centrifugal 15-30 minute, the sucking-off supernatant disperses again with deionized water again, and constant volume is to 100ml.The silver nano-grain solution of this 100ml is heated to rapidly boiling, is keeping adding 2mM HAuCl under backflow and the vigorous stirring
4(or NaAuCl
4/ KAuCl
4) 1-4ml, the reactant liquor color is changed into blackish green gradually by yellow, continuing room temperature cooling in the situation about stirring afterwards.
(2) the golden mark of colour developing antibody:
Use 0.05-0.20MK
2CO
3Regulate nm of gold shell solution to pH be 7.4-9.0.In 80-120ml nm of gold shell solution, dropwise add colour developing antibody (mouse-anti human neutrophil genatinase associated lipocalin antibody BOT05) 2.0-4.0mg, stirred 3-10 minute, left standstill 20-60 minute.Add again 15-35ml, 2-10% BSA and 20mM Tris-HClpH7.5-9.0,2-8 ℃ of sealing spent the night, and gets reactant liquor; Reactant liquor is in 2-8 ℃, 3000-5000g, and centrifugal 20-40 minute, precipitation was used 100-150ml, and 0.5-2%BSA and 20mM Tris-HCLpH8.0-9.0 are resuspended, and repeated centrifugation twice, centrifugal speed are followed successively by 4000-2000g and 3000-1000g must precipitate.(composition is the 20-40% trehalose, 0.5-2.0% BSA, 0.005-0.02%Tween-20,0.01-0.03% NaN with the resuspended liquid of 1000-1500 μ l
3, 10-30mMTris-HCl, pH8.0-9.0) and the precipitation that suspends, get golden labeling antibody;
(3) preparation of signal antibody BOT05 and 5-15nm and 40-60nm gold contracted payment nm of gold shell compound:
Use 0.1M K
2CO
3Regulate PH8.5-9.0;
NGAL signal monoclonal antibody BOT05 is adjusted into the g/mL into 4:100(μ with nanoshell than row);
Single stranded DNA 3' end is indicated (43BP(30-80BP) of biotin) SSDNA and the golden labeling antibody valency of nanoshell chain coupled reaction 6-8 hour, 25 ℃, its final concentration is 0.5-2.0 μ M.
It is 0.5% (0.2-1.0%) that SSDNA and nanoshell gold labeling antibody compound is added the PEG8000 final concentration, and 2MNaCl/20mMphosphate regulates SSDNA nanoshell complex PH to 7.4.
Above-mentioned PH7.4 complex and streptavidin, horseradish peroxidase (final concentration 0.4 μ M) under 25 ℃ of conditions, are hatched 4 hours, and centrifugal, precipitation is cleaned 3 times, preserve 4 ℃ for subsequent use.
The composition of cleaning and the punching of preservation liquid and PH:PH7.4,0.1%BSA, 0.1%Tween20,10mM phosphate sodium.
(4) golden shell nanometer material pad preparation
Nano material signal antibody compound is seen (3);
Glass film pre-service and for subsequent use:
Borate buffer: PH7.4,10mM boric acid, 0.5% sucrose, 1%BSA, 0.5Tween20,0.05% Sodium azide glass film (poLyecterfiter) were soaked in borate buffer 2 hours, changed damping fluid therebetween twice, 50 ℃ of oven dry 2 hours.
The glass film is combined with the functionalization signal antibody: pretreated glass film is soaked in the 0.05% sucrose Sodium azide liquid 1 hour, 50 ℃ of oven dry 2 hours.
Gold contracted payment shell antibody BOT05 is added on the glass film: functionalization BOT05 mark connects the BOT05 of Avidin-horseradish peroxidase, drips profit glass film, 37 ℃ of dryings 30 minutes, and kept dry is for subsequent use.
(5) nitrocellulose filter pre-service and for subsequent use
PBS buffer solution ph 7.4,10mMPBS, 3%BSA, 0.5%Tween20;
Nitrocellulose filter was soaked in the PBS damping fluid 4 hours, and 50 ℃ of dryings 2 hours are for subsequent use.
(6) prepare and assemble golden shell nanometer material
Assembling composition: sample pad, nano material pad, functionalization 10nm gold shell, signal antibody BOT05, functionalization 50nm signal antibody BOT05, nitrocellulose filter (catching the line of antibody BOT06 and sheep anti-mouse igg antibody) and 6 kinds of compositions of absorption pad.
37 ℃ of dryings of complete film after the assembling are stored for subsequent use.
(7) golden shell nanometer material pad preparation and spray pad:
Film treating fluid preparation: 0.15-0.35%Triton X-100,0.10-0.20mMNaCl, 0.01-0.1mM) TrispH7.5; The dacron film soaks (1-2h), vacuum drying in the film treating fluid; With 1.5-3.0 μ l/cm, with golden labeling antibody spray printing on golden shell nanometer material pad;
(8) detection line preparation:
Getting the anti-human NGAL monoclonal antibody of 1 strain BOT06(can form sandwich with mouse-anti human neutrophil genatinase associated lipocalin antibody BOT05 pairing) do detection line antibody, with damping fluid 5-20% trehalose, the dilution of 3-8% methyl alcohol is 0.5-2.0mg/ml; With drawing the film instrument antibody is drawn on nitrocellulose filter, concentration is that 1 μ l/cm forms detection line, vacuum drying;
(9) nature controlling line preparation:
Sheep anti-mouse igg is 0.2-1.0mg/ml with damping fluid 5-20% trehalose, 3-8% methyl alcohol dilution; With drawing the film instrument antibody is drawn on nitrocellulose filter, concentration is that 0.5 μ l/cm forms nature controlling line, vacuum drying;
(10) test strips assembling:
Sample pad 1, nano material pad 2, nitrocellulose filter 3 and thieving paper 4 are sticked on the plastic plate of single face pressure sensitive adhesive in order, be provided with detection line antibody 5 and nature controlling line antibody 6 at nitrocellulose filter 3; Plastic plate after cutting is pasted, the test card of packing into; Use with the heat-sealing Fresco Bag of drying agent and pack room temperature preservation;
(11) detect:
Sample drop is added well, observe in the time of 10-20 minute, interpretation ELISA test strip result's standard is: the positive, and all there is the colour developing band at detection line place and nature controlling line place; Feminine gender, the detection line place is without the colour developing band, and there is the colour developing band at the nature controlling line place; Test strips lost efficacy, and the nature controlling line place is without the colour developing band.
Embodiment 4
10ng/ml, 50ng/ml, 100ng/ml and the 200ng/ml NGAL standard items sample drop of 100 μ L preparation are added sample well, the 15min observations, band appears in the detection line position in the reference material 5min of testing result: 40ng/ml; Band appears in the detection line position in the reference material 10min of 20ng/ml; Band appears in the detection line position in the reference material 15min of 10ng/ml; The position does not appear in detection line in the reference material 30min of 5ng/ml.
Embodiment 5
1, golden shell nanometer material pad preparation
The preparation of signal antibody BOT05 and 10nm and 50nm gold contracted payment nm of gold shell compound:
Use 0.1M K
2CO
3Regulate PH8.5-9.0;
NGAL signal monoclonal antibody BOT05 is adjusted into the g/ml into 4:100(μ with nanoshell than row);
The 30-80BP SSDNA that single stranded DNA 3' end is indicated biotin and the golden labeling antibody valency of nanoshell chain coupled reaction 6-8 hour, 25 ℃, its final concentration is 0.5-2.0 μ M.
It is 0.5% (0.2-1.0%) that SSDNA and nanoshell gold labeling antibody compound is added the PEG8000 final concentration, and 2MNaCl/20mM phosphate regulates SSDNA nanoshell complex PH to 7.4.
Above-mentioned PH7.4 complex and streptavidin, horseradish peroxidase (final concentration 0.4 μ M) under 25 ℃ of conditions, are hatched 4 hours, and centrifugal, precipitation is cleaned 3 times, preserve 4 ℃ for subsequent use.
The composition of cleaning and the punching of preservation liquid and PH:PH7.4,0.1%BSA, 0.1%Tween20,10mM phosphate sodium.
Glass film pre-service and for subsequent use
Borate buffer: PH7.4,10mM boric acid, 0.5% sucrose, 1%BSA, 0.5Tween20,0.05% Sodium azide glass film were soaked in borate buffer 2 hours, changed damping fluid therebetween twice, 50 ℃ of oven dry 2 hours.
The glass film is combined with the functionalization signal antibody: pretreated glass film is soaked in the 0.05% sucrose Sodium azide liquid 1 hour, 50 ℃ of oven dry 2 hours.
Gold contracted payment shell antibody BOT05 is added on the glass film: functionalization BOT05 mark connects the BOT05 of Avidin-horseradish peroxidase, drips profit glass film, 37 ℃ of dryings 30 minutes, and kept dry is for subsequent use.
2, nitrocellulose filter pre-service and for subsequent use
PBS buffer solution ph 7.4,10mMPBS, 3%BSA, 0.5%Tween20;
Nitrocellulose filter was soaked in the PBS damping fluid 4 hours, and 50 ℃ of dryings 2 hours are for subsequent use.
3, prepare and assemble golden shell nanometer material
Assembling composition: sample pad, nano material pad, functionalization 10nm gold shell, signal antibody BOT05, functionalization 50nm signal antibody BOT05, nitrocellulose filter (catching the line of antibody BOT06 and sheep anti-mouse igg antibody) and 6 kinds of compositions of absorption pad.37 ℃ of dryings of complete film after the assembling are stored for subsequent use.
Embodiment 6
1, golden shell nanometer material pad preparation
The preparation of signal antibody BOT05 and 20nm and 40nm gold contracted payment nm of gold shell compound:
Use 0.1M K
2CO
3Regulate PH8.5-9.0; NGAL signal monoclonal antibody BOT05 is adjusted into the g/ml into 4:100(μ with nanoshell than row); The 30-80BP SSDNA that single stranded DNA 3' end is indicated biotin and the golden labeling antibody valency of nanoshell chain coupled reaction 6-8 hour, 25 ℃, its final concentration is 0.5-2.0 μ M.
It is 0.5% (0.2-1.0%) that SSDNA and nanoshell gold labeling antibody compound is added the PEG8000 final concentration, and 2MNaCl/20mM phosphate regulates SSDNA nanoshell complex PH to 7.4.
Above-mentioned PH7.4 complex and streptavidin, horseradish peroxidase (final concentration 0.4 μ M) under 25 ℃ of conditions, are hatched 4 hours, and centrifugal, precipitation is cleaned 3 times, preserve 4 ℃ for subsequent use.
The composition of cleaning and the punching of preservation liquid and PH:PH7.4,0.1%BSA, 0.1%Tween20,10mM phosphate sodium.
Glass film pre-service and for subsequent use
Borate buffer: PH7.4,10mM boric acid, 0.5% sucrose, 1%BSA, 0.5Tween20,0.05% Sodium azide glass film were soaked in borate buffer 2 hours, changed damping fluid therebetween twice, 50 ℃ of oven dry 2 hours.
The glass film is combined with the functionalization signal antibody: pretreated glass film is soaked in the 0.05% sucrose Sodium azide liquid 1 hour, 50 ℃ of oven dry 2 hours.
Gold contracted payment shell antibody BOT05 is added on the glass film: functionalization BOT05 mark connects the BOT05 of Avidin-horseradish peroxidase, drips profit glass film, 37 ℃ of dryings 30 minutes, and kept dry is for subsequent use.
2, nitrocellulose filter pre-service and for subsequent use
PBS buffer solution ph 7.4,10mMPBS, 3%BSA, 0.5%Tween20;
Nitrocellulose filter was soaked in the PBS damping fluid 4 hours, and 50 ℃ of dryings 2 hours are for subsequent use.
3, prepare and assemble golden shell nanometer material
Assembling composition: sample pad, nano material pad, functionalization 20nm gold shell, signal antibody BOT05, functionalization 40nm signal antibody BOT05, nitrocellulose filter (catching the line of antibody BOT06 and sheep anti-mouse igg antibody) and 6 kinds of compositions of absorption pad.
37 ℃ of dryings of complete film after the assembling are stored for subsequent use.
Embodiment 7
1, golden shell nanometer material pad preparation
The preparation of signal antibody BOT05 and 15nm and 45nm gold contracted payment nm of gold shell compound:
Use 0.1M K
2CO
3Regulate PH8.5-9.0;
NGAL signal monoclonal antibody BOT05 is adjusted into the g/ml into 4:100(μ with nanoshell than row);
The 30-80BP SSDNA that single stranded DNA 3' end is indicated biotin and the golden labeling antibody valency of nanoshell chain coupled reaction 6-8 hour, 25 ℃, its final concentration is 0.5-2.0 μ M.
It is 0.5% (0.2-1.0%) that SSDNA and nanoshell gold labeling antibody compound is added the PEG8000 final concentration, and 2MNaCl/20mM phosphate regulates SSDNA nanoshell complex PH to 7.4.
Above-mentioned PH7.4 complex and streptavidin, horseradish peroxidase (final concentration 0.4 μ M) under 25 ℃ of conditions, are hatched 4 hours, and centrifugal, precipitation is cleaned 3 times, preserve 4 ℃ for subsequent use.
The composition of cleaning and the punching of preservation liquid and PH:PH7.4,0.1%BSA, 0.1%Tween20,10mM phosphate sodium.
Glass film pre-service and for subsequent use
Borate buffer: PH7.4,10mM boric acid, 0.5% sucrose, 1%BSA, 0.5Tween20,0.05% Sodium azide glass film (poLyecterfiter) were soaked in borate buffer 2 hours, changed damping fluid therebetween twice, 50 ℃ of oven dry 2 hours.
The glass film is combined with the functionalization signal antibody: pretreated glass film is soaked in the 0.05% sucrose Sodium azide liquid 1 hour, 50 ℃ of oven dry 2 hours.
Gold contracted payment shell antibody BOT05 is added on the glass film: functionalization BOT05 mark connects the BOT05 of Avidin-horseradish peroxidase, drips profit glass film, 37 ℃ of dryings 30 minutes, and kept dry is for subsequent use.
2, nitrocellulose filter pre-service and for subsequent use
PBS buffer solution ph 7.4,10mMPBS, 3%BSA, 0.5%Tween20;
Nitrocellulose filter was soaked in the PBS damping fluid 4 hours, and 50 ℃ of dryings 2 hours are for subsequent use.
3, prepare and assemble golden shell nanometer material
Assembling composition: sample pad, nano material pad, functionalization 15nm gold shell, signal antibody BOT05, functionalization 45nm signal antibody BOT05, nitrocellulose filter (catching the line of antibody BOT06 and sheep anti-mouse igg antibody) and 6 kinds of compositions of absorption pad.37 ℃ of dryings of complete film after the assembling are stored for subsequent use.
＜110〉The Second Affiliated Hospital of Nanjing Medical University, Nanjing Botian Kezhi Biotechnology Co., Ltd.
＜120〉nano material test strip of a kind of hypersensitivity neutrophil leucocyte gelatinase relative carrier lipoprotein NGAL and preparation method thereof
<130> 20121123
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<170> PatentIn version 3.3
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Claims (10)
1. the nano material test strip of a hypersensitivity neutrophil leucocyte gelatinase relative carrier lipoprotein NGAL, it is characterized in that: comprise being located at the sample pad (1) that links to each other successively on the fixed head, nano material pad (2), nitrocellulose filter (3) and absorption pad (4), be provided with detection line antibody (5) and nature controlling line antibody (6) at nitrocellulose filter (3), be provided with golden contracted payment nm of gold shell compound at nano material pad (2).
2. the nano material test strip of described a kind of hypersensitivity neutrophil leucocyte gelatinase relative carrier lipoprotein NGAL according to claim 1 is characterized in that: described golden contracted payment nm of gold shell compound is the compound that 40-60nm and 10-20nm nm of gold contracted payment shell label is linked by specific single stranded DNA.
3. the nano material test strip of described a kind of hypersensitivity neutrophil leucocyte gelatinase relative carrier lipoprotein NGAL according to claim 2 is characterized in that: the nucleotide sequence of described specific single stranded DNA such as SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4.
4. the nano material test strip of described a kind of hypersensitivity neutrophil leucocyte gelatinase relative carrier lipoprotein NGAL according to claim 2 is characterized in that: described golden contracted payment nm of gold shell compound is the compound that 50nm and 10nm nm of gold contracted payment shell label is linked by specific single stranded DNA.
5. the nano material test strip of described a kind of hypersensitivity neutrophil leucocyte gelatinase relative carrier lipoprotein NGAL according to claim 1-3, it is characterized in that: include colour developing antibody mouse-anti human neutrophil genatinase associated lipocalin antibody BOT05 in the described golden contracted payment nm of gold shell compound, include anti-human NGAL monoclonal antibody in the detection line antibody (5) and be also referred to as seizure antibody BOT06, described nature controlling line antibody (6) is sheep anti-mouse igg.
6. the nano material test strip of described a kind of hypersensitivity neutrophil leucocyte gelatinase relative carrier lipoprotein NGAL according to claim 3, it is characterized in that: described specific single stranded DNA length is 30-80bp, specific single stranded DNA 3 ' end indicates biotin, and specific single stranded DNA 5 ' end is connected with sulfydryl (SH).
7. the nano material test strip of described a kind of hypersensitivity neutrophil leucocyte gelatinase relative carrier lipoprotein NGAL according to claim 4 is characterized in that: colour developing antibody is that two strains can be matched mutually and formed sandwich anti-human NGAL monoclonal antibody with catching antibody.
8. the nano material test strip of described a kind of hypersensitivity neutrophil leucocyte gelatinase relative carrier lipoprotein NGAL according to claim 1, it is characterized in that: the pad that described nano material pad (2) is made for the dacron film, the pad that described sample pad (1) is made for glass fibre membrane.
9. the preparation method of the nano material test strip of a hypersensitivity neutrophil leucocyte gelatinase relative carrier lipoprotein NGAL as claimed in claim 1 is characterized in that: may further comprise the steps:
(1) preparation of nm of gold shell solution;
(2) golden shell mark preparation and confirm golden contracted payment shell nanometer material (40-60nm and 10-20nm) size and purity is determined;
(3) preparation of golden contracted payment nm of gold shell compound is about to the compound that signal antibody BOT05 and 10-20nm and 40-60nm gold contracted payment shell label links;
Use 0.1M K
2CO
3Regulate PH8.5-9.0; NGAL signal monoclonal antibody BOT05 is adjusted into 4:100(μ g/ml with nanoshell than row); Single stranded DNA 3' end was indicated the 30-80bpSSDNA of biotin and nanoshell signal antibody valency chain coupled reaction 6-8 hour, and 25 ℃, its final concentration is 0.5-2.0 μ M;
It is 0.2-1.0% that SSDNA and nanoshell signal antibody compound are added the PEG8000 final concentration, and 2MNaCl/20mM phosphate regulates SSDNA nanoshell complex PH to 7.4;
Above-mentioned PH7.4 complex and streptavidin, horseradish peroxidase (final concentration 0.4 μ M) under 25 ℃ of conditions, are hatched 4 hours, and centrifugal, precipitation is cleaned 3 times, preserve 4 ℃ for subsequent use;
(4) golden shell nanometer material pad preparation;
(5) nitrocellulose filter pre-service and for subsequent use;
(6) prepare and assemble golden shell nanometer material pad;
(7) detection line preparation;
(8) nature controlling line preparation;
(9) test strips assembling;
(10) detect.
10. the preparation method of the nano material test strip of described a kind of hypersensitivity neutrophil leucocyte gelatinase relative carrier lipoprotein NGAL according to claim 2, it is characterized in that: single stranded DNA length is 43bp in the described step (3), and described SSDNA and nanoshell signal antibody compound are added the PEG8000 final concentration is 0.5%.
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|---|---|---|---|---|
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101059519A (en) * | 2007-05-23 | 2007-10-24 | 南京博天科智生物技术有限公司 | Cardiac troponin I nano gold test paper and its preparation method |
| CN102298055A (en) * | 2011-07-21 | 2011-12-28 | 南京博天科智生物技术有限公司 | Human blood H-FABP nano-gold labeled test paper and preparation method thereof |
| CN102323422A (en) * | 2011-05-30 | 2012-01-18 | 中国科学院上海微系统与信息技术研究所 | Immunochromatographic test strip for semi-quantitatively and simultaneously detecting cTnI and Myo and preparation method thereof |
| CN102759623A (en) * | 2012-07-06 | 2012-10-31 | 南京基蛋生物科技有限公司 | Colloidal gold test strip for detecting NGAL (Neutrophil Gelatinase Associated Lipocalin) and preparation method thereof |
-
2012
- 2012-11-27 CN CN201210489700.2A patent/CN102955036B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101059519A (en) * | 2007-05-23 | 2007-10-24 | 南京博天科智生物技术有限公司 | Cardiac troponin I nano gold test paper and its preparation method |
| CN102323422A (en) * | 2011-05-30 | 2012-01-18 | 中国科学院上海微系统与信息技术研究所 | Immunochromatographic test strip for semi-quantitatively and simultaneously detecting cTnI and Myo and preparation method thereof |
| CN102298055A (en) * | 2011-07-21 | 2011-12-28 | 南京博天科智生物技术有限公司 | Human blood H-FABP nano-gold labeled test paper and preparation method thereof |
| CN102759623A (en) * | 2012-07-06 | 2012-10-31 | 南京基蛋生物科技有限公司 | Colloidal gold test strip for detecting NGAL (Neutrophil Gelatinase Associated Lipocalin) and preparation method thereof |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103267854A (en) * | 2013-05-03 | 2013-08-28 | 西安交通大学 | Method for enhancing detection signal of test paper |
| CN103728452A (en) * | 2014-01-23 | 2014-04-16 | 湖南大学 | Colloidal gold immunoassay kit for detecting aflatoxin B1 and preparation method of kit |
| CN103728451A (en) * | 2014-01-23 | 2014-04-16 | 湖南大学 | Colloidal gold immunoassay kit for detecting ochracin A and preparation method of kit |
| CN103728451B (en) * | 2014-01-23 | 2015-06-24 | 湖南大学 | Colloidal gold immunoassay kit for detecting ochracin A and preparation method of kit |
| CN103728452B (en) * | 2014-01-23 | 2015-06-24 | 湖南大学 | Colloidal gold immunoassay kit for detecting aflatoxin B1 and preparation method of kit |
| CN104372089A (en) * | 2014-11-11 | 2015-02-25 | 南京博纳天元生物科技有限公司 | Detection card for rapidly detecting micro ribonucleic acid (miRNA) as well as preparation method and application of detection card |
| CN108152514A (en) * | 2018-03-19 | 2018-06-12 | 南京博天科智生物技术有限公司 | A kind of neutrophil leucocyte gelatinase correlation apolipoprotein NGAL efficient nano Test papers |
| CN108918857A (en) * | 2018-05-11 | 2018-11-30 | 南京博天科智生物技术有限公司 | Nanogold cage compound and its application in the kit of the cMyBP-C in preparation detection human urine or blood |
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