CN109975557A - IL-6/PCT joint-detection time resolution detection kit and method - Google Patents
IL-6/PCT joint-detection time resolution detection kit and method Download PDFInfo
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Abstract
The present invention relates to a kind of sensitivitys using time-resolved fluoroimmunoassay chromatographic technique, using rare-earth vanadate nano fluorescent marker material, in conjunction with dry type immunofluorescence analysis instrument realize it is highly sensitive, can accurate quantitative analysis, simple and efficient IL-6/PCT joint-detection time-resolved fluoroimmunoassay chromatography immue quantitative detection reagent box and detection method.Detection kit of the present invention can accurate quantitative analysis detect human serum, the content of interleukin 6 and Procalcitonin in blood plasma and whole blood sample, sample can be achieved without pretreatment and diluted by the processing of blood filter membrane sample pad, utilize time-resolved fluorescence microballoon detection technique, it can avoid the interference of sample fluorescence itself, rare-earth vanadate nano material has background low, fluorescence signal is strong, the advantages such as signal-to-noise ratio height, rare-earth vanadate nano particle is connect by label by covalent bond with antibody, marked product is stablized, it is wide with detection range, high sensitivity, accuracy is high, detect the features such as fast and convenient.
Description
(1) technical field
The present invention relates to a kind of IL-6/PCT joint-detection time-resolved fluorescence detection kit and methods.
(2) background technique
Interleukin 6 (IL-6) is a kind of cell factor, belongs to one kind of interleukins, and participation can be stimulated to be immunoreacted
Cell Proliferation, differentiation and improve its function.IL-6 and its receptor and diseases associated with inflammation it is related, it is multi-functional inflammatory cell
The factor plays an important role in inflammatory reaction.IL-6 level rises the early stage index that can be used as inflammatory reaction, and can be used for assessing
The severity (SIRS) of Systemic inflammatory syndrome.After inflammatory reaction occurs, the raising of IL-6 serum-concentration earlier than
Other biological marker, concentration induce the generation of PCT and CRP after increasing.The concentration level of IL-6 is early in infection in patients serum
Phase quickly increases, and reaches peak value in 2 hours, more sensitive compared to PCT and CRP, in infection early stage more favorable clinical prediction, dynamic
Observation IL-6 level helps to understand progression of disease and confirms curative effect.Meanwhile IL-6 is also the excellent of assessment sepsis patient prognosis
Elegant marker.
Procalcitonin is a kind of functional protein, is the intermediate product in calcitonin synthesis process, is the drop of no hormonal activity
Peptide material before calcium element.Human normal plasma PCT content is extremely low, in normal person's blood be lower than 0.5ng/ml, when serious bacterial, fungi,
Its horizontal in blood plasma increases when parasitic infection and pyemia and multiple organ failure.PCT reflects systemic inflammatorome
The active degree of reaction is the specific index of serious bacterial inflammation and fungal infection, and is also that pyemia and inflammation are living
Move the reliability index of related multiple organs failure.
In pyemic full course of disease management, IL-6 and PCT joint-detection can learn from other's strong points to offset one's weaknesses, and improve the accurate of diagnosis
Rate.IL-6 advantage in terms of early warning and assessment prognosis is prominent, and PCT is in auxiliary diagnosis, assessment detection antibiotic curative effect side
Face is highly effective.Joint-detection IL-6 and PCT can improve the morning of infection to avoid single index to the error of infection classification judgement
Phase diagnosis helps the clinical therapeutic scheme for determining patient in time, has important clinical value.
Inflammation infection and pyemia detection are detected only for single inflammation index in the prior art, and in addition IL-6 is examined
Test paper slip is less, and the method for measuring IL-6/PCT mainly has immunoturbidimetry, fluorescence immune chromatography method, colloidal gold method etc..
Wherein colloidal gold method has the advantages that easy to operate quick, but sensitivity is low and quantitative inaccurate；Immunoturbidimetry is more sensitive,
Accurately, it can be applied to full automatic biochemical apparatus, but need instrument and take a long time, be suitble to processing great amount of samples, be unable to satisfy quickly
The purpose of detection；Immunofluorescence technique is that IL-6/PCT antibody is covalently bonded on fluorescent microsphere surface active groups, by swashing
Whether detection line generates fluorescence and carrys out judging result after hair, rapid and convenient can accurate quantitative analysis have that highly sensitive, marker is steady simultaneously
The advantages that determining is widely applied in medical immunology detection field.
However many compounds and albumen in biofluid and serum can inherently fluoresce, therefore use tradition
The chromophories such as fluorescein carry out when fluorescence detection sensitivity will degradation, but most of background fluorescence signal is to deposit in short-term
, come labelled antibody or antigen as marker using rare earth nano material (lanthanide chelate).Rare earth nano material
Material is a kind of widely used biomarker, and compared with conventional tag object, rare earth mixing with nano material has stable physics
The advantages that chemical property, narrow emission, long-life, can eliminate background fluorescence by time resolution to exclude exciting light interference
Interference, is not easy affected by environment during biomarker.Vanadate is larger in the absorption cross-section of deep ultraviolet, is excellent glimmering
Signal material, rare earth ion doped vanadate luminescent material, can be in differences since rare earth ion has energy level abundant
Transition between energy level greatly improves the optical property of rare-earth vanadate nano material.Therefore rare-earth vanadate nano fluorescent is used
Marker material is as marker, and using time-resolved fluoroimmunoassay technology, while two parameters of Detection wavelength and time carry out letter
Number differentiate, can effectively exclude the interference of non-specific fluorescence, greatly improve sensitivity for analysis.
(3) summary of the invention
It is an object of the present invention to provide a kind of sensitivitys using time-resolved fluoroimmunoassay chromatographic technique, utilize rare earth vanadic acid
Salt nano fluorescent marker material, in conjunction with dry type immunofluorescence analysis instrument realize it is highly sensitive, can accurate quantitative analysis, simple and efficient
IL-6/PCT joint-detection time-resolved fluoroimmunoassay chromatographs immue quantitative detection reagent box and method.
The technical solution adopted by the present invention is that:
A kind of IL-6/PCT joint-detection time-resolved fluorescence detection kit, including time-resolved fluorescence test card and contain
There is the ID card of calibration curve:
The time-resolved fluorescence test card includes test strip, and the test strips are by bottom liner and are pasted on bottom liner
Blood filter membrane sample pad, glass fiber conjugate pad, nitrocellulose filter and blotting paper composition, the blood filter membrane sample pad, glass
Fiber conjugate pad, nitrocellulose filter and blotting paper are sequentially overlapped and are pasted on bottom liner；
The sample pad treatment fluid comprising heterophile antibody blocking agent, glass fibre knot are coated at the blood filter membrane sample pad
It closes and is provided with microballoon line at pad, be coated with monoclonal antibody IL-6 1, the monoclonal antibody PCT of time-resolved fluorescence microballoon label
1 and rabbit igg antibody (content is respectively 50~200 μ g antibody/200 μ l fluorescent microspheres), the time-resolved fluorescence microballoon is
(group becomes LnVO to rare-earth vanadate particle4: xEu, preferably GdVO4: 30%Eu), LnVO4For matrix, wherein Ln be lanthanum, yttrium,
One of gadolinium, samarium, terbium are a variety of, and matrix is preferably GdVO4, Doped ions are europium (Eu3+), colon ": " is expressed as europium doping；
X is mole percent, and range is 5%~50%, preferably 30%, partial size is 100nm~300nm；The time-resolved fluorescence
Microballoon is stablized under ground state, launches wave-length coverage in the glimmering of 600~620nm under the excitation light source effect of 250~350nm
Light；
Detection line 1,2 and nature controlling line are disposed on the nitrocellulose filter, detection line 1,2 and nature controlling line wrap respectively
By monoclonal antibody IL-6 2, monoclonal antibody PCT2 and goat anti-rabbit antibody, detection line 1,2 and nature controlling line are parallel to each other, interval
Distance is 3~5mm, and wherein detection line 2 is close to bonding pad, and nature controlling line is far from bonding pad；Detection line 1 is monoclonal antibody IL-6
2, peridium concentration is 0.3~2mg/ml, dosage is 0.5~1.5 μ l coating liquid measure/cm film, and detection line 2 is monoclonal antibody
PCT2, peridium concentration are 0.3~2mg/ml, dosage is 0.5~1.5 μ l coating liquid measure/cm film, and goat anti-rabbit igg is anti-in nature controlling line
Body peridium concentration is 0.3~2mg/ml, dosage is 0.5~1.5 μ l coating liquid measure/cm film；
The test strip is prepared by the following method:
(1) preparation method of rare-earth vanadate nano fluorescent marker material
In 50mL round-bottomed flask, example in molar ratio is added 2 parts of distilled water, the nitrate or acetate of (1-x) part Ln or
Chloride, the nitrate or acetate or chloride, x of x parts of Eu is mole percent, and range is 5%~50%, 1 part of ortho-vanadic acid,
(1~5 part) citric acid or sodium citrate adjust pH value 6~8 with sodium hydroxide, are passed through nitrogen, and 85 DEG C are reacted 24 hours,
10000rpm centrifugation, is washed with distilled water 3 times, is dried to obtain the water-soluble LnVO4:x Eu namo fluorescence probe of good dispersion.
LnVO4 is that matrix is preferably GdVO4, and Doped ions are europium (Eu3+), and mole percent is preferably 30%.The GdVO4:
30%Eu material structure is pure tetragonal phase structure, and size is about 250nm and pattern is uniform, good luminous performance.
(2) activation of time-resolved fluorescence microballoon
After ultrasonication fluorescent microsphere 2min, take 200 μ l fluorescent microspheres with 5~15min of 14000rpm high speed centrifugation after,
10~100mM of sediment, the MES solution that pH is 5.0~7.0 are washed to 1ml, ultrasonication 2min；10~100 μ l are added
20~100mg/ml carbodiimide, mix 5~10min, add 20~100mg/ml N- hydroxy of 50~200 μ l
Succinimide, mixes 14000rpm 5~15min of high speed centrifugation after 10~20min, and precipitating is molten with the MES that pH is 5.0~7.0
Liquid is washed to 1ml；
(3) time-resolved fluorescence microballoon marks IL-6, PCT monoclonal antibody IL-6 1, PCT1 and rabbit igg antibody respectively
Preparation
IL- is separately added into according to 50~200 μ g/200 μ l after the fluorescent microsphere ultrasonication 2min after above-mentioned activation
6, PCT monoclonal antibody IL-6 1, PCT1 and rabbit igg, mix 1~3 hour, with 10 containing 0.5%BSA, 0.1% glycine~
14000rpm 5~15min of high speed centrifugation after 50mM, pH 7.5~8.5Tris-HCl confining liquid are closed 0.5~1 hour, with containing
1%Nacl, 0.5%BSA, 0.5% sucrose, 10~50mM of 0.1%Tween20, pH7.5~8.5Tris-Hcl save liquid and wash
It washs and is resuspended to 200 μ l and be kept in dark place in 4 DEG C.
(4) preparation of sample pad
With sample pad treatment fluid (blocking agent of heterophile antibody containing 1mg/ml, 0.5%NaCl, 0.5%Twee20,0.1%
The Tris-HCl of 20mM, pH8.0 of BSA) parallel even application two lines, dosage are 2~4 μ l liquid measures/cm sample on the blood filtration membrane
Product pad.It is placed in baking oven, 37 DEG C of drying are overnight.
(5) preparation of bonding pad
IL-6, PCT monoclonal antibody IL-61, the PCT1 and rabbit igg for marking time-resolved fluorescence microballoon on bonding pad
Antibody dilutes 8~30 times of even applications with microballoon dilution (the 20mMTris-Hcl buffer containing 0.5%BSA, 25% sucrose)
One line, dosage are 2~4 μ l liquid measures/cm sample pad.It is placed in baking oven, 37 DEG C of drying are overnight.
(6) preparation of coated film
Use coating buffer (the 10mM PBS buffer solution containing 2.5% sucrose) by IL-6, PCT monoclonal antibody IL-6 respectively
2, PCT2 and goat anti-rabbit igg antibody adjust concentration to 0.3~2mg/ml, and dosage is that 0.5~1.5 μ l is coated with liquid measure/cm film, respectively
It draws in being coated on nitrocellulose filter as detection line 1,2 is parallel with nature controlling line, nature controlling line, detection line and microballoon line interval
3~7mm is placed in baking oven, and 37 DEG C of drying are overnight.
(7) sequentially mutually overlap joint blood filter membrane sample pad, glass fiber conjugate pad, coated film and water suction are pasted on bottom liner
Paper obtains test paper plate, and cutting obtains 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 well and observation window in the part of counter sample pad and nitrocellulose filter are reserved in card face respectively.
The ID card containing standard curve is the calibration object that various concentration is measured by time-resolved fluorescence test strips,
Using calibration object concentration as abscissa, using fluorescence signal ratio as ordinate, it is depicted as standard curve, be written and generates corresponding two
Dimension code information, which is stored in ID card, to be obtained.Corresponding two-dimensional barcode information on reagent card can be read by dry type fluorescence immunity analyzer
And measure respective concentration.
The invention further relates to the methods using kit quantification detection IL-6/PCT, which comprises
(1) it will test kit and sample be placed at room temperature, it is to be restored to using after room temperature；
(2) dry type fluorescence immunity analyzer is opened, is inserted into corresponding ID card after preheating 5min；
(3) 80 μ L blood to be measured are accurately drawn to be added in detection card well；
(4) it will test card insertion and enter detection slot, detected after 15min and read and print testing result.
Kit detection range PCT of the present invention is 0.05~100ng/mL, and IL-6 is 1.5~1000pg/mL.
The testing principle of this kit is double antibody sandwich method, IL-6 and PCT in detection human serum, blood plasma and whole blood sample
Content.Blood sample containing IL-6 and PCT is added dropwise in sample application zone, and blood filter membrane may filter that in whole blood in red blood cell and sample
Granule foreign simultaneously blocks non-specific binding albumen in sample, is chromatographed through capillary action after filtering to bonding pad, with the time point
It distinguishes that IL-6, PCT monoclonal antibody IL-6 1, the PCT1 of fluorescent microsphere label are combined, forms microballoon Antibody-antigen complex, layer
Analysis to detection zone on nitrocellulose filter, react compound be detected the coated IL-6 and PCT monoclonal antibody IL-6 2 of survey line,
PCT2 capture, and the continuation of extra Fluorescent microsphere marker chromatographs forward, rabbit igg be fixed in conjunction with nature controlling line goat-anti rabbit.Instead
After answering, with ultraviolet source (285nm) to detection zone Scanning Detction, time-resolved fluorescence microballoon hair in detection line and nature controlling line
Fluorescence (618nm) out, decay time is also longer.Using detection time is delayed, to short life abiogenous in sample substrate
After fluorescence (1~10ns) all decays, then the specificity fluorescent of rare earth element is measured, can thus exclude special background completely
The interference of fluorescence.By the power and its ratio of detection line and nature controlling line fluorescence intensity, determinand in sample can be analyzed
Concentration.
The beneficial effects are mainly reflected as follows: the present invention detects card and kit being capable of accurate quantitative analysis detection people's blood
Clearly, in blood plasma and whole blood sample IL-6 and PCT content, sample utilizes the time point without carrying out pretreatment and diluted
It distinguishes fluorescent microsphere detection technique, can avoid the interference of sample fluorescence itself, microballoon is connect by label by covalent bond with antibody, is marked
Product is stablized, and has detection range wide (PCT is 0.05~100ng/mL, and IL-6 is 1.5~1000pg/mL), high sensitivity
(PCT detection limit 0.05ng/mL, IL-6 detection is limited to 1.5pg/mL), accuracy is high, detects the features such as fast and convenient.
(4) Detailed description of the invention
Fig. 1 is test strip in the kit of time-resolved fluorescence quantitative detection IL-6/PCT joint-detection of the invention
Structural schematic diagram；
Wherein: 1, sample pad；2, bonding pad；3, coated film；4, blotting paper；5, detection line 2；6, detection line 1；7, Quality Control
Line；8, bottom liner.
Fig. 2 be time-resolved fluorescence quantitative detection IL-6/PCT joint-detection of the invention kit in detect the knot of card
Structure schematic diagram；
Wherein: 9, two dimensional code；10, well；11, observation window.
Fig. 3 is kit IL-6 standard curve prepared by the embodiment of the present invention 1.
Fig. 4 is kit PCT standard curve prepared by the embodiment of the present invention 1.
Fig. 5 is that kit prepared by the embodiment of the present invention 1 and Roche immunoturbidimetry detect the detection of IL-6 kit and tie
Fruit correlation compares.
Fig. 6 is that kit prepared by the embodiment of the present invention 1 and Roche immunoturbidimetry detect PCT kit test result
Correlation compares.
(5) specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
The preparation of embodiment 1:IL-6/PCT joint-detection time-resolved fluoroimmunoassay chromatography immue quantitative detection reagent box
IL-6/PCT joint-detection time-resolved fluoroimmunoassay chromatographs immue quantitative detection reagent box, is exempted from using double antibody sandwich method
Epidemic disease chromatographic theory, IL-6 and PCT in detection human serum, blood plasma and whole blood sample.Card is detected by time-resolved fluorescence and containing fixed
Mark the ID card composition of curve.
As shown in Figure 1, sequentially overlapping sample pad 1, bonding pad 2, coated film 3 and blotting paper 4 on bottom liner 7, sample pad 1 is
Sample application zone, for drawing detection sample to be measured, bonding pad 2 has microballoon line, and coated film 3 is equipped with detection line and nature controlling line.
The rare earth vanadic acid of specific exciting light (285nm)/transmitting light (618nm) wavelength is used on bonding pad 2 on microballoon line
Salt nano particle (GdVO4:30%Eu) (diameter about 250nm) label monoclonal antibody IL-6 1, monoclonal antibody PCT 1 with
And rabbit igg antibody (100 μ g antibody/200 μ l fluorescent microspheres), detection line coating are coated with monoclonal antibody IL-6 2 respectively
(1.5mg/ml), monoclonal antibody PCT2 (1mg/ml), nature controlling line peridium concentration are the goat anti-rabbit igg antibody (PCT1 of 1mg/ml
From Xiamen with benevolence Biotechnology Co., Ltd, 1 monoclonal antibody of IL-6 is purchased from the vast farsighted biology in Nanjing for monoclonal antibody buying
Science and Technology Ltd., the buying of 2 monoclonal antibody of PCT is from Xiamen with benevolence Biotechnology Co., Ltd, 2 monoclonal antibody of IL-6
Buying comes from Changsha Bo You Biotechnology Co., Ltd from Nanjing Han Rui Biotechnology Co., Ltd, goat anti-rabbit igg antibody).It is micro-
Ball line dosage is that 4 μ l coating liquid measure/cm sample pad, detection line and nature controlling line dosage are 1 μ l coating liquid measure/cm film.
In this embodiment, the preparation of the kit of time-resolved fluorescence quantitative detection IL-6/PCT joint-detection include with
Lower step:
1. the preparation method of rare-earth vanadate nano fluorescent marker material
In 50ml round-bottomed flask, water 20ml, 0.35mmol Gd (NO is added3)3, 0.15mmolEu (NO3)3, 2mmol lemon
Lemon acid sodium, 0.5mmol sodium orthovanadate are passed through nitrogen, and 85 DEG C are reacted 24 hours, and 10000rpm centrifugation is washed with distilled water 3 times,
It is dried to obtain the water-soluble GdVO of good dispersion4: 30%Eu namo fluorescence probe, the GdVO4: 30%Eu material structure is
Pure tetragonal phase structure, size is about 250nm and pattern is uniform, good luminous performance.The chemical formula of the rare-earth vanadate is GdVO4:
30%Eu, colon ": " are expressed as Eu3+Doping.Wherein, GdVO4 is matrix, Eu3+For active ions；Colon ": " is expressed as Eu3+
Doping；Its mole percent is 30%.
2. the activation of time-resolved fluorescence microballoon
After ultrasonication fluorescent microsphere 2min, take 200 μ l fluorescent microspheres with 14000rpm high speed centrifugation 15min after, sink
Starch 100mM, the MES solution that pH is 6.0 are washed to 1ml, ultrasonication 2min；The 100mg/ml carbon two that 50 μ l are added is sub-
Amine mixes 5min, adds the 100mg/ml N- hydroxy thiosuccinimide of 100 μ l, mixes 14000rpm high after 15min
Speed centrifugation 15min, precipitating are washed with the MES solution that pH is 6.0 to 1ml.
3. time-resolved fluorescence microballoon labeled monoclonal antibody IL-6 1 and monoclonal antibody PCT1 and rabbit igg antibody
Preparation
Monoclonal is separately added into according to 100 μ g/200 μ l after the fluorescent microsphere ultrasonication 2min after above-mentioned activation to resist
Body IL-6 1, monoclonal antibody PCT 1, rabbit igg monoclonal antibody mix 2 hours, with containing 0.5%BSA, 0.1% glycine
14000rpm high speed centrifugation 15min after 50mM, pH8.0Tris-Hcl confining liquid are closed 1 hour, with containing 1%Nacl, 0.5%
BSA, 0.5% sucrose, 0.1%Tween20 50mM, pH8.0 Tris-HCl save liquid in buffer wash twice and ultrasound
Processing is resuspended and is kept in dark place to 200 μ l in 4 DEG C.
4. the preparation of sample pad
With sample pad treatment fluid (blocking agent of heterophile antibody containing 1mg/ml (Fei Peng Biological Co., Ltd.), 0.5%
The Tris-HCl of 20mM, pH8.0 of NaCl, 0.5%Tween, 0.1%BSA) in the parallel even application in side close to sample pad
Two lines, dosage are 4 μ l liquid measures/cm sample pad, are placed in baking oven, and 37 DEG C of drying are overnight.
5. the preparation of bonding pad
The monoclonal antibody IL-6 1 and monoclonal antibody PCT 1 for marking time-resolved fluorescence microballoon on bonding pad with
And rabbit igg antibody with microballoon dilution (the 20mMTris-Hcl buffer containing 0.5%BSA, 25% sucrose) by 12%, 6%,
One line of even application (i.e. microballoon line) after 3% dilution proportion, dosage are 4 μ l liquid measures/cm sample pad.It is placed in baking oven, 37
DEG C drying overnight.
6. the preparation of coated film
Use coating buffer (the 10mM PBS buffer solution containing 2.5% sucrose) by monoclonal antibody IL-6 2, Dan Ke respectively
Grand antibody PCT 2 and goat anti-rabbit antibody adjust separately concentration to 1.5mg/ml, 1mg/ml, 1mg/ml dosage be 1 μ l be coated with liquid measure/
Cm film is drawn in being coated on nitrocellulose filter respectively as detection line 1,2 is parallel with nature controlling line, between nature controlling line, detection line
Every 4mm, dried 10 hours in humidity < 30%, 37 DEG C of baking ovens of temperature, envelope is spare；
7. sequentially mutually sample pad is pasted (having a size of 18*300mm, filter in overlap joint ground on bottom liner (having a size of 80*300mm)
Blood film), bonding pad (15*4mm, glass fibre material), coated film (having a size of 25*300mm, nitrocellulose material) and water suction
Paper (having a size of 28*300mm) obtains test paper plate, is cut into the test strips of 4mm width as requested.
Test strip of the present invention, when in use, test strips are assembled in plugged together as plastics upper casing and plastics lower casing made of
(card slot for upper casing insertion is provided on lower casing) in plastic shell, plastics upper casing is set there are two aperture, well 10 and observation
Window 11, well 10 correspond to the sample pad 1 of the detection IL-6/PCT test strips, are provided with fixed test on plastics lower casing
The bayonet of test strips, observation window 11 correspond to the detection line 5,6 and nature controlling line 7 of the detection IL-6/PCT test strips, the detection
IL-6/PCT test strips can be taken out from the plastic shell.
In kit of the present invention, ID card (with batch standard curve identical) of every box containing standard curve passes through the time
Resolved fluorometric detection card measurement various concentration quality-control product, using quality-control product concentration as abscissa, using fluorescence signal ratio as indulge
Coordinate is depicted as standard curve, and ID card is written and generates two dimensional code, can be read on reagent card by dry type fluorescence immunity analyzer
Corresponding two-dimensional barcode information simultaneously measures respective concentration.
The quantitative detection of embodiment 2:IL-6/PCT joint-detection time-resolved fluoroimmunoassay chromatography immue quantitative detection reagent box
1. the drafting of standard curve
Various concentration IL-6 antigen quality-control product (1000pg/ is added in the time-resolved fluorescence detection card prepared by embodiment 1
ML, 800pg/mL, 400pg/mL, 200pg/mL, 100pg/mL, 7pg/mL, 1.5pg/mL and PCT antigen quality-control product (100ng/
ML, 60ng/mL, 20ng/mL, 2ng/mL, 1ng/mL, 0.5ng/mL, 0.05ng/mL) totally 7 concentration, each concentration set three
It repeats, IL-6/PCT antigen calf serum dilutes respectively obtains the quality-control product mother liquor of 1000pg/mL and 100ng/mL, then with small
Cow's serum is diluted to different quality-control product concentration), after chromatographing 15min, produced by Guangzhou Lan Bo Biotechnology Co., Ltd
AFS-1000 type dry type fluorescence immunity analyzer reads C, T line fluorescence signal and C/T value, and experimental result and analysis are shown in Table 1:
Standard curve, curve are drawn respectively with IL-6 and PCT quality-control product concentration and sample signal T1/C and T2/C average value
Data are shown in Table 1, and standard curve is as shown in Figure 3,4.It is 0.9987 that wherein IL-6R1 value, which is 0.9963, PCT R2 value, passes through the mark
Line carries out concentration quantitative measurement to IL-6 and PCT concentration contained in sample.
2. time-resolved fluorescence test card performance test.
Minimum detectability: being carried out replication 20 times with zero sample, calculates the mean value M and standard deviation SD of 20 results,
The detection of twice of standard deviation method for reporting is added to limit (M+2SD) with blank mean value, IL-6 result is 1.38pg/mL, meets sensitivity
1.5pg/mL.PCT result is 0.04ng/mL, meets sensitivity 0.05ng/mL
The range of linearity: IL-6 takes 7 concentration values between 1.5~1000pg/mL, and each concentration duplicate measurements three times, will survey
Determine mean concentration and theoretical concentration carries out linear analysis, obtains linear equation y=1.0375x-9.4498, r=0.9949, table
Bright kit of the present invention correlation in 1.5~1000pg/mL range of linearity is fine.PCT takes between 0.05~100ng/mL 7
Concentration value, each concentration duplicate measurements carry out linear analysis three times, by measurement mean concentration and theoretical concentration, obtain linear side
Journey y=0.9657x+0.1272, r=0.9994 show that kit of the present invention is related in 0.05~100ng/mL range of linearity
Property is fine.
Precision: made time-resolved fluorescence quantitative detection IL-6/PCT kit, IL-6 Quality Control in three batches of embodiments 1 are taken
Product detect 800,5pg/mL repeatability quality-control product respectively, and every batch of kit uses repeated quality-control product Parallel testing 10 times, 800pg/
CV is respectively 8.98%, 6.55%, 7.43% in mL concentration three batches batches, and CV is 7.99%, 5pg/mL concentration three batches between three batches batches
CV is respectively 9.33%, 9.63%, 7.98% in batch, and CV is 8.92% between three batches batches, within 10%.PCT quality-control product point
Not Jian Ce 28,0.6ng/mL repeatability quality-control product, for every batch of kit with repeated quality-control product Parallel testing 10 times, 28ng/mL is dense
CV is respectively 8.17%, 8.94%, 9.53% in degree three batches batches, and CV is 9.07%, 0.6ng/mL concentration three batches batches between three batches batches
Interior CV is respectively 9.97%, 10.04%, 8.71%, and CV is 9.27% between three batches batches, within 10%.
Accuracy: it for the Quality Control object of 5pg/mL is detection sample that IL-6, which selects concentration, and it is 3 parts identical to be divided into volume, wherein 2
It is separately added into the accuracy quality-control product of 500pg/mL and 30pg/mL in part sample, 2 different recycling samples that concentration are added are made
This, calculates the concentration of the determinand of addition.The solution without measured object of same amount is added in another sample, basic sample is made
This.3 replicate analysis are carried out to sample, its mean value is taken to be calculated.Calculate the rate of recovery=(analysis sample concentration-basis sample
Concentration)/concentration × 100% is added.The rate of recovery of sample 500pg/mL, which is recycled, as the rate of recovery of 98.83%, 30pg/mL is
107.08%, average recovery rate 102.96%.Deviation is within 10%.PCT selects the concentration to be for the Quality Control object of 0.75ng/mL
Sample is detected, is divided into that volume is 3 parts identical, the accuracy Quality Control of 60ng/mL and 5ng/mL is separately added into wherein 2 parts of samples
Product are made 2 different recycling samples that concentration is added, calculate the concentration of the determinand of addition.Recycle the recycling of sample 60ng/mL
The rate of recovery that rate is 93.81%, 5ng/mL is 93.13%, average recovery rate 93.47%.Deviation is within 10%.
3. clinical sample detects
Each 200 parts of plasma sample of hospital detection IL-6 and PCT are acquired, it is electrochemical with Roche respectively with kit of the invention
It learns luminescence method detection IL-6, PCT kit and carries out detection comparison.In kit of the present invention, 80 μ l of plasma sample is taken to be added to inspection
It surveys in card well, chromatographs the AFS-1000 type dry type fluorescence produced after 15min by Guangzhou Lan Bo Biotechnology Co., Ltd
Immunity analysis instrument reads concentration, and comparison system Roche Electrochemiluminescince detection IL-6, PCT kit is respectively adopted in same sample
Carry out Concentration Testing.Two methods testing result carries out linear analysis, and as shown in Figure 5,6, correlation is fine, IL-6r=
0.9869, P > 0.05, mean relative deviation is less than 10%, PCTr=0.9850, P > 0.05, mean relative deviation less than 10%,
As a result meet clinical analysis requirement, be suitable for clinical detection.
Claims (5)
1. a kind of IL-6/PCT joint-detection time-resolved fluorescence detection kit, including time-resolved fluorescence test card and contain
The ID card of calibration curve, it is characterised in that:
The time-resolved fluorescence test card includes test strip, and the test strip is by bottom liner and is pasted on bottom liner
Blood filter membrane sample pad, glass fiber conjugate pad, nitrocellulose filter and blotting paper composition, the blood filter membrane sample pad, glass
Fiber conjugate pad, nitrocellulose filter and blotting paper are sequentially overlapped and are pasted on bottom liner；
The sample pad treatment fluid comprising heterophile antibody blocking agent is coated at the blood filter membrane sample pad；
It is provided with microballoon line at the glass fiber conjugate pad, is coated with the monoclonal antibody of time-resolved fluorescence microballoon label
IL-6 1, monoclonal antibody PCT 1 and rabbit igg antibody, content are respectively 50~200 μ g antibody/200 μ l fluorescent microspheres, institute
Stating time-resolved fluorescence microballoon is rare-earth vanadate nano particle, and partial size is 250 ± 50nm, consisting of LnVO4:xEu,
In, LnVO4 is matrix, and Ln is one of lanthanum, yttrium, gadolinium, samarium, terbium or a variety of；Colon ": " is expressed as europium doping；X is rubbing for Eu
That percentage, range is 5%~50%；
Detection line 1,2 and nature controlling line are disposed on the nitrocellulose filter, detection line 1,2 and nature controlling line are coated with list respectively
Clonal antibody IL-6 2, monoclonal antibody PCT2 and goat anti-rabbit antibody, detection line 1,2 and nature controlling line are parallel to each other, spacing distance
For 3~5mm, wherein detection line 2 is close to bonding pad, and nature controlling line is far from bonding pad；The coating of detection line 1 monoclonal antibody IL-6 2,
Peridium concentration is 0.3~2mg/ml, dosage is 0.5~1.5 μ l coating liquid measure/cm film, and detection line 2 is coated with monoclonal antibody
PCT2, peridium concentration are 0.3~2mg/ml, dosage is 0.5~1.5 μ l coating liquid measure/cm film, and goat anti-rabbit igg is anti-in nature controlling line
Body peridium concentration is 0.3~2mg/ml, dosage is 0.5~1.5 μ l coating liquid measure/cm film.
2. kit as described in claim 1, it is characterised in that the test strip is prepared by the following method:
(1) preparation method of rare-earth vanadate nano fluorescent marker material
In 50mL round-bottomed flask, example, is added 2 parts of distilled water, the nitrate or acetate of (1-x) part Ln or chlorination in molar ratio
Object, the nitrate or acetate or chloride, x of x parts of Eu is mole percent, and range is 5%~50%, 1 part of ortho-vanadic acid, (1~
5 parts) citric acid or sodium citrate, pH value 6~8 is adjusted with sodium hydroxide, is passed through nitrogen, 85 DEG C are reacted 24 hours, 10000rpm
Centrifugation, is washed with distilled water 3 times, is dried to obtain the water-soluble LnVO4:x Eu namo fluorescence probe of good dispersion；
(2) activation of time-resolved fluorescence microballoon
After ultrasonication fluorescent microsphere 2min, take 200 μ l fluorescent microspheres with 5~15min of 14000rpm high speed centrifugation after, precipitating
10~100mM of object, the MES solution that pH is 5.0~7.0 are washed to 1ml, ultrasonication 2min；It is added the 20 of 10~100 μ l
~100mg/ml carbodiimide mixes 5~10min, adds 20~100mg/ml N- hydroxy amber of 50~200 μ l
Acid imide, mixes 14000rpm 5~15min of high speed centrifugation after 10~20min, and precipitating is washed with the MES solution that pH is 5.0~7.0
It washs to 1ml；
(3) time-resolved fluorescence microballoon marks the preparation of IL-6, PCT monoclonal antibody IL-6 1, PCT1 and rabbit igg antibody respectively
IL-6, PCT are separately added into according to 50~200 μ g/200 μ l after the fluorescent microsphere ultrasonication 2min after above-mentioned activation
Monoclonal antibody IL-6 1, PCT1 and rabbit igg, mix 1~3 hour, with containing 0.5%BSA, 0.1% glycine 10~50mM,
14000rpm 5~15min of high speed centrifugation after the Tris-HCl confining liquid of pH7.5~8.5 is closed 0.5~1 hour, with containing 1%
Nacl, 0.5%BSA, 0.5% sucrose, 10~50mM of 0.1%Tween20, the Tris-Hcl of pH7.5~8.5 save liquid washing
And it is resuspended and is kept in dark place to 200 μ l in 4 DEG C；
(4) preparation of sample pad
With sample pad treatment fluid in the parallel even application two lines in side close to sample pad, dosage is 2~4 μ l liquid measures/cm sample
Product pad, is placed in baking oven, and 37 DEG C of drying are overnight；The sample pad treatment fluid is the blocking agent of heterophile antibody containing 1mg/ml, 0.5%
The Tris-HCl of 20mM, pH8.0 of NaCl, 0.5%Tween20,0.1%BSA；The microballoon dilution be containing 0.5%BSA,
The 20mM Tris-Hcl buffer of 25% sucrose；
(5) preparation of bonding pad
IL-6, PCT monoclonal antibody IL-61, the PCT1 and rabbit igg antibody for marking time-resolved fluorescence microballoon on bonding pad
With 8~30 times of the microballoon diluted line of even application one, dosage is 2~4 μ l liquid measures/cm bonding pad, is placed in baking oven, 37
DEG C drying overnight；
(6) preparation of coated film
IL-6, PCT monoclonal antibody IL-6 2, PCT2 and goat anti-rabbit igg antibody adjustment concentration are arrived with coating buffer respectively
0.3~2mg/ml, dosage are that 0.5~1.5 μ l is coated with liquid measure/cm film, are drawn respectively as detection line 1,2 is parallel with nature controlling line in nitre
It is coated on acid cellulose film, nature controlling line, detection line and microballoon line 3~7mm of interval are placed in baking oven, and 37 DEG C of drying are overnight；
The coating buffer is the 10mM PBS buffer solution containing 2.5% sucrose；
(6) sequentially mutually overlap joint blood filter membrane sample pad, glass fiber conjugate pad, coated film and blotting paper is pasted on bottom liner to obtain
To test paper plate, cutting obtains the test strips.
3. kit as claimed in claim 1 or 2, which is characterized in that the time-resolved fluorescence test card is by Test paper
Item is fixed on plastic bottom card, and test paper surface is tight with card face pressure, and card face is in the part of counter sample pad and nitrocellulose filter
Well and observation window are reserved respectively.
4. kit as claimed in claim 1 or 2, which is characterized in that the ID card containing standard curve, is to pass through the time
Resolved fluorometric test strips measure various concentration calibration object, using calibration object concentration as abscissa, using fluorescence signal ratio as indulge
Coordinate is depicted as standard curve, is written and generates respective two-dimensional code information and be stored in ID card and obtains.
5. the method for the quantitative detection IL-6/PCT using kit described in claim 1, which comprises
(1) it will test kit and sample be placed at room temperature, it is to be restored to using after room temperature；
(2) dry type fluorescence immunity analyzer is opened, is inserted into corresponding ID card after preheating 5min；
(3) 80 μ L blood to be measured are accurately drawn to be added in detection card well；
(4) it will test card insertion and enter detection slot, detected after 15min and read and print testing result.
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