CN106119340A

CN106119340A - Detection method, detectable and the detection kit of a kind of de-γ carboxyl thrombinogen

Info

Publication number: CN106119340A
Application number: CN201610519925.6A
Authority: CN
Inventors: 刘婕; 吴文强; 李国栋
Original assignee: Fujian Cosunter Pharmaceutical Co Ltd
Current assignee: Fujian Cosunter Pharmaceutical Co Ltd
Priority date: 2016-07-04
Filing date: 2016-07-04
Publication date: 2016-11-16
Anticipated expiration: 2036-07-04
Also published as: CN106119340B

Abstract

The invention discloses detection method, agents useful for same and the detection kit of a kind of de-γ carboxyl thrombinogen, the detection method of the present invention comprises the following steps: step 1, the preparation of the magnetic particle A of surface coupling anti-DCP antibody, and the preparation of the magnetic particle B of surface coupling DCP antigen；Step 2, the preparation of fluorescently-labeled anti-DCP antibody；Step 3, the detection of DCP content: blood plasma or blood serum sample and magnetic particle A reaction, react complete by DCP eluting, add fluorescently-labeled anti-DCP antibody incubation, add magnetic particle B and continue to hatch, then Magnetic Isolation, detect sample fluorescence value with spectrofluorophotometer, calculate DCP content in sample.

Description

A kind of de--γ-detection method of carboxyl thrombinogen, detectable and detection examination Agent box

Technical field

The present invention relates to a kind of medical detecting method, particularly to a kind of DCP magnetic particle immunofluorescent detection method and DCP magnetic particle immunofluorescence detection agent and detection kit.

Background technology

Hepatocarcinoma is a kind of common malignant tumor, and its mortality rate is the 2nd of China's malignant tumor, accounts for whole world hepatocarcinoma 53% (Bruix J, Llovet JM.Major achievements in hepatocellular of death toll carcinoma.Lancet.2009,373(9664):614-616).The generation of HCC is the most relevant with hepatitis b virus infected, Its development process is complicated, relates to multistage, multifactor, tumor cell and local microenvironment about jointly acting on (Altekruse SF,McGlynn KAJDickie LA,et al.Hepatocellular carcinoma confirmation,treatment,and survival in surveillance 5 epidemiology 9 and end results registries,1992-2008.Hepatology.2012,55(2):476-482).Clinical effectiveness shows, examines Measuring that to have the patient of typical clinical symptom be later period of hepatocarcinoma the most, therefore early diagnosis is the key for the treatment of HCC.Clinic is examined Disconnected blood serum tumor markers the most frequently used for HCC is AFP, and its detection sensitivity is only 60%～70%, but about 18% Hepatocarcinoma AFP do not raise.The false negative rate of early hepatocarcinoma AFP more may be up to 40%, and its reason is probably some type hepatocarcinoma early It is relevant with gross tumor volume size that phase does not express AFP or AFP content.Additionally, have under chronic viral hepatitis background in screening During hepatocarcinoma, specificity and the sensitivity of AFP are relatively low, and its false negative and false positive all can affect the correct diagnosis of hepatocarcinoma, and And AFP also can raise in the serum of some optimum hepatopathys, part reproductive system and gastrointestinal malignant tumor patient, thus lead Cause to produce false positive, affect clinical diagnosis result, there is certain defect (Gupta S, Bent S, Kohlwes J.Test characteristics of alpha-fetoprotein for detecting hepatocellular carcinoma in patients with hepatitis C.A systematic review and critical analysis.Ann Intern Med,2003；139(1):46-50)(Spangenberg HC,Thimme R,Blum HE.Serum markers of hepatocellular carcinoma.Setnin Liver Dis.2006；26(4):385-390).Therefore, find newly The HCC early diagnosis blood serum designated object focus that always research is paid close attention to.

I.e. de--γ-carboxyl the thrombinogen of DCP (Des-γ-carboxy prothrombin), can be described as again vitamin K and lacks The protein PIVKA-II (Protein induced by vita min K absence-II) that weary or antagonist II induces, also It is referred to as people abnormal prothrombin APT (Human abnormal prothrombin), is hepatocarcinoma (hepatocellular Carcinoma, HCC) product.Compared with normal coagulation proenzyme, the molecular structure territory of DCP has one or more Glu residue Do not become Gla by complete carboxylation, thus lose coagulation function.Normal coagulation proenzyme (Prothrombin) is micro-hepatocyte Gamma-glutamyl carboxylase (γ-glutamyl-carboxylase) and the coenzyme vitamin of vitamin K is relied primarily in plastochondria Under K-epoxide-reductase and vitamin K-reductase participates in, by the 6th in structure Gla domam, 7,14, 10 Glu residues (Glu residus) carboxylation of 16,19,20,25,26,29 and 32 is Gla, makes activated prothrombin.One or more Glu residue carboxylations in any of the above described site are not complete, all will be likely to be formed DCP, and lose The coagulation function of prothrombin.As the tumor marker of HCC, DCP has become and has assisted the important auxiliary of clinical diagnosis and prognosis Help index.It is relevant to multiple hepatocarcinoma malignant proliferation approach, it is in particular possible to HCC development, infiltrate and the aspects such as transfer Promote the malignant proliferation of hepatoma carcinoma cell.Reported first (Liebman HA, Furie BC, Tong MJ, the et such as Liebman in 1984 al.Des-gamma-carboxy(abnormal)prothrombin as a serum marker of primary hepatocellular carcinoma.N Engl J Med.1984；310:1427-1431) claim DCP 91% hepatocarcinoma suffer from In person's serum, level raises.Sakon reports that in 60 examples HCC, the positive rate of DCP is 58.4%, and its positive rate of AFP negative HCC is still It is 61.9%, points out it to contribute to the diagnosis of AFP negative HCC.The positive rate of DCP is also relevant with the growth of tumor, its positive rate It is 66.2% at the HCC of diameter<HCC of 3cm is 19.1%, and the HCC of diameter 3～5cm is 55.6%, diameter>5cm, Chronic Liver Sick or liver cirrhosis is 14.8%, therefore can diagnose as the auxiliary of AFP negative or low concentration AFP hepatocarcinoma.DCP is big with tumor Little, Intrahepatic metastasis, portal vein invade (portal venous invasion, PVI) and tumor tissues propagation is relevant.Khan exists 65 examples are treated first table after using the patient of anhydrous alcohol percutaneous injection single HCC carcinoma tuberosity diameter≤6cm to make to analyze Bright, positive (>20 μ g/L of DCP) relapse rate is respectively 22.5%, 31.4% in patient 1 year and 2 years livers, and negative patient (<20 μ g/ L) 8% and 17.8% then it is respectively.It is taken as that the blood plasma level of DCP can refer to as the prediction of early stage HCC recurrence before treatment Mark, and contribute to selecting suitable Therapeutic Method for patient.Sakon and Naka thinks that DCP positive compares raw with DCP negative patient Rate of depositing reduces, and the rate of transform and relapse rate increase, cancer diameter bigger (Khan KN, Yatsuhashi H, Yamasaki K, et al.Prospective analysis of risk factor for early intrahepatic recurrence of hepatocellular carcinoma following ethanol injection.J Hepatol,2000,32(2): 269-278).Koike to 227 example initial hospital admissions without PVI and the HCC patient that accepts Percutaneous Ethanol Injection or micro-wave therapeutic, do The follow-up evaluation of average 19 months, finds that the patient of 11% occurs that PVI, single factor analysis show: tumor number, histology's class Type, AFP and DCP level have significant correlation compared with when PVI occur；Entering part analysis to show, plasma D CP level is for HCC The later stage PVI that whether occurs together is the most frequently used prognostic indicator, monitors its level and contributes to the early diagnosis and therapy (Koike of PVI Y,Shiratori Y,Sato S,et al.Des-γ-carboxy prothrombin as a useful predisposing factor for development of portal venous in-vasion of hepatocellular carcinoma.Cancer,2001,91(3):561-569).During Utsunomiya operation former to 23 examples For accompanying the outer recidivist of liver to contrast with recidivist in 186 example livers after progressive stage HCC.After tumor size is limited, two groups of trouble Person's prognosis zero difference, and when diameter of tumor > 5cm time, plasma D CP content occurs to recur (62.5%) outside liver higher than 2000 μ g/L persons Be significantly more than recurrence (P < 0.05) in liver.Result, it is believed that once diameter of tumor is higher than more than 5cm and its plasma D CP level 2000 μ g/L, it is therefore necessary to take the most careful follow-up examination with the possibility of recurrence outside early discovery liver as far as possible (Utsunomiya T,Shimada M,Shirabe K,et al.Clinicopathological characteristics of patients with extrahepatic recurrence following a hepatectomy for hepatocellular carcinoma.Hepatogastroenterology,2001,48(40):1088-1093).DCP's Existing on aminoacid ad-hoc location without carboxylated glutaminic acid residue, when hepatocyte generation canceration, endoplasmic reticulum can not be by DCP Carboxylation becomes activated thrombinogen, so that DCP content raises.Having report display, serum DCP is favorably improved diagnosing cancer of liver Sensitivity and Specificity, it is possible to find part AFP negative small liver cancer.Therefore DCP be clinically one have and very well should With the HCC blood serum designated object of prospect, it is expected to improve the Sensitivity and Specificity of HCC diagnosis, it is achieved the early diagnosis (Lok of HCC AS,Sterling RK,EverhartJE,et al.Des-γ-carboxy prothrombin andα-fetoprotein as biomarkers for the early detection of hepatocellular carcinoma.Gastroenterology,2010,138(2):493-502)。

DCP is affirmative to the diagnostic value of disease, but the detection method of some routines lacks enough sensitivity, leads Cause testing result is inaccurate.The content of clinical common immunoassays method detection DCP, utilizes antigen and antibody specific association reaction to enter Row detection, immunolabelling technique carries out result judgement, by probe material traget antibodies (or antigen) such as fluorescein, isotope or enzymes Carry out antigen-antibody reaction, by the detection to the label in immune complex, reach the mesh that immunoreation is monitored 's.Conventional immunoassay labelling technique includes that EIA enzyme immunoassay, radioimmunoassay, RIA, luciferase immunoassay, gold colloidal are exempted from Epidemic disease technology, electrochemiluminescent immunoassay technology etc..Wherein EIA enzyme immunoassay sensitivity is relatively low and operation is complicated；The sensitive essence of radioimmunoassay, RIA Really, sample consumption few, but detection effect duration is shorter and have radioactive pollution；The preparation letter of colloid gold immune technical mark thing Just, sensitive intuitively, but the most impacted many factors；Electrochemiluminescent immunoassay technology for detection step is more complicated, background is higher and result not Stable.

In prior art, magnetic microparticle chemiluminescence immune assay technological synthesis magnetic particle carrier technique and chemiluminescence are exempted from Epidemic disease detection technique, makes measurement result more accurate, more stable.This technology includes:

● magnetic microparticle chemiluminescence--double antibody sandwich method: determined antigen is tied with fluorescein-labeled antibody and enzyme labelled antibody Close the complex forming " sandwich " structure.It is subsequently added the magnetic particle being connected with anti-fluorescein antibody, passes through anti-fluorescein antibody Specific binding with fluorescein makes antigen antibody complex be connected on magnetic particle, and Direct precipitation in externally-applied magnetic field will be exempted from The complex that epidemic disease reaction is formed separates with other material unconjugated.Clean the complex of precipitation after removing supernatant, add enzymatic Learn luminous substrate.Substrate by catalytic pyrolysis, forms unstable excited state intermediate, when excited state intermediate returns under enzyme effect To just sending photon during ground state, form luminescence-producing reaction, by light quantum reading system record photon energy, and by computer Light energy intensity is converted to the concentration of determined antigen on standard curve by reason system, and reports result.

Magnetic microparticle chemiluminescence--competition law: by determined antigen, be coated the antibody of magnetic particle and quantitative labelling by excess Antigen is simultaneously introduced reaction cup incubation, and its immunoreactive combining form has two kinds, and one is that labelled antigen is formed with antibodies Complex；Two is that determined antigen forms complex with antibodies.As specimen to be measured contained determined antigen, then with labelled antigen With same chance and the coated antibodies of magnetic particle, the labelled antigen that accounted for competitively is tied with the coated antibody of magnetic particle The chance closed, makes labelled antigen reduce with the binding capacity of the coated antibody of magnetic particle.Owing to the coated antibody of magnetic particle is excess , it is sufficient to be combined with determined antigen.Magnetic particle is Direct precipitation in externally-applied magnetic field, by immunoreation formed complex with do not tie Other material closed separates.Clean the complex of precipitation after removing supernatant, add enzyme-catalyzed chemical luminescence substrate.Substrate is under enzyme effect By catalytic pyrolysis, form unstable excited state intermediate, just send photon when excited state intermediate returns to ground state, formed and send out Photoreaction, by light quantum reading system record photon energy, and passes through computer processing system by light energy intensity in standard Be converted to the concentration of determined antigen on curve, and report result.

Magnetic microparticle chemiluminescence indirect method: test antibodies is combined with fluorescein-labeled antigen, is subsequently added and is coated The magnetic particle of anti-fluorescein antibody, makes antigen antibody complex be connected by anti-fluorescein antibody with the specific binding of fluorescein On magnetic particle, Direct precipitation in externally-applied magnetic field, cleans the complex of precipitation, adds enzyme labelled antibody, form magnetic after removing supernatant Microgranule-Ag-Ab-ELIAS secondary antibody sandwich immunoassay complex.After again cleaning, add enzyme-catalyzed chemical luminescence substrate.Substrate By catalytic pyrolysis under enzyme effect, form unstable excited state intermediate, just send when excited state intermediate returns to ground state Photon, forms luminescence-producing reaction, by light quantum reading system record photon energy, and by computer processing system by light energy Intensity is converted to the concentration of test antibodies on standard curve, and reports result.

But there are some defects in above-mentioned technology: such as:

Be interrupted, glitter luminescence instability,

Course of reaction is easily fissioned, unstable result,

With microwell plate as carrier, testing cost is high, the time is long,

Need to combining phase, free phase separates, operating procedure is many,

The chemiluminescence peak value that moment produces quickly is decayed,

Background is higher, and interference hinders application,

It is difficult to automatization etc..

Based on above not enough, the present invention establishes a kind of DCP magnetic particle immunofluorescent detection method, use magnetic particle A with The antibody coupling that DCP is specific binding, uses this antibody of fluorescein labelling as detection antibody, and the magnetic re-using coupling DCP is micro- Grain B removes the detection antibody of excess, is calculated the content of target detection thing DCP by fluorescence intensity, it is achieved to DCP content Easy quick, sensitive and accurate detection.

Summary of the invention

The present invention provides a kind of and detects the method for DCP content in blood plasma or serum: said method comprising the steps of:

Step 1, the preparation of the magnetic particle A of surface coupling anti-DCP antibody, and the system of the magnetic particle B of surface coupling DCP antigen Standby；

Step 2, the preparation of fluorescently-labeled anti-DCP antibody；

Step 3, the detection of DCP content: blood plasma or blood serum sample and magnetic particle A reaction, react complete by DCP eluting, add Enter fluorescently-labeled anti-DCP antibody incubation, add magnetic particle B and continue to hatch, then Magnetic Isolation, use spectrofluorophotometer Detection sample fluorescence value, calculates DCP content in sample；

Wherein said DCP is de--γ-carboxyl thrombinogen (Des-γ-carboxy prothrombin), can be described as again PIVKA-II (Protein induced by vita min K absence-II), vitamin K deficiency or antagonist II induction Protein, also referred to as APT (Human abnormal prothrombin), people's abnormal prothrombin.

Method of the present invention, wherein said magnetic particle A and the preparation of magnetic particle B, method is as follows: prepared by magnetic particle Become magnetic particle suspension, magnetic particle suspension and anti-DCP antibody or DCP antigen coupling: by anti-DCP antibody or DCP antigen and magnetic Coupling is hatched in microparticle suspending liquid mixing, closes with confining liquid after coupling, and the magnetic particle A and coupling DCP that obtain coupling DCP antibody are anti- Former magnetic particle B.

Method of the present invention, wherein, the described antibody specific binding with DCP is monoclonal antibody or Anti-TNF-α Body.

Method of the present invention, wherein, the coupling condition of magnetic particle and DCP antigen is selected from following condition:

Magnetic particle particle diameter 120nm, antigen concentration 20 μ g/mL, 22 DEG C hatch 2h, pH=11；

Magnetic particle particle diameter 180nm, antigen concentration 20 μ g/mL, 22 DEG C hatch 2h, pH=10；

Magnetic particle particle diameter 200nm, antigen concentration 25 μ g/mL, 25 DEG C hatch 4h, pH=11；

Magnetic particle particle diameter 300nm, antigen concentration 25 μ g/mL, 25 DEG C hatch 4h, pH=10；

Magnetic particle particle diameter 500nm, antigen concentration 30 μ g/mL, 28 DEG C hatch 6h, pH=11；

Or magnetic particle particle diameter 800nm, antigen concentration 30 μ g/mL, 28 DEG C hatch 6h, pH=10；

Method of the present invention, wherein, the coupling condition of magnetic particle and anti-DCP antibody is selected from following condition:

Magnetic particle particle diameter 120nm, antibody concentration 20 μ g/mL, 22 DEG C hatch 2h, pH=11；

Magnetic particle particle diameter 180nm, antibody concentration 20 μ g/mL, 22 DEG C hatch 2h, pH=10；

Magnetic particle particle diameter 200nm, antibody concentration 25 μ g/mL, 25 DEG C hatch 4h, pH=11；

Magnetic particle particle diameter 300nm, antibody concentration 25 μ g/mL, 25 DEG C hatch 4h, pH=10；

Magnetic particle particle diameter 500nm, antibody concentration 30 μ g/mL, 28 DEG C hatch 6h, pH=11；

Or magnetic particle particle diameter 800nm, antibody concentration 30 μ g/mL, 28 DEG C hatch 6h, pH=10.

Method of the present invention, step is as follows:

Step (1) takes magnetic particle, adds magnetic particle volume 2～the lavation buffer solution of 50 times, and mixing, then on magnetic frame Magnetic Isolation, abandons supernatant；Repeat previous step, then magnetic particle is being suspended in the coupling buffer of its equivalent, obtaining Magnetic particle suspension；

Step (2) adds the magnetic particle that the step (1) of volume 0.01～1 times processes in anti-DCP antibody or DCP antigen and hangs Supernatant liquid, adds coupling buffer, hatches, until magnetic particle coupling is complete；Magnetic Isolation, retains magnetic particle；Magnetic particle coupling is tied Shu Hou, adds confining liquid, mixing, hatches Magnetic Isolation, and the magnetic obtaining the magnetic particle A of coupling DCP antibody and coupling DCP antigen is micro- Grain B；

The preparation of the fluorescently-labeled anti-DCP antibody of step (3): anti-DCP antibody is dissolved in carbonate buffer solution, fluorescein It is dissolved in DMSO, fluorescein is dropwise slowly added in anti-DCP antibody-solutions, 4 DEG C of lucifuge stirrings 12～20h, use G-25 Portugal Polysaccharide gel removes free fluorescein；

Step (4) takes testing sample, adds sample volume 1～the magnetic particle A of 100 times, in room temperature rotate hatch 2～6h or Person 4 DEG C hatches 12～20h, makes DCP antigen-antibody fully react；It is placed in Magnetic Isolation on magnetic frame, abandons supernatant, slow with washing Rush liquid and clean magnetic particle complex, be placed in Magnetic Isolation on magnetic frame, abandon supernatant；Add the eluent with antibody equivalent, mixed Even, resuspended magnetic particle, incubated at room 5min, it is placed in Magnetic Isolation on magnetic frame, retains supernatant；Add sample volume 1～100 Times fluorescent labeling DCP antibody, in room temperature rotate hatch 2～6h or 4 DEG C hatch 12～20h；Add magnetic particle A volume 1～ The magnetic particle B of 100 times, in room temperature rotate hatch 2～6h or 4 DEG C hatch 12～20h；It is placed in Magnetic Isolation on magnetic frame, protects Stay supernatant, use spectrofluorophotometer detection sample fluorescence value, and reference standard curve calculates DCP content in sample.

Method of the present invention, preferred step is as follows:

(5) take magnetic particle to be placed in EP pipe, add magnetic particle volume 10～the lavation buffer solution of 20 times, mixing, then exist Magnetic Isolation on magnetic frame, abandons supernatant；Repeat previous step, then by magnetic particle outstanding with the coupling buffer of its equivalent Floating, obtain magnetic particle suspension；

(6) magnetic particle suspension vol 0.2～the anti-DCP antibody needing coupling of 0.3 times or DCP antigen are added in filling magnetic In the EP pipe of microgranule magnetic particle suspension, and add the coupling buffer of magnetic particle suspension vol 10 times, mixing；EP pipe is put On rotary mixer, incubated at room 2～6h or 4 DEG C hatch 12～20h, until magnetic particle coupling is complete；EP pipe is placed in magnetic Magnetic Isolation on power frame, retains magnetic particle；Surface coupling anti-DCP antibody for magnetic particle A, surface coupling DCP antigen for magnetic Particles B, supernatant is used for detecting coupling efficiency；

(7), after magnetic particle coupling terminates, add the confining liquid of magnetic particle suspension vol 10 times, mixing, EP pipe is placed in On rotary mixer, incubated at room 2～6h or 4 DEG C hatch 12～20h, EP pipe is placed in Magnetic Isolation on magnetic frame, abandons supernatant Liquid；Repeat previous step once；

(8) take test plasma or blood serum sample, add sample volume 1～the magnetic particle A of 100 times, rotate in room temperature and hatch 2 ～6h or 4 DEG C hatch 12～20h, DCP antigen-antibody is made fully to react；It is placed in Magnetic Isolation on magnetic frame, abandons supernatant, use Lavation buffer solution cleans magnetic particle complex, is placed in Magnetic Isolation on magnetic frame, abandons supernatant；Add the eluting with antibody equivalent Liquid, mixing, resuspended magnetic particle, incubated at room 5min, it is placed in Magnetic Isolation on magnetic frame, retains supernatant；Add sample volume 1 ～the fluorescent labeling DCP antibody of 100 times, in room temperature rotate hatch 2～6h or 4 DEG C hatch 12～20h；Add magnetic particle A body The magnetic particle B of long-pending 1～100 times, in room temperature rotate hatch 2～6h or 4 DEG C hatch 12～20h；It is placed in magnetic on magnetic frame to divide From, retain supernatant, use spectrofluorophotometer detection sample fluorescence value, and reference standard curve calculates DCP in sample and contains Amount.

Method of the present invention, wherein,

Coupling buffer is selected from: bicarbonate buffer, carbonate buffer solution, sulfate buffer one or more Combination, concentration is 1～100mM, and pH is 8.0～10.0；

Lavation buffer solution is selected from: glycine buffer, Tris-HCl buffer, phosphate buffer, borate buffer solution, Carbonate buffer solution, the combination of one or more of chlorate buffer, concentration is 2～200mM, and pH is 7.0～8.0；

Eluent is selected from: glycine, Tris Buffer, EDTA solution, IPTG solution, Pidolidone solution, MES The combination of one or more of Buffer, concentration is 0.01～10M, and pH is 2.0～3.0；

Confining liquid is selected from: BSA, casein, the combination of one or more of glycine, and concentration is 0.05%～5%, and pH is 8.0～9.0；

Preservation liquid is selected from: BST buffer, Tween-20, NaN₃, the combination of one or more of BSA, concentration be 1～ 50mM, pH are 8～10.

The present invention also provides for a kind of test kit using the inventive method detection DCP, including:

Magnetic particle, and optional following components: the magnetic particle A of coupling DCP antibody, the magnetic particle B of coupling DCP antigen, DCP antigen, anti-DCP antibody, fluorescein, coupling buffer, lavation buffer solution, eluent, confining liquid, preserve liquid, magnetic frame.

Preferably use the test kit of the inventive method detection DCP, including:

The magnetic particle A of coupling DCP antibody,

The magnetic particle B of coupling DCP antigen,

And optional following components: DCP antigen, anti-DCP antibody, fluorescein, coupling buffer, lavation buffer solution, wash De-liquid, confining liquid, preserve liquid, magnetic frame.

Test kit of the present invention, including: the reagent in box body, box body, reagent trough, description, described reagent is placed In reagent trough.

It is an object of the invention to provide: compared with conventional method, easy to be quick, high sensitivity, high accuracy, safety Nontoxic, recyclable DCP magnetic particle immunofluorescent detection method, and its detectable and detection kit.

The present inventor conducts in-depth research for the detection method of DCP, found that: by magnetic particle separation fluorescence Immuno analytical method, compared with conventional method, has easy to be quick, and highly sensitive, accuracy is high, and safety non-toxic is recyclable Etc. advantage, thus complete the present invention.

That is, the present invention provides the magnetic particle separating immune fluorescence analysis detection method of DCP, and this detection method includes: use Magnetic particle is as solid phase carrier, and the coupling antibody specific binding with DCP, as coated antibody, uses fluorescein labelling and DCP Specific binding antibody, as detection antibody, finally uses the magnetic particle of coupling DCP to remove the detection antibody of excess, by glimmering DCP in light intensity detection by quantitative sample.The present invention also provides for DCP magnetic particle immunofluorescence detection agent, and this immunofluorescence is examined Test agent comprises: magnetic particle, DCP antigen, anti-DCP antibody, fluorescein, coupling buffer, lavation buffer solution, eluent, closes Liquid, preserves liquid.The present invention further provides DCP magnetic particle immunofluorescence detection agent box, this detection kit includes the present invention Described DCP magnetic particle immunofluorescence detection agent.

Can provide easy to be quick according to the present invention, high sensitivity, high accuracy, safety non-toxic, the magnetic of callable DCP Separation of particles immunofluorescence analysis detection method and for the reagent of this detection and detection kit.The method of the present invention with Conventional method is compared, and detection efficiency is largely increased, and as DCP detection method, it has easy to be quick, highly sensitive, The advantages such as accuracy is high, safety non-toxic, recyclable.

In the detection method of the present invention, the anti-DCP using magnetic particle specific binding for coupling DCP with DCP respectively resists Body and fluorescently-labeled anti-DCP antibody carry out the detection of magnetic particle separating immune fluorescence analysis.By the method, can be in accordance with needing Strategic point simplifies operating procedure, reduces testing cost, improves the accuracy of detected value.

DCP antigen, i.e. DCP antigen protein standard substance, for commercial goods, can conventionally buy acquisition, its characteristic As follows: product design 1mg/ml, purification process is HPLC, purity >=98%.

Anti-DCP antibody is the antibody specific binding with DCP, can be monoclonal antibody, it is also possible to be polyclonal antibody. Angularly consider from the repeatability of immune detection, preferably use monoclonal antibody.It addition, these antibody can also keep with right The form answering the antibody fragment (antigen-binding fragment) of the associativity of antigen uses.

The preparation method of polyclonal antibody, monoclonal antibody and antigen-binding fragment itself is known conventional method, Anti-DCP antibody or and antigen-binding fragment can conventionally prepare.It addition, these antibody there is also commercial goods, because of This can also use commercially available antibody.

Anti-DCP monoclonal antibody such as can be obtained by known hybridoma: using DCP or its Partial Fragment as immunity Former Yu suitable adjuvant is mixed for immune animal (except people), gathers the antibody tormation such as splenocyte or lymphocyte from this animal Cell, by itself and myeloma cell fusion, prepares hybridoma, then selects to produce the hybridization of the antibody specific binding with DCP Tumor so that it is propagation, obtains anti-DCP monoclonal antibody from culture supernatant.In order to make antibody titer in immunized animal raise, exempt from Epidemic disease typically requires cost and carries out repeatedly several weeks.In the present invention, in order to improve the specificity that DCP combines, preferably monoclonal antibody.

The polypeptide used as immunogen or its Partial Fragment can pass through the routine side such as chemosynthesis, genetic engineering's method Prepared by method, or extract DCP from fresh human plasma etc. and purification obtains.The object lesson of chemical synthesis such as can be enumerated: Fmoc method (fluorenylmethyloxycarbonyl method), tBoc method (tert-butoxycarbonyl method) etc..Various commercially available peptide synthesizer can also be utilized, logical Cross conventional method with reference to the polypeptide needed for the DCP sequence information synthesis in the data bases such as GenBank.By genetic engineering's method The method preparing polypeptide is also known.Specifically, such as, can be prepared by method described below: first, by from people Cultivation cell etc. in extract RNA, synthesized cDNA by reverse transcription reaction by mRNA.With this cDNA as template, according to GenBank Go forward side by side performing PCR Deng the design primer such as the DCP sequence information in data base, the polynucleotide of preparation encoding D CP.Or, encoding D CP Polynucleotide can be prepared by the conventional method using commercially available nucleic acid synthesizer.It is known for encoding each amino acid whose codon , as long as therefore specific amino acid sequence, the base sequence of the polynucleotide encoding this aminoacid sequence also can determine that.Then, The polynucleotide of preparation are imported suitable carrier, makes expression of polypeptides by suitable expression system, reclaim this polypeptide, thus may be used Immunogen polypeptide needed for acquisition.The carrier used or various expression system (bacterial expression system, yeast cell to express system System, mammalian cell expression system, insect cell expression system, Cell free expression system etc.) also it is known, various carriers Or host cell, reagent class, detection kit are commercially available.

Immunoassay is known conventional method.As object lesson, competitive protein binding assay, receptor knot can be enumerated Close and analyze, and radioimmunoassay, RIA, EIA enzyme immunoassay, fluorescence immunoassay, colloid gold immune analysis, chemiluminescence and biology Chemiluminescence immunoassay methods etc., can use any means in the present invention.From the accuracy of immune detection and safety angularly Consider, preferably use fluoroimmunoassay.

Magnetic particle i.e. superparamagnetic nano particle, magnetic particle separating immune fluorescence analysis is affine by target molecule Group such as antibody, albumen etc. are coupled to magnetic nanoparticle surface, form the colloidal state can homodisperse with high stability Compound magnetic particle.After immunity magnetic particle mixes with the solution containing detection target molecule, due to target molecule and its group Affine combination and form magnetic particle-group-target molecule complex, then utilize externally-applied magnetic field (magnetic frame or bar magnet) micro-with magnetic Magnetic particle-group-target molecule complex is separated by the magnetic between Li, removes non-specific binding through lavation buffer solution miscellaneous Matter, then separates target molecule with magnetic particle with eluent, and then realizes the separation detection of target molecule, have low cost, Less energy consumption, safety non-toxic, the advantage such as recyclable, if supporting the use with full-automatic separation detection instrument, can further improve reaction Flux and work efficiency.The present invention is on the magnetic particle surface that particle diameter is distributed between 100～1000nm or by magnetic particle surface Functional group (amino, carboxyl, sulfydryl, epoxy radicals, NHS group etc.) coupling DCP and the anti-DCP specific binding with DCP resist Body, as solid phase carrier for immune detection.Magnetic particle surface in the present invention can use any one functional group and DCP Or anti-DCP antibody coupling.Suspension magnetic particle, as solid phase carrier, replaces traditional immunology solid phase carrier for detecting, There is bigger specific surface area, it is possible to sufficient and example reaction, in addition the flexible utilization of externally-applied magnetic field, have rapidly and efficiently, The advantage such as highly sensitive, reproducible.It addition, these magnetic particles there is also commercial goods, commercially available magnetic therefore can also be used Microgranule or magnetic bead.

As a example by using the anti-DCP antibody of use as the situation of coated antibody, illustrate the DCP detection method of the present invention.First First, anti-DCP antibody (coated antibody) is coupled on solid phase carrier magnetic particle, after being closed by unnecessary group, makes enough magnetic particles Being fully contacted with testing sample, the DCP that thus the anti-DCP antibody on magnetic particle is contained with sample is specific binding, then magnetic Property separate, with suitable buffer solution magnetic particle complex, remove other compositions in unconjugated sample, the most unnecessary load Body etc..Then eluting DCP, uses the fluorescein-labeled anti-DCP antibody of excess to be combined with DCP, hatches and make fully to react.Reaction After end, with suitable method detection from the fluorescence signal of fluorescein label, thus can detect the DCP content in sample.

Solid phase carrier is not particularly limited, can be identical with the solid phase carrier used in known immune detection system. The object lesson of the material of solid phase carrier can be enumerated: polystyrene, polrvinyl chloride, agarose, liposome, membrane carrier, macromolecule Magnetic particle etc., but it is not limited to these.The solid phase carrier used preferably antibody is firmly combined with its surface, and can be easily The material that the immune complex formed in detection is separated with unreacted composition.From operability, economy, safety and combination From the standpoint of efficiency etc., magnetic particle in described above material is preferably used.

Anti-DCP antibody that DCP with DCP is specific binding or its antigen-binding fragment can lead to the combination of solid phase carrier Cross conventional method well known in the art to carry out, as object lesson, covalent bond chemical coupling, non-covalent bond absorption or thing can be enumerated Reason absorption etc., the present invention can be attached to surface of solid phase carriers to use any one mode, but is not limited to these.

A kind of magnetic particle, it is characterised in that: described magnetic particle surface is coated with one layer of antibody with antigen recognition activity, Or it is coated with one layer of antigen with antibody recognition activity.

Label is not particularly limited, it is possible to use as the label used in known immune detection system Material.Object lesson can be enumerated: enzyme, fluorescent material, chemiluminescent substance, coloring matter, radioactive substance etc..In order to improve inspection Survey sensitivity, simplify operating procedure, reduce radiocontamination, fluorescein labelling is preferably used.Fluorescent dye for labelling does not has yet It is particularly limited to, it is possible to use with the material as the label used in known Immunofluorescence test system, concrete example Son can be enumerated: Fluorescein isothiocyanate (fluoresceinisothiocyanate, FITC), RB 200 (rhoda Mine, RIB200), Tetramethylrhodamine isothiocyanate (tetramethylrhoda mineisothiocyanate, TRITC), Lanthanide series (europium Eu₃, terbium Tb₃, cerium Ce₃Deng) chelate, phycoerythrin (phycoerythrin, PE) and other fluorescent materials (beta galactosidase, alkaline acid enzyme, horseradish peroxidase) etc..The present invention can use any one contaminate as fluorescence Material, but it is not limited to these.

When using biotin as label, it is possible to use combine enzyme, fluorescent material, chemiluminescent substance, product dyed thereby The streptavidin of matter or radioactive substance etc. or hapten antibody etc. detect.

The detection of signal suitably can select according to the kind of label.Such as signal if colour developing, then can use colorimetric Meter or extinction photometer, if fluorescence then can use spectrofluorophotometer, if luminescence then can use photon meter Number instrument, if lonizing radiation then can use radiation detecting apparatus.Mark known to the concentration of DCP for containing with various concentration Quasi-sample, detects DCP according to the method for the present invention, by from the concentration of DCP relevant in the semaphore and standard sample of labelling Relation is charted, and draws standard curve, the sample that DCP concentration is unknown is carried out detection operation equally, detects the signal from labelling Amount, substitutes into detected value this standard curve, thus can carry out DCP in sample quantitatively.

The sample that the method for the present invention is suitable for is the sample separated in subject, and preferably blood sample is the most excellent Select blood plasma or serum.According to the detection method of the present invention, no matter it is blood plasma or serum, all can stably detect DCP content.Root According to needs can suitable dilute sample, to guarantee to detect in the range of working concentration.

Antibody described in this DCP magnetic particle immunofluorescent detection method is not to be coated onboard, but is coated magnetic with antibody Microgranule, uses coated magnetic particle as reagent, more conducively quantitative work, conveniently stablizes and sensitivity is higher, resists at antibody Detection interference factor can be preferably minimized by former reaction.

The present invention also provides for DCP magnetic particle immunofluorescence detection agent, and this reagent comprises: magnetic particle, DCP antigen, anti-DCP Antibody, fluorescein, coupling buffer, lavation buffer solution, eluent, confining liquid, preserve liquid.

Above-mentioned DCP magnetic particle immunofluorescence detection agent can be appropriately combined, tries as DCP magnetic particle Immunofluorescence test Agent box provides.DCP magnetic particle immunofluorescence detection agent can also be appropriately combined, such as, except above-mentioned inspection with other reagent class etc. Outside test agent, the detection kit of the present invention can also contain EP pipe, Sample dilution etc. further.Wherein, EP pipe is i.e. Eppendorf centrifuge tube, for commercial goods, can conventionally buy acquisition.Immune detection other reagent class necessary It is known.

Sensitive quick DCP magnetic particle immunofluorescence detection agent box provided by the present invention, for detection and the disease of DCP Early diagnosis provide a kind of easy quickly, high sensitivity, high accuracy, safety non-toxic, callable approach.Detectable Box includes described DCP magnetic particle immunofluorescence detection agent, is fixed in the groove in box.

Accompanying drawing explanation

Fig. 1 is to represent the chart of the linear relationship of DCP standard concentration and absorbance in embodiment 1.

Detailed description of the invention

Below by way of specific instantiation, embodiments of the present invention are described, to use DCP as target detection thing As a example by situation, illustrate the DCP magnetic particle immunofluorescent detection method of the present invention.The present invention is not by these embodiments etc. Limit.Those skilled in the art can be understood other advantages and effect of the present invention easily by the content disclosed by this specification. The present invention can also be carried out by the most different detailed description of the invention or apply, and the every details in this specification also may be used With based on different viewpoints and application, under the spirit without departing from the present invention, carry out various modification or change.

In the present invention, when numerical ranges are given, it should be appreciated that unless the present invention is otherwise noted, each numerical range Between two end points and two end points, any one numerical value all can be selected for.Unless otherwise defined, use in the present invention is all The same meaning that technology and scientific terminology are generally understood that with those skilled in the art of the present technique.Except the concrete side used in embodiment Outside method, equipment, material, according to those skilled in the art's grasp to prior art and the record of the present invention, it is also possible to Use any method of prior art similar or equivalent with the method described in the embodiment of the present invention, equipment, material, equipment and Material realizes the present invention.

The preparation of embodiment 1.DCP magnetic particle separating immune fluorescence analysis detectable

1, the preparation of DCP antigen

(1) structure of pCold II-DCP expression vector

Design primer with reference to the gene order of people DCP in GenBank and synthesize DCP gene, after PCR amplification, 1% agarose Detected through gel electrophoresis DCP amplified production, cuts purpose band, uses glue to reclaim test kit and reclaims purpose fragment.To it with limiting Property restriction endonuclease Nde I and Hind III double digestion prokaryotic expression carrier pCold II connect, convert to E. coli .DH5 α Middle amplification, then proceed to E.coli.BL21 (DE3) competent cell, it is inoculated in LB solid medium, containing ampicillin (Amp) 100mg/L, extracts plasmid, carries out Nde I and Hind III enzyme action and identifies and order-checking.Recombiant plasmid pCold II-DCP is through 5' Nde I and 3'Hind III carries out double digestion, two specific bands, position and pCold II and the size one of purpose fragment occurs Cause.Sequencing result display target sequence is consistent with NCBI corresponding sequence, it was demonstrated that expression vector pCold II-DCP successfully constructs.

(2) expression of DCP fusion protein and purification

The picking colony inoculation containing recombiant plasmid pCold II-DCP-BL21 is in 10mL LB culture medium (Amp, 100mg/L) In, 220r/min 37 DEG C shakes bacterium 12h, is inoculated in the LB culture medium (Amp, 100mg/L) of 1000mL in the ratio of 1:100, phase Cultivate 3h under the conditions of Tong, treat that bacterium solution reaches A₆₀₀Time add IPTG to final concentration of 0.5mmol/L, 16h are cultivated in 15 DEG C of shakings, lure Lead after cultivation terminates, collect thalline to 50mL sterile centrifugation tube, 4 DEG C of centrifugal 10min of 5000r/min, abandon supernatant, be resuspended in In 20mL lysis buffer (20mmol/L Tris-HCl comprises 1mmol/L protease inhibitor cocktail, pH 8.0), warp Cell Ultrasonic Cell Disruptor broken (ultrasonic 2s, cooling 4s, power 100W), 12000r/min, 4 DEG C of centrifugal 15min.Precipitation is resuspended in Lysis buffer (containing 8mol/L carbamide), and through 0.22 μm membrane filtration, cross Ni-NTA chromatographic column and be purified；The post of 10 times Volume lysis buffer (containing 8mol/L carbamide+20mmol/L imidazoles) washs；With buffer C (20mmol/L Tris-HCl Buffer, pH 8.0, containing 150mmol/L NaCl, 8mol/L carbamide, 250mmol/L imidazoles) eluting destination protein.By eluting Albumen with containing finite concentration gradient carbamide (6,5,4,2,1mol/L) carry out renaturation, finally with PBS, after having dialysed Detection lyophilizing protein concentrate concentration is 1.5mg/mL.

2, the preparation of DCP antibody

(1) preparation of hybridoma cell strain

S p2/0 myeloma cell good for growth conditions is mixed with the ratio of 1:10 with immune mouse spleen cell, adds 50% Polyethylene Glycol merges, and fusion process is carried out according to a conventional method.With DCP expressing protein as detection antigen, melt having The supernatant closing cell hole carries out indirect ELISA detection, and one resists for cells and supernatant, and two resist for horseradish peroxidase-labeled Goat anti-mouse IgG (1:2000 dilution), TMB color developing detection screening positive cell clone strain.Positive cell gram by screening Grand strain limiting dilution assay carries out monoclonal screening, and indirect elisa method detects, until positive rate reaches 100%, filters out stable point The hybridoma cell strain secreting anti-DCP antibody is enlarged cultivating, and frozen in liquid nitrogen.

(2) preparation of monoclonal antibody

Selection BALb/c mice, every lumbar injection sterilized liquid paraffin 0.5mL is thin through intraperitoneal inoculation hybridoma after 1 week Born of the same parents 0.5mL (1 × 10⁶Cell/only).After 10～14d, mouse web portion obvious tumefaction collects ascites, and 12000r/min is centrifuged 10min, Remove the fat on upper strata, liquid paraffin and precipitation, draw faint yellow ascites, by ascites through Protein A agarose affinity chromatography Post obtain antibody purification, carry out in PBS afterwards 4 DEG C dialysis 12h, the next day use BCA method detection antibody concentration, ELISA method examine Survey antibody titer, be subsequently adding 50% glycerol mixing, in a small amount after subpackage-80 DEG C save backup.

DCP magnetic particle immunofluorescence analysis detection method and operating procedure

1, magnetic particle and the coupling of targeted biological specimen

(1) pretreatment of magnetic particle

By the reverse mixing repeatedly of magnetic particle suspension, take 50 μ L and be placed in 1.5mL EP pipe, add 0.5～1.0mL washing Buffer, lavation buffer solution is: pH is the 20mM sodium phosphate of 7.2～7.6,150mM sodium chloride, and mixing, then on magnetic frame Magnetic Isolation, abandons supernatant.Add 1mL lavation buffer solution suspension magnetic particle, mixing, Magnetic Isolation on magnetic frame, abandon supernatant Liquid.Repeating previous step, then suspended in 50 μ L coupling buffers by magnetic particle, coupling buffer is: pH is 8.0～8.2 25mM～50mM ammonium hydrogen carbonate, stand-by.

(2) coupling of targeted biological specimen

During by immune with preparation to targeted biological specimen and magnetic particle coupling magnetic particle, monoclonal antibody used or antigen bag Being to affect the direct factor of late detection by the concentration of liquid, its concentration directly affects the accuracy of testing result and linear model Enclose.If the concentration being coated liquid used by is too low, and in the immunoreation in later stage, the reaction efficiency of magnetic particle is poor, and antigen antibody reaction is not Completely, make testing result on the low side；It is coated the excessive concentration of liquid used by if, the waste of expensive reagent can be caused.Therefore below using Method carries out condition optimizing screening:

Taking 10～15 μ L needs the DCP antibody of coupling or antigen in filling the EP pipe that 2mL particle diameter is 100～1000nm magnetic particles In, and adding 500 μ L coupling buffers, coupling buffer is: pH is 25mM～the 50mM ammonium hydrogen carbonate of 8.0～8.2, mixing. EP pipe is placed on rotary mixer and hatches.Then EP pipe is placed in Magnetic Isolation on magnetic frame, retains magnetic particle and supernatant, The protein content of anti-DCP antibody standard solution before and after employing BCA method detection coupling.Described magnetic particle and anti-DCP antibody coupling bar Part is preferably:

Described magnetic particle is preferably with anti-DCP antigen coupling condition:

Or magnetic particle particle diameter 800nm, antigen concentration 30 μ g/mL, 28 DEG C hatch 6h, pH=10.

(3) unreacted radical is closed

After magnetic particle coupling terminates, adding 500 μ L confining liquids, confining liquid is: the BSA of 0.1%, is placed in by EP pipe after mixing On rotary mixer, incubated at room 2～6h or 4 DEG C hatch 12～20h, then EP pipe are placed in Magnetic Isolation on magnetic frame, abandon Supernatant.Repeat previous step once.

(4) preservation of magnetic particle

Adding the preservation liquid of 1mL in the magnetic particle completing biomolecule covalent coupling, preserving liquid is: pH is the 5mM of 9 BST buffer, 0.05%Tween-20,0.01%NaN₃, 0.1%BSA, mixing, save backup in 4 DEG C.

2, magnetic particle and the detection of anti-DCP antibody coupling efficiency

After anti-DCP antibody and magnetic particle coupling, the egg of anti-DCP antibody standard solution before and after employing BCA method detection coupling Bai Hanliang, calculates magnetic particle coupling efficiency.Result shows, after adding magnetic particle, solution protein concentration significantly reduces, magnetic particle table The group in face can be with anti-DCP antibody generation coupling, so that magnetic particle has biological activity, i.e. has capture antibody or the energy of antigen Power becomes immunity magnetic particle.Magnetic particle and anti-DCP antibody coupling efficiency are 84.78% ± 7.23.

3, the drafting of DCP standard curve

Take the anti-DCP antibody 100 μ L of FITC labelling in EP pipe, be separately added into each 100 μ L of DCP standard substance, use 0.01mol/L PBS be diluted to 10 Concentraton gradient (0ng/mL, 0.2ng/mL, 0.4ng/mL, 0.8ng/mL, 1.6ng/mL, 3.2ng/mL, 6.4ng/mL, 12.8ng/mL, 25.6ng/mL, 51.2ng/mL), in room temperature rotate hatch 2～6h or 4 DEG C incubate Educate more than 12h, make DCP antigen-antibody fully react.Return to zero by blank well, use spectrofluorophotometer examination criteria product fluorescence Value, draws standard curve according to DCP standard concentration and fluorescent value thereof, sees Fig. 1.

4, the detection of DCP content

Concrete operation step is as follows:

(1) take 10 parts of human serum sample 200 μ L, add the magnetic particle A of 2mL coupling DCP antibody, rotate in room temperature and hatch 2 ～6h or 4 DEG C hatch 12～20h, DCP antigen-antibody is made fully to react.

(2) being placed in Magnetic Isolation on magnetic frame, abandon supernatant, clean magnetic particle complex with lavation buffer solution, washing is slow Rushing liquid is: pH is the 20mM sodium phosphate of 7.2～7.6, and 150mM sodium chloride is placed in Magnetic Isolation on magnetic frame, abandons supernatant.

(3) adding 100 μ L eluents, eluent is: pH is the 0.1M glycine of 2.5, mixing, resuspended magnetic particle, room temperature Hatch 5min, be placed in Magnetic Isolation on magnetic frame, retain supernatant.

(4) add 2mL FITC labelling anti-DCP antibody, in room temperature rotate hatch 2～6h or 4 DEG C hatch 12～20h. Add 20mL coupling DCP antigen magnetic particle, in room temperature rotate hatch 2～6h or 4 DEG C hatch 12～20h.

(5) it is placed in Magnetic Isolation on magnetic frame, retains supernatant, use spectrofluorophotometer detection sample fluorescence value, And reference standard curve calculates DCP content in sample.

Comparing with existing method, advantages of the present invention shows:

Immunity magnetic particle has higher surface quality ratio, can participate in immunity in conjunction with more antibody or antigen anti- Should；

Using two set magnetic particle coupled antibody and antigens respectively, compared with conventional double antibody sandwich method, this method only needs The anti-DCP antibody wanting a kind of correspondence can complete reaction, and cost is relatively low；

Use fluorescent-labeled antibody technology, by the accuracy of the specificity of antigen antibody reaction and sensitivity with micro-spike Combining, high specificity, highly sensitive, accuracy good.

5, the stability of DCP detection method compares

Above-mentioned testing result being compareed with ELISA method, result shows that the two has good dependency: use magnetic micro- The range of linearity of grain separating immune fluorimetry detection DCP is 0.1～50.0ng/mL, and detection is limited to 0.1ng/mL, and standard is bent The equation of linear regression of line is y=2.1069+0.9766x, R²=0.9995, (wherein x is DCP concentration, and y is absorbance), Its Monitoring lower-cut is lower 50 times than ELISA method.Blood serum sample DCP content detection result is as follows:

Note :-represent and can not detect.

6, the response rate detection of magnetic particle

Being mixed with eluent by the magnetic particle of coupling DCP antibody or antigen, eluent is: pH is the sweet ammonia of 0.1M of 2.5 Acid, fully reaction are placed on Magnetic Isolation on magnetic frame, clean magnetic particle with lavation buffer solution immediately, and lavation buffer solution is: pH It is the 20mM sodium phosphate of 7.2～7.6,150mM sodium chloride, remove antibody or the antigen residuing in magnetic particle surface, be subsequently adding 1mL preserves liquid, preserves liquid and is: pH is the 5mM BST buffer of 9,0.05%Tween-20,0.01%NaN₃, 0.1%BSA, mixed Even, save backup in 4 DEG C.After the magnetic particle coupling with recovery of the anti-DCP antibody, before and after using the detection coupling of BCA method, anti-DCP resists The protein content of body standard solution, calculating the magnetic particle response rate is 83.99% ± 8.14.

7, full-automatic magnetic particle separating immune fluorescence analysis detection

The concrete methods of realizing of magnetic particle separation detection generally has manually and automatically two kinds of forms.Manually detection refers to operation Consumptive material and the instruments such as librarian use DCP magnetic particle immunofluorescence detection agent, magnetic frame, have been manually done whole testing process.Hands The magnetic frame used in dynamic sorting is relatively simple for structure, is mainly made up of the permanent magnet of support and generation magnetic field, plays support examination Manage and provide the effect of externally-applied magnetic field.The hatching of magnetic particle, external magnetic field add with remove, cleaning and the eluting etc. of adsorbate Each committed step is all completed by manual operation.Manually sorting need not the equipment of complexity, and flexible form, cost are relatively low, is suitable for few Amount experiment uses.

When needs frequently carry out operating and sample size is bigger, use that to be automatically separated detection method more convenient efficiently.Automatically Partition method mainly utilizes full-automatic magnetic particle sorter, and sample to be sorted is joined in sorter, by sorter by operator It is automatically performed separation process.The sorting flux of Full-automatic magnetic sorter is relatively big, and typically has the process of separation of multiple optimization Being available for calling, simple to operate, sorting reliability is high.The most representative commercialization Full-automatic magnetic sorter has U.S. sky Ni (MACS), the brand such as BD, R&D, StemCell RoboSep and Dynal Bead Retriever.

The detection method of magnetic particle separating immune fluorescence analysis DCP content of the present invention, can be used for full-automatic immunomagnetic beads and divides Select system, it is achieved automatical analysis detects, and concrete operation step is as follows:

(1) rush autoMACS pro sorter in advance, prepare test serum sample, magnetic particle A/B, DCP standard substance, FITC mark The anti-DCP antibody of note, coupling buffer, lavation buffer solution, eluent, confining liquid, preservation liquid；

(2) cell sorting policy selection " positive sorting strategy ", mark mode selects " direct labelling "；

(3) take 25 μ L DCP standard substance in S1-7 pipe, in 2 blank tubes, add 1mL lavation buffer solution, to centrifugal Pipe is separately added into 25 μ L magnetic particle A and 50 μ L fluorescent labeling anti-DCP antibody, response procedures condition, upper machine testing are set.Will inspection Surveying result to compare with manual detection method, result shows that the two has good dependency.

The composition of embodiment 2.DCP magnetic particle immunofluorescence detection agent box

The present invention devises a kind of test kit according to the method for the invention, and this test kit may be used for the detection of DCP, logical Cross and use this test kit, make simple to operate, time saving and energy saving, it is to avoid matching while using loaded down with trivial details, make operational standardization simultaneously.

Therefore the present invention provides a kind of test kit.

The test kit of the present invention, including magnetic particle, and optional following components: the magnetic particle A of coupling DCP antibody, even Magnetic particle B, the DCP antigen of connection DCP antigen, anti-DCP antibody, fluorescein, coupling buffer, lavation buffer solution, eluent, close Liquid, preserves liquid, magnetic frame.

The another kind of test kit of the present invention, including: the magnetic particle A of coupling DCP antibody, the magnetic particle B of coupling DCP antigen, And optional following components: DCP antigen, anti-DCP antibody, fluorescein, coupling buffer, lavation buffer solution, eluent, close Liquid, preserves liquid, magnetic frame.

Wherein said optionally for any of which component can not be selected, it is also possible to select wherein one or more component.

The test kit of the present invention, is different components to be contained respectively, another with being packaged in same packing box, during use Operate according to the method described in description.

In test kit, coupling buffer is selected from: bicarbonate buffer, carbonate buffer solution, the one of sulfate buffer Kind or multiple combination, concentration is 1～100mM, and pH is 8.0～10.0；

The detection kit of the present invention, including: the reagent in box body, box body, described reagent is DCP magnetic particle immunofluorescence Detectable, described tray interior is provided with some reagent troughs, places the EP pipe filling magnetic particle, in test kit in described reagent trough The amount of reagent can be a person-portion, it is also possible to be many person-portions.

Comparing with existing method, advantages of the present invention shows:

Using two set magnetic particle coupled antibody and antigens respectively, compared with conventional double antibody sandwich method, this method only needs The anti-immunological marker thing antibody wanting a kind of correspondence can complete reaction, and cost is relatively low；

In sum, DCP magnetic particle immunofluorescence detection agent box provided by the present invention have good accuracy and Specificity and highly sensitive, effectively overcomes shortcoming of the prior art and tool high industrial value.

The principle of above-described embodiment only illustrative present invention and effect thereof, not for limiting the present invention.Any ripe Above-described embodiment all can be modified under the spirit and the scope of the present invention or change by the personage knowing this technology.Cause This, have usually intellectual such as complete with institute under technological thought without departing from disclosed spirit in art All equivalences become are modified or change, and must be contained by the claim of the present invention.

Claims

1. one kind is detected the method for DCP content in blood plasma or serum: said method comprising the steps of:

Step 1, the preparation of the magnetic particle A of surface coupling anti-DCP antibody, and the preparation of the magnetic particle B of surface coupling DCP antigen；

Step 2, the preparation of fluorescently-labeled anti-DCP antibody；

Step 3, the detection of DCP content: blood plasma or blood serum sample and magnetic particle A reaction, react complete by DCP eluting, add glimmering The anti-DCP antibody incubation of signal, adds magnetic particle B and continues to hatch, and then Magnetic Isolation detects with spectrofluorophotometer Sample fluorescence value, calculates DCP content in sample.

Method the most according to claim 1, it is characterised in that described magnetic particle A and the preparation of magnetic particle B, method is as follows: Magnetic particle is prepared as magnetic particle suspension, magnetic particle suspension and anti-DCP antibody or DCP antigen coupling: by anti-DCP antibody or DCP antigen mixes with magnetic particle suspension hatches coupling, closes with confining liquid, obtain the magnetic particle A of coupling DCP antibody after coupling Magnetic particle B with coupling DCP antigen.

Method the most according to claim 1, it is characterised in that wherein, the described antibody specific binding with DCP is Dan Ke Grand antibody or polyclonal antibody.

Method the most according to claim 1, it is characterised in that wherein, magnetic particle is selected from such as with the coupling condition of DCP antigen Lower condition:

Method the most according to claim 1, it is characterised in that wherein, magnetic particle is selected from the coupling condition of anti-DCP antibody Following condition:

Method the most according to claim 1, it is characterised in that step is as follows:

Step (1) takes magnetic particle, adds magnetic particle volume 2～the lavation buffer solution of 50 times, mixing, then magnetic on magnetic frame Separate, abandon supernatant；Repeat previous step, then magnetic particle is being suspended in the coupling buffer of its equivalent, obtaining magnetic micro- Grain suspension；

Step (2) adds the magnetic particle that the step (1) of volume 0.01～1 times processes in anti-DCP antibody or DCP antigen and suspends Liquid, adds coupling buffer, hatches, until magnetic particle coupling is complete；Magnetic Isolation, retains magnetic particle；Magnetic particle coupling terminates After, add confining liquid mixing, hatch, Magnetic Isolation, obtain magnetic particle A and the magnetic particle of coupling DCP antigen of coupling DCP antibody B；

The preparation of the fluorescently-labeled anti-DCP antibody of step (3): anti-DCP antibody is dissolved in carbonate buffer solution, fluorescein dissolves In DMSO, fluorescein is dropwise slowly added in anti-DCP antibody-solutions, 4 DEG C of lucifuge stirrings 12～20h, uses G-25 glucosan Free fluorescein removed by gel；

Step (4) takes testing sample, adds sample volume 1～the magnetic particle A of 100 times, rotates in room temperature and hatches 2～6h or 4 DEG C hatch 12～20h, make DCP antigen-antibody fully react；It is placed in Magnetic Isolation on magnetic frame, abandons supernatant, use washing buffer Liquid cleans magnetic particle complex, is placed in Magnetic Isolation on magnetic frame, abandons supernatant；Add the eluent with antibody equivalent, mix, Resuspended magnetic particle, incubated at room 5min, it is placed in Magnetic Isolation on magnetic frame, retains supernatant；Add sample volume 1～100 times Fluorescent labeling DCP antibody, in room temperature rotate hatch 2～6h or 4 DEG C hatch 12～20h；Add magnetic particle A volume 1～100 Times magnetic particle B, in room temperature rotate hatch 2～6h or 4 DEG C hatch 12～20h；It is placed in Magnetic Isolation on magnetic frame, in reservation Clear liquid, uses spectrofluorophotometer detection sample fluorescence value, and reference standard curve calculates DCP content in sample.

(1) taking magnetic particle to be placed in EP pipe, add magnetic particle volume 10～the lavation buffer solution of 20 times, mixing, then at magnetic force Magnetic Isolation on frame, abandons supernatant；Repeat previous step, then magnetic particle suspended in the coupling buffer of its equivalent, Obtain magnetic particle suspension；

(2) magnetic particle suspension vol 0.2～the anti-DCP antibody needing coupling of 0.3 times or DCP antigen are added in filling magnetic particle In the EP pipe of suspension, and add the coupling buffer of magnetic particle suspension vol 10 times, mixing；EP pipe is placed in rotation mixing On instrument, incubated at room 2～6h or 4 DEG C hatch 12～20h, until magnetic particle coupling is complete；EP pipe is placed in magnetic on magnetic frame Separate, retain magnetic particle；Surface coupling anti-DCP antibody for magnetic particle A, surface coupling DCP antigen for magnetic particle B, supernatant Liquid is used for detecting coupling efficiency；

(3), after magnetic particle coupling terminates, add the confining liquid of magnetic particle suspension vol 10 times, mixing, EP pipe is placed in rotation On mixed instrument, incubated at room 2～6h or 4 DEG C hatch 12～20h, EP pipe is placed in Magnetic Isolation on magnetic frame, abandons supernatant； Repeat previous step once；

(4) take test plasma or blood serum sample, add sample volume 1～the magnetic particle A of 100 times, rotate in room temperature and hatch 2～6h Or hatch 12～20h for 4 DEG C, make DCP antigen-antibody fully react；It is placed in Magnetic Isolation on magnetic frame, abandons supernatant, with washing Buffer solution for cleaning magnetic particle complex, is placed in Magnetic Isolation on magnetic frame, abandons supernatant；Add the eluent with antibody equivalent, Mixing, resuspended magnetic particle, incubated at room 5min, it is placed in Magnetic Isolation on magnetic frame, retains supernatant；Add sample volume 1～ The fluorescent labeling DCP antibody of 100 times, in room temperature rotate hatch 2～6h or 4 DEG C hatch 12～20h；Add magnetic particle A volume 1 ～the magnetic particle B of 100 times, in room temperature rotate hatch 2～6h or 4 DEG C hatch 12～20h；It is placed in Magnetic Isolation on magnetic frame, Retain supernatant, use spectrofluorophotometer detection sample fluorescence value, and reference standard curve calculates DCP content in sample.

Method the most according to claim 1, it is characterised in that wherein,

Coupling buffer is selected from: bicarbonate buffer, carbonate buffer solution, the group of one or more of sulfate buffer Closing, concentration is 1～100mM, and pH is 8.0～10.0；

Lavation buffer solution is selected from: glycine buffer, Tris-HCl buffer, phosphate buffer, borate buffer solution, carbonic acid Salt buffer, the combination of one or more of chlorate buffer, concentration is 2～200mM, and pH is 7.0～8.0；

Eluent is selected from: glycine, Tris Buffer, EDTA solution, IPTG solution, Pidolidone solution, MES Buffer The combination of one or more, concentration is 0.01～10M, and pH is 2.0～3.0；

Confining liquid is selected from: BSA, casein, the combination of one or more of glycine, and concentration is 0.05%～5%, and pH is 8.0 ～9.0；

Preservation liquid is selected from: BST buffer, Tween-20, NaN₃, the combination of one or more of BSA, concentration is 1～50mM, pH It is 8～10.

9. use a test kit of claim 1 method detection DCP, including:

Magnetic particle, and optional following components: the magnetic particle A of coupling DCP antibody, the magnetic particle B, DCP of coupling DCP antigen resists Former, anti-DCP antibody, fluorescein, coupling buffer, lavation buffer solution, eluent, confining liquid, preserve liquid, magnetic frame.

10. use a test kit of claim 1 method detection DCP, including:

The magnetic particle A of coupling DCP antibody,

The magnetic particle B of coupling DCP antigen,

And optional following components: DCP antigen, anti-DCP antibody, fluorescein, coupling buffer, lavation buffer solution, eluent, Confining liquid, preserves liquid, magnetic frame.