CN109444422A - A kind of detection card and its construction method and application based on UPT-LF quantitative determination blood-serum P IVKA-II - Google Patents
A kind of detection card and its construction method and application based on UPT-LF quantitative determination blood-serum P IVKA-II Download PDFInfo
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Abstract
The present invention provides a kind of detection card based on UPT-LF quantitative determination blood-serum P IVKA-II and its construction method and applications, the detection card includes sample pad, bonding pad, nitrocellulose filter, blotting paper, up-conversion luminescence nanoparticle -1 conjugate of anti-PIVKA-II monoclonal antibody is fixed on the bonding pad, the nitrocellulose filter detection band and quality control band, the detection band is fixed with anti-PIVKA-II monoclonal antibody 2, and the quality control band is fixed with secondary antibody sheep anti-mouse igg.Detection card of the invention has good detection linear, sensitivity, stability, accuracy, precision and a specificity, and detection performance is good, it can be achieved that HCC patients serum PIVKA-II fast and efficiently POCT formula quantitative detection.
Description
Technical field
The invention belongs to field of biomedicine technology, and layer is immunized based on up-converting phosphor technology in particular to one kind
Analyse construction method and the application of standard measure measurement blood-serum P IVKA-II detection card.
Background technique
Vitamin K deficiency factor-II (PIVKA-II) also referred to as removes γ-carboxyl factor (DCP), is a kind of
Lose the abnormal prothrombin of normal coagulation function.There are ten complete carboxylated in normal prothrombin precursor structure
Glutaminic acid residue, glutamyl carboxylase of these glutaminic acid residues in liver cell under the action of, are transformed into γ-carboxyl paddy
Histidine residue.When the activity decline of vitamin K deficiency or glutamyl carboxylase, then it can lead to shortage one to ten not
Deng γ-carboxyglutamic acid residue and abnormal prothrombin (PIVKA-II) without normal coagulation function generate and discharge to HCC
Patient's blood circulation.
The study found that in HCC patient of the tumor nodule diameter greater than 5 centimetres, in about 95% patients serum
PIVKA-II level is more than 6000mAU/ml (S.Nakamura, K.Nouso, K.Sakaguchi, et al., Sensitivity
and specificity of des-gamma-carboxy prothrombin for diagnosis of patients
with hepatocellular carcinomas varies according to tumor size,
AM.J.GASTROENTEROL.Sci.101(2006)2038-43.).The totality that Yoon et al. discovery PIVKA-II diagnoses HCC
Sensibility and specificity is respectively 48%-62%, 81%-98%, 39%-64%, 76%-91% better than AFP
(Y.J.Yoon,K.H.Han,D.Y.Kim.,Role of serum prothrombin induced by vitamin K
absence or antagonist-II in the early detection of hepatocellular carcinoma
in patients with chronic hepatitis B virus infection,
Scand.J.Gastroenterol.Sci.44(2009)861-6.).When blood-serum P IVKA-II level is more than 90mAU/ml,
The sensibility and specificity of prediction HCC tumor cell invasion Hepatic Microvascular can respectively reach 70% and 63% (N.Pote,
F.Cauchy,M.Albuquerque,et al.,Performance of PIVKA-II for early
hepatocellular carcinoma diagnosis and prediction of microvascular invasion,
J.Hepatol.Sci.62(2015)848-854.).In addition, comparing the raised patient of AFP, blood-serum P IVKA-II increases HCC and suffers from
The gross tumor volume of person significantly increases, occur the low differentiation of tumour it is significantly raised with the Proportion of patients of portal vein embolization (B.X.Truong,
Y.Yano,VT.VAN,et al.,Clinical utility ofprotein induced by vitamin K absence
in patients with chronic hepatitis B virus infection,Biomed.Rep.1(2013)122-
128.).Therefore, PIVKA-II has very high clinical value to the early diagnosis of HCC, condition assessment, Index for diagnosis.
Currently, clinical labororatory mainly includes Chemiluminescence quantitative immunoassay for the detection method of blood-serum P IVKA-II
(CLEIA), chemiluminescence immunoassay (CLIA) and liquid phase binding analysis method (LBA).However these measuring methods all need
Relative complex detection device is wanted, using the central laboratory for being limited to macro-organism chemistry department or particularization, and it is uncomfortable
(POCT) is detected together in by bed.There is the condition assessment demand to patient by bed in view of clinic, POCT detection method is for serum
The detection of PIVKA-II will be a selection well.
Summary of the invention
In view of the deficiencies in the prior art, the purpose of the present invention is to provide one kind to be based on up-converting phosphor technology immunochromatography
The detection card and its construction method of standard measure measurement blood-serum P IVKA-II and application.
In order to achieve the object of the present invention, the present inventor uses rare earth Ln3+Series elements ytterbium (Yb3+) and erbium (Er3+) codope
Nano particle NaYF4:Yb3+,Er3+As biomarker probe luminous host material, using immunity test strip consolidating as reaction
Phase carrier, building are based on the detection card of up-conversion luminescence immunochromatographic method (UPT-LF), realize and suffer to primary hepatoma
The fast and efficiently POCT formula quantitative detection of person's blood-serum P IVKA-II effectively increases the early diagnostic rate of HCC, improves patient
Prognosis.
Specifically, technical solution of the present invention overview is as follows:
A kind of detection card based on up-converting phosphor technology immunochromatographic method quantitative determination blood-serum P IVKA-II, the detection card
Including sample pad, bonding pad, nitrocellulose filter, blotting paper, the sample pad and bonding pad are glass fibre element film, the nitre
Acid cellulose film is equipped with detection band and quality control band, the sample pad, bonding pad, nitrocellulose filter, blotting paper are successively pasted
On sticky bottom liner, the test strips band of 3-6mm wide is cut into using cutting machine, and is packaged in UPT and is got stuck；
Up-conversion luminescence nanoparticle -1 conjugate of anti-PIVKA-II monoclonal antibody is fixed on the bonding pad, it is described
Nitrocellulose filter detects band and quality control band, and the detection band is fixed with anti-PIVKA-II monoclonal antibody 2, and the quality control band is solid
Surely there is secondary antibody sheep anti-mouse igg.
Further, the sample pad, bonding pad, analyzing film, water absorption pad are successively with the spacing alternation of bed of 1~2mm of overlapping
Folded to paste in PVC board, the width of the test strips is 4mm.
Further, the up-conversion luminescence nanoparticle is the NaYF that TEOS and APES is modified4:Yb3+,Er3+UCPs。
It is modified by TEOS, so that NaYF4:Yb3+,Er3+UCPs particle surface is enclosed with one layer of SiO2；It is modified again by APES, point
Sub- one end is connected with silicon, and the other end then carries active carboxyl groups, and the UCPs of functionalization can be with multiple biological activities molecule knot
It closes, such as antibody.
A kind of preparation side of the detection card based on up-converting phosphor technology immunochromatographic method quantitative determination blood-serum P IVKA-II
Method, this method comprises the following steps:
(1)NaYF4:Yb3+,Er3+The preparation of UCPs；
(2)NaYF4:Yb3+,Er3+The functional modification of UCPs；
(3) building of up-conversion luminescence bioprobe；
(4) configuration and coating of conjugate solution；
(5) production of blocking agent sample pad；
(6) configuration and coating of Quality Control solution and detection solution；
(7) preparation of the detection card of immunochromatographic method is converted on.
Further, step (1) NaYF4:Yb3+,Er3+UCPs the preparation method is as follows:
By YCl3(0.2343g),YbCl3(0.0755g) and ErCl3The oleic acid of (0.0083g) three kinds of reagents and 4.5mL and
The 1- octadecylene of 26.0ml is mixed in the flask of 100mL, and 150-160 DEG C is heated in argon atmosphere and is mixed to solution, is held
It is cooled to room temperature after continuous 25min.15mL is contained to the NH of the NaOH and 0.222g of 0.015g4The methanol solution of F slowly adds
Enter in flask, and quickly form solid precipitation in the solution, reaction system stirs 30min at this time.Then, reaction solution is slowly added
Heat is to 100 DEG C and maintains 10min, and methanol is vapored away.Then it is heated rapidly to 300 DEG C and maintains 1h.It is naturally cold to solution
But after, product is precipitated from solution using ethyl alcohol.It is deposited under 12000rpm and is centrifuged 5min, and with water/ethyl alcohol (1:1 body
Product ratio) cleaning is three times.Prepared product is dissolved in 5mL hexamethylene and is saved for use.
Further, the step (2) the preparation method is as follows:
Weigh NaYF4:Yb3+,Er3+95mL hexamethylene, and ultrapure water under the conditions of ultrasonic vibration is added in UCPs 40mg
Middle dispersion simultaneously hangs particle completely, and CO-5202.5mL, ultrasonic 20min is then added；Addition ammonium hydroxide 0.35mL (30wt%),
TEOS 0.09mL after magnetic agitation reaction for 24 hours, is added 16ml ethyl alcohol and is centrifuged, discard supernatant liquid；Precipitating uses 90mL isopropyl
Alcohol dissolution, and APES 0.9mL is added thereto, magnetic agitation reacts 2.5h, spare after being centrifuged and washing 3 times.
Further, the probe construction method of the step (3) is as follows:
The UCPs of 5mg is added in the purified water of 1ml and dissolves under the conditions of ultrasonic vibration and mixes, and 20% Tween- is added
20 activating agent 5ul are mixed under ultrasound condition；The anti-PIVKA-II monoclonal antibody 1 of 75ug, ultrasound vibration are added into reaction system
20min is reacted under the conditions of dynamic；5ul BSA (20%) is added after after reaction thereto, closes 5min under the conditions of ultrasonic vibration；
Then in 4 DEG C, it is centrifuged 30min in the high speed refrigerated centrifuge of 12000r/min, take out and discards supernatant liquid is spare.
Further, the configuration of the conjugate solution of the step (4) and method for coating are as follows:
The configuration of conjugate store buffer liquid: being separately added into 0.1%BSA in the PBS buffer solution of 0.02mol/l, PH7.4,
0.3%TritonX-100,0.02%NaN3；Buffer configuration is lyophilized in conjugate: in the PBS buffer solution of 0.02mol/l, PH7.4
It is separately added into 5%trehalose, 1%BSA, 2%mannitol, 1%glycine, 0.01%TB, 0.5%casein, 0.1%
NaN3.It first is separately added into 100ul conjugate store buffer liquid in backward precipitating, buffer is lyophilized in 5.5ml conjugate, shakes in ultrasound
Dynamic condition mixes under water；The mixed liquor is uniformly coated on 3*19.33cm with 600ul/ sample loading gun2(S=58cm2) SB08 type
On glass fibre, and it is transferred quickly to carry out -60 DEG C of frozen drieds, cooling time 2h in freeze dryer；Then vacuumized
And at least 8h is maintained, it is spare to room temperature preservation to take out balance.
Further, the blocking agent sample pad of the step (5) the production method is as follows:
HBR blocking agent is diluted to concentration using TB buffer (0.01mol/L, PH8.0) to take out after 75ug/mL
75.0ul Casein (20%) is added in 10.0ml, then equably pours it in 6*19.33cm2(S=116cm2) SB08 glass
On glass fiber, room temperature is dried rear spare.
Further, the configuration of the Quality Control solution of the step (6) and detection solution and method for coating are as follows:
NC film is pasted on sticky bottom liner designated position, blotting paper one fixed width Chong Die with NC film and the sticky bottom liner upper end and
It flushes and is pasted.Then by test antibody (T is anti-, PIVKA-II monoclonal antibody 2) and Quality Control antibody (C is anti-, sheep anti-mouse igg)
It is diluted to 2.0mg/mL, 0.6mg/mL respectively with PB buffer (0.02mol/L, PH7.2)；Using continuous Film-cutting machine in NC film
The sticky place bottom liner bottom end 12mm and 15mm of distance (chromatography direction is from top to bottom) sprays that T is anti-and C resists with the speed of 1ul/cm respectively
As detection band (T line) and quality control band (C line)；The NC film being coated with is placed in 37 DEG C of baking ovens and dries 1.5h, is encapsulated spare.
Further, the upper conversion immunochromatographic method of the step (7) detection card the preparation method is as follows:
It is 0.5cm size that the glass fibre (bonding pad) of up-conversion luminescence bioprobe, which is cut into width, by the upper end
The width of 1mm Chong Die with NC film is pasted on sticky bottom liner；Glass fibre (blocking agent sample pad) containing blocking agent is cut
At 2.0cm wide, it is flushed with sticky bottom liner lower end and is pasted；Bonding pad and sample pad are fixed again with MAX film,
Sticky bottom liner is cut into the test strips band of 4mm wide by cutting machine；By test strips mounted in dedicated interior, the completion UPT- that gets stuck of UPT
The whole preparation process of LF detection card.
The present invention uses double-antibody sandwich mode UPT-LF analytic approach quantitative detection blood-serum P IVKA-II, the method is as follows: sample
The configuration of this dilution: 0.15%BSA, 0.1%NaN3 are separately added into the PBS buffer solution of 0.02mol/l, PH7.4；Sample to be tested
With Sample dilution mixed volume ratio 4:6, draw 100uL mixed liquor be added to detection card sample aperture, be immunoreacted 15min after,
Will test card be inserted on UPT-3A-1800 convert immunity analysis instrument detection hole be scanned, UCPs is close in instrument internal
Launching wavelength under 980nm NIR excitation is 541.5nm visible light, and instrument will be received from light of the test strips with T, C line
Signal is transformed into electric signal and with " T value " and " C value " display, and the measurement result of analyte is then with the ratio (T/C of T value and C value
Value) it indicates, while T/C value is converted into specific concentration (ng/ml) according to standard curve by instrument.
Compared with prior art, the detection card based on UPT-LF that the present invention constructs has good detection with property, sensitive
Degree, stability, accuracy, precision and specificity, while having that detection performance is good, detection time is short and use cost is opposite
Lower advantage, is not only suitable for laboratory testing, is also very applicable for detecting by bed, it can be achieved that HCC patients serum PIVKA-
II fast and efficiently POCT formula quantitative detection, to promote PIVKA-II detection in clinical (especially different medical unit) application
Promotion and popularization, meet the needs of clinical extensive HCC Susceptible population screening, detection, effectively improve the early diagnosis of HCC
Rate, the prognosis for improving patient.
Detailed description of the invention
Fig. 1 is rare earth Ln3+Metal Yb3+With Er3+The NaYF of codope4(NaYF4:Yb3+,Er3+) on conversion nano particle
(UCPs) schematic diagram, in which: (A) NaYF4:Yb3+,Er3+Transmission electron microscope picture；(B) UCPs swashs at 980nm near infrared light (NIR)
Give the visible light (VIS) for launching 541.5nm；(C) SiO of UCPs2Shell package, surface-functionalized modification and connection biology
Activated protein.
Fig. 2 is that UPT-LF detects card structure schematic diagram, in which: get stuck schematic diagram on (A) detection card；(B) immunity test strip
Structural schematic diagram；(C) get stuck exit orifice and each position corresponding relationship of internal test strips are detected.
Fig. 3 is the schematic diagram of UPT-LF detection card detection sample, in which: turns luminescence immunoassay on (A) UPT-3A-1800
Instrument shows testing result after forwarding light immunity analysis instrument detection sample on (B) UPT-3A-1800, and (C) UPT-LF detection card is internal
Test strips are immunoreacted schematic illustration.
Fig. 4 is the standard quantitative curve of UPT-LF analytic approach, in which: T/C value is each dilution water of PIVKA-II calibration object
Mean longitude measures the mean value of acquired results, mean twiceblankAnd CoRespectively level (the ng/ of background values and calibration object after diluting
ML), T/C-meanblankAnd CoStandard curve is drawn respectively as Y, X-axis using after same bottom logarithm.
Fig. 5 be 25 DEG C and 37 DEG C five time points of storage UPT-LF detection card to PIVKA-II recombinant antigen and HCC,
The measurement result of LC, CHB, HC blood-serum P IVKA-II, in which: after (A), (B) are 25 DEG C of room temperature 0,7,15,30 and 60d of storage
Detect card measurement result；(C), (D) is the detection card measurement result after 37 DEG C of high temperature 0,1,3,5 and 7d of storage.
Fig. 6 is 4 DEG C of storage 0d, conventional effect phase (18m) two batch UPT-LF of time point four detection card is to PIVKA-II
Measurement result, in which: (A) to (E) be followed successively by UPT-LF detection card to PIVKA-II recombinant antigen and HCC, LC, CHB and
The result of HC blood-serum P IVKA-II measurement.
Fig. 7 is UPT-LF detection card to the quantified results of each group blood-serum P IVKA-II and to the ROC curve of HCC,
In: (A) UPT-LF detection card to HCC group, LC group, CHB group, HC group blood-serum P IVKA-II quantified results scatter plot, cross
Line and vertical line respectively represent the median and quartile spacing of detection each group blood-serum P IVKA-II level；(B) UPT-LF detection card
To the ROC curve [vs LBL (including LC and CHB)+HC group] of HCC, area under the curve AUC is 0.85, P < 0.001.
Fig. 8 is UPT-LF and CLEIA analytic approach to the linear of 178 HCC patients serum's PIVKA-II quantified results
Regression curve, in which: UPT-LF and CLEIA analytic approach quantitative determines the coefficient R between blood-serum P IVKA-II result2For
0.901, equation of linear regression is Y=0.08X -4.50, P < 0.01.
Specific embodiment
Present invention building is based on the detection card of up-conversion luminescence immunochromatographic method (UPT-LF), realizes to primary liver cell
The fast and efficiently POCT formula quantitative detection of cancer (HCC) patients serum PIVKA-II, and is given by detection performance and is examined for detection card
The evaluation of disconnected performance.Method is to form label probe by up-conversion luminescence particle (UCPs) and in conjunction with anti-PIVKA-II antibody,
Building can measure the detection card of blood-serum P IVKA-II.Pass through detection limit, linear, stability, precision, determination of recovery rates and interference
Test, detection performance of detection card of the evaluation based on UPT-LF to PIVKA-II.By to 228 HCC patient groups, 170 livers
Dirty benign lesion (LBL) group [cirrhosis (LC) 80, chronic hepatitis B (CHB) 90], 100 normal controls (HC) organize blood
The quantitative determination of clear PIVKA-II is evaluated detection card and is divided to the diagnostic sensitivity and specificity of HCC, and with chemiluminescence immunoassay
The correlation of analysis method (CLEIA) quantitative detection result.The results show that modifying and the UCPs particle aqueous dispersion state activated is good
It is good, it can be firmly combined to form stable luminescence probe with PIVKA-II antibody.The UPT-LF detection card of building is to PIVKA-II's
Detection limit and the range of linearity are respectively 2.66ng/mL, 4-20000ng/mL.25 DEG C of thermal stability and 4 DEG C of effect phases have good stability,
The analyte rate of recovery of 93.1%-99.2%, batch in and batch between CV respectively within the scope of 2.6-5.8% and 5.4-8.9%, to 8
The strong antijamming capability of kind serum common analytical object.UPT-LF detection blocks is respectively with specificity to the diagnostic sensitivity of HCC
71.49%, 88.89% (vs LBL+HC group).Linear regression curves analysis shows that, UPT-LF and CLEIA analytic approach are to serum
The coefficient R of PIVKA-II quantitative detection result2=0.901.
In order to be clearer to understand technical concept and technological means of the invention, and can be according to the content of specification and this
The conventional technical means in field is practiced, and is described in further details below with reference to specific embodiment to the present invention, still
Test example is explanation of the invention rather than limits in detail below.
1. material and method
1.1 materials and detection device
Rare earth upconversion nano particle (UCPs, NaYF4:Yb3+,Er3+), UPT is dedicated to get stuck, sticky bottom liner, blotting paper,
Nitrocellulose filter (NC film, 503C type), glass fibre SB08, PIVKA-II (monoclonal antibody 1, mIgG1), PIVKA-II
(monoclonal antibody 2, mIgG2), sheep anti-mouse igg, PIVKA-II recombinant antigen and different biting property antibody blocking agent IgG (HBR-
IgG).The above material is provided by Beijing hot scape biology Co., Ltd.3-aminopropyltriethoxysilane (APES), nonionic
Emulsifier CO-520, tetraethoxysilane (TEOS) are purchased from Chemical Industry Co., Ltd. of earth of Hangzhou.G PIVKA-II
Detection kit is purchased from FUJIREBIO company, Japan.Continous way Film-cutting machine and high-speed induction cutting machine are Hangzhou Autokun science and technology
Co., Ltd's product；Up-conversion luminescence immunity analysis instrument (UPT-3A-1800 type) is the hot scape biology in Beijing Co., Ltd product；G1200 immunity analysis instrument is Japan's FUJIREBIO Products.
1.2 reagent
Reaction reagent needed for this research is prepared according to formula as below and condition: Sample dilution
(0.02mol/l PBS, 0.15%BSA, 0.1%NaN3；PH 7.4), the dilution buffer of PIVKA-II recombinant antigen and antibody
(0.02mol/l PB；PH 7.4), reaction system buffer (0.02mol/l Tris-HCl, 0.1%Tween-20,0.15%
BSA；PH 7.4), conjugate store buffer liquid (0.02mol/l PBS, 0.1%BSA, 0.3%TritonX-100,0.02%
NaN3；PH 7.4), conjugate freeze-drying buffer (5%trehalose, 1%BSA, 2%mannitol, 1%glycine,
0.01%TB, 0.5%casein, 0.1%NaN3；pH 7.4).All of above reagent is mentioned by Beijing hot scape biology Co., Ltd
For.
1.3 patients and sample
448 research objects that this research is related to from June, 2015 to Fujian Provincial Hospital during in December, 2017,
The use of selected research object serum sample obtains the approval of Fujian Provincial Hospital's Institutional Review Board, ethics examination & approval number:
2016[K2016-10-28]。
448 research objects include normal healthy controls (HC) 100 (the median age 44 years old, male 47, women 53)；Liver
Dirty benign disease (LBL) patient 170, containing chronic hepatitis B (CHB) patient 90 (the median age 48 years old, male 57, female
Property 33) and cirrhosis (LC) patient 80 (the median age 63 years old, male 54, women 26)；228 (middle positions of HCC patient
Age 56 years old, male 199, women 29).The hepatitis B surface antigen of HC is feminine gender, and liver function it is normal, in ultrasound or
Without hepatic neoplasms in CT/MRI imaging；The equal trans-abdominal ultrasound of LC patient or liver impedance rheograph confirmation；CHB patients serum's hepatitis B surface
Antigen positive continues more than half a year；HCC patient's pathology after row liver biopsy or liver surgery under ultrasound guidance
Confirmation.The serum sample of all research objects is strictly left and taken as claimed below: acquisition early morning empty stomach peripheric venous blood 5ml, room
Temperature is placed 2 hours, isolates within 5 minutes serum with 3000rpm centrifugation, 2 pipe of packing, -80 DEG C freeze it is spare.
The building of 1.4UPT-LF detection card
1.4.1UCPs shell package is surface modification activation
By YCl3(0.2343g),YbCl3(0.0755g) and ErCl3The oleic acid of (0.0083g) three kinds of reagents and 4.5mL and
The 1- octadecylene of 26.0ml is mixed in the flask of 100mL, and 150-160 DEG C is heated in argon atmosphere and is mixed to solution, is held
It is cooled to room temperature after continuous 25min.15mL is contained to the NH of the NaOH and 0.222g of 0.015g4The methanol solution of F slowly adds
Enter in flask, and quickly form solid precipitation in the solution, reaction system stirs 30min at this time.Then, reaction solution is slowly added
Heat is to 100 DEG C and maintains 10min, and methanol is vapored away.Then it is heated rapidly to 300 DEG C and maintains 1h.It is naturally cold to solution
But after, product is precipitated from solution using ethyl alcohol.It is deposited under 12000rpm and is centrifuged 5min, and with water/ethyl alcohol (1:1 body
Product ratio) it cleans three times, obtain UCPs.
UCPs 40mg is weighed, 95mL hexamethylene is added, and dissolve in ultrapure water under the conditions of ultrasonic vibration and make particle
It hangs completely, CO-5202.5mL, ultrasonic 20min is then added；Ammonium hydroxide 0.35mL (30wt%), TEOS 0.09mL, magnetic is added
After power is stirred to react for 24 hours, 16ml ethyl alcohol is added and is centrifuged, discards supernatant liquid；Precipitating is dissolved using 90mL isopropanol, and thereto
APES 0.9mL is added, magnetic agitation reacts 2.5h, spare after being centrifuged and washing 3 times.
1.4.2UPT-LF the building of card is detected
UCPs particle and labelled antibody mIgG1 (0.075mg/ after 5mg is modified and activated with protein binding activity
Ml luminescent marking probe) is combined and formed in reaction system buffer, by UCPs-mIgG1 compound conjugate buffer
And conjugate freeze-drying liquid is diluted, and is then attached on blank glass fiber SB08 it with the volume of 0.1ml/cm and is formed
Bonding pad.Bonding pad is put in freeze-drying 8h or more in the freeze drier at -60 DEG C.Blank glass fiber SB08 is used into HBR
Blocking agent forms blocking agent sample pad after being handled；By test antibody (T is anti-, mIgG2), (C is anti-, sheep anti mouse with Quality Control antibody
IgG after) distinguishing diluted concentration to 2.0mg/mL, 0.6mg/mL with antibody dilution buffer, using continuous Film-cutting machine on NC film
The detection band (T band) and quality control band (C band) at a distance of 3mm are marked, NC film is then put into the 2h into 37 DEG C of baking ovens；Bonding pad and sample
Pad cuts width 0.5cm, 2.0cm respectively, blotting paper, bonding pad, sample pad is successively pasted on sticky bottom liner, using slitting
Machine is cut into the test strips band (Fig. 2 B) of 4mm wide, and is packaged in that UPT is dedicated to get stuck in (Fig. 2A), completes UPT-LF detection
The entire assembling process of card.Detection blocks internal test strips structure and the hole location relationship that gets stuck is as shown in Figure 2 C.
The continuous mode of 1.5UPT-LF detection method
It is 100uL that UPT-LF, which detects card injection volume, and sample to be examined volume and Sample dilution volume mixture ratio are 4:
6, sample is added from detection card sample aperture, and sample liquid successively passes through sample pad, bonding pad, NC under the siphonage of blotting paper
Film finally reaches blotting paper (Fig. 3 C).Reaction is to 15min after sample-adding, will test card be put on UPT-3A-1800 convert it is immune
The detection hole of analyzer (such as Fig. 3 A) is detected, and label probe is launched under the irradiation of instrument internal 980nmNIR exciting light
The visible light (Figure 1B) of 541.5nm, the optical signal that instrument will receive from T, C line be transformed into electric signal and with " T value " and
" C value " indicates, and the measurement result of checking matter is then indicated with the ratio (T/C value) of T value and C value, such as Fig. 3 B.T/C value and analysis
The level of object has positive correlation.
1.6.UPT-LF the evaluation of analytic approach
1.6.1 detection limits (LoD) and linear
The appraisal procedure of blank detection limit (LoB) is the Sample dilution of the not analyte-containing of replication 60 times, LoB=
meanblank+2(SD blank).The appraisal procedure of the detection limit LoD of UPT-LF detection card is the sample of 60 low concentrations of replication
This (0.5ng/ml), LoD=LoB+2 (SDlowlevelsample)。
PIVKA-II calibration object (PIVKA-II recombinant antigen) is serially diluted to 40000,20000,10000,5000,
2500,1250,625,312,156,78,39,19.5,9.7,4.8 and 2.4ng/ml, each horizontal calibration object repeat to survey
Determine twice, to calculate each horizontal analysis object average value (C of measuremento), by CoLogarithm log (Co) it is used as X-axis, T/C-
meanblankLogarithm log (T/C-meanblank) Y-axis is used as to draw linear regression curves, phase is obtained by being fitted the curve
The equation of linear regression answered: " Y=a × X+b ", coefficient a and b in formula be the parameter of detection card, determine T/C value and analyze
The conversion relation of object serum levels.
1.6.2 estimation of stability
Use the UPT-LF detection card measurement PIVKA- that 0,7,15,30,60 day (d) totally five time points are stored at 25 DEG C
The serum of II recombinant antigen and HCC, LC, CHB and HC；Use storage 0,1,3,5, the inspection at 7d totally five time points at 37 DEG C
It surveys card and measures above-mentioned five kinds of samples；Using storing at 4 DEG C, 0d, (with reference to Beijing, Re Jing company is based on conventional effect 18 months phases (m)
The effect phase of the sundry item detection card of UPT-LF) the detection card at totally two time points measure above-mentioned five kinds of samples, every part of sample is equal
Detection takes mean value afterwards twice.Detection card stores the measurement result at each time point compared with 0d, calculates relative deviation δ, δ < 15%
For tolerance interval.
2.6.3 determination of recovery rates
PIVKA-II recombinant antigen is configured to the 55ng/mL of low concentration, the 1000ng/mL of middle concentration and highly concentrated respectively
The 15000ng/mL of degree is added with the ratio of 1:9 into normal human serum matrix, and mixing sample and serum matrix repeat to survey to determine
It takes mean value afterwards three times, calculates the rate of recovery according to the following formula:
PIVKA-II recombinant antigen volume is V in formula0, concentration C0；Serum matrix volume is V1, measurement concentration is C1；
It is C that mixing sample, which measures concentration,；The rate of recovery is R.85%~115% is the tolerance interval of R.
1.6.4 precision is evaluated
Select in the linear range low (26.3ng/mL), in three kinds of (1052.9ng/mL) and high (15925.9ng/ml)
Horizontal serum sample assesses object as batch interior and betweenrun precision.Three kinds of horizontal samples repeat detection 20 times respectively with assessment
Withinrun precision；Above-mentioned serum sample is measured in different 20 days respectively, the equal replication two of every kind of horizontal samples
It is secondary to obtain betweenrun precision.Precision is assessed by batch interior/interassay coefficient of variation (CV%),It is respectively lower than 10%, 15% with the CV% of betweenrun precision in batch and can receive range.
1.6.5 interference experiment
In the serum sample that PIVKA-II level is respectively 7.7ng/ml (HC group), 527.8ng/ml (HCC group) respectively
The rheumatoid factor (RF), dense that CEA, concentration that AFP, concentration that concentration is 5mg/dL are 0.2ug/ml are 750.0IU/mL is added
Spending vitamin C, concentration that the bilirubin for being 65.0mg/dL, the albumin that concentration is 7.0g/dL, concentration are 12.3mg/dL is
The hemoglobin (HB) of 0.35g/dL, the triglycerides (TG) that concentration is 500.0mg/dL, and using distilled water as control, 8 kinds
Mixture with the equal replication of serum sample 3 times.The interference free performance of UPT-LF analytic approach is assessed by deviation (δ %), δ %
Analyte level × 100% of=(analyte level-serum sample analyte level of mixture)/serum sample.δ % <
15% is tolerance interval.
1.6.6UPT-LF the Cutoff that analytic approach diagnoses HCC, sensitivity and specificity
Using UPT-LF detection card to HCC group 228, LBL group 170 (LC patient 80, CHB patient 90) and HC
The serum sample of group 100 carries out the quantitative determination of PIVKA-II.HCC group and the analyte level of control group (LBL+HC group) are poor
Different comparison is examined using the Mann-Whitney U of nonparametric statistics；Analyte level comparison in difference in control group between each group is adopted
With the Kruskal-Wallis H rank sum test of nonparametric statistics；Receiver Operating Characteristics are used to the diagnosis performance analysis of HCC
Curve (ROC) is analyzed, by maximum youden index (YI) as the cutoff value foundation for determining diagnosis HCC.
1.6.7UPT-LF compared with the detection performance of CLEIA analytic approach
Randomly select HCC group 178, LBL group 60 (each 30 of LC patient, CHB patient), HC group 35 serum samples
This, be respectively adopted UPT-LF detection card withG PIVKA-II kit (CLEIA) carries out quantitative detection, compares two
Kind of method draws two methods to 178 HCC patients to the difference of HCC diagnostic sensitivity and specific (vs LBL+HC group)
The linear regression curves of blood-serum P IVKA-II quantified results carry out correlation analysis.G PIVKA-II inspection
Detection limit (LoD), the range of linearity and the diagnosis cutoff value of test agent box are respectively 1.37mAU/ml, 5-75000mAU/mL
With 40mAU/mL.
2. statistical analysis
Otherness between the continuous variable of two groups of Non-Gaussian Distributions compares to be examined using Mann-Whitney U, and is compareed
Otherness between the continuous variable of three groups of Non-Gaussian Distributions of group compares to be examined using Kruskal-Wallis.The detection point of two methods
The relevance evaluation analysed between the result of object uses Pearson correlation coefficients (R).Statistical analysis involved in this research uses
SPSS 20.0, GraphPad Prism 5.0 and Excel is carried out, and P < 0.05 is thought with statistical significance.
3. experimental result
The surface modification and functionalization of 3.1UCPs
For UCPs because being influenced by synthetic method, surface often has one layer of hydrophobic oleic acid ligand.Nonionic emulsifier CO-
520 can reduce particle surface tension, improve water solubility, particle is made to be in monodisperse status as far as possible.The modification and activation of UCPs
Principle is as shown in Figure 1 C.In the Nano-meter SiO_2 for TEOS being added with after ammonium hydroxide, TEOS is formed under ammonium hydroxide effect2It is attached to particle table
Face, so that particle surface is enclosed with one layer of SiO2.When in solution there are when silane coupling A PES, molecule one end and silicon phase
Even, the other end then carries active carboxyl groups, and the UCPs of functionalization can be in conjunction with multiple biological activities molecule, such as antibody.
The detection architecture of 3.2UPT-LF analytic approach
The detection architecture is sent out by converting on PIVKA-II Sample dilution, detection card and detection device UPT-3A-1800
Light immunity analysis instrument forms (Fig. 3).Detection card is formed by getting stuck with internal test strips.It gets stuck including above getting stuck and bottom case, upper card
Shell successively has sample-adding window, detection window and chromatography termination instruction window, test strips then to deposit in the groove of bottom case by chromatography direction.Exempt from
Epidemic disease test strips chromatography direction is followed successively by HBR-IgG blocking agent sample pad, the bonding pad containing bioprobe, has T line (2.0mg/
) and the NC film and blotting paper of C line (0.6mg/ml) ml.Sample pad and bonding pad overall width are 2.5cm, and bonding pad is covered on NC
Width on film is 1mm.Cheap infrared light supply and Rechargeable battery, lightweight built in portable UPT-3A-1800 analyzer
It is small, it is very suitable for the detection of POCT formula.
The detection limit and linear evaluation of 3.3UPT-LF analytic approach
The blank sample dilution mean value Mean of measurementblank(T/C value) and standard variance SDblank, low concentration sample
Standard variance SDlowlevelsampleRespectively 0.112,0.0064 and 0.0041.The T/C value of LoB and LoD be respectively 0.125 with
0.129, it is 2.25ng/ml, 2.66ng/ml that linear regression equation (as follows), which converses corresponding concentration value,.It is quantitative to survey
The linear regression curve for determining the UPT-LF analytic approach of PIVKA-II is shown in Fig. 4.The visible horizontal section in 4.8-20000ng/ml Fig. 4
With significant linear relationship (R2=0.984, P < 0.01), but when analyte level be more than 20000ng/mL or be lower than 4.8ng/
When mL, do not have apparent linear relationship (data are not shown), thus this research is determined using 4.8-20000ng/mL as detection
The range of linearity of card quantitative determination blood-serum P IVKA-II concentration.By the Log (C for calculating 13 respective concentration pointso) and Log (T/
C-mean blank) matched curve obtains equation of linear regression: Y=0.827X -1.871 (N=13, P < 0.01).
3.4 estimation of stability
25 DEG C of storages 0,7,15,30, the detection card sample measures result of 60d are shown in Fig. 5 A and Fig. 5 B, as can be seen from the figure
25 DEG C of storages in two months influence very little to the detection performance of detection card, and the measurement result of different time points detection card is compared with 0d
Compared with bias δ is respectively less than 15%.The detection card sample measures result of 37 DEG C of 0,1,3,5 and 7d of storage is shown in Fig. 5 C and Fig. 5 D, can in figure
To find out that 37 DEG C of storages can cause to detect a degree of decline of card detection performance appearance, store to the measurement result and 0d of 7d
It compares, δ > 15%.Research finally assesses the detection card effect phase, and 4 DEG C of storage 0d, conventional effect phase 18m are (based on Beijing warm
The effect phase that scape biology Co., Ltd is recommended) detection card sample measures result see Fig. 6.It is computed analysis, 4 DEG C of storages to 18m
Four batches detection card testing result with 0d compared with, bias δ is equal < 15%.
3.5 determination of recovery rates
The accuracy of the measurement analyte of UPT-LF detection method is assessed by determination of recovery rates.By low middle high three kinds of levels
PIVKA-II recombinant antigen be added to healthy normal human serum after be measured, through formula calculate analysis obtain it is low middle three kinds high
The rate of recovery of horizontal analysis object is respectively 93.1%, 97.7% and 99.2%, all in the tolerance interval of 85%-115%.
The evaluation of 3.6 precision
Batch interior and betweenrun precision assessment result that PIVKA-II serum sample by measuring three kinds of different levels obtains
It is shown in Table 1.Withinrun precision CV% is less than 5% as can be seen from the table, and betweenrun precision CV% is small by 10%.
Batch interior and betweenrun precision of 1 UPT-LF of table detection card measurement blood-serum P IVKA-II
The experiment of 3.7 interference
The specificity of UPT-LF analytic approach is assessed by interference experiment.Eight kinds of conventional chaff interferents are added separately to HC group
It is measured in HCC group serum sample, the interference free performance evaluation result of UPT-LF detection method is shown in Table 2, can from table
See that the δ (%) of eight kinds of chaff interferents is both less than 10%.
The interference of 2 UPT-LF analytic approach of table is evaluated
AFP: AFP5 mg/dl, CEA: carcinomebryonic antigen 0.20ug/ml, RF: rheumatoid arthrosis factor 750IU/ml,
Bilirubin: bilirubin 65mg/dl, Albumin: albumin 7g/dl, Vitamin C: vitamin C 12.30mg/dl, Hb:
Hemoglobin 0.35g/dl, TG: triglycerides 500mg/dl.
The cutoff value that 3.8UPT-LF detection method diagnoses HCC, sensibility and specificity
The result of the quantitative determination serum sample of UPT-LF detection method is as shown in Fig. 7 A.The blood-serum P IVKA-II water of HCC group
Flat (median 53.2ng/ml, quartile spacing 20.6-322.3ng/ml) will be significantly higher than control group (LBL+HC group) (middle position
Number 9.2ng/ml, quartile spacing 4.8-15.4ng/ml) (Z=-13.7, P < 0.01) level；The LC group serum of control group
PIVKA-II level (median 10.8ng/ml, quartile spacing 7.0-20.1ng/ml) and CHB group (median 10.7ng/ml,
Quartile spacing 5.4-16.3ng/ml) and HC group (median 6.7ng/ml, quartile spacing 3.1-14.2ng/ml) between
Difference be not statistically significant (X2=5.67, P > 0.05).ROC curve analyzes (HCC group vs LBI+HC group) display, detection
The area under the curve AUC of card measurement blood-serum P IVKA-II is 0.85 (P < 0.001), sees that the big youden index YI=0.60 institute of Fig. 7 B is right
The diagnosing cancer of liver cutoff value answered is 25.3ng/ml (value=0.29 T/C), the sensitivity that UPT-LF detection method diagnoses HCC at this time
Property with specificity be respectively 71.49%, 88.89%.
3.9UPT-LF analytic approach is compared with CLEIA analytic approach is to HCC diagnosis performance and the correlation of quantitative detection result
Property analysis
When the horizontal 25.3ng/ml of analyte is diagnosed cutoff value to HCC as UPT-LF analytic approach, HCC is measured
Group 178, LBL group 60 (each 30 of LC group, CHB group), HC group 35 serum samples testing result statistical analysis obtain
It is respectively 70.2% and 90.5% to diagnostic sensitivity and specificity, and it is acquired when the above-mentioned sample of CLEIA assay
Diagnostic sensitivity and specificity are respectively 67.4% and 91.6%.It can be seen that the diagnosis performance otherness of two kinds of analysis methods
It is smaller.In addition, in our study it has also been found that both measurement blood-serum P IVKA-II also have significant linear relationship (P <
0.01).Fig. 8 is as it can be seen that equation of linear regression is Y=0.08X -4.50, R2=0.901.
4. discussing
In our current research, we are reported for the first time using the detection card quantitative determination blood-serum P IVKA- based on UPT-LF analytic approach
II.In the detection card, the anti-PIVKA-II monoclonal antibody of UCPs label is used as bioprobe, and test strips are then used as and exempt from
The solid phase carrier of epidemic disease reaction.Result of study shows that the detection card of the UPT-LF constructed by us not only detects blood-serum P IVKA-II
Wider detection band property range with 4.8-20000ng/mL, and minimum detection limit LoD is down to 2.66ng/mL, it is seen that detection spirit
Sensitivity is high.Estimation of stability is the result shows that UPT-LF detection is stuck in 4 DEG C and can save steadily in the long term, in 25 DEG C of preferable thermostabilizations of tool
Property, it shows as not occurring obviously in 4 DEG C of storages to conventional effect phase (18 months), 25 DEG C of storages to 60 days detection card detection performances
Change；But 25 DEG C are compared, the thermostable effect that detection is stuck in 37 DEG C is bad, shows as the measurement result of 37 DEG C of storages to 7d
Bias δ > 15% compared with 0d, thus it is speculated that this may be with the coated antibody on the labelled antibody or NC film in bonding pad in high temperature
Under have occurred it is a degree of degradation it is related.In addition, UPT-LF detection card is for the low middle three kinds high of PIVKA-II recombinant antigen
The rate of recovery acquired by the analyte determination of concentration between 93.1%-99.2%, batch in batch variation CV be respectively lower than 5% with
10%, the bias δ (%) of 8 kinds of serum common interference object measurement results is below 10%, shows UPT-LF detection card while having
Good detection accuracy, precision and specificity, result above prompt the UPT-LF detection card of building to serum
The detection performance of PIVKA-II is good.Especially, light is forwarded on the detection device UPT-3A-1800 in UPT-LF detection system
Immunity analysis instrument, as excitation light source, meets the detector bar of POCT formula using relatively inexpensive near-infrared semiconductor laser
Part, having the characteristics that small in size, light-weight, easy to carry, detection time is short only needs 15min, illustrates making for UPT-LF detection card
With can be achieved to blood-serum P IVKA-II fast and efficiently POCT formula quantitative detection.Currently in clinical labororatory, blood-serum P IVKA-II
Measurement is usually to be conducted batch-wise in such as CLEIA analyzer equipment complicated in this way, while measuring is not all can daily yet
Carry out；And use this research and establishment UPT-LF test and analyze method, then can to avoid large-scale clinical labororatory's biochemistry department or specially
The dependence that the Code in Hazardous Special Locations such as Ye Hua central laboratory transport sample, realize to detection by the bed of patients serum PIVKAII with
And the implementation of clinic diagnosis decision-making in-situ.
Further blood-serum P IVKA-II detection of the research based on UPT-LF blocks the diagnosis performance to HCC, as the result is shown HCC group
Blood-serum P IVKA-II level be significantly higher than control group (LBL+HC group) (P < 0.01), and the LC group as control, CHB group and HC
The poor opposite sex of blood-serum P IVKA-II level is not statistically significant (P > 0.05) between group.In addition, UPT-LF method detects serum
PIVKA-II reaches 0.85 to area (AUC) under the ROC curve of HCC, shows that it has good diagnosis performance to HCC；As general
When the cutoff value that the 25.3ng/ml level of analyte is diagnosed as HCC, UPT-LF method can obtain optimal diagnosis performance,
Its diagnostic sensitivity and specificity are respectively 70.2% and 90.5%, have the commercial CLEIA method kit phase with PIVKA-II
Similar diagnosis performance.Linear regression curves are analyzed the result shows that two methods of UPT-LF and CLEIA are to 178 HCC patient's blood
The coefficient R of clear PIVKA-II quantified results2=0.901, show good linear relationship.
In conclusion this research and establishment can be achieved based on the detection card of UPT-LF analytic approach to HCC patients serum PIVKA-
Quick, the efficient and convenient POCT formula of II quantitative determines.The realization of this research achievement will be expected to promote PIVKA-II clinical real
The popularization and application for testing room further increase HCC early detective rate, reduce mortality.
Claims (10)
1. a kind of detection card based on up-converting phosphor technology immunochromatographic method quantitative determination blood-serum P IVKA-II, feature exist
In the detection card includes sample pad, bonding pad, nitrocellulose filter, blotting paper, and the sample pad and bonding pad are glass fibre
Plain film, the nitrocellulose filter are equipped with detection band and quality control band, the sample pad, bonding pad, nitrocellulose filter, water suction
Paper is successively pasted on sticky bottom liner, and the test strips band of 3-6mm wide is cut into using cutting machine, and is packaged in UPT and is got stuck；
Up-conversion luminescence nanoparticle -1 conjugate of anti-PIVKA-II monoclonal antibody is fixed on the bonding pad, the detection band is solid
Surely there is anti-PIVKA-II monoclonal antibody 2, the quality control band is fixed with secondary antibody sheep anti-mouse igg.
2. the detection according to claim 1 based on up-converting phosphor technology immunochromatographic method quantitative determination blood-serum P IVKA-II
Card, which is characterized in that the sample pad, bonding pad, nitrocellulose filter and blotting paper are successively handed over the spacing for being overlapped 1~2mm
Mutually stacking is pasted on sticky bottom liner, and the width of the test strips is 4mm.
3. the detection according to claim 1 based on up-converting phosphor technology immunochromatographic method quantitative determination blood-serum P IVKA-II
Card, which is characterized in that the up-conversion luminescence nanoparticle is the NaYF that TEOS and APES is modified4:Yb3+,Er3+UCPs。
4. a kind of preparation method of the detection card based on up-converting phosphor technology immunochromatographic method quantitative determination blood-serum P IVKA-II,
This method comprises the following steps:
(1)NaYF4:Yb3+,Er3+The preparation of UCPs；
(2)NaYF4:Yb3+,Er3+The functional modification of UCPs；
(3) building of up-conversion luminescence bioprobe；
(4) configuration and coating of conjugate solution；
(5) production of blocking agent sample pad；
(6) configuration and coating of Quality Control solution and detection solution；
(7) preparation of the detection card of immunochromatographic method is converted on；
(8) double-antibody sandwich mode UPT-LF analytic approach quantitative detection blood-serum P IVKA-II；
The method of modifying of step (2) is as follows: taking NaYF4:Yb3+,Er3+Hexamethylene is added in UCPs, super under the conditions of ultrasonic vibration
Disperse in pure water and hang particle completely, CO-520, ultrasonic 10-40min is then added；Ammonium hydroxide, TEOS, magnetic agitation is added
After reacting 16-32h, ethyl alcohol is added and is centrifuged, discards supernatant liquid；Precipitating is dissolved using isopropanol, and APES is added thereto, magnetic
Power is stirred to react 2-4h, after being centrifuged and washing, obtains the NaYF of functional modification4:Yb3+,Er3+UCPs。
5. the detection according to claim 4 based on up-converting phosphor technology immunochromatographic method quantitative determination blood-serum P IVKA-II
The preparation method of card, which is characterized in that NaYF in the step (1)4:Yb3+,Er3+UCPs's the preparation method is as follows: will
0.2343gYCl3,0.0755g YbCl3With 0.0083g ErCl3The 1- 18 of the oleic acid and 26.0ml of three kinds of reagents and 4.5mL
Alkene is mixed in flask, and 150-160 DEG C is heated in argon atmosphere and is mixed to solution, is cooled to room temperature after continuing 25min；It will
15mL contains the NH of the NaOH and 0.222g of 0.015g4The methanol solution of F is added in flask, and quickly forms in the solution solid
State precipitating, reaction system stirs 20-40min at this time；Then, reaction solution is slowly heated to 100 DEG C and maintains 8-15min, with
Vapor away methanol；Then it is heated rapidly to 300 DEG C and maintains 1h；After solution natural cooling, using ethyl alcohol by product from
It is precipitated in solution；It is deposited under 12000rpm and is centrifuged 5-10min, and cleaned 2-4 times with water/ethanol solution of 1:1 volume ratio, obtained
To NaYF4:Yb3+,Er3+UCPs。
6. the detection according to claim 4 based on up-converting phosphor technology immunochromatographic method quantitative determination blood-serum P IVKA-II
The preparation method of card, which is characterized in that the method for modifying of the step (2) is as follows: weighing NaYF4:Yb3+,Er3+UCPs 40mg,
95mL hexamethylene is added, and disperses in ultrapure water under the conditions of ultrasonic vibration and hangs particle completely, CO- is then added
5202.5mL, ultrasonic 20min；30wt% ammonium hydroxide 0.35mL, TEOS 0.09mL is added to be added after magnetic agitation reaction for 24 hours
16ml ethyl alcohol is simultaneously centrifuged, and discards supernatant liquid；Precipitating is dissolved using 90mL isopropanol, and APES 0.9mL, magnetic force are added thereto
It is stirred to react 2.5h, after being centrifuged and washing 3 times, obtains the NaYF of functional modification4:Yb3+,Er3+UCPs。
7. the detection according to claim 6 based on up-converting phosphor technology immunochromatographic method quantitative determination blood-serum P IVKA-II
The preparation method of card, which is characterized in that the construction method of the up-conversion luminescence bioprobe of the step (3) is as follows: by 5mg's
The NaYF of functional modification4:Yb3+,Er3+UCPs is added in the purified water of 1ml and dissolves under the conditions of ultrasonic vibration and mixes, and is added
20% Tween-20 activating agent 5ul is mixed under ultrasound condition；The anti-PIVKA-II monoclonal of 75ug is added into reaction system
Antibody 1 reacts 20min under the conditions of ultrasonic vibration；5ul 20%BSA, ultrasonic vibration condition is added after after reaction thereto
Lower closing 5min；Then with 4 DEG C, it is centrifuged 30min in the high speed refrigerated centrifuge of 12000r/min, takes and precipitates and to discard supernatant liquid standby
With.
8. the detection according to claim 7 based on up-converting phosphor technology immunochromatographic method quantitative determination blood-serum P IVKA-II
The preparation method of card, which is characterized in that the configuration of the conjugate solution of the step (4) and method for coating are as follows:
A. conjugate store buffer liquid configures: it is added 0.1%BSA in the PBS buffer solution of 0.02mol/l, pH7.4,0.3%
TritonX-100,0.02%NaN3；
B. conjugate freeze-drying buffer configuration: being added 5%trehalose in the PBS buffer solution of 0.02mol/l, pH7.4, and 1%
BSA, 2%mannitol, 1%glycine, 0.01%TB, 0.5%casein, 0.1%NaN3；
C. it first is separately added into 100ul conjugate store buffer liquid in precipitating obtained by backward step (3), the freeze-drying of 5.5ml conjugate is slow
Fliud flushing mixes under water in ultrasonic vibration condition；The mixed liquor is uniformly coated on 3*19.33cm with 600ul/ sample loading gun2's
On SB08 type glass fibre, and it is transferred quickly to carry out -60 DEG C of frozen drieds, cooling time 2h in freeze dryer；Then by it
At least 8h is vacuumized and maintained, it is spare to room temperature preservation to take out balance.
9. the detection according to claim 4 based on up-converting phosphor technology immunochromatographic method quantitative determination blood-serum P IVKA-II
The preparation method of card, which is characterized in that the blocking agent sample pad of the step (5) the production method is as follows: HBR blocking agent is made
With 0.01mol/L, pH8.0TB buffer is diluted to concentration and 75.0ul 20% is added after 75ug/mL, to take out 10.0ml
Casein then equably pours it in 6*19.33cm2SB08 glass fibre on, room temperature is dried rear spare.
10. the inspection according to claim 4 based on up-converting phosphor technology immunochromatographic method quantitative determination blood-serum P IVKA-II
Survey the preparation method of card, which is characterized in that the configuration of the Quality Control solution and detection solution of the step (6) and method for coating are such as
Under: nitrocellulose filter is pasted on sticky bottom liner designated position, blotting paper 1-2mm Chong Die with nitrocellulose filter and and viscosity
Bottom liner upper end, which flushes, is pasted, and then uses test antibody PIVKA-II monoclonal antibody 2 and Quality Control antibody sheep anti-mouse igg
0.02mol/L, pH7.2PB buffer are diluted to 2.0mg/mL, 0.6mg/mL respectively；Using continuous Film-cutting machine in nitrocellulose
PIVKA-II monoclonal antibody 2 and sheep are sprayed respectively with the speed of 1ul/cm at distance viscosity bottom liner the bottom end 12mm and 15mm of film
Anti- mouse IgG is as detection band and quality control band；The nitrocellulose filter being coated with is placed in 37 DEG C of baking ovens and dries 1.5h, is encapsulated
It is spare.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110007096A (en) * | 2019-05-06 | 2019-07-12 | 江苏硕世生物科技股份有限公司 | A kind of dengue virus IgG/IgM antibody test strip, kit and preparation method thereof |
| CN110007095A (en) * | 2019-05-06 | 2019-07-12 | 江苏硕世生物科技股份有限公司 | A kind of dengue virus NS 1 antigen test strip, kit and preparation method thereof |
| CN113372447A (en) * | 2021-05-26 | 2021-09-10 | 重庆中元汇吉生物技术有限公司 | anti-PIVKA-II monoclonal antibody and application thereof |
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| CN113372447A (en) * | 2021-05-26 | 2021-09-10 | 重庆中元汇吉生物技术有限公司 | anti-PIVKA-II monoclonal antibody and application thereof |
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