CN105842464B - Joint based on up-converting phosphor technology quantitatively detects uNGAL and uCr device and preparation method thereof - Google Patents

Joint based on up-converting phosphor technology quantitatively detects uNGAL and uCr device and preparation method thereof Download PDF

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CN105842464B
CN105842464B CN201610404263.8A CN201610404263A CN105842464B CN 105842464 B CN105842464 B CN 105842464B CN 201610404263 A CN201610404263 A CN 201610404263A CN 105842464 B CN105842464 B CN 105842464B
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蔡林君
陈雷
杨津
赵冰
姜春来
孔维
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Jilin University
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Abstract

Joint of the one kind based on up-converting phosphor technology (UPT) quantitatively detects device for immunochromatography of the related apolipoprotein (uNGAL) of disease marker urine neutrophil leucocyte gelatinase and urine dilution influence correction factor urine creatinine (uCr) and preparation method thereof, belongs to technical field of immunoassay.It is made up of sample diluting liquid freeze-dried powder, NGAL and Cr standard items freeze-dried powder, immuno-chromatographic test paper strip；Immuno-chromatographic test paper strip is made up of liner plate, sample pad or UCP pads, analyzing film and absorption pad；Analyzing film is provided with two parallel detection line T1s, T2 and nature controlling line C, two detection line T1s, T2 are coated with anti-NGAL antibody and anti-Cr antibody respectively, nature controlling line is coated with the antibody of anti-UCP labelled antibodies source animal immunoglobulin, and the quantitative uNGAL and uCr of UCP readout instruments can be used in the test strips.The present invention can combine quantitative different molecular form uNGAL and uCr, have the advantages that quick, easy.UNGAL can improve the ability of the diseases such as NGAL early diagnosis injury of kidney after being corrected through uCr, therefore with great scientific research and clinical value.

Description

Based on up-converting phosphor technology joint quantitatively detection uNGAL and uCr device and its Preparation method
Technical field
The invention belongs to technical field of immunological detection, and in particular to the joint based on up-converting phosphor technology (UPT) is determined The related apolipoprotein (uNGAL) of amount detection disease biomarkers urine neutrophil leucocyte gelatinase and urine dilution influence correction because Device for immunochromatography of sub- urine creatinine (uCr) and preparation method thereof.
Background technology
Neutrophil gelatinase-associated lipocalin (neutrophil gelatinase associated Lipocalin, NGAL) it is also known as human neutrophil lipocalin protein (human neutrophil lipocalin, HNL) and is A kind of glycoprotein that the beginning of the nineties finds in the particle of neutrophil leucocyte second, category lipocalin protein (lipocalin) surpasses house Race 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 forms different two with monomer, homodimer and with MMP9 The different kinds of molecules such as aggressiveness form is present, and different molecular form NGAL has difference correlation from different pathologic processes.It is wherein single Body NGAL has of a relatively high correlation with renal tubule acute injury, and homodimer NGAL can effectively reflect urinary tract infection and thin Bacterium infection conditions.In addition to neutrophil leucocyte can secrete NGAL, NGAL has expression in people's Various Tissues but expressed in physiological conditions Level is low；Such as inflammation, infection, tumour, ischemic under pathological conditions, 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 as the mark of the diseases such as acute bacterial infection, tumour, renal dysfunction by people Concern.Particularly NGAL is increasingly paid attention to as the early diagnosis biomarker of renal dysfunction by people.Have at present Serum, blood plasma and urine NGAL are used for the report of the early diagnosis of renal dysfunction.The serum creatinine (sCr) used now with clinic Compare, NGAL shows excellent properties when early diagnosing renal dysfunction.Report NGAL in early diagnosis because of ischemic (J.Endourol.2013,27:1510-1515), antineoplastic cis-platinum (Am.J.Nephrol.2004,24:307-315)、 Pyemia (Biomed.Res.Int.2015,2009,186:48-51；Chinese practical medicine 2015,24:46-47), department of cardiac surgery Perform the operation (Lancet 2005,365:1231-1238；Clin.Chim.Acta.2009,403:Acute kidney caused by 121-125) etc. During function damage (AKI), shift to an earlier date whether 1 to 2 days anticipation patients will occur AKI than sCr, this is that clinical intervention treatment AKI strives for To the quality time.
Compared with serum and blood plasma NGAL, the biomarker that urine NGAL (uNGAL) is used as diagnosis AKI especially attracts people Notice, main cause has following three points：First, animal model and Cell culture invitro result of study show acute injury of kidney When renal cells up-regulated expression NGAL and urine is directly contacted with diseased region, therefore uNGAL can be directly rapid anti- Reflect injury of kidney situation；Second, urine sampling mode is Non-Invasive, is readily obtained the cooperation of patient, is the body for being easiest to obtain Liquid sample；3rd, current uroscopy is a kind of most common necessary inspection project for judging kidney trouble.On the other hand, Although uNGAL has the advantages that its own is unique as renal function biomarker, NGAL concentration is drunk by patient in urine Urine dilution influence caused by the factors such as water, intravenous drip infusion medicine, so that can not the highly effective real urine of reflection The dynamic change situation of NGAL concentration, it is therefore desirable to corrected on influence caused by being diluted because of urine.At present clinically, UCr levels are long-standing to the influence that detectable substance concentration is brought in urine for correcting urine dilution.Creatinine (Cr) is phosphorus in muscle The metabolite of sour creatinine, body is generally produced with constant speed；In addition, creatinine is small-molecule substance, kidney can be passed freely through Bead is filtered and in renal tubule seldom or not by reabsorption.Therefore, the creatinine almost all that body is produced daily with urine ejection, UCr daily output is very stable, is not influenceed substantially by food protein content and urine volume.So can be by determining uCr water Put down to correct urine because diluting the influence to uNGAL concentration dynamic changes.
Although current laboratory research and clinical practice have the method for individually testing NGAL and uCr respectively, but do not have so far In same sample while the report of the method and apparatus of both marks of joint-detection.And future, NGAL was likely to become AKI Clinical floor inspection (Point of Care Testing, POCT) project immediately.Therefore, design and develop uNGAL and uCr connection Quantitative approach and equipment are closed with huge laboratory science research and clinical practice in future demand.
NGAL quantitative approach have RIA (international monopoly WO/1995/029404A1 and China ZL 200980155194 etc.), ELISA (European patent EP 2225268 and Chinese patent ZL200980155194 etc.), Western-blotting, latex are immunized Turbidimetry (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.To NGAL For detection demand, most significantly have the disadvantage that detection cycle is long, it is difficult to meet for the requirement of POCT detection projects.Immunochromatography skill Art is a kind of fast diagnosis method, is usually used in POCT projects.NGAL immunochromatography technique (the Chinese patents developed in recent years ZL201220323587, ZL201420423962, ZL201220392399, ZL201220323586 and ZL201220497401) Detection time can be shortened.Although NGAL immunochromatography techniques disclosed above have the advantages that easy to operate and detect quick, But the intrinsic shortcoming of various report molecules itself limits the performance of Related product.Such as fluorescence immune chromatography because fluorescent dye is easily sent out Life is quenched and causes product stability poor；Fluorescent latex particles are formed because of its fluorescent dye system physical doping used, easily hair Raw to leak, non-specific adsorption easily occurs for such material to cause the low problem of signal to noise ratio in addition；Immune colloidal gold technique is used The method of physical absorption combines label and report molecule, because the weak label of physical absorption power is easily from report molecule gold nano Particle surface comes off and influences to detect performance.In recent years, what is be doped in the lattice of crystal and constituted with thulium is upper Changing luminous material (up-converting phosphor, UCP) is the immunochromatography up-converting phosphor technology of report molecule (up-converting phosphor technology, UPT) is a research and development weight of current clinic POCT detection fields Point and focus.UCP is by main matrix (host matrix), absorption sub (absorber) and sub (emitter) the three part group of transmitting Into with upper forwarding optical phenomenon i.e. in infrared ray excited lower transmitting visible ray.The signal that UCP is produced can be read by UCP readout instruments Take.Because this kind of material is artificial synthesized and in nature and internal not similar substance presence, therefore asked without ambient interferences Topic；In addition, the excellent properties, the material conduct such as also there is UCP the good, of stability to go out Wu temper, environment-friendly and good biocompatibility Tracer is applied in a variety of POCT detecting systems.Chinese patent (ZL201220497401) discloses a kind of NGAL detections Upper forwarding light fast quantification device, the technology segment overcomes the deficiency of current NGAL detection techniques, but the technology does not have Consider to influence NGAL quantitative so as to influence the fact that NGAL diagnoses renal dysfunction ability because urine dilutes；In addition the disclosure Technology do not consider the difference of different molecular form NGAL clinical manifestations when diagnosing the illness.Therefore ZL201220497401 Simply a kind of method of measure NGAL concentration, not can determine that whether its uNGAL determined truly reflects uNGAL dynamic change feelings Condition and the NGAL that cannot distinguish between measure different molecular form, so as to limit uNGAL as the ability of renal dysfunction mark And then limit following clinical practice of this method.
UCr quantitative approach is mainly had the enzyme process based on Creatinine deiminase and Creatininase, made based on creatinine and picrate With generation yellowish red color picric acid creatinine compound chemical assay (Jaffe methods) and based on creatinine in weak acid environment band High performance liquid chromatography (HPLC) that positive charge can be separated by cation-exchange chromatography post etc..Although enzyme process has high specificity sensitivity High the advantages of, but the activity of enzyme is affected by many factors so as to influenceing the stabilization of this method, and also cost is higher than other method.Change Learn determination method with low cost, it is easy to operate, it is to determine one of creatinine most common method both at home and abroad at present.Due to vitamin C, one A little antibiotic such as material such as penicillin, acetone, acetoacetate and high concentration glucose also can be red with alkaline picric acid reaction generation Color product, therefore the specificity of this method is not high.The precision that high performance liquid chromatography (HPLC) determines uCr is high, specific good, but the method Require that instrument and equipment high and complex operation is unsuitable for the analysis of high-volume clinical samples.The enzyme process and chemical method of current creatinine can expire The clinical measure individually to creatinine of foot, creatinine is micromolecular compound in addition, prepares antibody relatively difficult；Therefore people lack at present The immunological assay method of weary creatinine of the exploitation based on antigen-antibody reaction and the power accordingly equipped.With antibody production techniques Progressive and development, by small haptens Cr and carrier mass such as seralbumin, ovalbumin, keyhole azurin, carboxymethyl The coupling of the carrier molecule such as cellulose and poly-D-lysine, which is constituted, completes the corresponding anti-Cr antibody of antigen-immunized animal acquisition into reality. Because immunology detection technology has the advantages that high specificity, species are more, setting up and develop Cr immunoassay technologies and equipment is The following area research and a focus of exploitation.But disclosed so far still without technical scheme related to the present invention.
The content of the invention
In order to overcome influence and the specific detection difference point caused by urine dilutes to uNGAL concentration dynamic changes Sub- form uNGAL difficulty, the present invention design and be developed based on up-converting phosphor technology joint quantitatively detect uNGAL and UCr device and preparation method thereof, wherein uCr are used to correct influence of the urine dilution to uNGAL concentration.The present invention can be same Sample synchronization simultaneous determination uNGAL and uCr concentration, can additionally quantify different molecular form uNGAL, with place before sample Reason is simple, amount of samples is few, detection process conveniently and quickly, the advantages of sensitivity and specificity are high.In renal dysfunction and its His disease such as acute bacterial infection etc., which early diagnoses laboratory research and clinical examination field, very big application potential.
In order to realize above-mentioned target, the present invention is adopted the following technical scheme that：
Joint based on up-converting phosphor technology quantitatively detects uNGAL and uCr devices, by sample diluting liquid freeze-dried powder, NGAL and Cr standard items freeze-dried powder, immuno-chromatographic test paper strip composition, wherein immuno-chromatographic test paper strip is by liner plate, sample pad or UCP Pad, analyzing film and absorption pad composition containing two detection lines and a nature controlling line.The liner plate be used for support sample pad or The assembling of UCP pads, analyzing film and absorption pad, the UCP pads are used to combine the anti-NGAL bodies of UCP marks and UCP marks Cr, the analyzing film is used for the setting of detection line and nature controlling line so as to realize NGAL and Cr quantitative and immuno-chromatographic test paper strip Quality Control, the absorption pad is used for the sample solution after collecting and surveying.1) the sample diluting liquid freeze-dried powder
Preparation method is as follows：By 20mL, pH=7.2~7.5, containing 150~250mmol/L NaCl, 0.25~0.5% (vol/vol) the 50 of Tween-20,0.5%~2% (wt/vol) BSA or 2.5%~5% (wt/vol) skimmed milk power~ After 100mmol/L HEPES buffer solutions are stirred fully through mechanically or magnetically power, filtered out using 0.22 μm or 0.45 μm of filter Bacterium and removing insoluble impurities, freeze-dried rear room temperature preservation.
Sample diluting liquid freeze-dried powder obtains sample diluting liquid after fully being dissolved using preceding addition 20mL deionized waters.
2) totally 4 sets of the NGAL and Cr standard items freeze-dried powder
A) standard items A：It is the standard items determined for monomer uNGAL and uCr, often manages the NGAL of monomer containing 60ng and 6 μm of ol Cr；Preparation method is that 60ng monomers NGAL and 6 μm of ol Cr are dissolved in into 300 μ L, pH=7.4 and containing 5~10% trehalose (wt/ Vol the monomer containing the 200ng/mL NGAL and 20mmol/L Cr that are obtained in PBS solution) solution, using conventional after filtration sterilization Room temperature preservation after method is lyophilized.
B) standard items B：For homodimer uNGAL and uCr determine standard items, often manage the NGAL of homodimer containing 60ng with 6μmol Cr；Preparation method is that 60ng homodimers NGAL and 6 μm of ol Cr are dissolved in into 300 μ L, pH=7.4 and containing 5~10% seas Homodimer containing 200ng/mL NGAL and 20mmol/L Cr that the PBS solution of algae sugar (wt/vol) is obtained solution, filtration sterilization Afterwards using the lyophilized rear room temperature preservation of conventional method.
C) standard items C：The standard items determined for different dimerization uNGAL and uCr, often manage heterodimer containing 60ng NGAL and 6 μ molCr；Preparation method is that 60ng heterodimers NGAL and 6 μm of ol Cr are dissolved in into 300 μ L, pH=7.4 and containing 5~10% marine algas Heterodimer containing 200ng/mL NGAL and 20mmol/L Cr that the PBS solution of sugared (wt/vol) is obtained solution, after filtration sterilization Using the lyophilized rear room temperature preservation of conventional method.
D) standard items D：The standard items determined for three kinds of molecular forms uNGAL and uCr, often manage NGAL containing 60ng (wherein 20%) and 6 μm of ol Cr monomer NGAL, homodimer NGAL and heterodimer NGAL respectively account for 40%, 40% and；Preparation method is By 24ng monomers NGAL, 24ng homodimer NGAL, 12ng heterodimer NGAL and 6 μm of ol Cr be dissolved in 300 μ L, pH=7.4 and NGAL containing 200ng/mL that PBS solution containing 5~10% trehaloses (wt/vol) is obtained (wherein monomer NGAL, homodimer NGAL and heterodimer NGAL respectively account for 40%, 40% and 20%) solution with 20mmol/L Cr, using conventional after filtration sterilization Room temperature preservation after method is lyophilized.
Above standard items are mixed using preceding addition deionized water 300 μ L and are prepared into standard items working solution.Ground according to laboratory Study carefully the different purposes with clinical research and select one or more sets more than uses in 4 sets of standard items.By the above standard items when using Working solution carries out 2 times of dilution methods with sample diluting liquid and is diluted, the formulation for standard curve.
NGAL and Cr standard items are prepared using this area routine techniques and/or obtained by commercial sources.Monomer NGAL is marked Quasi- product purify the NGAL for obtaining or be commercialization using prokaryotic expression carrier and carrier for expression of eukaryon in corresponding host cell expression (Biochem.Biophys.Res.Commun.1994,202:1468-1475；Abcam ab188461).Homodimer NGAL Separate and obtain from human blood with heterodimer NGAL, be specifically to be obtained after blood buffy coat natural subsidence is removed into red blood cell Granulocyte is obtained, granulocyte obtains granulocyte particle through nitrogen cavitation is broken, then isolated and purified after acid cleavage particle (Scand.J.Clin.Lab.Invest.1994,54:365-376).Cr (CAS 60-27-5) be commercial prod (such as The C4255 of Sigma companies or other company's similar clauses), purity should be greater than being equal to 98%.
3) the report molecule of the immuno-chromatographic test paper strip is UCP, and UCP particle diameters are 200~600nm, and UCP is marked at respectively Anti- NGAL is marked on Cr.
UCP marks anti-NGAL to include UCP and marks anti-monomer NGAL antibody, the anti-homodimer NGAL antibody of UCP marks, UCP Anti-mm P-9 antibody or UCP marks is marked to resist above-mentioned three kinds of molecular forms NGAL antibody.
Above-mentioned UCP marks anti-NGAL or mark Cr are coupled by covalent bond to be realized, coupling method is published for routine Technology.UCP and antibody molecule by the conventional coupling mode formation UCP labelled antibodies in this area (Anal.Biochem.2001, 293:307-318；J.Clin.Micro.2008,46:171-187).UCP passes through the conventional coupling mode in this area with Cr molecules UCP mark Cr molecules are formed, UCP is first such as subjected to 3- aminopropyls-trimethoxy silane (APTMS) modification and then using double work( Energy connection molecule glutaraldehyde makees crosslinking agent and realizes Cr and UCP covalent bonds.
Anti- NGAL antibody and UCP is marked to mark Cr to be dissolved in containing 0.05% (vol/vol) Triton-100,0.1% UCP (wt/vol)NaN3, pH=8.0 50mmol/L glycine buffers in, UCP marks anti-NGAL concentration to be 1mg/mL, UCP The concentration for marking Cr is 10mol/L.
4) immuno-chromatographic test paper strip is divided to A and the classes of B two, and different size can be set according to the actual requirements per class quantity.
A) A para-immunities chromatograph test strip：It is made up of liner plate, sample pad, analyzing film and absorption pad, further can be by plastics Shell is encapsulated.A para-immunity chromatograph test strip optional equipments UCP marks anti-NGAL antibody-solutions and UCP to mark Cr solution compositions (UCP that every 100 A para-immunities chromatograph test strip is equipped with 10 μ L marks anti-NGAL antibody-solutions and UCP marks Cr molten to mixed solution The mixed solution of liquid composition), using preceding by 200~500 times of the sample diluting liquid dilution of above-mentioned mixed solution.
The application method of A para-immunity chromatograph test strips is as follows：Standard items working solution is carried out 2 times with sample diluting liquid first Gradient dilution method is diluted, and obtains standard concentration gradient solution；Then by urine sample with sample diluting liquid dilution 10~ 100 times；25~100 μ L above-mentioned standards product concentration gradient solution or urine sample dilution multiple holes are taken to add corresponding 96 orifice plate In hole or among the container of similar structures；Then each diluted UCP marks of 25~100 μ L that add resist again in above-mentioned 96 orifice plate NGAL antibody-solutions and UCP mark the mixed solution of Cr solution compositions, and suspension is made in mixing；Then by A para-immunity chromatographic test papers The sample pad end of bar is dipped vertically into above-mentioned suspension, waits and progress UCP letters after 5~20min of horizontal positioned are taken out after 5~30min Number collection.
B) B para-immunities chromatograph test strip：It is made up of liner plate, UCP pads, analyzing film and absorption pad, further can be by Plastic casing is encapsulated.Mark anti-NGAL antibody and UCP mark Cr containing UCP in UCP pads, specific preparation method be by 0.5mL/cm2Consumption mark anti-NGAL antibody-solutions and UCP to mark the mixed solution of Cr solution compositions to be uniformly added dropwise UCP to exist On UCP pads, it is made after 30 DEG C~35 DEG C dryings.
The application method of B para-immunity chromatograph test strips is as follows：After immunity test strip horizontal positioned, plus 20~100 μ L On standard concentration gradient solution or urine sample dilution to UCP pads, progress UCP signals after 10~30min are waited to adopt Collection.
The sample pad or UCP pads of the para-immunity chromatograph test strip of above A, B two, analyzing film, the width of absorption pad and liner plate Degree is consistent, is 4~5.5mm, and the length of sample pad and UCP pads is 5~10mm, and the length of analyzing film is 25~40mm, The length of absorption pad is 20~30mm, the length of liner plate be equal to after front and rear overlap joint assembling sample pad or UCP pads, analyzing film and The length summation of absorption pad；Wherein the material of sample pad and UCP pads is the plain film of glass fibre 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 absorbent filter Or the material with similar functions, the material of liner plate is the material with Self-adhesive property polyvinyl chloride plastic sheet or with similar functions Material.
The assembling mode of UCP immuno-chromatographic test paper strips is realized using ability convenient technical process.
UCP be average grain diameter synthesized by this area routine techniques in laboratory or commercialization be 200~ The 600nmol/L crystalline material being made up of thulium through silicon dioxide coated and surface-functionalized modification is (such as NaYF4：Er, Yb).
5) analyzing film is provided with parallel two detection lines (T1 and T2) and a nature controlling line (C), described two inspections Survey line T1 and T2 are coated with anti-NGAL antibody and anti-Cr antibody respectively, and both can mutually exchange.Contain in UCP pads The anti-NGAL antibody of UCP marks and the coated anti-NGAL antibody identification NGAL different epitopes of analyzing film, described anti-NGAL antibody For antigen behaviour source NGAL, the people source NGAL is monomer, homodimer, heterodimer or the mixture of three, described UCP marks the uCr energy of Cr and urine sample competitive and the coated anti-Cr antibody bindings of analyzing film, and the nature controlling line coating is anti- The antibody of UCP labelled antibody source animal immunoglobulins.The test strips can carry out data acquisition with UCP readout instruments.
Further, detection line T1 from sample pad or UCP pads 5~10mm of end, between T1, T2 and C at intervals of 5 ~10mm；Wherein T1 is coated with anti-NGAL antibody, and T2 is coated with anti-Cr antibody or T1 is coated with anti-Cr antibody and T2 is coated with anti-NGAL Antibody, nature controlling line C is coated with the antibody of anti-UCP labelled antibodies source animal immunoglobulin；Method for coating is to utilize manual method Or special point sample instrument on analyzing film (from UCP pads toward absorption pad direction) successively the anti-NGAL antibody-solutions of even application, The antibody of anti-Cr antibody-solutions and anti-UCP labelled antibodies source animal immunoglobulin；Antibody-solutions are that antibody is dissolved in into antibody Obtained after buffer solution, antibody concentration is 1mg/mL, antibodies buffer is the 100mM containing 1% (vol/vol) methanol, pH=8.0 Tris-HCL solution liquid；Final quantity for spray is 0.025~0.05mL/mm, equivalent to every millimeter T1, T2 or C wire spraying 25ng~ 50ng antibody；Room temperature can long-term room-temperature preservation after drying (properties of product are not significantly changed at least 12 months).
Anti- NGAL antibody, anti-Cr antibody and nature controlling line antibody prepare or passed through commercial channel using this area routine techniques Purchase is obtained.Anti- NGAL antibody be the antibody of commercialization or by antigen of NGAL by conventional antibody technology of preparing prepare it is anti- NGAL antibody (Current protocols in molecular biology.New York:John Wiley, 2008 11.10.1-11.10.10；Ab23477, ab70287, ab188551 of Abcam companies etc.)；What above method was prepared or bought Antibody obtains the antibody for specific molecular form NGAL through monomer, homodimer or heterodimer NGAL antigen selections.Anti- Cr The access approaches of antibody：Cr haptens is passed through and carrier such as seralbumin, ovalbumin, keyhole azurin, carboxymethyl cellulose The carrier molecule such as element and poly-D-lysine is made up of routine techniques coupling completes the corresponding anti-Cr antibody of antigen-immunized animal acquisition Or (there are the sale of such product in such as Creative Diagnostics companies or other companies) is bought through commercial channel, anti-Cr resists Body can be combined with the free Cr and UCP Cr marked.The coated antibody of nature controlling line is determined according to UCP labelled antibodies type, can be Dynamics or anti-rabbit IgG antibody of rabbit anti-mouse igg antibody or sheep anti-mouse igg antibody or other animal origins etc., by business Channel purchase obtains (such as abcam companies corresponding antibodies product).
Due to taking above technical scheme, make the present invention that there is notable beneficial effect.
Currently without joint-detection uNGAL and uCr technology, with immediate prior art (ZL201220497401) phase Than the present invention has following two aspects creative：First, based on because of urine dilution influence, individually measure uNGAL can not be effectively anti- The dynamic change reflected the dynamic mapping situation of the biomarker and determine uNGAL is the most important approach of anticipation renal dysfunction This basic theories and the fact, the introduction of the invention simultaneously realize simultaneous determination uNGAL and urine simultaneously in same sample The correction factor uCr of dilution, and ZL201220497401 is to disclose a kind of NGAL assay methods based on UPT not relate to And uCr；Second, the present invention can for determine whole molecular forms uNGAL may also be used for measure monomer or homodimer or Heterodimer uNGAL, the molecular forms for the NGAL that ZL201220497401 is determined are simultaneously indefinite, and difference NGAL molecule shape Formula has significant correlation with it as the clinical manifestation of the diagnostic biomarkers of different pathological (Clin.J.Am.Soc.Nephrol.2010,12:2229-2235).
In addition, the present invention is also creative in following tripartite's mask：First, by double-antibody sandwich immunoassay technology and list Antibody competition immunoassay technology is applied to same test system, so as to realize to macro-molecular protein (NGAL) and small molecule The joint of compound (Cr) is quantified.Second, using determination of immunological methods Cr concentration, enrich the assay method of current Cr concentration. 3rd, using UCP immunochromatography techniques NGAL and Cr concentration mensuration being allow to complete in a short time, (apparatus of the present invention exist Test is completed in 30~70min clocks, and conventional ELISA needs 3~5h) so that NGAL can be used for POCT visual inspections of clinic Survey.
To sum up, the motivation that is improved to prior art is not present in the present invention and claimed technical scheme not shows and It is clear to, and claimed technical scheme can produce significant beneficial effect, therefore is one there is novelty and wound The innovation and creation for the property made.
Brief description of the drawings
Fig. 1 is that the joint of the invention based on up-converting phosphor technology quantitatively detects A para-immunities layer in uNGAL and uCr devices Analyse the diagrammatic cross-section of test strips.Wherein 1 sample pad, 2 be analyzing film, 3 be absorption pad, 4 be liner plate, 5 and 6 be detection line T1 and (wherein T1 is coated with anti-NGAL antibody to T2 and T2 is coated with anti-Cr antibody, or T1 is coated with anti-Cr antibody and T2 is coated with anti-NGAL and resisted Body), 7 be the nature controlling line C (antibody of the anti-UCP labelled antibodies source animal immunoglobulin of coating, according to the kind of UCP labelled antibodies Class is determined).
Fig. 2 is that the joint of the invention based on up-converting phosphor technology quantitatively detects B para-immunities in NGAL and urine creatinine device The diagrammatic cross-section of chromatograph test strip.Wherein 21 be UCP pads (antibody of coating UCP marks and the Cr of UCP marks, UCP marks Note antibody can mark anti-NGAL antibody or UCP to be marked at anti-mm P9 antibody for UCP, and UCP marks anti-NGAL antibody to be UCP marks Anti- monomer NGAL antibody, UCP mark anti-homodimer NGAL antibody or UCP mark anti-above-mentioned three kinds of molecular forms NGAL Antibody), 2 be analyzing film, 3 be absorption pad, 4 be liner plate, 5 and 6 be detection line T1 and T2 (wherein T1 is coated with anti-NGAL antibody, And T2 is coated with anti-Cr antibody or T1 is coated with anti-Cr antibody and T2 is coated with anti-NGAL antibody), 7 be nature controlling line C (the anti-UCP marks of coating Remember the antibody of antibody sources animal immune globulin, determined according to the species of UCP labelled antibodies).
Fig. 3 is that the joint of the invention based on up-converting phosphor technology quantitatively detects A para-immunities layer in uNGAL and uCr devices Analyse the supporting test strips rack schematic diagram of test strips.Rack is hollow, bottom surface missing a cuboid, and long and wide size is omited It is highly 40mm~50mm more than the size (such as a length of 130mm, a width of 87mm) of the orifice plate of standard 96；Material is plastics or stationery； Wherein 31 have slot corresponding with 96 orifice plates thereon for placement top of support, and (size of slot is with slightly larger than A para-immunities The width and thickness of chromatograph test strip are defined), 32 be rack side, and 33 be that before rack, 34 be the examination for placing top of support Paper slip putting hole.
Fig. 4 is that embodiment 3 quantitatively detects the preparation of total uNGAL and uCr devices based on up-converting phosphor technology joint and made With the standard curve of method.Fig. 4 left figures are the average value and total NGAL standard items according to table 1uNGAL peak areas AUC1 and AUC2 The standard curve that concentration is drawn, the calculating for total uNGAL in urine sample；Fig. 4 right figures are according to table 2uCr peak areas AUC1 The standard curve drawn with AUC2 average value with Cr standard concentrations, the calculating for uCr in urine sample.
Fig. 5 is that joint of the embodiment 4 based on up-converting phosphor technology quantitatively detects uNGAL and uCr device joint-detection lists The standard curve of body uNGAL and uCr concentration.Fig. 5 left figures are the average value and list according to table 2uNGAL peak areas AUC1 and AUC2 The standard curve that body NGAL standard concentrations are drawn, the calculating for monomer uNGAL in urine sample；Fig. 5 right figures are according to table The standard curve that 2uCr peak areas AUC1 and AUC2 average value are drawn with Cr standard concentrations, for uCr in urine sample Calculate.
Fig. 6 is that joint of the embodiment 5 based on up-converting phosphor technology quantitatively detects that uNGAL and uCr device joint-detections are different The canonical plotting of dimer uNGAL and uCr concentration.Fig. 6 left figures are being averaged according to table 3uNGAL peak area AUC1 and AUC2 The standard curve that value is drawn with heterodimer NGAL standard concentrations, the calculating for heterodimer uNGAL in urine sample；Figure 6 right figures are the standard curves drawn according to table 4uCr peak areas AUC1 and AUC2 average value with Cr standard concentrations, for urinating UCr calculating in liquid sample.
Embodiment
The present invention will be further illustrated with embodiment below.The embodiment of the present invention is for being explained further for the present invention It is not limitation of the invention.The scope of protection of present invention is belonged to according to the specific improvement that the essence of the present invention is carried out.
Embodiment 1：Total uNGAL and uCr devices composition is quantitatively detected based on up-converting phosphor technology joint
1) 1 part of sample diluting liquid freeze-dried powder (reagent I)
2) standard items solution D freeze-dried powder 1 is managed (reagent II)
3) UCP marks resist three kinds of molecular forms NGAL antibody and UCP mark Cr solution 1 to manage (reagent III)
4) A para-immunities chromatograph test strip 96
5) the supporting test strips rack of A para-immunities chromatograph test strip 1
6) 1 piece of 96 orifice plate, 96 orifice plate shrouding films one
Embodiment 2：Total NGAL and urine creatinine device composition are quantitatively detected based on up-converting phosphor technology joint
1) 1 part of sample diluting liquid freeze-dried powder (reagent I)
2) standard items solution D freeze-dried powder 1 is managed (reagent II)
3) B para-immunities chromatograph test strip 100
Embodiment 3：It is a kind of that the preparation of total uNGAL and uCr devices is quantitatively detected based on up-converting phosphor technology joint and made Use method
1) preparation of device
A) sample diluting liquid freeze-dried powder (reagent I) is prepared
Sample diluting liquid 20mL, by 100mmol/L HEPES, pH 7.2,200mmol/L NaCl, 0.25% (vol/ Vol) Tween-20,0.5% (wt/vol) BSA compositions；Through magnetic agitation it is abundant after, filtered out using 0.45 μm of filter Freezed after bacterium and removing insoluble impurities；Room temperature preservation.
B) standard items solution D freeze-dried powder (reagent II) is prepared
By 300 μ L NGAL containing 200ng/mL, (monomer, homodimer and heterodimer NGAL respectively account for 40%, 40% and 20%), the pH=7.4 of 20mmol/L Cr and 10% trehalose (wt/vol) 10mmol/L PBS solutions are filled through magnetic agitation After point, carry out filtration sterilization using 0.22 μm of filter and remove to freeze after insoluble impurities；Room temperature preservation.
C) UCP marks resist three kinds of molecular forms NGAL antibody and UCP mark Cr solution 1 to manage (reagent III) preparation
UCP marks resist three kinds of molecular forms NGAL antibody and the μ L of UCP mark Cr solution 10, and solution composition is the sweet ammonia of 50mM Acid, 0.05%Triton-100,0.1%NaN3, 1mg/mL UCP mark three kinds of molecular forms NGAL antibody of rabbit-anti (22264-1-AP of Proteintech companies) and 10mol/mL UCP mark the Cr aqueous solution；4 DEG C of preservations.
D) 96 preparations of A para-immunities chromatograph test strip
A para-immunities chromatograph test strip is made up of liner plate, sample pad, analyzing film and absorption pad；Sample cushion material is glass fibers The plain film of dimension, analysis membrane material is nitrocellulose filter, and absorbent pad material is absorbent filter, and the material of liner plate is with Self-adhesive Matter polyvinyl chloride plastic sheet.Liner plate, sample pad, the width of analyzing film and absorption pad are 4.5mm, length be respectively 56mm, 10mm, 30mm and 20mm.T1, T2 and C line spray goat-anti NGAL antibody-solutions (the Abcam companies of the antibody containing 100ng respectively on analyzing film Ab166677), goat-anti Cr antibody (ab30719 of Abcam companies) solution and goat anti-rabbit igg antibody (Abcam companies Ab97091) solution.Sample pad end is mounted on analyzing film, and both overlap span access location length for 2mm, and analyzing film rear end is mounted in Under absorption pad, both overlap span access location length for 2mm；Sample pad, analyzing film and absorption pad are assembled on liner plate successively, and room temperature is protected Deposit.
Above-mentioned goat-anti NGAL antibody-solutions used, goat-anti Cr antibody-solutions and goat anti-rabbit igg antibody solution are as follows Prepare：Anti- NGAL antibody, anti-Cr antibody or dynamics are dissolved in pH8.0 100mmol/L Tris-HCl, matter respectively Fraction is measured in 1% methanol solution, the solubility of antibody is 1mg/mL.
2) device uses step
A) working solution of reagent preparation I：Plus 20mL deionized waters are into reagent I container, the concussion that is vortexed mixes 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) it is each in above S0~S7 centrifuge tubes to add the μ L of reagent I working solution 150 prepared by step a).
D) add 300 μ L deionized waters to obtain reagent II solution into reagent II container, all taken out after mixing and add S0 pipes； Then 150 μ L are taken out from S0 and adds S1 pipes；150 μ L are taken out from S1 pipes add S2 pipes after mixing；After mixing 150 μ L are taken out from S2 pipes Add S3 pipes；150 μ L are taken out from S3 pipes add S4 pipes after mixing；150 μ L are taken out from S4 pipes add S5 pipes after mixing；After mixing from S5 pipes take out 150 μ L and add S6 pipes；Mix S6 pipes.
E) sample solution after dilution is prepared：Clinical urine utilizes reagent I working solution after centrifuging (10000rpm, 1min) Make the dilution of 10~100 multiples, whether centrifuging should determine according to actual urine sample quality.
F) UCP labelled antibody working solutions are prepared
Add 4990 μ L reagent I working solutions into the container of reagent III, water bath sonicator (20kHz, 320W, each ultrasound 15s, Stop 15s, 3 times altogether；Or similar ultrasound intensity).
G) sample solution added to every hole of 96 orifice plates after 50 μ L S0-S7 solution or 50 μ L dilutions；Then again toward per hole 50 μ L UCP labelled antibody working solutions of middle addition.
H) cover and be fixed on after shrouding film on 96 plate shakers, 800rpm, be incubated 30min at 37 DEG C.
I) remove shrouding film, 96 orifice plates are placed under immuno-chromatographic test paper strip rack.
J) A para-immunity chromatograph test strips are sequentially inserted into every hole, 20min is waited.
K) take out A para-immunities chromatograph test strip and data acquisition is carried out on UCP readout instruments
L) normative reference curve is drawn
Table 1 is that the present embodiment NGAL is corresponding with Cr standard solutions (S0~S6) and placebo solution (S7) various concentrations Peak area (AUC).Wherein AUC1 and AUC2 refer to that two of a concentration gradient standard solution or placebo solution are multiple The AUC readings in hole (parallel hole).
Table 1：Corresponding UCP peak areas (AUC) readings of various concentrations standard items NGAL and Cr
Fig. 4 left figures are to be drawn according to uNGAL the peak areas AUC1 and AUC2 of table 1 average value with NGAL standard concentrations Standard curve；Fig. 4 right figures are the marks drawn according to uCr the peak areas AUC1 and AUC2 of table 1 average value with Cr standard concentrations Directrix curve.
Embodiment 4：The quantitatively preparation of detection monomer uNGAL and uCr device is combined based on up-converting phosphor technology and used Method
1) preparation of device
A) sample diluting liquid freeze-dried powder (reagent I) is prepared
Sample diluting liquid 20mL, by 100mmol/L HEPES, pH 7.2,200mmol/L NaCl, 0.25% (vol/ Vol) Tween-20,0.5% (wt/vol) BSA compositions；Through magnetic agitation it is abundant after, filtered out using 0.45 μm of filter Freezed after bacterium and removing insoluble impurities；Room temperature preservation.
B) standard items solution A freeze-dried powder (reagent II) is prepared
By the 300 μ L NGAL and 20mmol/L Cr 20mmol/L of monomer containing 200ng/mL and 10% trehalose (wt/vol) PH=7.4 10mmol/L PBS solution through magnetic agitation it is abundant after, filtration sterilization is carried out using 0.22 μm of filter and removed not Freezed after solubility impurity；Room temperature preservation.
C) UCP marks anti-monomer NGAL antibody and UCP mark Cr solution 1 to manage (reagent III) preparation
UCP marks anti-monomer NGAL antibody and the μ L of UCP mark Cr solution 10, the solution composition be 50mM glycine, 0.05%Triton-100,0.1%NaN3, 1mg/mL UCP mark the anti-monomer NGAL monoclonal antibodies (Bioporto of mouse No. 009 product of BW of diagnostocsA/S companies) and 10mol/mL UCP mark Cr the aqueous solution；4 DEG C of preservations.
D) 96 preparations of A para-immunities chromatograph test strip
A para-immunities chromatograph test strip is made up of liner plate, sample pad, analyzing film and absorption pad；Sample cushion material is glass fibers The plain film of dimension, analysis membrane material is nitrocellulose filter, and absorbent pad material is absorbent filter, and the material of liner plate is with Self-adhesive Matter polyvinyl chloride plastic sheet or the material with similar functions.Liner plate, sample pad, the width of analyzing film and absorption pad are 4.5mm, Length is respectively 56mm, 10mm, 30mm and 20mm.T1, T2 and C line spray the sheep of the antibody containing 100ng respectively on every analyzing film Anti- NGAL antibody-solutions (ab166677 of Abcam companies), goat-anti Cr antibody (ab30719 of Abcam companies) solution and goat-anti Mouse IgG antibody (ab6710 of Abcam companies) solution.Sample pad end is mounted on analyzing film, and both overlap span access location length and are 2mm, analyzing film rear end is mounted under absorption pad, and both overlap span access location length for 2mm；Sample pad, analyzing film and absorption pad are successively It is assembled on liner plate, room temperature preservation.
Above-mentioned goat-anti NGAL antibody-solutions used, goat-anti Cr antibody-solutions and rabbit anti-mouse igg antibody-solutions are as follows Prepare：Goat-anti NGAL antibody, goat-anti Cr antibody or rabbit anti-mouse igg antibody are dissolved in pH=8.0 100mmol/L respectively Tris-HCl, mass fraction is in 1% methanol solution, the concentration of antibody are 1mg/mL.
2) device uses step
A) working solution of reagent preparation I：Plus 20mL deionized waters are into reagent I container, the concussion that is vortexed mixes 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) it is each in above S0~S7 centrifuge tubes to add the μ L of reagent I working solution 150 prepared by step a).
D) add 300 μ L deionized waters to obtain reagent II solution into reagent II container, all taken out after mixing and add S0 pipes； Then 150 μ L are taken out from S0 and adds S1 pipes；150 μ L are taken out from S1 pipes add S2 pipes after mixing；After mixing 150 μ L are taken out from S2 pipes Add S3 pipes；150 μ L are taken out from S3 pipes add S4 pipes after mixing；150 μ L are taken out from S4 pipes add S5 pipes after mixing；After mixing from S5 pipes take out 150 μ L and add S6 pipes；Mix S6 pipes.
E) sample solution after dilution is prepared：Clinical urine utilizes reagent I working solution after centrifuging (10000rpm, 1min) Make the dilution of 10~100 multiples, whether centrifuging should determine according to actual urine sample quality.
F) UCP labelled antibody working solutions are prepared
Add 4990 μ L reagent I working solutions into the container of reagent III, water bath sonicator (20kHz, 320W, each ultrasound 15s, Stop 15s, 3 times altogether；Or similar ultrasound intensity).
G) sample solution added to every hole of 96 orifice plates after 50 μ L S0-S7 solution or 50 μ L dilutions；Then again toward per hole 50 μ L UCP labelled antibody working solutions of middle addition.
H) cover and be fixed on after shrouding film on 96 plate shakers, 800rpm, be incubated 30min at 37 DEG C.
I) remove shrouding film, 96 orifice plates are placed under immuno-chromatographic test paper strip rack.
J) A para-immunity chromatograph test strips are sequentially inserted into every hole, 20min is waited.
K) take out A para-immunities chromatograph test strip and data acquisition is carried out on UCP readout instruments.
L) normative reference curve is drawn.
Table 2 is that the present embodiment NGAL is corresponding with Cr standard solutions (S0~S6) and placebo solution (S7) various concentrations Peak area (AUC).Wherein AUC1 and AUC2 refer to that two of a concentration gradient standard solution or placebo solution are multiple The AUC readings in hole (parallel hole).
Table 2：Corresponding UCP peak areas (AUC) readings of various concentrations standard items monomer NGAL and Cr
Fig. 5 left figures are to be painted according to uNGAL the peak areas AUC1 and AUC2 of table 2 average value with monomer NGAL standard concentrations The standard curve of system；Fig. 5 right figures are to be drawn according to uCr the peak areas AUC1 and AUC2 of table 2 average value with Cr standard concentrations Standard curve.
Embodiment 5：Based on up-converting phosphor technology combine quantitatively detection heterodimer uNGAL and uCr agent box preparation and Application method
1) preparation of device
A) sample diluting liquid freeze-dried powder (reagent I) is prepared
Sample diluting liquid 20mL, by 100mmol/L HEPES, pH 7.2,200mmol/L NaCl, 0.25% (vol/ Vol) Tween-20,0.5% (wt/vol) BSA compositions；Through magnetic agitation it is abundant after, filtered out using 0.45 μm of filter Freezed after bacterium and removing insoluble impurities；Room temperature preservation.
B) standard items C solution freeze-dried powder (reagent II) is prepared
By 300 μ L heterodimers containing 200ng/mL NGAL and 20mmol/L Cr, 20mmol/L and 10% trehalose (wt/ Vol the 10mmol/L of pH=7.4) PBS solution through magnetic agitation it is abundant after, using 0.22 μm of filter carry out filtration sterilization and Remove and freezed after insoluble impurities；Room temperature preservation.
E) prepared by B para-immunities chromatograph test strip 96 (test strips used in the embodiment have plastic casing)
B para-immunity chromatograph test strips liner plate, UCP pads, analyzing film and absorption pad composition；UCP combinations cushion material is glass Glass cellulose membrane, analysis membrane material is nitrocellulose filter, and absorbent pad material is absorbent filter, and the material of liner plate is with self-adhesion Paste property polyvinyl chloride plastic sheet or the material with similar functions.Liner plate, UCP pads, analyzing film and absorption pad width it is equal For 4mm, length is respectively 56mm, 10mm, 10mm, 30mm and 20mm.Pad contains the rabbit-anti MMP9 antibody of UCP marks The Cr of (ab38898 of Abcam companies) and UCP mark；Specific preparation method is by 0.5mL/cm2Amount by concentration be 1mg/mL The rabbit-anti MMP-9 antibody-solutions of UCP marks and the 10mol/L Cr solution of UCP marks be uniformly added on UCP pads, 35 DEG C are dry It is made after dry.On every analyzing film T1, T2 and C line spray respectively the antibody containing 100ng goat-anti NGAL antibody (Abcam companies Ab166677) solution, the solution of goat-anti Cr antibody (ab30719 of Abcam companies) and goat anti-rabbit igg antibody (Abcam companies Ab97091) solution.UCP pads are mounted on analyzing film, and both overlap span access location length for 2mm；Analyzing film rear end is mounted in suction Receive under pad, both overlap span access location length for 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 goat anti-rabbit igg antibody solution are as follows Prepare：Goat-anti NGAL antibody, goat-anti Cr antibody or sheep anti-mouse igg antibody are dissolved in pH=8.0 100mmol/L respectively Tris-HCl, mass fraction is in 1% methanol solution, the solubility of antibody are 1mg/mL.
2) device uses step
A) working solution of reagent preparation I：Plus 20mL deionized waters are into reagent I container, the concussion that is vortexed mixes 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) it is each in above S0~S7 centrifuge tubes to add the μ L of reagent I working solution 150 prepared by step a).
D) add 300 μ L deionized waters to obtain reagent II solution into reagent II container, all taken out after mixing and add S0 pipes； Then 150 μ L are taken out from S0 and adds S1 pipes；150 μ L are taken out from S1 pipes add S2 pipes after mixing；After mixing 150 μ L are taken out from S2 pipes Add S3 pipes；150 μ L are taken out from S3 pipes add S4 pipes after mixing；150 μ L are taken out from S4 pipes add S5 pipes after mixing；After mixing from S5 pipes take out 150 μ L and add S6 pipes；Mix S6 pipes.
E) sample solution after dilution is prepared：Clinical urine utilizes reagent I working solution after centrifuging (10000rpm, 1min) Make the dilution of 10~100 multiples, whether centrifuging should determine according to actual urine sample quality.
F) by B para-immunity test strips horizontal positioned on the table, added on UCP pads 100 μ L S0-S7 solution or Sample solution after dilution, waits 20min；
G) data acquisition is carried out on UCP readout instruments；
H) normative reference curve is drawn.
Table 2 is that the present embodiment NGAL is corresponding with Cr standard solutions (S0~S6) and placebo solution (S7) various concentrations Peak area (AUC).Wherein AUC1 and AUC2 refer to that two of a concentration gradient standard solution or placebo solution are multiple The AUC readings in hole (parallel hole).
Table 3：Corresponding UCP peak areas (AUC) readings of various concentrations standard items heterodimer NGAL and Cr
Fig. 6 left figures are dense according to uNGAL the peak areas AUC1 and AUC2 of table 3 average value and heterodimer NGAL standard items Spend the standard curve drawn；Fig. 6 right figures are the average value and Cr standard concentrations according to uCr the peak areas AUC1 and AUC2 of table 3 The standard curve of drafting.

Claims (8)

1. a kind of joint based on up-converting phosphor technology quantitatively detects uNGAL and uCr devices, it is characterised in that：It is dilute by sample Release liquid freeze-dried powder, NGAL and Cr standard items freeze-dried powder, immuno-chromatographic test paper strip composition；
(1) sample diluting liquid freeze-dried powder be by 20mL, pH=7.2~7.5, containing 150~250mmol/L NaCl, 0.25~ The 50 of 0.5% (vol/vol) Tween-20,0.5%~2% (wt/vol) BSA or 2.5%~5% (wt/vol) skimmed milk power After~100mmol/L HEPES buffer solutions are stirred fully through mechanically or magnetically power, filtered using 0.22 μm or 0.45 μm of filter It is degerming and remove insoluble impurities, it is freeze-dried after obtain；
(2) totally 4 sets of NGAL and Cr standard items freeze-dried powder, wherein,
Standard items A：It is the standard items determined for monomer uNGAL and uCr, often manages the NGAL of monomer containing 60ng and 6 μm of ol Cr；
Standard items B：The standard items determined for homodimer uNGAL and uCr, often manage the NGAL of homodimer containing 60ng and 6 μm of ol Cr；
Standard items C：The standard items determined for different dimerization uNGAL and uCr, often manage the NGAL of heterodimer containing 60ng and 6 μm of olCr；
Standard items D：The standard items determined for three kinds of molecular forms uNGAL and uCr, often manage NGAL containing 60ng and 6 μm of ol Cr, Wherein monomer NGAL, homodimer NGAL and heterodimer NGAL respectively account for 40%, 40% and 20%；
(3) immuno-chromatographic test paper strip is divided to A and the classes of B two, and A para-immunities chromatograph test strip is by liner plate, sample pad, analyzing film and suction Receive pad composition；B para-immunities chromatograph test strip is made up of liner plate, UCP pads, analyzing film and absorption pad；Contain in UCP pads UCP marks anti-NGAL antibody and UCP marks Cr；
(4) analyzing film is provided with two parallel detection line T1s, T2 and nature controlling line C, and two detection line T1s, T2 are coated with respectively Anti- NGAL antibody and anti-Cr antibody, and both can mutually exchange；Nature controlling line is coated with anti-UCP labelled antibodies source animal and ball is immunized The antibody of albumen；
The liner plate is used for the assembling for supporting sample pad or UCP pads, analyzing film and absorption pad, and the UCP pads are used for Anti- NGAL bodies and UCP mark Cr are marked with reference to UCP, the analyzing film is used to the setting of detection line and nature controlling line realize NGAL With the Quality Control of Cr quantitative and immuno-chromatographic test paper strip, the absorption pad is used for the sample solution after collecting and surveying.
2. a kind of joint based on up-converting phosphor technology as claimed in claim 1 quantitatively detects uNGAL and uCr devices, its It is characterised by：The sample pad or UCP pads of the para-immunity chromatograph test strip of A, B two, analyzing film, the width one of absorption pad and liner plate Cause, be 4~5.5mm, the length of sample pad and UCP pads is 5~10mm, and the length of analyzing film is 25~40mm, is absorbed The length of pad is 20~30mm, and the length of liner plate is equal to sample pad or UCP pads, analyzing film and absorption after front and rear overlap joint assembling The length summation of pad.
3. a kind of joint based on up-converting phosphor technology as claimed in claim 1 quantitatively detects uNGAL and uCr devices, its It is characterised by：Wherein the material of sample pad and UCP pads is the plain film of glass fibre or the membrane material with similarity, analysis The material of film is nitrocellulose filter or the membrane material with similarity, and the material of absorption pad is for absorbent filter or with similar The material of function, the material of liner plate is the material with Self-adhesive property polyvinyl chloride plastic sheet or with similar functions.
4. a kind of joint based on up-converting phosphor technology as claimed in claim 1 quantitatively detects uNGAL and uCr devices, its It is characterised by：The use of A para-immunity chromatograph test strips is that standard items working solution is carried out into 2 times of gradient dilution methods with sample diluting liquid It is diluted, obtains standard concentration gradient solution；Urine sample is diluted 10~100 times with sample diluting liquid；Take 25~100 It is in the hole of μ L above-mentioned standards product concentration gradient solution or corresponding 96 orifice plate of urine sample dilution difference multiple holes addition or similar Among the container of structure, then each diluted UCP of 25~100 μ L that add mark anti-NGAL antibody molten again in above-mentioned 96 orifice plate Liquid and UCP mark the mixed solution of Cr solution compositions, and suspension is made in mixing；Then by the sample pad of A para-immunity chromatograph test strips End is dipped vertically into above-mentioned suspension, waits and progress UCP signal acquisitions after 5~20min of horizontal positioned are taken out after 5~30min.
5. a kind of joint based on up-converting phosphor technology as claimed in claim 1 quantitatively detects uNGAL and uCr devices, its It is characterised by：The use of B para-immunity chromatograph test strips is that standard items working solution is carried out into 2 times of gradient dilution methods with sample diluting liquid It is diluted, obtains standard concentration gradient solution；Then urine sample is diluted 10~100 times with sample diluting liquid；Then After B para-immunity chromatograph test strip horizontal positioneds, plus 20~100 μ L standard concentration gradient solution or urine sample dilution To UCP pads, wait and UCP signal acquisitions are carried out after 10~30min.
6. a kind of joint based on up-converting phosphor technology as claimed in claim 1 quantitatively detects uNGAL and uCr devices, its It is characterised by：UCP is average grain diameter for 200~600nmol/L through silicon dioxide coated and surface-functionalized modification by dilute The crystalline material that earth metal element is constituted.
7. a kind of joint based on up-converting phosphor technology as claimed in claim 1 quantitatively detects uNGAL and uCr devices, its It is characterised by：It is anti-that UCP marks anti-NGAL antibody to include the anti-monomer NGAL antibody of UCP marks, the anti-homodimer NGAL of UCP marks Body, UCP mark anti-mm P-9 antibody or UCP marks resist above-mentioned three kinds of molecular forms NGAL antibody.
8. a kind of joint based on up-converting phosphor technology described in claim 1 quantitatively detects the preparation of uNGAL and uCr devices Method, its step is as follows：
1) preparation of the sample diluting liquid freeze-dried powder, be 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 After 50~100mmol/L HEPES buffer solutions of powder are stirred fully through mechanically or magnetically power, entered using 0.22 μm or 0.45 μm of filter Row filtration sterilization and removing insoluble impurities, freeze-dried rear room temperature preservation；Sample diluting liquid freeze-dried powder is gone using preceding addition 20mL Ionized water obtains sample diluting liquid after fully dissolving；
2) preparation of the NGAL and Cr standard items freeze-dried powder, standard items A is that 60ng monomers NGAL and 6 μm of ol Cr are dissolved in into 300 In μ L, pH=7.4 and PBS solution containing 5~10% trehaloses (wt/vol) the obtained NGAL of monomer containing 200ng/mL with 20mmol/L Cr solution, using the lyophilized rear room temperature preservation of conventional method after filtration sterilization；Standard items B is by the same dimerization of 60ng Body NGAL and 6 μm of ol Cr are dissolved in 300 μ L, pH=7.4 and PBS solution containing of obtaining containing 5~10% trehaloses (wt/vol) 200ng/mL homodimer NGAL and 20mmol/L Cr solution, using the lyophilized rear room temperature preservation of conventional method after filtration sterilization； Standard items C is that 60ng heterodimers NGAL and 6 μm of ol Cr are dissolved in into 300 μ L, pH=7.4 and containing 5~10% trehalose (wt/ Vol heterodimer containing 200ng/mL NGAL and 20mmol/L Cr that PBS solution) is obtained solution, using normal after filtration sterilization Room temperature preservation after rule method is lyophilized；Standard items D is by 24ng monomer NGAL, 24ng homodimer NGAL, 12ng heterodimers NGAL and 6 μm of ol Cr is dissolved in 300 μ L, pH=7.4 and PBS solution containing of obtaining containing 5~10% trehaloses (wt/vol) 200ng/mL NGAL and 20mmol/L Cr solution, using the lyophilized rear room temperature preservation of conventional method after filtration sterilization；With subscript Quasi- product freeze-dried powder is mixed using preceding addition deionized water 300 μ L and is prepared into standard items working solution；
3) UCP marks anti-NGAL antibodies Antibodies and UCP mark Cr, and specific preparation method is by 0.5mL/cm2Consumption UCP is marked Remember that anti-NGAL antibody-solutions and the mixed solution of UCP mark Cr solution compositions are uniformly added dropwise on UCP pads, 30 DEG C~35 DEG C It is made after drying；
4) T1, T2 and C method for coating are that even application resists successively on analyzing film using manual method or special point sample instrument The antibody of NGAL antibody-solutions, anti-Cr antibody-solutions and anti-UCP labelled antibodies source animal immunoglobulin；Antibody-solutions be by Antibody, which is dissolved in after antibodies buffer, to be obtained, and antibody concentration is 1mg/mL, and antibodies buffer is containing 1% (vol/vol) methanol, pH= 8.0 100mM Tris-HCL solution liquid；Final quantity for spray is 0.025~0.05mL/mm, equivalent to every millimeter T1, T2 or C line Spray 25ng~50ng antibody；
5) liner plate, sample pad or UCP pads, analyzing film and absorption pad are sticked on liner plate after mutually overlapping.
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