CN105954522B - The detection of active urokinase receptor - Google Patents

The detection of active urokinase receptor Download PDF

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Publication number
CN105954522B
CN105954522B CN201610541379.6A CN201610541379A CN105954522B CN 105954522 B CN105954522 B CN 105954522B CN 201610541379 A CN201610541379 A CN 201610541379A CN 105954522 B CN105954522 B CN 105954522B
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upar
auca
activity
active
sample
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CN105954522A (en
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黄明东
周晓雷
许明明
袁彩
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Fujian Yitong Biotechnology Co ltd
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Fujian Institute of Research on the Structure of Matter of CAS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Abstract

The detection of active urokinase receptor.The invention discloses preparing the application in the kit for detecting active uPAR, and the kit comprising AUCA or its fusion protein using the method and AUCA or its fusion protein of AUCA or its fusion protein detection activity uPAR.The detection method includes: to contact capture reagent under conditions of activity uPAR in being suitable for the capture reagent capture sample to be tested with sample to be tested, the compound of capture reagent and activity uPAR is formed, the capture reagent includes can the specific AUCA or its fusion protein for capturing activity uPAR；2) captured activity uPAR is detected.Method of the invention has preferable accuracy and reproducibility, and Monitoring lower-cut reaches 15ng/L.

Description

The detection of active urokinase receptor
Technical field
The present invention relates to field of biological medicine, and in particular to the detection to active urokinase receptor.
Background technique
Urokinase receptor (urokinase type plasminogen acitivator receptor, uPAR) is a kind of Cell surface receptor.This receptor is equal to 1985 by Stoppelli to be found, Roldan in 1989 etc. has cloned the cDNA, 1990 Year Behrendt etc. is purified into the albumen from the cell membrane extract of human lymphoma cell U937 with affinity chromatography.The 5th In secondary world leukocyte differentiation antigen meeting, uPAR is named as (the cluster of of cluster of differentiation 87 Differentiation 87, CD87).UPAR is a kind of surface membrane protein of high glycosylation, and wide expression is in immunocyte Surface, such as the neutrophil cell of activation, monocyte, the T lymphocyte of activation, macrophage and a variety of pernicious swollen Oncocyte surface, but it is lower [1-5] in most normal cell surface expression amounts.
UPAR is a kind of flexible molecule, can be with multiple ligands or acceptor interaction.Such as vitronectin (vitronectin), plasma urokinase-type plasminogen activator (urokinase-type plasminogen activator, UPA), (the low density lipoprotein receptor-related of LDH receptor related protein 1 Protein 1, LRP1), integrin (integrin), g protein coupled receptor (G protein coupled receptor, GPCR) etc..These different interactions play extensive important function in a variety of physiology of human body and pathologic process, including Activation, cell adhesion and the migration of plasminogen, cell differentiation, chemokinesis, chemokine receptors adjust, are immunized and answer It answers, inflammatory reaction etc. [4,6].UPAR is by three rich in cysteine, the Ly6/uPAR structural domain that size is 81-87 amino acid It forms (D1, D2, D3), molecular weight is about 55kDa.UPAR passes through the glycolsyl-phosphatidylinositol of its C-terminal (glycosylphosphatidylinositol, GPI) is anchored on cell membrane surface [4,6].
Urokinase receptor is there is a variety of forms, the membrane receptor including overall length, the soluble urokinase without transmembrane domains Receptor (soluble uPAR, suPAR) and various degradation fragments.Water of the uPAR membrane receptor of overall length vulnerable to phosphatidyl enzyme C Solution, makes uPAR fall off from cell membrane surface, forms the Monomeric Soluble Human Urokinase Receptor (soluble for being free of glycolsyl-phosphatidylinositol UPAR, suPAR).In addition uPAR is also sensitive to a variety of hydrolases, can be further hydrolyzed to D1 and D2-D3 segment [3].
We early period crystal structure studies have shown that uPAR by it three structural domains formed a bowl structure, and Exactly this bowl structure can efficiently combine its ligand uPA [7], and uPA is enriched in cell surface, and by Plasminogen activation It for fibrinolysin, and then degrades to extracellular matrix, is playing a significant role in cell migration.Our structure also turns out simultaneously The combination of uPA and uPAR is greatly improved the combination [8] of uPAR and vitronectin, and a large amount of document also demonstrate uPA with Importance [6,7] of the combination of uPAR to itself and integrin interaction.These albumen and other albumen such as caveolin etc. exist Lesion is locally sticked, is gathered, signal in integrin receptor active cell, so that signal is passed to intracellular, activation from extracellular Intracellular protein kinase promotes cell division and cell migration.This uPAR is defined as active uPAR [7].It is only active UPAR can carry out the conduction of signal, it is and closely related with the invasion of tumour and transfer, and other uPAR segments are without activity.Mesh The preceding method still without measuring activity uPAR.
Since uPAR content and disease are closely related, there are many diagnostic methods for being directed to uPAR currently on the market, they lead If being detected by double antibody sandwich method, that generally measure is active and inactive total amount suPAR in blood.Such as Enzyme-linked Immunosorbent Assay (the enzyme-linked immuno sorbent for the detection suPAR that ViroGates company, Denmark releases Assay, ELISA) kitCE-IVD (external diagnosis reagent) certification for having passed through Europe, for referring to Lead clinic.This method is to increase to imply patient's immune system by sustained activation based on the concentration of total suPAR in blood [3], the concentration level of total suPAR in blood also can be used as the deliberated index of disease progression and for patient's risk status Clinical decision [9-11].In addition, suPAR can also be used for the screening of Patients of Intensive Care Unit coincident with severity degree of condition and patient controls The evaluation [12,13] of therapeutic effect.Specifically, the concentration range of suPAR 0.1-4.0 μ g/L indicate subject be it is normal, do not have Infection or inflammatory reaction；4-6 μ g/L shows that subject may have infection or the improper activation of immune system to need further inspection It looks into；> 6 μ g/L show disease in rapid progress, need tightly to monitor and follow-up is treated.However it is similar at presentWhat the double sandwich methods of these antibody detected is all the total value of diversified forms suPAR in blood, can not be directly Detect the content of activity uPAR in blood.
Summary of the invention
The object of the present invention is to provide the detection methods of activity uPAR a kind of.
The content of present invention is as follows: using activity uPAR capture reagent (active uPAR capture agent, AUCA) or Fusion protein containing it captures activity uPAR, to realize the detection to uPAR.AUCA is the truncation of urokinase N-terminal segment Body retains its segment that is related and keeping structural stability, sequence such as SEQ ID NO:1 in conjunction with uPAR by structural analysis It is shown.As ATF, it is capable of the active pocket of specific binding activity uPAR, and to inactive uPAR (such as D1 and D2D3) Only with respect to very weak combination.
The amino acid sequence of the activity uPAR capturing agent AUCA is as follows:
PSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWN SATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCA(SEQ ID NO.1)。
Term used in the present invention " uPA " refers to plasma urokinase-type plasminogen activator.
Term used in the present invention " uPAR " refers to uPAR Research, alternatively referred to as urokinase Receptor.
Term used in the present invention " suPAR " refers to the solvable shape of uPAR Research (uPAR) Formula.
Term used in the present invention " AUCA " refers to one section of protein sequence of specificity capture activity uPAR, ammonia Base acid sequence is defined in SEQ ID NO.1.AUCA is the abbreviation of active uPAR capture agent.
Term used in the present invention " active uPAR " refers in structure containing three complete domain D1 and D2D3, functions On can in conjunction with native ligand uPA and vitronectin uPAR." active uPAR " as described herein include overall length uPAR film by Body, the Monomeric Soluble Human Urokinase Receptor (soluble uPAR, suPAR) without transmembrane domains, but do not include the various degradation pieces of suPAR Section.
According to an aspect of the present invention, the detection method of active uPAR is provided, this method comprises:
1) make the item for capturing reagent and sample to be tested the activity uPAR in being suitable for the capture reagent capture sample to be tested It is contacted under part, forms the compound of capture reagent and activity uPAR；
2) captured activity uPAR is detected；
Wherein the capture reagent includes AUCA or the fusion protein containing AUCA.
In preferred embodiments, detection method of the invention is non-diagnostic purpose, and is carried out in vitro. In preferred embodiments, the fusion protein containing AUCA is AUCA-HSA fusion protein." it is suitable for the capture reagent to catch Obtain the condition of activity uPAR in sample to be tested " it include suitable temperature, reaction time and solution etc., to avoid protein denaturation, And make to capture reagent and activity uPAR specific binding.Suitable condition depends on the specific detection system of selection and/or specific Detection method, those skilled in the art based on its general knowledge grasped can select to be suitable for different detection systems and/or The condition of specific detection method.
It can be by specific detection system well known to those skilled in the art and/or specific detection method to captured work Property uPAR is detected.Selected specific detection method can be heterogeneous or homogeneity measuring method.Heterogeneous measuring method be include one Measuring method secondary or that step is cleaned multiple times, and in homogeneity measuring method, such cleaning step is not required, and only will test reagent It mixes and measures with sample to be tested.The measuring method includes but is not limited to be based on ELISA (enzyme-linked immunosorbent assay) Measuring method, DELFIA (dissociation enhancing lanthanide series fluorescence immunoassay), SPA (scintillation proximity assay), (time-resolved fluorescence resonance energy turns by Flashplate measuring method, FRET (fluorescence resonance energy transfer) measuring method, TR-FRET Move) measuring method, FP (fluorescence polarization) measuring method, ALPHA (amplification chemiluminescence affinity homogeneously detects), EFC (enzyme fragment complementation) Measuring method, two-hybrid assay or co-immunoprecipitation measuring method.
In a further embodiment, in the detection method of above-mentioned activity uPAR, step 2) may include making institute's shape At compound combined with the reagent for specifically binding captured activity uPAR, and be caught by described specifically bind of detection The reagent of the active uPAR obtained detects captured activity uPAR.
It, can be by realizing to the measurement of ingredient is detected included in reaction system to quilt during said determination The active uPAR of capture is detected.The detection ingredient include capture reagent (such as capture reagent contained in AUCA or Its fusion protein) and/or with captured activity uPAR specific binding reagent.It can be by making to the measurement of detection ingredient It is marked with detectable marked member and detects marked member to carry out.Detectable marked member is according to specifically used Measuring method and it is different.In the present invention, biotin, enzyme, fluorescent dye such as, but not limited to can be used in many ways (such as FITC, fluorescein, rhodamine, Cy dyestuff or Alexa fluor), to mark detection ingredient, also can be used radioactivity mark Remember object (such as3H、 32P、35S、125I or14C) and common enzyme marker such as horseradish peroxidase and alkaline phosphatase carry out Label.For example, marked member can be marked on the reagent specifically bound with captured activity uPAR, marked by detection Remember that ingredient realizes the detection to captured activity uPAR；One in pairs of marked member can also be marked and be tried in capture In agent, another label is on the reagent specifically bound with captured activity uPAR, by between pairs of marked member The detection of interaction realize detection to captured activity uPAR；Marked member can also be marked in capture reagent On, the marked member that the combination of capture reagent and activity uPAR will lead on capture reagent changes, and is marked as by detection The detection to captured activity uPAR is realized in the variation divided；These marked members and its detection method are all those skilled in the art Known to member.
In some preferred embodiments, the reagent for specifically binding captured activity uPAR is to be incorporated into activity Monoclonal antibody on the outside of uPAR.This kind of antibody can by obtaining polyclonal antibody using active uPAR protein immune animal, It captures and obtains through uPAR-AUCA affinity column after and.
In a more preferred embodiment, the monoclonal antibody being incorporated on the outside of active uPAR is with uPAR D2D3 Mouse is immunized in (amino acid 88-283), the anti-uPAR antibody of monoclonal obtained from screening through hybridoma technology.
In more preferred embodiment, the monoclonal antibody being incorporated on the outside of active uPAR is antibody A TN- 658。
Terms used herein " monoclonal antibody " refer to the antibody obtained from a kind of substantially uniform group, i.e., in the group The single antibody for including be it is identical, other than a small number of mutation that may be present naturally occurred, monoclonal antibody is high special Property be directed to single epitope.The method for preparing the monoclonal antibody of specific antigen is it is known in the art that for example by miscellaneous Tumor method is handed over to obtain." being incorporated into the monoclonal antibody on the outside of active uPAR " of the present invention refers to targeted epitope position Monoclonal antibody on the outside of active uPAR." on the outside of the active uPAR " is the work being inserted on active uPAR relative to AUCA The site on outside for the inside of property hydrophobic pocket.In the case of active uPAR is in conjunction with AUCA, site is tied with its outside The monoclonal antibody of conjunction can be combined with being formed by compound, so as to realize the inspection to captured activity uPAR It surveys.The method of the targeted epitope of identification monoclonal antibody is it is known in the art that therefore those skilled in the art can be with Determine whether monoclonal antibody combines on the outside of active uPAR by the method known to it.
The measurement of the monoclonal antibody can be marked simultaneously detection ingredient by using detectable marked member Marked member is detected to carry out.Detection ingredient includes capture reagent (containing AUCA or containing the fusion protein of AUCA), is incorporated into Monoclonal antibody on the outside of active uPAR and/or the secondary antibody in conjunction with the monoclonal antibody.For example, marked member can be marked In monoclonal antibody, the detection to captured activity uPAR is realized by detection marked member；Monoclonal can also be made anti- Body and two anti-bindings are realized by detection marked member to captured activity uPAR's by marked member label on secondary antibody Detection；One in pairs of marked member can also be marked on capture reagent, another label is in the monoclonal antibody Or on the secondary antibody in conjunction with the monoclonal antibody, realized by the detection of the interaction between pairs of marked member to being caught The detection of the active uPAR obtained；Marked member can also be marked on capture reagent, capture the combination of reagent and activity uPAR The marked member that will lead on capture reagent changes, and the variation by detecting marked member is realized to captured activity The detection of uPAR；These marked members and its detection method are all well known to those skilled in the art.Detectable marked member Including but not limited to biotin, enzyme, fluorescent dye (such as FITC, fluorescein, rhodamine, Cy dyestuff or Alexa fluor), radiation Property marker is (such as3H、32P、35S、125I or14) and common enzyme marker such as horseradish peroxidase and alkaline phosphatase C.
In more preferred embodiment, specific step is as follows for detection method of the invention:
(1) be added into the hole of porous plate 100 μ l be coated the diluted AUCA-HSA protein solution of liquid (140 μ g/ml) into Row coating, 4 DEG C overnight, washs and dry；
(2) 100 μ l confining liquids are added into hole to be closed, 80 revs/min of room temperature, are incubated for 1 hour, wash and dry；
(3) the 100 μ l of active uPAR standard items of various concentration is added into a some holes, the various concentration is respectively 1 μ g/ L,0.5μg/L,0.25μg/L,0.125μg/L,0.0625μg/L,0.03125μg/L；The sample of 100 μ l is added into another some holes Product dilution does not contain uPAR as blank control in blank control；Being diluted with sample for measuring samples is added into other holes Liquid dilutes 10 times of dilution；It 80 revs/min of room temperature, is incubated for 1 hour, washs and dry；
(4) it is anti-that monoclonal of 100 μ l, the 9 μ g/ml through the anti-human source uPAR of the diluted mouse of sample diluting liquid is added in the hole Xiang Suoyou Body, is incubated for 1 hour by 80 revs/min of room temperature, is washed and dry, wherein the monoclonal antibody is with uPAR D2D3 (amino acid Mouse, the anti-uPAR antibody of monoclonal obtained from screening through hybridoma technology 88-283) is immunized；
(5) anti-mouse IgG of the 100 μ l through 500 times of diluted alkali phosphatase enzyme marks of sample diluting liquid is added in the hole Xiang Suoyou, It 80 revs/min of room temperature, is incubated for 1 hour, washs and dry；
(6) 100 μ l developing solutions are added into every hole, then ELISA Plate is placed in microplate reader and reads absorbance in 405nm, 60min is read altogether, and every 1min reads an absorbance value；
(7) standard curve is made, the concentration of activity uPAR in sample is calculated.
According to another aspect of the present invention, AUCA or the fusion protein containing AUCA are provided in preparation for test sample Application in the reagent of middle activity uPAR.
In the present invention, " sample to be tested " includes but is not limited to from the blood of subject, serum, plasma cell culture Liquid, saliva and urine.
Term used in the present invention " fusion protein " refers to the amino acid sequence comprising two or more different proteins The chimeric protein of column.The method for preparing fusion protein is it is known in the art that usually fusion protein is by well known in the art external Recombinant technique generates." fusion protein containing AUCA " of the invention is AUCA and another protein or polypeptide or its segment Chimeric protein.The another kind protein or polypeptide or its segment can be seralbumin, such as human serum albumins (HSA), bovine serum albumin(BSA) (BSA) is also possible to ovalbumin (OVA).In preferred embodiments, melting containing AUCA Hop protein is AUCA-HSA fusion protein.
In the present invention, by various methods known in the art AUCA can be prepared or containing the fusion protein of AUCA, example It can such as be prepared by chemical synthesis or recombination method.In preferred embodiments, AUCA of the invention or contain AUCA's Fusion protein is recombinant protein.
The AUCA or fusion protein containing AUCA can be fixed on solid matrix.The solid matrix include but It is not limited to porous plate (such as 96 orifice plates), protein-chip counterdie (such as nitrocellulose filter, nylon membrane), magnetic bead etc..
According to another aspect of the present invention, AUCA or the fusion protein containing AUCA are provided in preparation for test sample Application in the kit of middle activity uPAR.In preferred embodiments, the kit includes capture reagent, the capture Reagent includes can the specific AUCA for capturing activity uPAR or the fusion protein containing AUCA.
According to an aspect of the present invention, the kit for detecting active uPAR is provided, the kit includes capture Reagent, the capture reagent includes can the specific AUCA or its fusion protein for capturing activity uPAR.
In some embodiments, the capture reagent is marked by detectable marked member.Capture reagent and activity The marked member that the combination of uPAR will lead on capture reagent changes, and passes through the marked member on detection capture reagent The detection to captured activity uPAR may be implemented in variation.
In other embodiments, the kit also includes one kind or more for detecting captured activity uPAR Kind reagent.In preferred embodiments, one or more reagents for detecting captured activity uPAR include knot Together in the monoclonal antibody on the outside of active uPAR and/or the secondary antibody in conjunction with the monoclonal antibody.In some embodiments, institute Monoclonal antibody is stated to be marked by detectable marked member.In other embodiments, the monoclonal antibody can not examined The marked member of survey marks.In some embodiments, the secondary antibody is marked by detectable marked member.In other implementations In scheme, the secondary antibody is not marked by detectable marked member.
In preferred embodiments, the monoclonal antibody being incorporated on the outside of active uPAR is uPAR D2D3 (amino Sour 88-283) mouse, the anti-uPAR antibody of monoclonal obtained from screening through hybridoma technology is immunized.
In other embodiments, the kit also includes one kind or more for detecting captured activity uPAR Kind reagent, wherein the capture reagent is marked by an ingredient in detectable pairs of marked member, and for detecting quilt One or more reagents of the active uPAR of capture are by another label in pairs of marked member.It is marked as in pairs by detection / interaction the detection to captured activity uPAR may be implemented.In preferred embodiments, described to be used for The one or more reagents for detecting captured activity uPAR include be incorporated into monoclonal antibody on the outside of active uPAR and/or with The secondary antibody that the monoclonal antibody combines.
In other embodiments, the kit also includes any activity for being suitable for detecting captured reagent capture The detectable marked member of uPAR.
Above-described detectable marked member includes but is not limited to biotin, enzyme, fluorescent dye (such as FITC, fluorescence Element, rhodamine, Cy dyestuff or Alexa fluor), radioactively labelled substance (such as3H、32P、35S、125I or14) and common enzyme C Marker such as horseradish peroxidase and alkaline phosphatase.
In some embodiments, in kit of the invention, capture reagent can be fixed on solid-phase matrix, example Such as it is fixed on porous plate (such as 96 orifice plates), protein-chip counterdie (such as nitrocellulose filter, nylon membrane), on magnetic bead. It can also include coating buffer, confining liquid, cleaning solution, sample diluting liquid and the reagent for detecting marked member in kit One of or it is a variety of.
Mentioned reagent box can also include operation instructions.
In preferred embodiments, kit of the invention includes: ELISA Plate；AUCA-HSA protein solution；Activity UPAR standard items；Mouse, monoclonal obtained from screening through hybridoma technology is immunized in employment uPAR D2D3 (amino acid 88-283) Anti- uPAR antibody；The anti-mouse IgG of alkali phosphatase enzyme mark；Coating buffer；Cleaning solution/sample diluting liquid；Confining liquid and developing solution.
In a more preferred embodiment, kit of the invention includes: ELISA Plate；The AUCA-HSA of 1ml 1.4mg/ml Protein solution；1ml activity uPAR standard items (1mg/ml)；Mouse, warp is immunized in 100 μ l uPAR D2D3 (amino acid 88-283) The anti-uPAR antibody (0.9mg/ml) of monoclonal obtained from hybridoma technology screening；25 μ 500 × alkali phosphatase enzyme marks of l resist Mouse IgG；Coating buffer 10ml；30 × cleaning solution/sample diluting liquid 20ml；BSA confining liquid 10ml；PNPP developing solution 10ml.
In the present invention, coating buffer preferably contains 40mM NaHCO3, 10mM Na2CO3, the aqueous solution of pH 9.6.
In the present invention, confining liquid is preferably the 5%BSA solution prepared with coating buffer.
In the present invention, cleaning solution/sample diluting liquid is preferably the PBS solution for containing 0.5 ‰ Tween-20pH 7.4, wherein PBS formula is NaCl 8g/L, KCl 0.2g/L, Na2HPO4·12H2O 3.58g/L, KH2PO4 0.27g/L。
In the present invention, developing solution is preferably comprised 0.1mM TrisHCl (pH 9.5), 0.1mM NaCl, 5mM MgCl2, 2mg/ml PNPP。
Using detection method and detection kit of the invention, the active uPAR in various samples can be specifically detected.
"comprising" used in the present invention, " comprising " or " containing " can refer to " including but not limited to ", " substantially by ... Composition " or " mainly by ... form ".
Technical solution that "comprising", " comprising " or " containing " is used in the present invention can also be to be made of ingredients listed Technical solution.
Detailed description of the invention
Fig. 1 is the design principle schematic diagram of the ELISA detection method of the active uPAR of embodiment 1.
Fig. 2 is 96 hole ELISA detection plate typical case used in embodiment 1.
Fig. 3 is the dynamic process figure that the absorbance of various concentration activity uPAR standard items changes over time.
Fig. 4 is the standard curve that active uPAR concentration corresponds to enzyme reaction rate.
Fig. 5 is the rate of recovery calculated result of the ELISA detection method of activity uPAR of the invention.
Specific embodiment
The present invention is further elaborated combined with specific embodiments below, but provides these embodiments and be to help understanding The present invention, the present invention is not limited to these Examples.
The following are the reagent used involved in embodiment and experimental materials:
AUCA-HSA: AUCA-HSA fusion protein, warp are prepared using the method similar with patent 201410034942.1 SDS-PAGE electrophoresis verifies purity and is greater than 90%.Can also be prepared by other methods well known to those skilled in the art AUCA and The fusion protein of HSA, for example, in pichia pastoris yeast X-33 expression and it is pure through nickel-nitrilotriacetic acid (Ni-NTA) column Change.
Active uPAR standard items: the recombination suPAR expressed in S2 drosophila embryos cell is pure through affinity column coupled ion column Change.
Antibody A TN-658: it is provided by Attenuon, LLC (San Diego, Calif), is uPAR D2D3 (amino acid 88- 283) mouse, the anti-uPAR antibody of monoclonal obtained from screening through hybridoma technology is immunized.
Alkali phosphatase enzyme mark secondary antibody (AP-antimouse-IgG): it chooses in Cell Signaling Technology Company is the ELIAS secondary antibody being exclusively used in Western blotting and ELISA.
96 hole elisa Plates: choosing is transparent Maxisorp type ELISA Plate in Thermo scientific company.
Coating buffer: NaHCO containing 40mM3, 10mM Na2CO3, the aqueous solution of pH 9.6.
Confining liquid: the 5%BSA solution configured with coating buffer, wherein BSA chooses Yu Shenggong bioengineering Co., Ltd.
Cleaning solution/sample diluting liquid: the PBS solution containing 0.5 ‰ Tween-20pH 7.4, wherein PBS formula is NaCl 8g/L, KCl 0.2g/L, Na2HPO4·12H2O 3.58g/L, KH2PO4 0.27g/L。
Developing solution: 0.1mM TrisHCl (pH 9.5), 0.1mM NaCl, 5mM MgCl2, 2mg/ml PNPP.
Embodiment one: the active uPAR in human blood sample is detected using AUCA-HSA
The principle of the ELISA detection method of activity uPAR is as shown in Figure 1 in the present embodiment.
The collection and preservation of plasma sample: the whole blood of collector is in the vacuum blood collection tube of the anti-coagulants containing EDTA, in 1200* G is centrifuged 30min.By the blood plasma number of different patients and 10 times are diluted with sample diluting liquid, is sub-packed in 1.5ml centrifuge tube, protects It is stand-by in the presence of -80 DEG C.
Specific operating procedure is as follows:
1 coating: 100 μ l are added into all holes 1A-12H (referring to fig. 2) of ELISA Plate and are coated the diluted AUCA- of liquid HSA protein solution (140 μ g/ml), it is with sealant tape that 96 pore plate by sealing are good to prevent liquid evaporation, and overnight in 4 DEG C.It opens Sealant tape dries the liquid in ELISA Plate hole, and draws cleaning solution with the volley of rifle fire, washs every hole 6 times, washs in last time Afterwards, ELISA Plate is gently patted dry on blotting paper and guarantees to retain in hole without bubble.
2 closings: 100 μ l confining liquids are added in all holes 1A-12H into ELISA Plate, and with sealant tape that 96 orifice plates are close It seals to prevent liquid evaporation, in being incubated for 1 hour on 80 revs/min of room temperature of shaking table.Sealant tape is opened, is dried in ELISA Plate hole Liquid, and with the volley of rifle fire draw cleaning solution wash every hole 6 times, after the final washing, on blotting paper gently by ELISA Plate It pats dry and guarantees to retain in hole without bubble.
3 sample-addings: being added the 100 μ l of active uPAR standard items of various concentration to ELISA Plate 1A-1F respectively, and each concentration has two A multiple holes (A1, A2:1 μ g/L；B1, B2:0.5 μ g/L；C1, C2:0.25 μ g/L；D1, D2:0.125 μ g/L；E1, E2:0.0625 μ g/L；F1, F2:0.03125 μ g/L).The sample diluting liquid of 100 μ l is separately added into as blank control to G1, G2.Then to enzyme The dilute serum sample (diluting 10 times with sample diluting liquid) of patient to be detected, each blood serum sample 2 is added in other holes in target A repetition, it is then with sealant tape that 96 pore plate by sealing are good, and in being incubated for 1 hour on 80 revs/min of room temperature of shaking table.It opens close Sealed adhesive tape dries the liquid in ELISA Plate hole, and draws cleaning solution with the volley of rifle fire, washs every hole 6 times, after the final washing, ELISA Plate is gently patted dry on blotting paper and guarantees to retain in hole without bubble.
4 add primary antibody: it is anti-through the diluted mouse of sample diluting liquid that 100 μ l, 9 μ g/ml being added into all holes ELISA Plate 1A-12H The ATN-658 antibody of source of people uPAR, and it is with sealant tape that 96 pore plate by sealing are good, and incubated on 80 revs/min of room temperature of shaking table It educates 1 hour.Sealant tape is opened, the liquid in ELISA Plate hole is dried, and draw cleaning solution with the volley of rifle fire, every hole is washed 6 times, most Afterwards after once washing, ELISA Plate is gently patted dry on blotting paper and guarantees to retain in hole without bubble.
5 add ELIAS secondary antibody: 100 μ l being added into all holes ELISA Plate 1A-12H through 500 times of diluted alkali of sample diluting liquid The anti-mouse IgG (antimouse-AP conjugate IgG) of acid phosphatase label, and it is with sealant tape that 96 pore plate by sealing are good, And in being incubated for 1 hour on 80 revs/min of room temperature of shaking table.Sealant tape is opened, dries the liquid in ELISA Plate hole, and use the volley of rifle fire Cleaning solution is drawn, washs every hole 6 times, is then washing every hole 3 times with volley of rifle fire absorption deionized water.By ELISA Plate on blotting paper It gently pats dry and guarantees to retain in hole without bubble.
6 add developing solution: and then 100 μ l developing solutions are rapidly joined into every hole with the volley of rifle fire, ELISA Plate is then placed in enzyme mark Absorbance is read in 405nm on instrument, reads 60min altogether, every 1min reads an absorbance value.
7 production standard curves, calculate the concentration of activity uPAR in blood sample.
Firstly, establishing a series of activity uPAR concentration (X-axis) and corresponding enzyme reaction by the method for linear regression analysis The standard curve of speed-Rate (Y-axis).Fig. 3 is exactly the extinction that microplate reader reads various concentration activity uPAR standard items in 405nm The dynamic process changed over time is spent, wherein abscissa is time (monitoring 60 minutes), and ordinate is the extinction at 405nm Value, from top to bottom corresponding activity uPAR concentration is successively for kinetic curve are as follows: 1 μ g/L, 0.5 μ g/L, 0.25 μ g/L, 0.125 μ g/ L, 0.0625 μ g/L, 0.03125 μ g/L, 0 μ g/L.At the corresponding 60th minute OD 405nm of different activities uPAR concentration Light absorption value subtracts the light absorption value at the 1st minute OD405nm, obtains the changing value X of the OD 405nm in 60 minutes (milliAbsorbance, milliAbs).The corresponding enzyme reaction speed of different activities uPAR concentration was obtained divided by 60 minutes with X It spends (milliAbs/min).Fig. 4 is a series of activity uPAR concentration standard curve done to enzyme reaction rate.It will by conversion The corresponding enzyme reaction rate of patient's plasma sample, which brings standard curve into, can measure active uPAR content in corresponding plasma sample.
Embodiment two: the performance characterization of active uPAR detection method
The present embodiment is the examination to the ELISA detection method of active uPAR of the invention, if not specified, specifically Experimental method and agents useful for same with 1 the method for embodiment.
(1) Precision Experiment
Precision usually indicates with within-run and between-run analysis coefficient (CV%), the ELISA method pair of Application Example one Active uPAR content in the same plasma sample obtains accordingly averagely for duplicate measurements 16 times in same plate on the same day It is worth (AVE) and standard deviation (SD), corresponding variation within batch coefficient (%) is obtained by formula 100%* (SD/AVE), as a result such as Shown in table one.In addition the active uPAR content in continuous 4 days in the daily same plasma sample of duplicate measurements, obtains corresponding 4 It average value (AVE') and standard deviation (SD'), obtains corresponding batch variation system by formula 100%* (SD'/AVE') Number (%), as a result as shown in Table 2.By table one and table two it can be seen that variation within batch coefficient is 8.27%, less than 10%, become between batch Different coefficient is 9.52%, less than 15%, illustrates that established ELISA new method reproducibility is good.
One withinrun precision measurement result of table:
Average value (AVE) (μ g/L) Standard deviation (SD) Variation within batch coefficient (%)
1.33 0.11 8.27%
Two betweenrun precision measurement result of table:
Average value (AVE') (μ g/L) Standard deviation (SD') Interassay coefficient of variation (%)
1.47 0.14 9.52%
(2) recovery experiment
The recovering effect of recovery experiment is generally indicated with the rate of recovery (%).By a series of activity of 10 known concentrations of μ l UPAR standard sample is added separately in 100 μ l sample diluting liquids or the blood plasma of 10 times of dilution, and calculates separately out 110 μ l bodies The active uPAR concentration finally added in system (this concentration is denoted as active uPAR addition concentration).Then using this ELISA method point Activity uPAR concentration in the sample diluting liquid and diluted blood plasma of activity uPAR Jian Ce not be added, and (this concentration is denoted as active uPAR inspection Survey concentration)).Active uPAR addition concentration and activity uPAR detectable concentration for sample diluting liquid are drawn respectively and are directed to The linear relationship chart of active uPAR the addition concentration and activity uPAR detectable concentration of diluting plasma, as a result as shown in Figure 5.By right Rate of recovery R of the standard sample in blood plasma is calculated than two slope of a curve values, wherein being directed to the active uPAR of sample diluting liquid The slope of the linear equation of addition concentration and activity uPAR detectable concentration is considered as 100% rate of recovery.Therefore activity uPAR Rate of recovery R=1.0102/1.0165=99.4% in blood plasma also shows this ELISA method with preferable accuracy.
(3) detection limit and the range of linearity of standard curve
In order to confirm the Monitoring lower-cut of this ELISA method detection activity uPAR content, (added by measuring 4 groups of blank controls Entering sample is sample diluting liquid, and active uPAR concentration is corresponding enzyme reaction rate in 0 μ g/L), is then brought into active uPAR In the standard curve of concentration and enzyme reaction rate, the content of corresponding activity uPAR is obtained.It calculates active in four groups of blank controls The average value (AVE ") of uPAR content and corresponding standard deviation (SD ").The Monitoring lower-cut of the method can pass through formula AVE "+3* SD " is obtained, and the Monitoring lower-cut thus to obtain the active uPAR content of this ELISA method is about 15ng/L.Additionally by drafting 0- 128 μ g/L activity uPAR concentration correspond to the linear relationship chart of enzyme reaction rate, it has been found that the range of linearity of this method is 0-8 μ G/L (R in linear equation2> 0.99 to be considered linear relationship preferable).
Bibliography:
[1]A.Estreicher,J.Muhlhauser,J.L.Carpentier,L.Orci,J.D.Vassalli,The receptor for urokinase type plasminogen activator polarizes expression of the protease to the leading edge of migrating monocytes and promotes degradation of enzyme inhibitor complexes,The Journal of cell biology 111(2)(1990)783-92.
[2]C.Pyke,E.Ralfkiaer,E.Ronne,G.Hoyer-Hansen,L.Kirkeby,K.Dano, Immunohistochemical detection of the receptor for urokinase plasminogen activator in human colon cancer,Histopathology 24(2)(1994)131-8.
[3]M.Thuno,B.Macho,J.Eugen-Olsen,suPAR:the molecular crystal ball, Disease markers27(3)(2009)157-72.
[4]F.Blasi,P.Carmeliet,uPAR:a versatile signalling orchestrator, Nature reviews.Molecular cell biology 3(12)(2002)932-43.
[5]T.Plesner,N.Behrendt,M.Ploug,Structure,function and expression on blood and bone marrow cells of the urokinase-type plasminogen activator receptor,uPAR,Stem cells 15(6)(1997) 398-408.
[6]H.W.Smith,C.J.Marshall,Regulation of cell signalling by uPAR, Nature reviews.Molecular cell biology 11(1)(2010)23-36.
[7]C.Yuan,M.Huang,Does the urokinase receptor exist in a latent Form?, Cellular and molecular life sciences:CMLS 64 (9) (2007) 1033-7.
[8]Q.Huai,A.Zhou,L.Lin,A.P.Mazar,G.C.Parry,J.Callahan,D.E.Shaw, B.Furie,B.C.Furie,M.Huang,Crystal structures of two human vitronectin, urokinase and urokinase receptor complexes,Nature structural&molecular biology 15(4)(2008)422-3.
[9]O.Slot,N.Brunner,H.Locht,P.Oxholm,R.W.Stephens,Soluble urokinase plasminogen activator receptor in plasma of patients with inflammatory rheumatic disorders:increased concentrations in rheumatoid arthritis,Annals of the rheumatic diseases 58(8)(1999)488-92.
[10]M.Persson,G.Engstrom,H.Bjorkbacka,B.Hedblad,Soluble urokinase plasminogen activator receptor in plasma is associated with incidence of CVD.Results from the Malmo Diet and Cancer Study,Atherosclerosis 220(2)(2012) 502-5.
[11]N.Sidenius,C.F.Sier,H.Ullum,B.K.Pedersen,A.C.Lepri,F.Blasi, J.Eugen-Olsen,Serum level of soluble urokinase-type plasminogen activator receptor is a strong and independent predictor of survival in human immunodeficiency virus infection,Blood 96(13)(2000)4091-5.
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Claims (22)

1. the detection method of the active uPAR of non-disease diagnostic purpose, this method comprises:
1) make to capture reagent and sample to be tested in being suitable for the capture reagent capture sample to be tested under conditions of activity uPAR Contact forms the compound of capture reagent and activity uPAR；
2) captured activity uPAR is detected；
Wherein the capture reagent includes AUCA or the fusion protein containing AUCA, the amino acid sequence of the AUCA such as SEQ ID Shown in NO:1.
2. the detection method of claim 1, the capture reagent includes AUCA-HSA fusion protein.
3. the detection method of claims 1 or 2, wherein step 2) includes making to be formed by compound and being incorporated into outside active uPAR The monoclonal antibody of side combines, and detects captured activity uPAR by detecting the monoclonal antibody；The activity It is the site on the outside on active uPAR for the inside for the active hydrophobic pocket that AUCA is inserted on the outside of uPAR.
4. the detection method of claim 3, the monoclonal antibody being incorporated on the outside of active uPAR is immune with uPAR D2D3 Mouse, the anti-uPAR antibody of monoclonal obtained from being screened through hybridoma technology.
5. detection method according to claim 4, wherein specific step is as follows:
(1) it 100 μ l is added into the hole of porous plate is coated the AUCA-HSA protein solution of the diluted 140 μ g/ml of liquid and wrapped Quilt, 4 DEG C overnight, washs and dry；
(2) 100 μ l confining liquids are added into hole to be closed, 80 revs/min of room temperature, are incubated for 1 hour, wash and dry；
(3) into a some holes be added various concentration 100 μ l of active uPAR standard items, the various concentration be respectively 1 μ g/L, 0.5μg/L,0.25μg/L,0.125μg/L,0.0625μg/L,0.03125μg/L；The sample of 100 μ l is added into another some holes Dilution is free of uPAR as blank control in blank control；The dilute with sample diluting liquid of measuring samples is added into other holes Release 10 times of dilution；It 80 revs/min of room temperature, is incubated for 1 hour, washs and dry；
(4) monoclonal antibody of 100 μ l, the 9 μ g/ml through the anti-human source uPAR of the diluted mouse of sample diluting liquid is added in the hole Xiang Suoyou, It 80 revs/min of room temperature, is incubated for 1 hour, washs and dry, wherein the monoclonal antibody is that mouse is immunized with uPAR D2D3, through miscellaneous Hand over the anti-uPAR antibody of monoclonal obtained from tumor technology screening；
(5) anti-mouse IgG of the 100 μ l through 500 times of diluted alkali phosphatase enzyme marks of sample diluting liquid, room temperature are added in the hole Xiang Suoyou It 80 revs/min, is incubated for 1 hour, washs and dry；
(6) 100 μ l developing solutions are added into every hole, then ELISA Plate is placed in microplate reader and reads absorbance in 405nm, is read altogether 60min, every 1min is taken to read an absorbance value；
(7) standard curve is made, the concentration of activity uPAR in sample is calculated.
6. the detection method of claims 1 or 2, wherein sample to be tested be selected from blood, serum, blood plasma, cell culture fluid, saliva and Urine.
7. the detection method of claim 3, wherein sample to be tested is selected from blood, serum, blood plasma, cell culture fluid, saliva and urine Liquid.
8. the detection method of claim 4, wherein sample to be tested is selected from blood, serum, blood plasma, cell culture fluid, saliva and urine Liquid.
9. the detection method of claim 5, wherein sample to be tested is selected from blood, serum, blood plasma, cell culture fluid, saliva and urine Liquid.
The application of 10.AUCA or fusion protein containing AUCA in reagent of the preparation for activity uPAR in test sample, institute The amino acid sequence of AUCA is stated as shown in SEQ ID NO:1.
The application of 11.AUCA or fusion protein containing AUCA in kit of the preparation for activity uPAR in test sample, The amino acid sequence of the AUCA is as shown in SEQ ID NO:1.
12. 0 or 11 application according to claim 1, wherein the fusion protein containing AUCA is AUCA-HSA fusion protein.
13. the kit for detecting active uPAR, the kit includes capture reagent, and the capture reagent includes can be special Property capture activity uPAR AUCA or fusion protein containing AUCA, the amino acid sequence of the AUCA such as SEQ ID NO:1 institute Show.
14. the kit of claim 13, the capture reagent includes AUCA-HSA fusion protein.
15. the kit of claim 13 or 14, wherein the kit also includes the monoclonal being incorporated on the outside of active uPAR Antibody and/or the secondary antibody in conjunction with the monoclonal antibody；It is to be inserted on active uPAR relative to AUCA on the outside of the activity uPAR The site on outside for the inside of the active hydrophobic pocket entered.
16. the kit of claim 15, it is small that the monoclonal antibody being incorporated on the outside of active uPAR is that uPAR D2D3 is immunized Mouse, the anti-uPAR antibody of monoclonal obtained from being screened through hybridoma technology.
17. the kit of claim 13 or 14, wherein the kit includes: ELISA Plate；AUCA-HSA protein solution；Activity UPAR standard items；Mouse, the anti-uPAR antibody of monoclonal obtained from screening through hybridoma technology is immunized in employment uPAR D2D3；Alkali The anti-mouse IgG of acid phosphatase label；Coating buffer；Cleaning solution/sample diluting liquid；Confining liquid and developing solution.
18. the kit of claim 17, wherein the kit includes: ELISA Plate；The AUCA-HSA egg of 1ml 1.4mg/ml White solution；1ml concentration is the active uPAR standard items of 1mg/ml；100 μ l 0.9mg/ml's is immunized mouse, warp with uPAR D2D3 The anti-uPAR antibody of monoclonal obtained from hybridoma technology screening；The anti-mouse IgG of 25 μ 500 × alkali phosphatase enzyme marks of l；Coating Liquid 10ml；30 × cleaning solution/sample diluting liquid 20ml；BSA confining liquid 10ml, PNPP developing solution 10ml.
19. the application of claim 11, wherein the kit also include be incorporated into monoclonal antibody on the outside of active uPAR and/ Or the secondary antibody in conjunction with the monoclonal antibody；It is the activity being inserted on active uPAR relative to AUCA on the outside of the activity uPAR The site on outside for the inside of hydrophobic pocket.
20. the application of claim 19, it is small that the monoclonal antibody being incorporated on the outside of active uPAR is that uPAR D2D3 is immunized Mouse, the anti-uPAR antibody of monoclonal obtained from being screened through hybridoma technology.
21. the application of claim 11, wherein the kit includes: ELISA Plate；AUCA-HSA protein solution；Active uPAR mark Quasi- product；Mouse, the anti-uPAR antibody of monoclonal obtained from screening through hybridoma technology is immunized in employment uPAR D2D3；Alkaline phosphatase The anti-mouse IgG of enzyme label；Coating buffer；Cleaning solution/sample diluting liquid；Confining liquid and developing solution.
22. the application of claim 21, wherein the kit includes: ELISA Plate；The AUCA-HSA albumen of 1ml 1.4mg/ml Solution；1ml concentration is the active uPAR standard items of 1mg/ml；100 μ l 0.9mg/ml's is immunized mouse with uPAR D2D3, through miscellaneous Hand over the anti-uPAR antibody of monoclonal obtained from tumor technology screening；The anti-mouse IgG of 25 μ 500 × alkali phosphatase enzyme marks of l；Coating buffer 10ml；30 × cleaning solution/sample diluting liquid 20ml；BSA confining liquid 10ml, PNPP developing solution 10ml.
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