CN106872420A - The kit and method of a kind of time-resolved fluorescence quantitative determination microdose urine protein - Google Patents
The kit and method of a kind of time-resolved fluorescence quantitative determination microdose urine protein Download PDFInfo
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- CN106872420A CN106872420A CN201611230229.XA CN201611230229A CN106872420A CN 106872420 A CN106872420 A CN 106872420A CN 201611230229 A CN201611230229 A CN 201611230229A CN 106872420 A CN106872420 A CN 106872420A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The present invention relates to the kit and detection method of a kind of time-resolved fluorescence quantitative determination microdose urine protein.The kit includes time-resolved fluorescence test card, Sample dilution and the SD card containing calibration curve, the time-resolved fluorescence test card includes test strip, the test strips are made up of end liner and the sample pad being pasted onto on end liner, nitrocellulose filter and blotting paper, microballoon line, detection line and nature controlling line are disposed with the nitrocellulose filter, the microballoon line is made up of the time-resolved fluorescence microballoon for being marked with human albumin monoclonal antibody, detection line is coated with human albumin recombinant antigen, and nature controlling line is coated with sheep anti-mouse igg.Kit of the present invention can accurate quantitative analysis detection human urine in microalbumin content, mark microballoon is connected with antibody by covalent bond, marked product stably.With detection range (2~300mg/ml) wide, sensitivity (detection limit 2mg/ml) high, the degree of accuracy are high, detect fast and convenient the features such as.
Description
(1) technical field
The present invention relates to the kit and detection method of a kind of time-resolved fluorescence quantitative determination microdose urine protein.
(2) background technology
Microdose urine protein refers to occur microalbumin in urine.Albumin is normal protein in a kind of blood,
Under normal circumstances, albumin is few in urine for body metabolism, every liter of Analysis of urine albumin no more than 20mg (<20mg/L), so crying
Microalbumin.But meeting when the early stage such as diabetic nephropathy, hypertensive nephropathy compromised kidneys and angiocardiopathy complication etc. occur
The intrinsic cellular damage of kidney is caused, the intrinsic eucaryotic cell structure of kidney and function is changed and is caused albumin content in urine
Raise.Microalbumin just belongs to microalbuminuria in the range of 20~200mg/L in urine, when Microalbunin in urine
When in vain more than 200mg/L, it was demonstrated that patient has a large amount of albumin to spill, in fact it could happen that Hypoproteinemia, can develop into ephrosis, if not
Treatment in time can enter Uremic.Microdose urine protein sees diabetic nephropathy, hypertension, pre-eclampsia, is that kidney is damaged
The early stage sensitive indicator of wound.
Determining the method for microdose urine protein in the prior art mainly has immunoturbidimetry, fluorescence immune chromatography method, enzyme-linked
Immunization, chemoluminescence method, colloidal gold method etc..Wherein colloidal gold method has a quick advantage simple to operate, but sensitivity it is low and
It is quantitative inaccurate；ELISA sensitivity is high, sample size greatly can quantitative determination, but the operating time it is long and automate it is low；It is immune
Turbidimetry and chemoluminescence method are more sensitive, accurate, can be applied to full automatic biochemical apparatus, but need instrument and time-consuming more long, fit
Close treatment great amount of samples, it is impossible to meet the purpose of quick detection；Immunofluorescence technique is by microdose urine protein antibody covalent bond
In on fluorescent microsphere surface active groups, by exciting whether rear detection line produces fluorescence come judged result, rapid and convenient can be accurate
It is determined that amount has the advantages that high sensitivity, label stabilization simultaneously, in medical immunology detection field extensive use.
But many compounds and albumen in biofluid and serum can inherently fluoresce, therefore use tradition
The chromophore such as fluorescein carry out during fluoroscopic examination sensitivity will degradation, but most of background fluorescence signal is to deposit in short-term
, come labelled antibody or antigen as label using rare-earth fluorescent microballoon (lanthanide chelate).Because of lanthanide series
Chelate stoke displacements are easy to differentiate exciting light and launching light greatly, so that exciting light interference is excluded, and decay time is long leads to
Cross time resolution and eliminate background fluorescence interference, in addition lanthanide chelate exciting light spectrum width and emission spectrum is narrow can further carry
High sensitivity and reduction background fluorescence.Therefore time-resolved fluoroimmunoassay technology is used, while two ginsengs of Detection wavelength and time
Number carries out signal resolution, can effectively exclude the interference of non-specific fluorescence, drastically increases sensitivity for analysis.
(3) content of the invention
The present invention seeks to provide a kind of using time-resolved fluoroimmunoassay chromatography with defect in view of the shortcomings of the prior art
The sensitivity of technology, with reference to dry type immunofluorescence analysis instrument realize high sensitivity, can accurate quantitative analysis, simple and efficient time resolution
The kit and detection method of fluorogenic quantitative detection microdose urine protein.
The technical solution adopted by the present invention is：
A kind of kit of time-resolved fluorescence quantitative determination microdose urine protein, including time-resolved fluorescence test card,
Sample dilution and the SD card containing calibration curve；
The time-resolved fluorescence test card includes test strip, and the test strips are by end liner and are pasted onto on end liner
Sample pad, nitrocellulose filter and blotting paper composition, the sample pad, nitrocellulose filter and blotting paper sequentially overlap stickup
On end liner；
Microballoon line, detection line and nature controlling line, microballoon line, detection line and Quality Control are disposed with the nitrocellulose filter
Line is parallel to each other, and spacing distance is 3~5mm, wherein microballoon line near well, nature controlling line away from well, the microballoon line
It is made up of the time-resolved fluorescence microballoon for being marked with human albumin monoclonal antibody, it is anti-that detection line is coated with human albumin restructuring
Original, nature controlling line is coated with sheep anti-mouse igg antibody.
Doped with the rare-earth europium element of solid content 1% (w/w), and microspherulite diameter is the time-resolved fluorescence microballoon
100nm~300nm, the stabilization under ground state, launch under the excitation source effect of 350~380nm wave-length coverage 600~
The fluorescence of 620nm.
The content of human albumin monoclonal antibody is 50~200 μ g antibody/200 μ l fluorescent microspheres, detection line in microballoon line
Middle human albumin recombinant antigen coating concentration is 0.5~2mg/ml, consumption for 0.5~1.5 μ l are coated with liquid measure/cm films, nature controlling line
Middle sheep anti-mouse igg antibody coating concentration is 0.5~2mg/ml, consumption for 0.5~1.5 μ l are coated with liquid measure/cm films.
The test strip is prepared by the following method：
(1) activation of time-resolved fluorescence microballoon
After ultrasonication fluorescent microsphere 2min, after taking 200 μ l fluorescent microspheres with 5~15min of 14000rpm high speed centrifugations,
With 10~100mM, pH is that 5.0~7.0 MES solution is washed to 1ml, ultrasonication 2min to sediment；Add 10~100 μ l
20~100mg/ml carbodiimides, mix 5~10min, add 20~100mg/ml N- hydroxies of 50~200 μ l
Succinimide, mixes 14000rpm 5~15min of high speed centrifugation after 10~20min, precipitates molten with the MES that pH is 5.0~7.0
Liquid is washed to 1ml；
(2) time-resolved fluorescence microballoon marks the preparation of human albumin monoclonal antibody
After fluorescent microsphere ultrasonication 2min after above-mentioned activation human albumin is added according to 50~200 μ g/200 μ l
Monoclonal antibody, mixes 1~3 hour, is closed with 10~50mM containing 0.5%BSA, 7.5~8.5Tris-HCl of pH confining liquids
5~15min of 14000rpm high speed centrifugations after 0.5~1 hour, with containing 1%Nacl, 0.5%BSA, 0.1%Tween20 10~
50mM, pH7.5~8.5Tris-Hcl preserve liquid washing and resuspended are kept in dark place in 4 DEG C to 200 μ l.
(3) preparation of coated film
Coating buffer solution (the 10mM PBSs containing 2.5% sucrose) is used respectively by human albumin recombinant antigen and goat-anti
Mouse IgG antibody adjusts concentration to 0.5~2mg/ml, and consumption is that 0.5~1.5 μ l are coated with liquid measure/cm films, respectively as detection line and
Parallel stroke of nature controlling line in being coated with nitrocellulose filter, with microballoon dilution by mark human albumin monoclonal antibody when
Between the dilution of 3~6 times of resolved fluorometric microballoon, consumption is that 0.5~1.5 μ l are coated with liquid measure/cm films, is drawn in cellulose nitrate as microballoon line
It is coated near sample pad direction on plain film, nature controlling line, detection line and microballoon line are spaced 3~7mm, are placed in baking oven, 45 DEG C
Drying is overnight.
(4) sequentially mutually pasting sample pad, coated film and blotting paper obtains test paper plate overlap joint on end liner, and cutting is obtained
The test strips.
The time-resolved fluorescence test card is fixed on plastic bottom card by test strip, test paper surface card face pressure
Tightly, and card face the part of counter sample pad and nitrocellulose filter respectively reserve well and observation window.
The Sample dilution composition is as follows：NaCl 0.5%, BSA 0.5%, Tween 1%, solvent be 10~50mM,
PH7.5~8.5Tris-HCl buffer solutions, are sub-packed in centrifuge tube with 270 μ l/ pipes.Sample buffer is used for chromatography samples.
The SD card containing standard curve, is the calibration object that various concentrations are determined by time-resolved fluorescence test strips,
With calibration object concentration as abscissa, using fluorescence signal ratio as ordinate, standard curve is depicted as, writes and generate corresponding two
Dimension code information Store is obtained in SD card.Corresponding 2 D code information on reagent card can read by dry type fluorescence immunity analyzer
And measure respective concentration.
The invention further relates to the method for the quantitative determination microdose urine protein using the kit, methods described includes：
(1) detection kit and sample are placed at room temperature, it is to be restored to using after room temperature；
(2) dry type fluorescence immunity analyzer is opened, correspondence SD card is inserted after preheating 5min；
(3) during 30 μ L of accurate absorption urines to be measured add Sample dilution, mix；
(4) the μ L of sample 80 after mixing are drawn, is added in detection card well；
(5) by detection card insertion detect tank, detected after 10min and read and print testing result.Detection range 2~
300mg/L。
The Cleaning Principle of the kit of time-resolved fluorescence quantitative determination microdose urine protein of the present invention is competition law,
The content of microalbumin in detection human urine.Urine specimen dilution containing microdose urine protein is added dropwise in sample application zone,
Chromatographed to nitrocellulose filter microballoon line by capillarity, resisted with the human albumin monoclonal that time-resolved fluorescence microballoon is marked
Body is combined, and forms microballoon Antibody-antigen complex, chromatography to detection zone, the human albumin recombinant antigen fixed by detection line and
The human albumin monoclonal antibody competition binding that microalbumin is marked with microballoon in sample, therefore albumin is more in sample,
The microballoon antibody combined at detection line is fewer, and unnecessary Fluorescent microsphere marker continues to chromatograph forward, and is fixed on nature controlling line
Sheep anti mouse is combined.After reaction terminates, with ultraviolet source (365nm) to detection zone Scanning Detction, detection line and Quality Control time on the wire
Resolved fluorometric microballoon sends fluorescence (615nm), and its decay time is also more long.Using time of measuring is delayed, treat in sample substrate from
The short life fluorescence (1~10ns) for so occurring is all after decay, then measures the specificity fluorescent of rare earth element, thus can be with complete
The interference of special background fluorescence is excluded entirely.By detection line and the power and its ratio of nature controlling line fluorescence intensity, you can analyze
The concentration of determinand in sample.
The beneficial effects are mainly as follows：Kit of the present invention can be micro white in accurate quantitative analysis detection human urine
The content of albumen, using time-resolved fluorescence microballoon detection technique, fluorescence is disturbed in itself can to avoid sample, and mark passes through covalent bond
Microballoon is connected with antibody, marked product stabilization, with detection range (2~300mg/L) wide, sensitivity (detection limit 2mg/ high
L the features such as), the degree of accuracy is high, detection is fast and convenient.
(4) illustrate
Fig. 1 be time-resolved fluorescence quantitative determination microdose urine protein of the invention kit in test strip knot
Structure schematic diagram；
Wherein：1st, sample pad；2nd, coated film；3rd, blotting paper；4th, microballoon line；5th, detection line；6th, nature controlling line；7th, end liner.
Fig. 2 is the kit standard curve prepared by the embodiment of the present invention 1.
Fig. 3 is the kit and Siemens's immunoturbidimetry detection microdose urine protein examination prepared by the embodiment of the present invention 1
Agent box testing result correlation compares.
(5) specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1：The preparation of time-resolved fluorescence quantitative determination microdose urine protein kit
Time-resolved fluorescence quantitative determination microdose urine protein kit, using competition law immunochromatography principle, detects people
Microalbumin in urine.SD card by time-resolved fluorescence test card, Sample dilution and containing calibration curve is constituted.
As shown in figure 1, sequentially overlapping sample pad 1, coated film 2 and blotting paper 3, sample in this embodiment, on end liner 7
Pad 1 is sample application zone, and for drawing urine detection sample to be measured, coated film 2 is provided with microballoon line, detection line and nature controlling line.
Rare-earth europium on coated film 2 on microballoon line using specific exciting light (365nm)/launching light (615nm) wavelength is glimmering
(100 μ g antibody/200 μ l fluorescence are micro- for light microballoon (diameter about 200nm, europium solid content 1%) mark human albumin monoclonal antibody
Ball), detection line is coated with human albumin recombinant antigen (1mg/ml), and nature controlling line coating concentration is the sheep anti-mouse igg antibody of 1mg/ml
(from Bangs Lab companies of the U.S., the buying of human albumin monoclonal antibody is visited from Chongqing and given birth to for wherein time-resolved fluorescence microballoon buying
Science and Technology Ltd., human albumin recombinant antigen buying comes from the Tai Ke Bioisystech Co., Ltd of Luoyang one hundred, sheep anti-mouse igg antibody
From Changsha Bo You bio tech ltd).Microballoon line, detection line and nature controlling line consumption are that 1 μ l are coated with liquid measure/cm films.
In this embodiment, the preparation of the kit of time-resolved fluorescence quantitative determination microdose urine protein includes following step
Suddenly：
1. the activation of time-resolved fluorescence microballoon
After ultrasonication fluorescent microsphere 2min, after taking 200 μ l fluorescent microspheres with 14000rpm high speed centrifugations 15min, sink
Starch 100mM, pH are that 6.0 MES solution is washed to 1ml, ultrasonication 2min；Add the 100mg/ml carbon two of 50 μ l sub-
Amine, mixes 5min, adds the 100mg/ml N- hydroxy thiosuccinimides of 100 μ l, and 14000rpm is high after mixing 15min
Speed centrifugation 15min, precipitation is washed to 1ml with the MES solution that pH is 6.0.
2. time-resolved fluorescence microballoon marks the preparation of human albumin monoclonal antibody
After fluorescent microsphere ultrasonication 2min after above-mentioned activation human albumin Dan Ke is added according to 100 μ g/200 μ l
Grand antibody, mixes 2 hours, and 14000rpm is high after being closed 1 hour with 50mM, pH8.0Tris-Hcl confining liquid containing 0.5%BSA
Speed centrifugation 15min, delays in preserving liquid with the Tris-HCl of 50mM, pH8.0 containing 1%Nacl, 0.5%BSA, 0.1%Tween20
Fliud flushing is washed twice and ultrasonically treated resuspended kept in dark place in 4 DEG C to 200 μ l.
3. the preparation of coated film
Coating buffer solution (the 10mM PBSs containing 2.5% sucrose) is used respectively by human albumin recombinant antigen and goat-anti
To 1mg/ml, consumption is 1 μ l coating liquid measure/cm films to mouse IgG antibody adjustment concentration, respectively as parallel with nature controlling line stroke of detection line
In being coated with nitrocellulose filter, will with microballoon dilution (the 10mM PBSs containing 0.5%BSA and 20% sucrose)
5 times of dilutions of time-resolved fluorescence microballoon of human albumin monoclonal antibody are marked, consumption is that 1 μ l are coated with liquid measure/cm films, used as micro-
Ball line is drawn in being coated near sample pad direction on nitrocellulose filter, and nature controlling line, detection line and microballoon line are spaced 4mm,
Humidity<30%, dried in temperature 45 C baking oven 10 hours, envelope is standby；
4. sequentially mutually paste sample pad on end liner (size is 80*300mm) (size is 30*300mm, glass overlap joint
Glass cellucotton material), coated film (size is 25*300mm, nitrocellulose material) and blotting paper (size is 28*300mm)
To test paper plate, the test strips of 4mm width are cut into as requested.
The time-resolved fluorescence test card of present invention detection microdose urine protein includes test strip, when in use, examination
Paper slip is assembled in the plastic casing fastened by plastics upper casing and plastics lower casing, and plastics upper casing is provided with two perforates, sample-adding
Hole and observation window, well correspond to the test strips sample pad 1 of described detection microdose urine protein, and as a result observation window corresponds to
The detection line 5 and nature controlling line 6 of the test strips of the detection microdose urine protein, the detection detect the test strips of microdose urine protein
Can be taken out from the plastic casing.
It is containing 0.5%Nacl, 0.5%BSA and 1%Tween also including Sample dilution in kit of the present invention
50mM, pH8.0Tris-Hcl buffer solution, are sub-packed in centrifuge tube with 270 μ l/ pipes.Sample buffer is used for chromatography samples.
In kit of the present invention, per SD card (with batch standard curve identical) of the box containing standard curve, by the time
Resolved fluorometric test strips determine the calibration object of various concentrations, with calibration object concentration as abscissa, using fluorescence signal ratio as vertical
Coordinate, is depicted as standard curve, writes SD card and generates Quick Response Code, be can read on reagent card by dry type fluorescence immunity analyzer
Corresponding 2 D code information simultaneously measures respective concentration.
Embodiment 2：The quantitative determination of time-resolved fluorescence quantitative determination microdose urine protein kit
1. the drafting of standard curve
The time-resolved fluorescence test card prepared by embodiment 1 adds various concentrations albumin antigen quality-control product
(302.5mg/L, 247.5mg/L, 192.5mg/L, 137.5mg/L, 82.5mg/L, 38.5mg/L, 11.0mg/L, 2.0mg/L are common
Eight concentration, each concentration sets three repetitions, is obtained with 0.85% normal saline dilution by 10mg/ml human albumins recombinant antigen
To), Sample dilution is added, after chromatography 10min, the AFS-1000 types produced by Guangzhou Lan Bo bio tech ltd are done
Formula fluorescence immunity analyzer reads C, T line fluorescence signal and C/T values, experimental result and analysis in table 1：
LOG values are taken with microalbumin quality-control product concentration and sample signal T/C average values take LOG values and draw standard curve,
Curve data is shown in Table 1, and standard curve is as shown in Figure 2.Wherein R values are 0.9943, by the graticule to contained micro white in sample
Protein concentration carries out concentration quantitative measure.
2. time-resolved fluorescence test card performance test.
Minimum detectability：Replication is carried out with null value sample 20 times, calculates 20 the average M and standard deviation SD of result,
Add the test limit (M+2SD) of twice standard deviation method for reporting with blank average, be as a result 1.63mg/L, meet sensitivity 2mg/L.
The range of linearity：Eight concentration values between 2~300mg/L are taken, each concentration duplicate measurements three times will determine concentration and put down
Average carries out linear analysis in theoretical concentration, obtains linear equation y=1.0199x+0.2911, r=0.9965, shows the present invention
Kit correlation in 2~300mg/L ranges of linearity is fine.
Precision：Made time-resolved fluorescence quantitative determination microdose urine protein kit in three batches of embodiments 1 is taken, respectively
Detection 247.5,38.5mg/L repeatability quality-control product, every batch of kit uses repeated quality-control product Parallel testing 10 times, 247.5mg/L
CV is respectively 5.25%, 7.05%, 5.33% in concentration three batches batches, and CV is 6.17%, 38.5mg/L concentration three batches between three batches batches
CV is respectively 8.31%, 9.55%, 9.71% in batch, and CV is 9.97% between three batches batches, within 10%.
The degree of accuracy:Selection concentration is detection sample for the Quality Control thing of 22mg/L, and it is identical 3 parts to be divided into volume, wherein 2 parts of samples
The degree of accuracy quality-control product of 220mg/L and 38.5mg/L is separately added into this, 2 recovery samples of different addition concentration, meter are made
Calculate the concentration of the determinand for adding.The solution without measured object of same amount is added in another sample, basic sample is made.It is right
Sample carries out 3 replicate analysis, takes its average and is calculated.The calculating rate of recovery=(analysis sample concentration-basis sample concentration)/
Add concentration × 100%.The rate of recovery of sample 220mg/L is reclaimed for the rate of recovery of 106.03%, 28.5mg/L is 97.58%,
Average recovery rate is 90.92%.Deviation is within 10%.
3. clinical sample detection
200 parts of the urine specimen of microdose urine protein detects in collection hospital, immune with kit of the invention and Siemens
Two methods of turbidimetry detection microdose urine protein kit carry out detection comparing.In kit of the present invention, take the μ l of urine 30 and add
Enter Sample dilution, 80 μ l are taken after mixing and is added in detection card well, by the blue vigorous biotechnology in Guangzhou after chromatography 10min
The AFS-1000 type dry types fluorescence immunity analyzer of Co., Ltd's production reads concentration, and same urine uses comparison system Siemens
Immunoturbidimetry detection microdose urine protein kit carries out Concentration Testing.Two methods testing result carries out linear analysis, such as
Shown in Fig. 3, its correlation fine r=0.9893, P>0.05, mean relative deviation is less than 10%, and as a result meeting clinical analysis will
Ask, be suitable for clinical detection.
Claims (6)
1. a kind of kit of time-resolved fluorescence quantitative determination microdose urine protein, including time-resolved fluorescence test card, sample
This dilution and the SD card containing calibration curve, it is characterised in that：
The time-resolved fluorescence test card includes test strip, and the test strip is by end liner and is pasted onto on end liner
Sample pad, nitrocellulose filter and blotting paper composition, the sample pad, nitrocellulose filter and blotting paper sequentially overlap stickup
On end liner；
Microballoon line, detection line and nature controlling line, microballoon line, detection line and nature controlling line phase are disposed with the nitrocellulose filter
Mutually parallel, spacing distance is 3~5mm, and wherein, near well, nature controlling line is away from well for microballoon line；
The microballoon line is made up of the time-resolved fluorescence microballoon for being marked with human albumin monoclonal antibody；The time resolution is glimmering
Doped with the rare-earth europium element of solid content 1%, particle diameter is 100nm~300nm to light microballoon；The content of human albumin monoclonal antibody
It is 50~200 μ g antibody/200 μ l fluorescent microspheres；
The detection line is coated with human albumin recombinant antigen, and nature controlling line is coated with sheep anti-mouse igg antibody；Human albumin restructuring is anti-
Primordial covering concentration is 0.5~2mg/ml, consumption is that 0.5~1.5 μ l are coated with liquid measure/cm films, and sheep anti-mouse igg antibody coating concentration is
0.5~2mg/ml, consumption are that 0.5~1.5 μ l are coated with liquid measure/cm films.
2. kit as claimed in claim 1, it is characterised in that the test strip is prepared by the following method：
(1) activation of time-resolved fluorescence microballoon
After ultrasonication fluorescent microsphere 2min, after taking 200 μ l fluorescent microspheres with 5~15min of 14000rpm high speed centrifugations, precipitation
With 10~100mM, pH is that 5.0~7.0 MES solution is washed to 1ml, ultrasonication 2min to thing；Add the 20 of 10~100 μ l
~100mg/ml carbodiimides, mix 5~10min, add 20~100mg/ml N- hydroxy ambers of 50~200 μ l
Acid imide, mixes 14000rpm 5~15min of high speed centrifugation after 10~20min, and precipitation is washed with the MES solution that pH is 5.0~7.0
Wash to 1ml；
(2) time-resolved fluorescence microballoon marks the preparation of human albumin monoclonal antibody
After fluorescent microsphere ultrasonication 2min after above-mentioned activation human albumin Dan Ke is added according to 50~200 μ g/200 μ l
Grand antibody, is mixed 1~3 hour, and 0.5~1 is closed with 10~50mM containing 0.5%BSA, pH7.5~8.5Tris-Hcl confining liquids
Hour after 14000rpm 5~15min of high speed centrifugation, with 10~50mM containing 1%Nacl, 0.5%BSA, 0.1%Tween20,
PH7.5~8.5Tris-Hcl preserves liquid washing and resuspended is kept in dark place in 4 DEG C to 200 μ l；
(3) preparation of coated film
Human albumin recombinant antigen and sheep anti-mouse igg antibody are adjusted into concentration to 0.5~2mg/ml with coating buffer solution respectively, is used
It is 0.5~1.5 μ l coating liquid measure/cm films to measure, respectively as parallel with nature controlling line stroke of detection line in being carried out on nitrocellulose filter
Coating, 3~6 times of dilutions of time-resolved fluorescence microballoon of human albumin monoclonal antibody will be marked with microballoon dilution, and consumption is
0.5~1.5 μ l are coated with liquid measure/cm films, are drawn as microballoon line and are coated with close sample pad direction on nitrocellulose filter, matter
Control line, detection line and microballoon line are spaced 3~7mm, are placed in baking oven, and 45 DEG C of drying are overnight；
(4) sample pad, coated film and blotting paper are sequentially mutually pasted on end liner obtains test paper plate to overlap joint, cutting obtains described
Test strips.
3. kit as claimed in claim 1 or 2, it is characterised in that the time-resolved fluorescence test card is by Test paper
Bar is fixed on plastic bottom card, and test paper surface is tight with card face pressure, and card face is in counter sample pad and the part of nitrocellulose filter
Well and observation window are reserved respectively.
4. kit as claimed in claim 1 or 2, it is characterised in that the Sample dilution composition is as follows：NaCl
0.5%, BSA 0.5%, Tween 1%, solvent is 10~50mM, pH7.5~8.5Tris-HCl buffer solutions, is managed with 270 μ l/
It is sub-packed in centrifuge tube.
5. kit as claimed in claim 1 or 2, it is characterised in that the SD card containing standard curve, is by the time
Resolved fluorometric test strips determine the calibration object of various concentrations, with calibration object concentration as abscissa, using fluorescence signal ratio as vertical
Coordinate, is depicted as standard curve, writes and generates respective two-dimensional code information Store and obtained in SD card.
6. the method for utilizing the quantitative determination microdose urine protein of kit described in claim 1, methods described includes：
(1) detection kit and sample are placed at room temperature, it is to be restored to using after room temperature；
(2) dry type fluorescence immunity analyzer is opened, correspondence SD card is inserted after preheating 5min；
(3) during 30 μ L of accurate absorption urines to be measured add Sample dilution, mix；
(4) the μ L of sample 80 after mixing are drawn, is added in detection card well；
(5) by detection card insertion detect tank, detected after 10min and read and print testing result.
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| CN108195815A (en) * | 2018-01-31 | 2018-06-22 | 李翀 | Time-resolved fluoroimmunoassay chromatography detects test strips, kit and the method for AMH |
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