CN105842464A - Device for jointly quantitatively detecting uNGAL (urinary Neutrophil Gelatinase Associated Lipocalin) and uCr (urinary Creatinine) based on up-converting phosphor technology and preparation method of device - Google Patents
Device for jointly quantitatively detecting uNGAL (urinary Neutrophil Gelatinase Associated Lipocalin) and uCr (urinary Creatinine) based on up-converting phosphor technology and preparation method of device Download PDFInfo
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Abstract
The invention discloses an immunochromatographic device for jointly quantitatively detecting urinary neutrophil gelatinase associated lipocalin (uNGAL) in a disease marker and urinary creatinine (uCr) serving as a dilution influence correcting factor for urine based on up-converting phosphor technology (UPT) and a preparation method of the device, and belongs to the technical field of immunodetection. The immunochromatographic device is prepared from sample diluent freeze-dried powder, NGAL (Neutrophil Gelatinase Associated Lipocalin) and Cr (Creatinine) standard substance freeze-dried powder and an immunochromatographic test paper strip, wherein the immunochromatographic test paper strip consists of a lining board, a sample pad or a UCP (Up-Converting Phosphor) conjugate pad, an analytical film and an absorbent pad; two parallel detecting lines T1 and T2 and a quality control line C are arranged on the analytical film; the two detecting lines T1 and T2 encrust an anti-NGAL antibody and an anti-Cr antibody respectively; the quality control line encrusts an antibody, which comes from animal immune globulin, of an anti-UCP labeled antibody; the test paper strip can quantify the uNGAL and the uCr by using a UCP readout instrument. The immunochromatographic device can be used for jointly quantifying the uNGAL and the uCr in different molecular forms, and has the advantages of quickness, simpleness, convenience and the like. After the uNGAL is corrected by the uCr, the capacity, for the early diagnosis of diseases of renal injury and the like, of the NGAL can be improved, and therefore, the immunochromatographic device has enormous scientific-research and clinical-application value.
Description
Technical field
The invention belongs to technical field of immunological detection, be specifically related to associating based on up-converting phosphor technology (UPT)
Detection by quantitative disease biomarkers urine neutrophilic granulocyte gelatinase relative carrier lipoprotein (uNGAL) and urine dilution shadow
Ring device for immunochromatography of correction factor urine creatine (uCr) and preparation method thereof.
Background technology
Neutrophil gelatinase-associated lipocalin NGAL (neutrophil gelatinase associated lipocalin,
NGAL) it it is 90 years also known as human neutrophil lipocalin protein (human neutrophil lipocalin, HNL)
A kind of glycoprotein that generation just finds in neutrophilic granulocyte the second granule, belongs to lipocalin protein (lipocalin) superfamily
Member, and other lipocalin proteins have similar spatial structure (J.Biol.Chem.1993,268:10425-10432；
Scand.J.Clin.Lab.Invest.1994,54:365-376).NGAL is formed with monomer, homodimer and with MMP9
The different kinds of molecules forms such as heterodimer exist, and different molecular form NGAL has difference and is correlated with from different pathological processes
Property.Wherein monomer NGAL and renal tubules acute injury have of a relatively high dependency, and homodimer NGAL can have
Effect reflection urinary tract infection and antibacterial infection conditions.In addition to neutrophilic granulocyte can secrete NGAL, NGAL in physiological conditions
People's Various Tissues have expression but expression is low；Under pathological conditions such as inflammation, infection, tumor, ischemia etc., NGAL
Can in Various Tissues up-regulated expression (Histochem.J.1999,31:433-441；Mol.Med.Rep.2012,
6:716-722)。
Over nearly more than 20 years, NGAL is subject to as the mark of the diseases such as acute bacterial infection, tumor, renal dysfunction
The concern of people.Particularly NGAL is increasingly heavier by people as the early diagnosis biomarker of renal dysfunction
Depending on.There is serum, blood plasma and the urine NGAL report for the early diagnosis of renal dysfunction at present.Adopt now with clinic
Serum creatinine (sCr) compare, NGAL shows excellent properties when diagnosing renal dysfunction in early days.Report
NGAL diagnoses in early days because of ischemia (J.Endourol.2013,27:1510-1515), antitumor drug cisplatin
(Am.J.Nephrol.2004,24:307-315), sepsis (Biomed.Res.Int.2015,2009,186:48-51；In
State's practicality medicine 2015,24:46-47), cardiac operation (Lancet 2005,365:1231-1238；
Clin.Chim.Acta.2009,403:121-125) etc. cause acute kidney injury (AKI) time, shift to an earlier date 1 than sCr
Whether AKI will occur to 2 days anticipation patients, this has won the quality time for clinical interventional therapy AKI.
Compared with serum and blood plasma NGAL, the biomarker that urine NGAL (uNGAL) is used as diagnosis AKI is outstanding
It attracts the attention of people, and main cause has following three points: first, animal model and Cell culture invitro research knot
Fruit shows renal cells up-regulated expression NGAL during acute injury of kidney and urine directly contacts with diseased region,
Therefore uNGAL can reflect the most rapidly injury of kidney situation；Second, urine sampling mode is Non-Invasive, easy
To the cooperation of patient, it it is the body fluid sample being easiest to obtain；3rd, current uroscopy is judge kidney disease one
Plant modal necessary inspection project.On the other hand, although uNGAL has it certainly as renal function biomarker
The advantage that body is unique, but in urine, the concentration of NGAL is caused by factors such as patient's drinking-water, intravenous drip infusion medicines
Urine dilution impact, thus highly effective reflection can not urinate the dynamic situation of change of NGAL concentration really, because of
This needs the impact on causing because of urine dilution to correct.The most clinically, uCr level is used for correcting urine
Dilute the impact that detectable substance concentration in urine is brought long-standing.Creatinine (Cr) is the metabolite of fosfocreatinine in muscle,
Body generally produces with constant speed；Additionally, creatinine is small-molecule substance, can pass freely through glomerular filtration and
With little or no heavily being absorbed in renal tubules.Therefore, the creatinine almost all that body every day produces with urine ejection, uCr's
Daily output is the most stable, basic unable to take food thing protein content and the impact of urine volume.So can be by measuring uCr water
Put down and correct the impact that uNGAL concentration is dynamically changed because of dilution by urine.
Although laboratory research at present and clinical practice have the method testing NGAL and uCr the most respectively, but do not have so far
There is the report at the same sample method and apparatus of joint-detection both marks simultaneously.And NGAL in future is likely
Become clinical instant floor inspection (Point of Care Testing, the POCT) project of AKI.Therefore, design and develop
UNGAL and uCr associating quantitative approach and equipment have huge laboratory science research and clinical practice demand in the future.
NGAL quantitative approach has RIA (international monopoly WO/1995/029404A1 and China ZL 200980155194
Deng), ELISA (European patent EP 2225268 and Chinese patent ZL200980155194 etc.), Western-blotting,
Latex immunoturbidimetry (Chinese patent ZL 201410431182), chemiluminescence immunoassay (Chinese Patent Application No.
201510184818) etc..Any of the above NGAL quantitative approach has respective advantage and purposes, but there is also some shortcomings.
From the point of view of NGAL detection demand, the most significant shortcoming is that the detection cycle is long, is difficult to meet and detects project for POCT
Requirement.Immunochromatography technique is a kind of fast diagnosis method, is usually used in POCT project.The NGAL of development in recent years
Immunochromatography technique (Chinese patent ZL201220323587, ZL201420423962, ZL201220392399,
ZL201220323586 and ZL201220497401) the detection time can be shortened.NGAL immunity layer disclosed above
Although analysis technology has easy to operate and detects the advantages such as quick, but the intrinsic shortcoming of various reporter molecules self limits
The performance of Related product.As fluorescence immune chromatography easily occurs cancellation because of fluorescent dye and cause product stability poor；Fluorescence
Latex particle forms because of its fluorescent dye system physical doping used, and is susceptible to leakage, and additionally such material easily occurs
Non-specific adsorption thus cause the low problem of signal to noise ratio；Immune colloidal gold technique uses the method for physical absorption to make label
Combine with reporter molecules, affect because the weak label of physical absorption power easily comes off from reporter molecules gold nano grain surface
Detection performance.In recent years, the up-conversion luminescent material constituted in the lattice of crystal it is doped in thulium
(up-converting phosphor, UCP) is the immunochromatography up-converting phosphor technology (up-converting of reporter molecules
Phosphor technology, UPT) it is a research and development emphasis and the focus of at present clinical POCT detection field.
UCP is made up of main matrix (host matrix), absorption (absorber) and transmitting son (emitter) three part, tool
There is upper forwarding optical phenomenon i.e. at infrared ray excited lower transmitting visible ray.The signal that UCP produces can be read by UCP readout instrument
Take.Owing to this kind of material is synthetic and exists at nature and internal not similar substance, therefore do without background
Disturb problem；Additionally, UCP also have good stability, without going out, the excellent properties such as environmental friendliness and good biocompatibility,
This material has been applied in multiple POCT detecting system as tracer.Chinese patent (ZL201220497401) is public
Having opened the upper forwarding light fast quantification device of a kind of NGAL detection, this technology segment overcomes current NGAL and detects skill
The deficiency of art, but this technology does not consider to affect NGAL quantitatively thus affect NGAL diagnosis because of urine dilution
The fact that of renal dysfunction ability；In addition the technology of the disclosure does not consider that different molecular form NGAL is in diagnosis
The difference of clinical manifestation during disease.Therefore ZL201220497401 is a kind of method measuring NGAL concentration, and
Not can determine that its uNGAL measured the most truly reflects the dynamic situation of change of uNGAL different with cannot distinguish between mensuration points
The NGAL of sub-form, thus limit uNGAL and as the ability of renal dysfunction mark and then limit the party
The clinical practice that method is following.
UCr quantitative approach mainly has based on Creatinine deiminase and the enzyme process of Creatininase, makees with picrate based on creatinine
With generate yellowish red color picric acid creatinine complex chemical assay (Jaffe method) and based on creatinine in weak acid environment
The positively charged high performance liquid chromatography (HPLC) etc. separated by cation-exchange chromatography post.Although enzyme process has high specificity
Sensitivity advantages of higher, but the activity of enzyme is affected by many factors thus affects stablizing of the method, and also cost is than it
His method is high.Chemical assay is with low cost, easy and simple to handle, be measure at present both at home and abroad creatinine most common method it
One.Due to materials such as vitamin C, some antibiotic such as penicillin, acetone, acetoacetic acid and high concentration glucoses
Also can react generation red product with alkaline picric acid, therefore the specificity of the method is the highest.High performance liquid chromatography (HPLC) is surveyed
The precision determining uCr is high, specificity is good, but this method requires that to instrument and equipment high and operation complexity is unsuitable for high-volume and faces
Bed analyzing specimen.Enzyme process and the chemical method of creatinine can meet the clinical independent mensuration to creatinine at present, and additionally creatinine is little
Molecular compound, prepares antibody relatively difficult；Therefore people lack exploitation creatinine based on antigen antibody reaction at present
Immunological assay method and the power of corresponding equipment.Along with antibody production techniques progress and development, by small haptens
Cr and carrier mass such as serum albumin, ovalbumin, keyhole azurin, carboxymethyl cellulose and poly-D-lysine etc.
Carrier molecule coupling has constituted the antigen-immunized animal corresponding anti-Cr antibody of acquisition and has become reality.Due to immunology detection skill
Art has the advantages such as high specificity, kind are many, sets up and develops Cr immunoassay technology and equipment is that this field following is ground
The focus studied carefully and develop.But it is open still without technical scheme related to the present invention so far.
Summary of the invention
In order to overcome the impact that uNGAL concentration is dynamically changed caused because of urine dilution and specific detection different
The difficulty of molecular forms uNGAL, the present invention designs and is developed based on the associating detection by quantitative of up-converting phosphor technology
Device of uNGAL and uCr and preparation method thereof, wherein uCr is for correcting the urine dilution shadow to uNGAL concentration
Ring.The present invention can be in the concentration of same sample synchronization simultaneous determination uNGAL and uCr, additionally can be quantitatively different
Molecular forms uNGAL, have that sample pre-treatments is simple, amount of samples is few, detection process conveniently and quickly, sensitivity
With specificity advantages of higher.In early diagnosis laboratory researches such as renal dysfunction and other diseases such as acute bacterial infections
Very big application potential is had with Clinical Laboratory field.
In order to realize above-mentioned target, the present invention adopts the following technical scheme that:
Associating detection by quantitative uNGAL based on up-converting phosphor technology and uCr device, by sample diluting liquid lyophilized powder,
NGAL and Cr standard substance lyophilized powder, immuno-chromatographic test paper strip form, and wherein immuno-chromatographic test paper strip is by liner plate, sample
Pad or UCP pad, containing two detection lines and the analyzing film of a nature controlling line and absorption pad composition.Described liner plate is used for
Supporting sample pad or the assembling of UCP pad, analyzing film and absorption pad, described UCP pad is used for combining UCP
Labelling anti-NGAL body and UCP labelling Cr, described analyzing film realizes for detecting line and the setting of nature controlling line
Quantitative and the Quality Control of immuno-chromatographic test paper strip of NGAL and Cr, described absorption pad sample solution after collecting and surveying.
1) described sample diluting liquid lyophilized powder
Preparation method is as follows: by 20mL, pH=7.2～7.5, containing 150～250mmol/L NaCl, 0.25～0.5% (vol/vol)
Tween-20,0.5%～2% (wt/vol) BSA or the 50～100mmol/L of 2.5%～5% (wt/vol) defatted milk powder
HEPES buffer after the stirring fully of mechanically or magnetically power, use 0.22 μm or 0.45 μm filter carry out filtration sterilization and
Remove insoluble impurities, lyophilised rear room temperature preservation.
Sample diluting liquid lyophilized powder adds acquisition sample diluting liquid after 20mL deionized water fully dissolves before using.
2) described NGAL and Cr standard substance lyophilized powder totally 4 set
A) standard substance A: be the standard substance measured for monomer uNGAL and uCr, often pipe monomer NGAL Han 60ng
With 6 μm ol Cr；Preparation method is that 60ng monomer NGAL and 6 μm ol Cr is dissolved in 300 μ L, pH=7.4 and is contained
5～10% trehalose (wt/vol) PBS solution in obtain containing 200ng/mL monomer NGAL and 20mmol/L Cr
Solution, use room temperature preservation after conventional method lyophilizing after filtration sterilization.
B) standard substance B: the standard substance measured for homodimer uNGAL and uCr, often pipe same dimerization Han 60ng
Body NGAL and 6 μm ol Cr；Preparation method be 60ng homodimer NGAL and 6 μm ol Cr is dissolved in 300 μ L,
PH=7.4 and containing 5～10% the PBS solution of trehalose (wt/vol) obtain containing 200ng/mL homodimer NGAL
With the solution of 20mmol/L Cr, after filtration sterilization, use room temperature preservation after conventional method lyophilizing.
C) standard substance C: the standard substance measured for different dimerization uNGAL and uCr, often pipe heterodimer Han 60ng
NGAL and 6 μm olCr；Preparation method is that 60ng heterodimer NGAL and 6 μm ol Cr is dissolved in 300 μ L, pH
=7.4 and containing 5～10% the PBS solution of trehalose (wt/vol) obtain containing 200ng/mL heterodimer NGAL
With the solution of 20mmol/L Cr, after filtration sterilization, use room temperature preservation after conventional method lyophilizing.
D) standard substance D: the standard substance measured for three kinds of molecular forms uNGAL and uCr, often pipe NGAL Han 60ng
(wherein monomer NGAL, homodimer NGAL and heterodimer NGAL respectively account for 40%, 40% and 20%) and 6 μm ol
Cr；Preparation method is by 24ng monomer NGAL, 24ng homodimer NGAL, 12ng heterodimer NGAL
With 6 μm ol Cr be dissolved in 300 μ L, pH=7.4 and containing 5～10% the PBS solution of trehalose (wt/vol) obtain
Containing 200ng/mL NGAL (wherein monomer NGAL, homodimer NGAL and heterodimer NGAL respectively account for 40%,
40% and 20%) and the solution of 20mmol/L Cr, room temperature preservation after conventional method lyophilizing is used after filtration sterilization.
Above standard substance add deionized water 300 μ L mixing and are prepared as standard substance working solution before using.Grind according to laboratory
Study carefully that different purposes with clinical research select to use in above 4 set standard substance one or more sets.Will be with subscript during use
Quasi-product working solution sample diluting liquid carries out 2 times of dilution methods and is diluted, for the formulation of standard curve.
NGAL and Cr standard substance use this area routine techniques preparation and/or by being either commercially available.Monomer NGAL
Standard substance utilize prokaryotic expression carrier and carrier for expression of eukaryon to obtain at corresponding host cell expression purification or are commercialization
NGAL(Biochem.Biophys.Res.Commun.1994,202:1468-1475；The ab188461 of Abcam).
Homodimer NGAL and heterodimer NGAL separates from human blood and obtains, specifically by blood buffy coat certainly
Obtaining granulocyte after so erythrocyte is removed in sedimentation, granulocyte crushes through nitrogen cavitation and obtains granulocyte granule, and then acid is split
Isolated and purified (Scand.J.Clin.Lab.Invest.1994,54:365-376) is carried out after solving granule.Cr (No. CAS
Being 60-27-5) commercial prod (such as C4255 or other company's similar clauses of Sigma company), purity should be greater than
Equal to 98%.
3) reporter molecules of described immuno-chromatographic test paper strip be UCP, UCP particle diameter be 200～600nm, UCP is respectively
It is marked at anti-NGAL or is marked on Cr.
The anti-NGAL of UCP labelling includes UCP labelling anti-monomer NGAL antibody, UCP labelling anti-homodimer NGAL
Antibody, UCP labelling anti-mm P-9 antibody or UCP labelling resist above-mentioned three kinds of molecular forms NGAL antibody.
The above-mentioned anti-NGAL of UCP labelling or labelling Cr is realized by covalent bond coupling, and coupling method is conventional public affairs
The technology opened.UCP forms UCP traget antibody (Anal. with antibody molecule by the coupling mode that this area is conventional
Biochem.2001,293:307-318；J.Clin.Micro.2008,46:171-187).UCP Yu Cr molecule passes through ability
The coupling mode formation UCP labelling Cr molecule that territory is conventional, as first carried out 3-aminopropyl-trimethoxy silicon by UCP
Then alkane (APTMS) modification uses difunctional connection molecule glutaraldehyde to make cross-linking agent and realizes Cr Yu UCP covalent bond.
Anti-for UCP labelling NGAL antibody and UCP labelling Cr are dissolved in containing 0.05% (vol/vol) Triton-100,
0.1% (wt/vol) NaN3, pH=8.0 50mmol/L glycine buffer in, the anti-NGAL's of UCP labelling is dense
Degree is 10mol/L for the concentration of 1mg/mL, UCP labelling Cr.
4) described immuno-chromatographic test paper strip divides A and B two class, every class quantity can set different size according to the actual requirements.
A) A para-immunity chromatograph test strip: be made up of liner plate, sample pad, analyzing film and absorption pad, further can be by
Cabinet encapsulates.A para-immunity chromatograph test strip optional equipment UCP labelling anti-NGAL antibody-solutions and UCP mark
(every 100 A para-immunity chromatograph test strips are equipped with the UCP labelling of 10 μ L and resist the mixed solution of note Cr solution composition
NGAL antibody-solutions and the mixed solution of UCP labelling Cr solution composition), by above-mentioned mixed solution sample before using
Diluted 200～500 times.
The using method of A para-immunity chromatograph test strip is as follows: first standard substance working solution sample diluting liquid is carried out 2
Times gradient dilution method is diluted, it is thus achieved that standard concentration gradient solution；Then by dilute for urine sample sample diluting liquid
Release 10～100 times；Take 25～100 μ L above-mentioned standard concentration gradient solutions or the multiple hole of urine sample diluent adds phase
In the hole of 96 orifice plates answered or in the middle of the container of similar structures；Then in above-mentioned 96 orifice plates, respectively add 25～100 μ L
Diluted UCP labelling anti-NGAL antibody-solutions and the mixed solution of UCP labelling Cr solution composition, mixing is made
Suspension；Then the sample pad end of A para-immunity chromatograph test strip is dipped vertically in above-mentioned suspension, waits 5～30min
UCP signals collecting is carried out after rear taking-up horizontal positioned 5～20min.
B) B para-immunity chromatograph test strip: be made up of liner plate, UCP pad, analyzing film and absorption pad, further may be used
To be encapsulated by cabinet.Containing UCP labelling anti-NGAL antibody and UCP labelling Cr in UCP pad, specifically
Preparation method is by 0.5mL/cm2Consumption by anti-for UCP labelling NGAL antibody-solutions and UCP labelling Cr solution
The mixed solution of composition uniformly drips on UCP pad, and 30 DEG C～35 DEG C prepare after drying.
The using method of B para-immunity chromatograph test strip is as follows: after immunity test strip horizontal positioned, adds 20～100 μ L
Standard concentration gradient solution or urine sample diluent on UCP pad, carry out after waiting 10～30min
UCP signals collecting.
Above A, B two para-immunity chromatograph test strip sample pad or UCP pad, analyzing film, absorption pad and liner plate
Width consistent, be 4～5.5mm, sample pad and a length of the 5 of UCP pad～10mm, analyzing film
A length of 25～40mm, a length of the 20 of absorption pad～30mm, the length of liner plate is equal to sample after front and back overlap joint assembles
The length summation of product pad or UCP pad, analyzing film and absorption pad；Wherein sample pad and the material of UCP pad
For glass fibre element film or the membrane material with similarity, the material of analyzing film is nitrocellulose filter or has similar
The membrane material of character, the material of absorption pad is absorbent filter or the material with similar functions, and the material of liner plate is for having
Self-adhesive character polyvinyl chloride plastic sheet or there is the material of similar functions.
The assembling mode of UCP immuno-chromatographic test paper strip uses ability convenient technical process to realize.
UCP be by this area routine techniques laboratory synthesize or the mean diameter of commercialization be 200～600
Nmol/L through silicon dioxide coated and surface-functionalized modification be made up of thulium crystalline material (as
NaYF4:Er, Yb).
5) described analyzing film is provided with parallel two detection line (T1 and T2) and a nature controlling line (C), and described two
Individual detection line T1 and T2 is coated anti-NGAL antibody and anti-Cr antibody respectively, and both can mutually exchange.UCP
The anti-NGAL antibody of the UCP labelling contained in pad and coated anti-NGAL antibody recognition NGAL of analyzing film
Different epi-positions, described anti-NGAL antibody for antigen behaviour source NGAL, described people source NGAL be monomer,
The mixture of homodimer, heterodimer or three, the uCr of described UCP labelling Cr and urine sample can be competitive
Anti-Cr antibodies coated with analyzing film, described nature controlling line is coated anti-UCP traget antibody source animal immune globulin
White antibody.Described test strips can carry out data acquisition with UCP readout instrument.
Further, detection line T1 is between sample pad or UCP pad end 5～10mm, T1, T2 and C
Be spaced apart 5～10mm；Wherein T1 is coated anti-NGAL antibody, and T2 is coated anti-Cr antibody or T1 is coated
Anti-Cr antibody and T2 is coated anti-NGAL antibody, nature controlling line C is coated anti-UCP traget antibody source animal immunity ball
The antibody of albumen；Method for coating is to utilize manual method or special point sample instrument (past from UCP pad on analyzing film
Absorption pad direction) even application anti-NGAL antibody-solutions, anti-Cr antibody-solutions and anti-UCP traget antibody come successively
The antibody of source animal immune globulin；Antibody-solutions be antibody is dissolved in antibodies buffer after obtain, antibody concentration is 1
Mg/mL, antibodies buffer is containing 1% (vol/vol) methanol, the 100mM Tris-HCL solution liquid of pH=8.0；
Final quantity for spray is 0.025～0.05mL/mm, is equivalent to every millimeter of T1, T2 or C wire spraying 25ng～50ng and resists
Body；Room temperature can preserve (at least 12 months, properties of product do not significantly change) by long-term room-temperature after drying.
Anti-NGAL antibody, anti-Cr antibody and nature controlling line antibody utilize this area routine techniques preparation or pass through business
Channel is bought and is obtained.Anti-NGAL antibody is the antibody of commercialization or prepares skill for antigen by conventional antibody with NGAL
Anti-NGAL antibody prepared by art (Current protocols in molecular biology.New York:John Wiley,
2008 11.10.1-11.10.10；Ab23477, ab70287, ab188551 etc. of Abcam company)；Prepared by above method
Or the antibody bought obtains for specific molecular form through monomer, homodimer or heterodimer NGAL antigen selection
The antibody of NGAL.The access approaches of anti-Cr antibody: by Cr hapten warp and carrier such as serum albumin, egg white egg
In vain, the carrier molecule such as keyhole azurin, carboxymethyl cellulose and poly-D-lysine has been consisted of routine techniques coupling
Antigen-immunized animal obtain corresponding anti-Cr antibody or through commercial channel buy (as Creative Diagnostics company or its
His company all has this series products to sell), anti-Cr antibody can be combined with the Cr of free Cr and UCP labelling.Nature controlling line
Coated antibody according to UCP traget antibody type decided, can be rabbit anti-mouse igg antibody or sheep anti-mouse igg antibody or
The dynamics of other animal origins or anti-rabbit IgG antibody etc., bought by commercial channel and obtain (such as abcam company
Corresponding antibodies product).
Owing to taking above technical scheme, the present invention is made to have notable beneficial effect.
Currently without joint-detection uNGAL and uCr technology, with immediate prior art (ZL201220497401)
Comparing, it is creative that the present invention has following two aspects: first, individually measures uNGAL based on because of urine dilution impact
Can not effectively reflect the dynamic mapping situation of this biomarker and measure the dynamically change of uNGAL is anticipation renal function
This basic theories of most important approach of damage and the fact, the introduction of the invention also realizes simultaneously at same sample
Simultaneous determination uNGAL and correction factor uCr of urine dilution, and ZL201220497401 simply discloses a kind of base
NGAL assay method in UPT does not relate to uCr；Second, the present invention can be used to measure whole molecular forms
UNGAL may also be used for measuring monomer or homodimer or heterodimer uNGAL, and ZL201220497401 is measured
The molecular forms of NGAL indefinite, and the molecular forms of different NGAL is raw as the diagnosis of different pathological with it
The clinical manifestation of thing mark has significant correlation (Clin.J.Am.Soc.Nephrol.2010,12:2229-2235).
Additionally, the present invention is also creative at following tripartite's mask: first, by double-antibody sandwich immunoassay technology and
Single antibody competition immunoassay technology is applied to same test system, thus realizes macro-molecular protein (NGAL)
Associating with micromolecular compound (Cr) is quantitative.Second, use determination of immunological methods Cr concentration, enrich at present
The assay method of Cr concentration.3rd, use UCP immunochromatography technique that the concentration of NGAL and Cr can be made to measure short
Complete in time (apparatus of the present invention can complete in 30～70min clocks test, and the ELISA of routine need 3～
5h), so that NGAL can be used for clinical POCT project detection.
To sum up, the present invention do not exist the motivation that prior art is improved and claimed technical scheme not aobvious and
It is clear to, and claimed technical scheme can produce significant beneficial effect, be therefore one to there is novelty
With creative innovation and creation.
Accompanying drawing explanation
Fig. 1 is that in present invention associating based on up-converting phosphor technology detection by quantitative uNGAL and uCr device, A class is exempted from
The generalized section of epidemic disease chromatograph test strip.Wherein 1 sample pad, 2 be analyzing film, 3 for absorption pad, 4 for liner plate, 5
For detection line T1 and T2, (wherein T1 is coated anti-NGAL antibody and T2 is coated anti-Cr antibody, or T1 bag with 6
By anti-Cr antibody, T2 is coated anti-NGAL antibody), 7 (be coated anti-UCP traget antibody source dynamic for nature controlling line C
The antibody of thing immunoglobulin, determines according to the kind of UCP traget antibody).
Fig. 2 is that in present invention associating based on up-converting phosphor technology detection by quantitative NGAL and urine creatine device, B class is exempted from
The generalized section of epidemic disease chromatograph test strip.Wherein 21 is that UCP pad (is coated antibody and the UCP of UCP labelling
The Cr of labelling, UCP traget antibody can be UCP labelling anti-NGAL antibody or UCP is marked at anti-mm P9 antibody,
UCP labelling anti-NGAL antibody can be the anti-monomer NGAL antibody of UCP labelling, the anti-homodimer of UCP labelling
The antibody of anti-above-mentioned three kinds of molecular forms NGAL of NGAL antibody or UCP labelling), 2 be analyzing film, 3 for inhale
Receive pad, 4 for liner plate, 5 and 6 for detection line T1 and T2 (wherein T1 is coated anti-NGAL antibody, and T2 is coated
Anti-Cr antibody or T1 are coated anti-Cr antibody and T2 is coated anti-NGAL antibody), 7 (be coated anti-for nature controlling line C
The antibody of UCP traget antibody source animal immunoglobulin, determines according to the kind of UCP traget antibody).
Fig. 3 is that in present invention associating based on up-converting phosphor technology detection by quantitative uNGAL and uCr device, A class is exempted from
The test strips rack schematic diagram that epidemic disease chromatograph test strip is supporting.Rack is hollow a, cuboid for bottom surface disappearance, long
With the wide size (such as a length of 130mm, a width of 87mm) being slightly larger in dimension than standard 96 orifice plate, height be 40mm～
50mm；Material is plastics or stationery；Wherein 31 is that rack end face has the slot corresponding with 96 orifice plates on it
(size of slot is to be as the criterion slightly larger than the width of A para-immunity chromatograph test strip and thickness), 32 is rack side
Face, 33 is before rack, and 34 is the test strips putting hole of rack end face.
Fig. 4 is embodiment 3 preparation based on up-converting phosphor technology associating detection by quantitative total uNGAL and uCr device
And the standard curve of using method.The left figure of Fig. 4 is average according to table 1uNGAL peak area AUC1 and AUC2
The standard curve that value is drawn with total NGAL standard concentration, the calculating of total uNGAL in urine sample；Fig. 4
Right figure is the standard curve that the meansigma methods according to table 2uCr peak area AUC1 and AUC2 is drawn with Cr standard concentration,
The calculating of uCr in urine sample.
Fig. 5 is embodiment 4 associating based on up-converting phosphor technology detection by quantitative uNGAL and uCr device combines inspection
Survey monomer uNGAL and the standard curve of uCr concentration.The left figure of Fig. 5 be according to table 2uNGAL peak area AUC1 and
The standard curve that the meansigma methods of AUC2 is drawn with monomer NGAL standard concentration, monomer in urine sample
The calculating of uNGAL；The right figure of Fig. 5 is the meansigma methods according to table 2uCr peak area AUC1 and AUC2 and Cr standard
The standard curve that product concentration is drawn, the calculating of uCr in urine sample.
Fig. 6 is embodiment 5 associating based on up-converting phosphor technology detection by quantitative uNGAL and uCr device combines inspection
Survey heterodimer uNGAL and the canonical plotting of uCr concentration.The left figure of Fig. 6 is according to table 3uNGAL peak area
The standard curve that the meansigma methods of AUC1 and AUC2 is drawn with heterodimer NGAL standard concentration, for urine sample
The calculating of heterodimer uNGAL in product；The right figure of Fig. 6 is putting down according to table 4uCr peak area AUC1 and AUC2
The standard curve that average is drawn with Cr standard concentration, the calculating of uCr in urine sample.
Detailed description of the invention
The present invention will be further illustrated below by embodiment.The embodiment of the present invention is for being explained further of the present invention
It it not limitation of the invention.The concrete improvement that essence according to the present invention is carried out broadly falls into claimed model
Enclose.
Embodiment 1: based on up-converting phosphor technology associating detection by quantitative total uNGAL and uCr device composition
1) sample diluting liquid lyophilized powder 1 part (reagent I)
2) standard substance solution D lyophilized powder 1 manages (reagent II)
3) UCP labelling resists three kinds of molecular forms NGAL antibody and UCP labelling Cr solution 1 to manage (reagent III)
4) A para-immunity chromatograph test strip 96
5) A para-immunity chromatograph test strip supporting test strips rack 1
6) 96 orifice plate 1 piece, 96 orifice plate shrouding films one
Embodiment 2: based on the up-converting phosphor technology associating total NGAL of detection by quantitative and urine creatine device composition
1) sample diluting liquid lyophilized powder 1 part (reagent I)
2) standard substance solution D lyophilized powder 1 manages (reagent II)
3) B para-immunity chromatograph test strip 100
Embodiment 3: a kind of based on the up-converting phosphor technology associating preparation of detection by quantitative total uNGAL and uCr device and make
Use method
1) preparation of device
A) sample diluting liquid lyophilized powder (reagent I) preparation
Sample diluting liquid 20mL, by 100mmol/L HEPES, pH 7.2,200mmol/L NaCl,
The BSA composition of 0.25% (vol/vol) Tween-20,0.5% (wt/vol)；After magnetic agitation is abundant, adopt
Lyophilizing after carrying out filtration sterilization with 0.45 μm filter and remove insoluble impurities；Room temperature preservation.
B) standard substance solution D lyophilized powder (reagent II) preparation
By 300 μ L, containing 200ng/mL NGAL, (monomer, homodimer and heterodimer NGAL respectively account for
40%, 40% and 20%), the 10mmol/L of the pH=7.4 of 20mmol/L Cr and 10% trehalose (wt/vol)
PBS solution, after magnetic agitation is abundant, uses 0.22 μm filter carry out filtration sterilization and remove insoluble miscellaneous
Lyophilizing after matter；Room temperature preservation.
C) UCP labelling resists three kinds of molecular forms NGAL antibody and UCP labelling Cr solution 1 to manage (reagent III) preparation
UCP labelling resists three kinds of molecular forms NGAL antibody and UCP labelling Cr solution 10 μ L, solution group
Become 50mM glycine, 0.05%Triton-100,0.1%NaN3, 1mg/mL UCP labelling rabbit resist
Three kinds of molecular forms NGAL antibody (22264-1-AP of Proteintech company) and 10mol/mL UCP
The aqueous solution of labelling Cr；4 DEG C of preservations.
D) A para-immunity chromatograph test strip 96 preparation
A para-immunity chromatograph test strip is made up of liner plate, sample pad, analyzing film and absorption pad；Sample pad material
For glass fibre element film, analyzing film material is nitrocellulose filter, and absorbent pad material is absorbent filter, liner plate
Material for having Self-adhesive character polyvinyl chloride plastic sheet.The width of liner plate, sample pad, analyzing film and absorption pad
Degree is 4.5mm, and length is respectively 56mm, 10mm, 30mm and 20mm.T1 on analyzing film,
T2 and C line spray respectively the goat-anti NGAL antibody-solutions containing 100ng antibody (Abcam company
Ab166677), goat-anti Cr antibody (ab30719 of Abcam company) solution and goat anti-rabbit igg antibody (Abcam
The ab97091 of company) solution.Sample pad end is mounted on analyzing film, and both coincidence part bit lengths are 2mm,
Analyzing film rear end is mounted under absorption pad, and both coincidence part bit lengths are 2mm；Sample pad, analyzing film and suction
Receive pad to be assembled in successively on liner plate, room temperature preservation.
Above-mentioned goat-anti NGAL antibody-solutions used, goat-anti Cr antibody-solutions and goat anti-rabbit igg antibody solution
Prepare as follows: anti-NGAL antibody, anti-Cr antibody or dynamics are dissolved in pH8.0 respectively
100mmol/L Tris-HCl, mass fraction be 1% methanol solution in, the solubility of antibody is 1mg/mL.
2) device uses step
A) reagent I working solution is prepared: adding 20mL deionized water in reagent I container, vortex concussion mixing is standby.
B) 8 1.5mL centrifuge tubes, the number of putting on (such as S0, S1, S2, S3, S4, S5, S6 and S7) are taken.
C) the reagent I working solution 150 μ L that in above S0～S7 centrifuge tube prepared by each addition step a).
D) add 300 μ L deionized waters and obtain reagent II solution to reagent II container, all take out after mixing and add S0
Pipe；Then take out 150 μ L from S0 and add S1 pipe；Take out 150 μ L from S1 pipe after mixing and add S2 pipe；
Take out 150 μ L from S2 pipe after mixing and add S3 pipe；Take out 150 μ L from S3 pipe after mixing and add S4 pipe；
Take out 150 μ L from S4 pipe after mixing and add S5 pipe；Take out 150 μ L from S5 pipe after mixing and add S6 pipe；
Mixing S6 pipe.
E) sample solution after preparation dilution: utilize reagent I after clinical urine (10000rpm, 1min) by centrifugation
Working solution makees 10～100 multiple dilutions, whether is centrifuged and should determine according to actual urine sample quality.
F) UCP traget antibody working solution is prepared
4990 μ L reagent I working solutions are added in reagent III container, and water bath sonicator (20kHz, 320W, every time
Ultrasonic 15s, stops 15s, 3 times altogether；Or similar ultrasound intensity).
G) sample solution after every hole of 96 orifice plates adds 50 μ L S0-S7 solution or 50 μ L dilution；The most again toward every
Hole adds 50 μ L UCP traget antibody working solutions.
H) it is fixed on after covering shrouding film on 96 plate shaker, 800rpm, hatches 30min at 37 DEG C.
I) remove shrouding film, 96 orifice plates are placed under immuno-chromatographic test paper strip rack.
J) in every hole, it is sequentially inserted into A para-immunity chromatograph test strip, waits 20min.
K) take out A para-immunity chromatograph test strip on UCP readout instrument, carry out data acquisition
L) reference standard curve is drawn
Table 1 is the present embodiment NGAL and Cr standard solution (S0～S6) and placebo solution (S7) are different
The peak area (AUC) that concentration is corresponding.Wherein AUC1 and AUC2 refers to a Concentraton gradient standard solution or blank
The AUC reading in the multiple hole (parallel hole) of two of contrast solution.
Table 1: UCP peak area (AUC) reading corresponding for variable concentrations standard substance NGAL with Cr
The left figure of Fig. 4 is meansigma methods and the NGAL standard substance of uNGAL peak area AUC1 and AUC2 according to table 1
The standard curve that concentration is drawn；The right figure of Fig. 4 be uCr peak area AUC1 and AUC2 according to table 1 meansigma methods with
The standard curve that Cr standard concentration is drawn.
Embodiment 4: combine detection by quantitative monomer uNGAL and the preparation of uCr device and use based on up-converting phosphor technology
Method
1) preparation of device
A) sample diluting liquid lyophilized powder (reagent I) preparation
Sample diluting liquid 20mL, by 100mmol/L HEPES, pH 7.2,200mmol/L NaCl,
The BSA composition of 0.25% (vol/vol) Tween-20,0.5% (wt/vol)；After magnetic agitation is abundant, adopt
Lyophilizing after carrying out filtration sterilization with 0.45 μm filter and remove insoluble impurities；Room temperature preservation.
B) standard substance solution A lyophilized powder (reagent II) preparation
By 300 μ L containing 200ng/mL monomer NGAL and 20mmol/L Cr 20mmol/L and 10% sea
The PBS solution of the 10mmol/L of the pH=7.4 of algae sugar (wt/vol), after magnetic agitation is abundant, uses
0.22 μm filter carries out filtration sterilization and removes lyophilizing after insoluble impurities；Room temperature preservation.
C) UCP labelling anti-monomer NGAL antibody and UCP labelling Cr solution 1 manage (reagent III) preparation
UCP labelling anti-monomer NGAL antibody and UCP labelling Cr solution 10 μ L, this solution composition is
50mM glycine, 0.05%Triton-100,0.1%NaN3, the mouse-anti monomer of 1mg/mL UCP labelling
NGAL monoclonal antibody No. 009 product of BW of company (Bioporto diagnostocsA/S) and 10mol/mL
The aqueous solution of the Cr of UCP labelling；4 DEG C of preservations.
D) A para-immunity chromatograph test strip 96 preparation
A para-immunity chromatograph test strip is made up of liner plate, sample pad, analyzing film and absorption pad；Sample pad material
For glass fibre element film, analyzing film material is nitrocellulose filter, and absorbent pad material is absorbent filter, liner plate
Material be to there is Self-adhesive character polyvinyl chloride plastic sheet or there is the material of similar functions.Liner plate, sample pad,
The width of analyzing film and absorption pad is 4.5mm, and length is respectively 56mm, 10mm, 30mm and 20mm.
On every analyzing film, T1, T2 and C line sprays the goat-anti NGAL antibody-solutions containing 100ng antibody respectively
(ab166677 of Abcam company), goat-anti Cr antibody (ab30719 of Abcam company) solution and sheep
Dynamics (ab6710 of Abcam company) solution.Sample pad end is mounted on analyzing film, and two
Person's coincidence part bit length is 2mm, and analyzing film rear end is mounted under absorption pad, and both are at coincidence part bit length
2mm；Sample pad, analyzing film and absorption pad are assembled on liner plate successively, room temperature preservation.
Above-mentioned goat-anti NGAL antibody-solutions used, goat-anti Cr antibody-solutions and rabbit anti-mouse igg antibody-solutions
Prepare as follows: by the most molten to goat-anti NGAL antibody, goat-anti Cr antibody or rabbit anti-mouse igg antibody
Solution in the methanol solution that the 100mmol/L Tris-HCl of pH=8.0, mass fraction are 1%, antibody dense
Degree is 1mg/mL.
2) device uses step
A) reagent I working solution is prepared: adding 20mL deionized water in reagent I container, vortex concussion mixing is standby.
B) 8 1.5mL centrifuge tubes, the number of putting on (such as S0, S1, S2, S3, S4, S5, S6 and S7) are taken.
C) the reagent I working solution 150 μ L that in above S0～S7 centrifuge tube prepared by each addition step a).
D) add 300 μ L deionized waters and obtain reagent II solution to reagent II container, all take out after mixing and add S0
Pipe；Then take out 150 μ L from S0 and add S1 pipe；Take out 150 μ L from S1 pipe after mixing and add S2 pipe；
Take out 150 μ L from S2 pipe after mixing and add S3 pipe；Take out 150 μ L from S3 pipe after mixing and add S4 pipe；
Take out 150 μ L from S4 pipe after mixing and add S5 pipe；Take out 150 μ L from S5 pipe after mixing and add S6 pipe；
Mixing S6 pipe.
E) sample solution after preparation dilution: utilize reagent I after clinical urine (10000rpm, 1min) by centrifugation
Working solution makees 10～100 multiple dilutions, whether is centrifuged and should determine according to actual urine sample quality.
F) UCP traget antibody working solution is prepared
4990 μ L reagent I working solutions are added in reagent III container, water bath sonicator (20kHz, 320W,
Each ultrasonic 15s, stops 15s, 3 times altogether；Or similar ultrasound intensity).
G) sample solution after every hole of 96 orifice plates adds 50 μ L S0-S7 solution or 50 μ L dilution；The most past
Every hole adds 50 μ L UCP traget antibody working solutions.
H) it is fixed on after covering shrouding film on 96 plate shaker, 800rpm, hatches 30min at 37 DEG C.
I) remove shrouding film, 96 orifice plates are placed under immuno-chromatographic test paper strip rack.
J) in every hole, it is sequentially inserted into A para-immunity chromatograph test strip, waits 20min.
K) take out A para-immunity chromatograph test strip on UCP readout instrument, carry out data acquisition.
L) reference standard curve is drawn.
Table 2 is the present embodiment NGAL and Cr standard solution (S0～S6) and placebo solution (S7) is the denseest
The peak area (AUC) that degree is corresponding.Wherein AUC1 and AUC2 refers to a Concentraton gradient standard solution or blank right
AUC reading according to two multiple holes (parallel hole) of solution.
Table 2: UCP peak area (AUC) reading corresponding for variable concentrations standard substance monomer NGAL with Cr
The left figure of Fig. 5 is that the meansigma methods of uNGAL peak area AUC1 and AUC2 according to table 2 is marked with monomer NGAL
The standard curve that quasi-product concentration is drawn；The right figure of Fig. 5 is the average of uCr peak area AUC1 and AUC2 according to table 2
The standard curve that value is drawn with Cr standard concentration.
Embodiment 5: based on up-converting phosphor technology associating detection by quantitative heterodimer uNGAL and the preparation of uCr agent box and
Using method
1) preparation of device
A) sample diluting liquid lyophilized powder (reagent I) preparation
Sample diluting liquid 20mL, by 100mmol/L HEPES, pH 7.2,200mmol/L NaCl,
The BSA composition of 0.25% (vol/vol) Tween-20,0.5% (wt/vol)；After magnetic agitation is abundant, adopt
Lyophilizing after carrying out filtration sterilization with 0.45 μm filter and remove insoluble impurities；Room temperature preservation.
B) standard substance C solution lyophilized powder (reagent II) preparation
By 300 μ L containing 200ng/mL heterodimer NGAL and 20mmol/L Cr, 20mmol/L and 10%
The PBS solution of the 10mmol/L of the pH=7.4 of trehalose (wt/vol), after magnetic agitation is abundant, uses
0.22 μm filter carries out filtration sterilization and removes lyophilizing after insoluble impurities；Room temperature preservation.
E) prepared by B para-immunity chromatograph test strip 96 (test strips used by this embodiment has cabinet)
B para-immunity chromatograph test strip liner plate, UCP pad, analyzing film and absorption pad composition；UCP combines
Cushion material is glass fibre element film, and analyzing film material is nitrocellulose filter, and absorbent pad material is absorbent filter,
The material of liner plate is to have Self-adhesive character polyvinyl chloride plastic sheet or have the material of similar functions.Liner plate, UCP
The width of pad, analyzing film and absorption pad is 4mm, length be respectively 56mm, 10mm, 10
Mm, 30mm and 20mm.Pad contains rabbit anti-mm P9 antibody (the Abcam public affairs of UCP labelling
The ab38898 of department) and the Cr of UCP labelling；Concrete preparation method is by 0.5mL/cm2Amount by concentration
UCP labelling for the rabbit anti-mm P-9 antibody-solutions of UCP labelling of 1mg/mL and 10mol/L
Cr solution is uniformly added on UCP pad, and 35 DEG C prepare after drying.T1, T2 and C line on every analyzing film
Respectively spraying containing 100ng antibody goat-anti NGAL antibody (ab166677 of Abcam company) solution,
(Abcam is public for the solution of goat-anti Cr antibody (ab30719 of Abcam company) and goat anti-rabbit igg antibody
The ab97091 of department) solution.UCP pad is mounted on analyzing film, and both coincidence part bit lengths are 2mm；
Analyzing film rear end is mounted under absorption pad, and both coincidence part bit lengths are 2mm.Sample pad, analyzing film and
Absorption pad is assembled on liner plate successively.Room temperature preservation.
Above-mentioned goat-anti NGAL antibody-solutions used, goat-anti Cr antibody-solutions and goat anti-rabbit igg antibody solution
Prepare as follows: by the most molten to goat-anti NGAL antibody, goat-anti Cr antibody or sheep anti-mouse igg antibody
Solution in the methanol solution that the 100mmol/L Tris-HCl of pH=8.0, mass fraction are 1%, antibody molten
Degree is 1mg/mL.
2) device uses step
A) reagent I working solution is prepared: adding 20mL deionized water in reagent I container, vortex concussion mixing is standby.
B) 8 1.5mL centrifuge tubes, the number of putting on (such as S0, S1, S2, S3, S4, S5, S6 and S7) are taken.
C) the reagent I working solution 150 μ L that in above S0～S7 centrifuge tube prepared by each addition step a).
D) add 300 μ L deionized waters and obtain reagent II solution to reagent II container, all take out after mixing and add S0
Pipe；Then take out 150 μ L from S0 and add S1 pipe；Take out 150 μ L from S1 pipe after mixing and add S2 pipe；
Take out 150 μ L from S2 pipe after mixing and add S3 pipe；Take out 150 μ L from S3 pipe after mixing and add S4 pipe；
Take out 150 μ L from S4 pipe after mixing and add S5 pipe；Take out 150 μ L from S5 pipe after mixing and add S6 pipe；
Mixing S6 pipe.
E) sample solution after preparation dilution: utilize reagent I after clinical urine (10000rpm, 1min) by centrifugation
Working solution makees 10～100 multiple dilutions, whether is centrifuged and should determine according to actual urine sample quality.
F) by B para-immunity test strips horizontal positioned on the table, UCP pad adds 100 μ L S0-S7 molten
Liquid or the sample solution after dilution, wait 20min；
G) on UCP readout instrument, data acquisition is carried out；
H) reference standard curve is drawn.
Table 2 is the present embodiment NGAL and Cr standard solution (S0～S6) and placebo solution (S7) are different
The peak area (AUC) that concentration is corresponding.Wherein AUC1 and AUC2 refers to a Concentraton gradient standard solution or blank
The AUC reading in the multiple hole (parallel hole) of two of contrast solution.
Table 3: UCP peak area (AUC) reading corresponding for variable concentrations standard substance heterodimer NGAL with Cr
The left figure of Fig. 6 is meansigma methods and the heterodimer NGAL of uNGAL peak area AUC1 and AUC2 according to table 3
The standard curve that standard concentration is drawn；The right figure of Fig. 6 is the flat of uCr peak area AUC1 and AUC2 according to table 3
The standard curve that average is drawn with Cr standard concentration.
Claims (8)
1. an associating detection by quantitative uNGAL based on up-converting phosphor technology and uCr device, it is characterised in that: by sample
Diluent lyophilized powder, NGAL and Cr standard substance lyophilized powder, immuno-chromatographic test paper strip form；
(1) sample diluting liquid lyophilized powder is by 20mL, pH=7.2～7.5, containing 150～250mmol/L NaCl, 0.25～0.5%
(vol/vol) Tween-20,0.5%～2% (wt/vol) BSA or 2.5%～5% (wt/vol) defatted milk powder
50～100mmol/L HEPES buffer, after the stirring fully of mechanically or magnetically power, use 0.22 μm or 0.45 μm filter
Carry out filtration sterilization and remove insoluble impurities, lyophilised after obtain；
(2) NGAL and Cr standard substance lyophilized powder totally 4 set, wherein,
Standard substance A: be the standard substance measured for monomer uNGAL and uCr, often pipe monomer NGAL Han 60ng
With 6 μm ol Cr；
Standard substance B: the standard substance measured for homodimer uNGAL and uCr, often pipe homodimer Han 60ng
NGAL and 6 μm ol Cr；
Standard substance C: the standard substance measured for different dimerization uNGAL and uCr, often pipe heterodimer NGAL Han 60ng
With 6 μm olCr；
Standard substance D: the standard substance measured for three kinds of molecular forms uNGAL and uCr, often pipe NGAL Han 60ng
With 6 μm ol Cr, wherein monomer NGAL, homodimer NGAL and heterodimer NGAL respectively account for 40%, 40%
With 20%；
(3) described immuno-chromatographic test paper strip divides A and B two class, A para-immunity chromatograph test strip by liner plate, sample pad, point
Analysis film and absorption pad composition；B para-immunity chromatograph test strip is by liner plate, UCP pad, analyzing film and absorption pad group
Become；Containing UCP labelling anti-NGAL antibody and UCP labelling Cr in UCP pad；
(4) analyzing film is provided with parallel two detection line T1, a T2 and nature controlling line C, two detections line T1, T2
It is coated anti-NGAL antibody and anti-Cr antibody respectively, and both can mutually exchange；Nature controlling line is coated anti-UCP mark
The antibody of note antibody sources animal immune globulin；
Described liner plate is for supporting sample pad or the assembling of UCP pad, analyzing film and absorption pad, and described UCP ties
Closing pad to be used for combining UCP labelling anti-NGAL body and UCP labelling Cr, described analyzing film is used for detecting line and Quality Control
Line arrange thus realize NGAL and Cr quantitatively and the Quality Control of immuno-chromatographic test paper strip, described absorption pad is used for receiving
Sample solution after set analysis.
A kind of associating detection by quantitative uNGAL based on up-converting phosphor technology and uCr device,
It is characterized in that: A, B two para-immunity chromatograph test strip sample pad or UCP pad, analyzing film, absorption pad and
The width of liner plate is consistent, is 4～5.5mm, and sample pad and a length of the 5 of UCP pad～10mm analyze
A length of the 25 of film～40mm, a length of the 20 of absorption pad～30mm, the length of liner plate is equal to front and back overlapping assembling
The length summation of rear sample pad or UCP pad, analyzing film and absorption pad.
A kind of associating detection by quantitative uNGAL based on up-converting phosphor technology and uCr device,
It is characterized in that: wherein the material of sample pad and UCP pad is glass fibre element film or the film with similarity
Material, the material of analyzing film is nitrocellulose filter or the membrane material with similarity, and the material of absorption pad is water suction
Filter paper or have the material of similar functions, the material of liner plate is for having Self-adhesive character polyvinyl chloride plastic sheet or having similar
The material of function.
A kind of associating detection by quantitative uNGAL based on up-converting phosphor technology and uCr device,
It is characterized in that: the use of A para-immunity chromatograph test strip is that standard substance working solution sample diluting liquid is carried out 2 times of ladders
Degree dilution method is diluted, it is thus achieved that standard concentration gradient solution；By urine sample with sample diluting liquid dilution 10～
100 times；Take 25～100 μ L above-mentioned standard concentration gradient solutions or urine sample diluent is answered hole respectively and added corresponding
96 orifice plates hole in or the container of similar structures in the middle of, then in above-mentioned 96 orifice plates respectively add 25～100 μ L
Diluted UCP labelling anti-NGAL antibody-solutions and the mixed solution of UCP labelling Cr solution composition, mixing system
Become suspension；Then the sample pad end of A para-immunity chromatograph test strip is dipped vertically in above-mentioned suspension, waits 5～30min
UCP signals collecting is carried out after rear taking-up horizontal positioned 5～20min.
A kind of associating detection by quantitative uNGAL based on up-converting phosphor technology and uCr device,
It is characterized in that: the use of B para-immunity chromatograph test strip is that standard substance working solution sample diluting liquid is carried out 2 times of ladders
Degree dilution method is diluted, it is thus achieved that standard concentration gradient solution；Then urine sample sample diluting liquid is diluted
10～100 times；Then by after B para-immunity chromatograph test strip horizontal positioned, add 20～the standard concentration of 100 μ L ladder
Degree solution or urine sample diluent, on UCP pad, carry out UCP signals collecting after waiting 10～30min.
A kind of associating detection by quantitative uNGAL based on up-converting phosphor technology and uCr device,
It is characterized in that: UCP be mean diameter be 200～600nmol/L repair through silicon dioxide coated and surface-functionalized
The crystalline material being made up of thulium of decorations.
A kind of associating detection by quantitative uNGAL based on up-converting phosphor technology and uCr device,
It is characterized in that: UCP labelling anti-NGAL antibody includes UCP labelling anti-monomer NGAL antibody, UCP labelling
Anti-homodimer NGAL antibody, UCP labelling anti-mm P-9 antibody or UCP labelling resist above-mentioned three kinds of molecular forms
NGAL antibody.
8. a kind of based on up-converting phosphor technology the associating detection by quantitative uNGAL described in claim 1 and the system of uCr device
Preparation Method, its step is as follows:
1) preparation of described sample diluting liquid lyophilized powder, is by 20mL, pH=7.2～7.5, containing 150～250mmol/L
NaCl, 0.25～0.5% (vol/vol) Tween-20,0.5%～2% (wt/vol) BSA or 2.5%～5% (wt/vol)
50～100mmol/L HEPES buffer of defatted milk powder, after the stirring fully of mechanically or magnetically power, use
0.22 μm or 0.45 μm filter carry out filtration sterilization and remove insoluble impurities, lyophilised rear room temperature preservation；
Sample diluting liquid lyophilized powder adds acquisition sample diluting liquid after 20mL deionized water fully dissolves before using；
2) preparation of described NGAL and Cr standard substance lyophilized powder, standard substance A be by 60ng monomer NGAL and
6 μm ol Cr be dissolved in 300 μ L, pH=7.4 and containing 5～10% trehalose (wt/vol) PBS solution in
Arrive containing 200ng/mL monomer NGAL and the solution of 20mmol/L Cr, after filtration sterilization use routine side
Room temperature preservation after method lyophilizing；Standard substance B is to be dissolved in by 60ng homodimer NGAL and 6 μm ol Cr
300 μ L, pH=7.4 and containing 5～10% the PBS solution of trehalose (wt/vol) obtain containing 200ng/mL
Homodimer NGAL and the solution of 20mmol/L Cr, use room temperature after conventional method lyophilizing after filtration sterilization
Preserve；Standard substance C is that 60ng heterodimer NGAL and 6 μm ol Cr is dissolved in 300 μ L, pH=7.4
And containing 5～10% the PBS solution of trehalose (wt/vol) obtain containing 200ng/mL heterodimer NGAL
With the solution of 20mmol/L Cr, after filtration sterilization, use room temperature preservation after conventional method lyophilizing；Standard substance D
It is by 24ng monomer NGAL, 24ng homodimer NGAL, 12ng heterodimer NGAL and 6 μm ol
Cr is dissolved in 300 μ L, pH=7.4 and containing 5～10% PBS solution of trehalose (wt/vol) containing of obtaining
200ng/mL NGAL and the solution of 20mmol/L Cr, use room temperature after conventional method lyophilizing after filtration sterilization
Preserve；Above standard substance lyophilized powder adds deionized water 300 μ L mixing and is prepared as standard substance working solution before using；
3) UCP labelling anti-NGAL antibodies Antibodies and UCP labelling Cr, concrete preparation method is by 0.5mL/cm2's
The mixed solution of anti-for UCP labelling NGAL antibody-solutions and UCP labelling Cr solution composition is uniformly dripped by consumption
Being added on UCP pad, 30 DEG C～35 DEG C prepare after drying；
4) method for coating of T1, T2 and C is to utilize manual method or special point sample instrument the most uniformly to spray on analyzing film
It is coated with anti-NGAL antibody-solutions, anti-Cr antibody-solutions and anti-UCP traget antibody source animal immunoglobulin
Antibody；Antibody-solutions be antibody is dissolved in antibodies buffer after obtain, antibody concentration is 1mg/mL, anti-
Body buffer is containing 1% (vol/vol) methanol, the 100mM Tris-HCL solution liquid of pH=8.0；Finally
Quantity for spray is 0.025～0.05mL/mm, is equivalent to every millimeter of T1, T2 or C wire spraying 25ng～50ng
Antibody；
5) liner plate, sample pad or UCP pad, analyzing film and absorption pad stick on liner plate after mutually overlapping.
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