CN105974132A

CN105974132A - Detection method, detection reagent and detection kit for alpha-fetoprotein variant

Info

Publication number: CN105974132A
Application number: CN201610519524.0A
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: 2016-09-28

Abstract

The invention discloses a detection method, a detection reagent and a detection kit for an alpha-fetoprotein variant. The detection method comprises the following steps: preparing magnetic particles A of which the surface is coupled with anti-AFP-L3 antibodies and preparing magnetic particles B of which the surface is coupled with AFP-L3 antigens; step 2, preparing fluorescently-labeled anti-AFP-L3 antibodies; and step 3, detecting the content of AFP-L3 content, reacting plasma or serums with magnetic particles A, eluting AFP-L3 after reaction is finished, adding the fluorescently-labeled anti-AFP-L3 antibodies, and performing hatching; and then adding the magnetic particles B, further performing hatching, then performing magnetic separation, detecting the sample fluorescence value of a fluorescence spectrophotometer, and calculating the content of AFP-L3 in the sample.

Description

The detection method of a kind of alpha-fetoprotein variant, detectable and detection kit

Technical field

The present invention relates to a kind of medical detecting method, particularly to a kind of AFP-L3 magnetic particle immunity Fluorescence detection method and AFP-L3 magnetic particle immunofluorescence detection agent and detection kit.

Background technology

Primary hepatoma (HCC) is one of common malignant tumor of China, and mortality rate is high, Mortality of malignant tumors cis-position is only second to gastric cancer, esophageal carcinoma and occupy the 3rd, in some areas Rural area then accounts for second, is only second to gastric cancer.China is hepatitis B big country, about 1.2 hundred million hepatitis B Virus carrier, has 130,000 patients to die from hepatocarcinoma every year, accounts for the 45% of whole world PLC mortality number. Hepatocarcinoma is mostly based on chronic hepatitis, the liver cirrhosis that long-term hepatitis B virus causes.

As other cancer screenings, hepatocarcinoma preventing and treating essentially consists in " three early ", i.e. early discovery, Early diagnosis and early treatment.Early hepatocarcinoma, unless position is special, many without the biggest difficulty in treatment, Excision, thromboembolism, melt and the method such as liver transplantation all can be proved effective, as obtained Scientific Treatment, life in 5 years Rate of depositing is 50～more than 70%, and major part patient can be with long-term surviving.But mid and late liver cancer is usual With the transfer inside and outside liver, operation cannot be excised, and also cannot implement liver transplantation, intervention embolization Less effective, Drug therapy also difficulty has effective, and postoperative complication is many, weak curative effect, and life cycle is short, After general morbidity, life span is only 6 months.

Near late period when clinical therapeutic efficacy for hepatic carcinoma main reason not fully up to expectations is clinical definite, because of In the subclinical stage, this, for hepatocarcinoma, can show that diagnosis is extremely important.Alpha-fetoprotein (AFP) at present It is widely used in clinical diagnosis.Although AFP has in a lot of clinical diseases accordingly Raising, but it is primarily as the tumor markers of clinical diagnosis HCC, its serum-concentration may Relevant to the size of the differentiation degree of HCC and tumor.In high-risk group, 15～58% chronic Hepatitis and 11～47% its serum of liver cirrhosis patient in AFP all can raise, clinical On ALT is not raised, AFP raises as the index of liver cell regeneration.HCC and hepatopath In serum, AFP all has rising, causes the explanation to AFP testing result to produce and obscures.From clinic Empirical angle is observed, the examination of AFP patient suspicious to HCC (especially in high-risk group) It is effective, but AFP specificity deficiency limits the value of HCC early diagnosis.

AFP is a kind of glycoprotein, according to glycoprotein forms divide three classes: AFP-L1, AFP-L2, AFP-L3.AFP-L1 is primarily present in optimum hepatopathy, and AFP-L2 is from anemia of pregnant woman, AFP-L3 It is that hepatoma carcinoma cell institute is peculiar, can be as a new tumor markers of detection HCC.

Research shows, expressing the hepatoma carcinoma cell of AFP-L3 has in early days vessel invasion and Intrahepatic metastasis Tendency, generally can find Ki67, its cancerated for hepatocyte after dyeing in karyon Mark.If α-catenin disappearance, then HCC usually has the transfer outside liver.Iconography Research finds that HCC positive for AFP-L3 generally has abundant hepatic arterial blood supply, tumor again The increasing time is shorter, and HCC process positive for prompting AFP-L3 is very fast and is susceptible to shift in early days. Research shows, if diameter accounts for the 10% of total AFP less than AFP-L3 in the HCC patients serum of 2cm Above, then point out this tumor to have aggressivity canceration.In optimum liver chronic disease, liver Cell does not express AFP-L3, and therefore the grade malignancy of the content of AFP-L3 the highest prompting tumor is more High.The experimental results shows, AFP-L3 is relevant with its histoorgan source, different physiology and Pathological condition can produce different sugar chain structures, has related neoplasms specificity；Its individually or and its The use in conjunction of his Tumor invasion, diagnosis curative effect and Index for diagnosis for HCC have important meaning Justice.

Chang Binxia etc. report that expressing the cell of AFP-L3 has in early days involvement of blood vessel and Intrahepatic metastasis Tendency, < the small liver cancer focus patient of 2cm may occur in which that the rising of AFP-L3 is (often to the diameter of about 35% Refined rosy clouds, Xin Shaojie. alpha-fetoprotein and clinical application research progress thereof. world Chinese digests magazine, 2010；18:576-580).This index was applied by food and medicine Surveillance Authority in approval in 2005 In diagnosing cancer of liver, and the positive of AFP-L3 diagnosing liver cancer is defined value is 10%.AFP-L3 exists The warning aspect of early hepatocarcinoma also has remarkable effect, and research shows its Index for diagnosis after Liver Cancer Operation There is important clinical meaning (Taketa K, Ichikawa E, Sakuda H, et al.Lectin reactivity of alpha-fetoprotein in a case of renal cell carcinoma.Tumour Biol, 1989；10:275-280) (Wang Shouming, Gao Lei, Yu Lecheng, He Changlun. alpha-fetoprotein variant Diagnosing cancer of liver and curative effect evaluation value research are in progress. practical hepatopathy magazine, 2011；14: 479-481) (Chen Yan, yellow wren Oriolus chinensis diffusus, Hu Minhua, Chen Yansong. alpha-fetoprotein variant is at early hepatocarcinoma Diagnosis and forewarning function are studied. modern medical oncology, and 2012；20:1652-1654).Due to AFP-L3 derives from the secretion of hepatoma carcinoma cell, and therefore along with the increase of diameter of tumor, it is secreted into AFP-L3 content in blood increases that (Liu Yanfen, etc. Primary Hepatic for Li Bin, Liu Zhiwei the most therewith The change of alpha-fetoprotein variant and clinical meaning after cancer operation in patients. Kweiyang Chinese medicine journal, 2014； 36:52-54).Research shows, AFP-L3 >=10% as the diagnostic criteria sensitivity of hepatocarcinoma is 82.6%, it is 76.8% with the specificity of chronic hepatopathy Differential Diagnosis, the AFP-L3 of liver cancer patient is equal Value and positive rate are above liver cirrhosis group and chronic hepatitis group, and difference has statistical significance, prompting Detection AFP-L3 contributes to differentiating primary hepatocarcinoma and non-Patients with Primary.Especially at first tire Albumen low concentration group AFP<when 400ng/ml, AFP-L3>=10% are for Differential Diagnosis index, hepatocarcinoma Substantially differentiation available with optimum hepatopathy (Xiong Biao, Cai Qihao cover triumphant. three biochemical indicators associating first The research of fetoprotein diagnosing primary hepatocarcinoma. contemporary medical science, 2008；14(14):37-38).Bae etc. Pointing out, the mensuration of AFP-L3 content contributes to the Differential Diagnosis of liver benign and malignant diseases.AFP-L3 The lowest in optimum patients with chronic liver, and the highest hepatocarcinoma (HCC) patient.On the other hand, The patient that AFP-L3 content raises, its HCC incidence rate is also significantly greater than the patient that AFP raises (BAE JS,PARK SJ,PARK KB,et al.Acute exacerbation of hepatitis in liver cirrhosis with very high levels of alpha-fetoprotein but no occurrence of hepatocellular carcinoma.Korean J Intern Med,2005；20(1):80-85).Blood Clear AFP content between 10～200ng/mL patient, HCC is diagnosed by AFP-L3 ＞ 10% Sensitivity is 71%, and specificity is 63% (Wang Yongzhong, Luo Libo, Wu Guoxiang, etc. microcentrifugation Post method separation detection alpha-fetoprotein variant (AFP-L3) and clinical meaning thereof. China tests and faces Bed Journal of Virology, 2007；21(2):135-137).It is quick that HCC is diagnosed by AFP-L3 ＞ 35% Sensitivity is 35%, and specificity is up to 100% (STERLING RK, IEFFERS L, GORDON F,et al.Utility of Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein and des-gamma-carboxy prothrombin,alone or in combination,as biomarkers for hepatocellular carcinoma.Clin Gastroenterol Hepatol,2009；7(1):104-113).Clinic is general accounts for the 15% of AFP aggregate level with AFP-L3 As judging the marginal value of HCC, thus obtain between preferable specificity and sensitivity flat Weighing apparatus (LEERAPUN A, SURAVARAPU SV, B IDA JP, et al.The utility of Lens culinaris agglutinin2 reactive alpha2-fetoprotein in the diagnosis of hepatocellular carcinoma:evaluation in a United States refrral population. Clin Gastroenterol Hepatol,2007；5(3):394-402).Research both at home and abroad shows, AFP-L3 is hepatocarcinoma high degree of specificity index, checks that total AFP can significantly improve accuracy rate than simple, Can not be limited by AFP >=400ng standard.But the difference due to AFP-L3 Yu AFP Being present on sugar chain, therefore AFP-L3 index, cannot grind in routine clinical use for a long time Study carefully with new method detection AFP-L3 ten points be necessary (blue or green willow crack. the clinical diagnosis meaning of tumor markers Justice and prospect alpha-fetoprotein (AFP) L3 component. Japan medical science introduction, 2005；26:49-50) (HU KQ,Kyulo NL.Clinical significance of elevated alpha-fetoprotein (AFP)in patients with chronic hepatitis C,but not hepatocellular carcinoma.Am J Gastroenterol,2004；99:860-865).

AFP-L3 is affirmative to the diagnostic value of disease, but the detection method of some routines lacks Weary enough sensitivity, causes testing result inaccurate.Clinical common immunoassays method detection The content of AFP-L3, utilizes antigen and antibody specific association reaction to detect, immune labeled skill Art 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 immunity The purpose that reaction is monitored.Conventional immunoassay labelling technique includes EIA enzyme immunoassay, radiation Immunoassay, luciferase immunoassay, colloid gold immune technology, electrochemiluminescent immunoassay technology etc..Wherein EIA enzyme immunoassay sensitivity is relatively low and operation is complicated；Radioimmunoassay, RIA is sensitive accurately, sample consumption Few, but detection effect duration is shorter and has radioactive pollution；The system of colloid gold immune technical mark thing Standby easy, sensitive intuitively, but the most impacted many factors；Electrochemiluminescent immunoassay technology for detection step is relatively Complexity, background is higher and unstable result.

In prior art, magnetic microparticle chemiluminescence immune assay technological synthesis magnetic particle carrier technique With chemiluminescence immunoassay detection technique, make measurement result more accurate, more stable.This technology includes:

Magnetic microparticle chemiluminescence--double antibody sandwich method: determined antigen with fluorescein-labeled antibody and Enzyme labelled antibody combines the complex forming " sandwich " structure.Be subsequently added be connected with anti-fluorescein resist The magnetic particle of body, makes antigen-antibody be combined by anti-fluorescein antibody with the specific binding of fluorescein Thing is connected on magnetic particle, Direct precipitation in externally-applied magnetic field, complex immunoreation formed Separate with other material unconjugated.Clean the complex of precipitation after removing supernatant, add enzymatic chemistry Luminous substrate.Substrate by catalytic pyrolysis, forms unstable excited state intermediate under enzyme effect, Just send photon when excited state intermediate returns to ground state, form luminescence-producing reaction, read by light quantum Read apparatus record photon energy, and pass through computer processing system by light energy intensity at standard curve On be converted to the concentration of determined antigen, and report result.

Magnetic microparticle chemiluminescence--competition law: by determined antigen, be coated the antibody of magnetic particle by excess Being simultaneously introduced reaction cup incubation with quantitative labelled antigen, its immunoreactive combining form has two Kind, one is that labelled antigen forms complex with antibodies；Two is determined antigen and antibodies shape Become complex.As in specimen to be measured containing determined antigen, then with labelled antigen with as chance with The coated antibodies of magnetic particle, accounted for labelled antigen and the coated antibody of magnetic particle competitively In conjunction with chance, make the binding capacity of labelled antigen and the coated antibody of magnetic particle reduce.Owing to magnetic is micro- The coated antibody of grain is excessive, it is sufficient to be combined with determined antigen.Magnetic particle is straight in externally-applied magnetic field Connect precipitation, the complex that immunoreation is formed is separated with other material unconjugated.After removing supernatant Clean the complex of precipitation, add enzyme-catalyzed chemical luminescence substrate.Substrate is split by catalysis under enzyme effect Solve, form unstable excited state intermediate, just send light when excited state intermediate returns to ground state Son, forms luminescence-producing reaction, by light quantum reading system record photon energy, and passes through computer Light energy intensity is converted to the concentration of determined antigen on standard curve by processing system, and reports knot Really.

Magnetic microparticle chemiluminescence indirect method: test antibodies is combined with fluorescein-labeled antigen, with Rear addition is coated the magnetic particle of anti-fluorescein antibody, by the spy of anti-fluorescein antibody Yu fluorescein Anisogamy antigen antibody complex is made to be connected on magnetic particle, Direct precipitation in externally-applied magnetic field, Clean the complex of precipitation after removing supernatant, add enzyme labelled antibody, form magnetic particle-Ag-Ab -ELIAS secondary antibody sandwich immunoassay complex.After again cleaning, add enzyme-catalyzed chemical luminescence substrate.The end Thing by catalytic pyrolysis, forms unstable excited state intermediate, in the middle of excited state under enzyme effect Just send photon when body returns to ground state, form luminescence-producing reaction, by light quantum reading system recording light Sub-energy, and light energy intensity is converted on standard curve to be measured by computer processing system The concentration of antibody, and report 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 AFP-L3 magnetic particle Immunofluorescence test side Method, uses antibody coupling specific binding for magnetic particle A with AFP-L3, uses fluorescein labelling This antibody removes the detection of excess as detection antibody, the magnetic particle B re-using coupling AFP-L3 Antibody, calculates the content of target detection thing AFP-L3, it is achieved right by fluorescence intensity The detection that the simplicity of AFP-L3 content is quick, sensitive and accurate.

Summary of the invention

The present invention provides a kind of and detects the method for AFP-L3 content in blood plasma or serum: described method includes Following steps:

Step 1, the preparation of the magnetic particle A of surface coupling anti-AFP-L3 antibody, and surface coupling AFP-L3 The preparation of the magnetic particle B of antigen；

Step 2, the preparation of fluorescently-labeled anti-AFP-L3 antibody；

Step 3, the detection of AFP-L3 content: blood plasma or blood serum sample and magnetic particle A reaction, reaction Complete by AFP-L3 eluting, add fluorescently-labeled anti-AFP-L3 antibody incubation, add magnetic Particles B continues to hatch, then Magnetic Isolation, detects sample fluorescence value with spectrofluorophotometer, Calculate AFP-L3 content in sample；

Wherein said AFP-L3 is alpha-fetoprotein variant (Alpha-fetoprotein-L3).

Method of the present invention, wherein said 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-AFP-L3 antibody or AFP-L3 antigen coupling: anti-AFP-L3 antibody or AFP-L3 antigen are mixed with magnetic particle suspension Coupling is hatched in conjunction, closes with confining liquid, obtain the magnetic particle A of coupling AFP-L3 antibody after coupling Magnetic particle B with coupling AFP-L3 antigen.

Method of the present invention, wherein, the described antibody specific binding with AFP-L3 is monoclonal Antibody or polyclonal antibody.

Method of the present invention, wherein, magnetic particle and the coupling condition of AFP-L3 antigen are selected from as follows 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, magnetic particle is selected from such as with the coupling condition of anti-AFP-L3 antibody Lower 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, mixes, so After on magnetic frame Magnetic Isolation, abandon supernatant；Repeat previous step, then by magnetic particle with The coupling buffer of its equivalent suspends, obtains magnetic particle suspension；

Step (2) adds volume 0.01～the step of 1 times in anti-AFP-L3 antibody or AFP-L3 antigen (1) the magnetic particle suspension processed, adds coupling buffer, hatches, until magnetic particle coupling Completely；Magnetic Isolation, retains magnetic particle；After magnetic particle coupling terminates, add confining liquid, mix, Hatch Magnetic Isolation, obtain magnetic particle A and the coupling AFP-L3 antigen of coupling AFP-L3 antibody Magnetic particle B；

The preparation of the fluorescently-labeled anti-AFP-L3 antibody of step (3): anti-AFP-L3 antibody is dissolved in carbon In phthalate buffer, fluorescein is dissolved in DMSO, is dropwise slowly added to fluorescein resist In AFP-L3 antibody-solutions, 4 DEG C of lucifuge stirrings 12～20h, remove trip with G-25 polydextran gel From fluorescein；

Step (4) takes testing sample, adds sample volume 1～the magnetic particle A of 100 times, revolves in room temperature Turn hatch 2～6h or 4 DEG C hatch 12～20h, make AFP-L3 antigen-antibody fully react；Put Magnetic Isolation on magnetic frame, abandons supernatant, cleans magnetic particle complex with lavation buffer solution, puts Magnetic Isolation on magnetic frame, abandons supernatant；Add the eluent with antibody equivalent, mixing, weight Outstanding magnetic particle, incubated at room 5min, it is placed in Magnetic Isolation on magnetic frame, retains supernatant；Add Enter the fluorescent labeling AFP-L3 antibody of sample volume 1～100 times, rotate in room temperature and hatch 2～6h Or hatch 12～20h for 4 DEG C；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, retains Supernatant, uses spectrofluorophotometer detection sample fluorescence value, and reference standard curve calculates sample AFP-L3 content in product.

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 washing buffer of 20 times Liquid, mixing, then Magnetic Isolation on magnetic frame, abandons supernatant；Repeat previous step, then Magnetic particle is being suspended in the coupling buffer of its equivalent, is obtaining magnetic particle suspension；

(6) magnetic particle suspension vol 0.2～the anti-AFP-L3 antibody needing coupling of 0.3 times are added Or AFP-L3 antigen is in the EP pipe filling magnetic particle magnetic particle suspension, and add magnetic particle The coupling buffer that suspension vol is 10 times, mixing；EP pipe is placed in room temperature on rotary mixer Hatch 2～6h or 4 DEG C hatch 12～20h, until magnetic particle coupling is complete；EP pipe is placed in Magnetic Isolation on magnetic frame, retains magnetic particle；Surface coupling anti-AFP-L3 antibody for magnetic particle A, surface coupling AFP-L3 antigen for magnetic particle B, supernatant is used for detecting coupling efficiency；

(7), after magnetic particle coupling terminates, the confining liquid of magnetic particle suspension vol 10 times is added, Mixing, EP pipe is placed 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；Repeat previous step once；

(8) take test plasma or blood serum sample, add sample volume 1～the magnetic particle A of 100 times, In room temperature rotate hatch 2～6h or 4 DEG C hatch 12～20h, make AFP-L3 antigen-antibody abundant Reaction；It is placed in Magnetic Isolation on magnetic frame, abandons supernatant, clean magnetic particle with lavation buffer solution multiple Compound, 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, in reservation Clear liquid；Add sample volume 1～the fluorescent labeling AFP-L3 antibody of 100 times, rotate in room temperature and incubate Educate 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 meter Calculate AFP-L3 content in sample.

Method of the present invention, wherein,

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；

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

Eluent is selected from: glycine, Tris Buffer, EDTA solution, IPTG solution, Pidolidone Solution, the combination of one or more of MES 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, concentration is 0.05%～5%, pH are 8.0～9.0；

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

The present invention also provides for a kind of test kit using the inventive method detection AFP-L3, including:

Magnetic particle, and optional following components: the magnetic particle A of coupling AFP-L3 antibody, coupling Magnetic particle B, the AFP-L3 antigen of AFP-L3 antigen, anti-AFP-L3 antibody, fluorescein, even Connection buffer, lavation buffer solution, eluent, confining liquid, preserve liquid, magnetic frame.

Preferably use the test kit of the inventive method detection AFP-L3, including:

The magnetic particle A of coupling AFP-L3 antibody,

The magnetic particle B of coupling AFP-L3 antigen,

And optional following components: AFP-L3 antigen, anti-AFP-L3 antibody, fluorescein, coupling is delayed Rush liquid, lavation buffer solution, eluent, 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 non-toxic, recyclable AFP-L3 magnetic particle immunofluorescent detection method, and Its detectable and detection kit.

The present inventor conducts in-depth research for the detection method of AFP-L3, found that: By magnetic particle separation fluorescence immunoassay technology, compared with conventional method, have easy to be quick, Highly sensitive, the advantages such as accuracy is high, safety non-toxic, recyclable, thus complete the present invention.

That is, the present invention provides the magnetic particle separating immune fluorescence analysis detection method of AFP-L3, should Detection method includes: using magnetic particle as solid phase carrier, coupling is specific binding with AFP-L3 Antibody as coated antibody, use the antibody work that fluorescein labelling is specific binding with AFP-L3 For detection antibody, the magnetic particle of coupling AFP-L3 is finally used to remove the detection antibody of excess, logical Cross the AFP-L3 in fluorescence intensity detection by quantitative sample.The present invention also provides for AFP-L3 magnetic particle and exempts from Epidemic disease luciferase assay reagent, this immunofluorescence detection agent comprises: magnetic particle, AFP-L3 antigen, Anti-AFP-L3 antibody, fluorescein, coupling buffer, lavation buffer solution, eluent, confining liquid, Preserve liquid.The present invention further provides AFP-L3 magnetic particle immunofluorescence detection agent box, this inspection Test agent box includes AFP-L3 magnetic particle immunofluorescence detection agent of the present invention.

Can provide easy to be quick according to the present invention, high sensitivity, high accuracy, safety non-toxic, can Reclaim AFP-L3 magnetic particle separating immune fluorescence analysis detection method and for this inspection The reagent surveyed and detection kit.The method of the present invention is compared with conventional method, and detection efficiency obtains To being greatly improved, as AFP-L3 detection method, it has easy to be quick, highly sensitive, accurate The advantages such as really property is high, safety non-toxic, recyclable.

In the detection method of the present invention, use magnetic particle coupling AFP-L3 and AFP-L3 respectively It is micro-that specific binding anti-AFP-L3 antibody and fluorescently-labeled anti-AFP-L3 antibody carry out magnetic Grain separating immune fluorescence analysis detection.By the method, can desirably simplify operating procedure, Reduce testing cost, improve the accuracy of detected value.

AFP-L3 antigen, i.e. AFP-L3 antigen protein standard substance, for commercial goods, can be according to often Rule method is bought and is obtained, and its characteristic is as follows: product design 1mg/ml, and purification process is HPLC, Purity >=98%.

Anti-AFP-L3 antibody is the antibody specific binding with AFP-L3, can be monoclonal anti Body, it is also possible to be polyclonal antibody.Angularly consider from the repeatability of immune detection, preferably make Use monoclonal antibody.It addition, these antibody can also keep the antibody of the associativity with corresponding antigen The form of fragment (antigen-binding fragment) uses.

The preparation method of polyclonal antibody, monoclonal antibody and antigen-binding fragment itself is known Conventional method, anti-AFP-L3 antibody or and antigen-binding fragment can conventionally prepare. It addition, these antibody there is also commercial goods, commercially available antibody therefore can also be used.

Anti-AFP-L3 monoclonal antibody such as can be obtained by known hybridoma: by AFP-L3 Or its Partial Fragment is mixed for immune animal (except people) as immunogen and suitable adjuvant, Gather the antibody-producting cell such as splenocyte or lymphocyte from this animal, it is melted with myeloma cell Close, prepare hybridoma, then select to produce the hybridoma of the antibody specific binding with AFP-L3, Make it breed, from culture supernatant, obtain anti-AFP-L3 monoclonal antibody.In order to make to be moved by immunity In thing, antibody titer raises, and immunity typically requires cost and carries out repeatedly several weeks.In the present invention, in order to Improve the specificity that AFP-L3 combines, preferably monoclonal antibody.

The polypeptide used as immunogen or its Partial Fragment can pass through chemosynthesis, genetic engineering Prepared by the conventional methods such as method, or extract AFP-L3 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, by routine Method is with reference to the polypeptide needed for the AFP-L3 sequence information synthesis in the data bases such as GenBank.Logical It is also known for cross genetic engineering's method preparing the method for polypeptide.Specifically, such as can pass through Prepared by method described below: first, by middle RNA that extracts such as the cultivation cells from people, logical Cross reverse transcription reaction and synthesized cDNA by mRNA.With this cDNA as template, according to GenBank Go forward side by side performing PCR Deng the design primer such as the AFP-L3 sequence information in data base, preparation coding The polynucleotide of AFP-L3.Or, the polynucleotide of coding AFP-L3 can be commercially available by using Prepared by the conventional method of nucleic acid synthesizer.It is known for encoding each amino acid whose codon, therefore only Wanting specific amino acid sequence, the base sequence of the polynucleotide encoding this aminoacid sequence also can be true Fixed.Then, the polynucleotide of preparation are imported suitable carrier, is made by suitable expression system Expression of polypeptides, reclaims this polypeptide, thus can obtain required immunogen polypeptide.The carrier used Or various expression system (bacterial expression system, yeast expression system, mammalian cell table Reach system, insect cell expression system, Cell free expression system etc.) also it is known, various loads Body or host cell, reagent class, detection kit are commercially available.

Immunoassay is known conventional method.As object lesson, competition albumen knot can be enumerated Close analyze, receptor binding assay, and radioimmunoassay, RIA, EIA enzyme immunoassay, fluorescence immunoassay divide Analysis, colloid gold immune analysis, CL and BL immune analysis method etc., in the present invention Any means can be used.Angularly consider, preferably from accuracy and the safety of immune detection Use fluoroimmunoassay.

Magnetic particle i.e. superparamagnetic nano particle, magnetic particle separating immune fluorescence analysis is by mesh Affinity groups such as antibody, the albumen etc. of mark molecule are coupled to magnetic nanoparticle surface, and formation can be equal The even scattered colloidal state with high stability is combined magnetic particle.When immunity magnetic particle detects with containing After the solution mixing of target molecule, form magnetic due to target molecule and the affine combination of its group micro- Grain-group-target molecule complex, then utilizes 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, goes through lavation buffer solution Unless specific binding impurity, then with eluent, target molecule is separated with magnetic particle, Jin Ershi The separation detection of existing target molecule, has low cost, less energy consumption, safety non-toxic, recyclable etc. excellent Point, 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 micro-by magnetic Functional group (amino, carboxyl, sulfydryl, epoxy radicals, the NHS group etc.) coupling on grain surface AFP-L3 and the anti-AFP-L3 antibody specific binding with AFP-L3, be used for as solid phase carrier Immune detection.Magnetic particle surface in the present invention can use any one functional group with AFP-L3 or anti-AFP-L3 antibody coupling.Suspension magnetic particle, as solid phase carrier, replaces and passes The immunology solid phase carrier of system is used for detecting, and has bigger specific surface area, it is possible to sufficient and sample Product react, in addition the flexible utilization of externally-applied magnetic field, have rapidly and efficiently, highly sensitive, repeated The advantage such as good.It addition, these magnetic particles there is also commercial goods, therefore can also use commercially available Magnetic particle or magnetic bead.

As a example by using the anti-AFP-L3 antibody of use as the situation of coated antibody, illustrate the present invention AFP-L3 detection method.First, anti-AFP-L3 antibody (coated antibody) is coupled to admittedly On phase carrier magnetic particle, after being closed by unnecessary group, enough magnetic particles are made fully to connect with testing sample Touch, the AFP-L3 specificity knot that thus the anti-AFP-L3 antibody on magnetic particle is contained with sample Close, then Magnetic Isolation, with suitable buffer solution magnetic particle complex, remove unconjugated Other compositions in sample, the most unnecessary carrier etc..Then eluting AFP-L3, uses excess Fluorescein-labeled anti-AFP-L3 antibody is combined with AFP-L3, hatches and makes fully to react.Reaction After end, with suitable method detection from the fluorescence signal of fluorescein label, thus can detect AFP-L3 content in sample.

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

Anti-AFP-L3 antibody that AFP-L3 with AFP-L3 is specific binding or its antigen-binding Fragment can be carried out by conventional method well known in the art, as concrete example with the combination of solid phase carrier Son, can enumerate covalent bond chemical coupling, non-covalent bond absorption or physical absorption etc., and the present invention is permissible Use any one mode to be attached to surface of solid phase carriers, but be not limited to these.

A kind of magnetic particle, it is characterised in that: described magnetic particle surface is coated with one layer and has antigen knowledge The not antibody of activity, or it is coated with one layer of antigen with antibody recognition activity.

Label is not particularly limited, it is possible to use with use in known immune detection system The material that label is same.Object lesson can be enumerated: enzyme, fluorescent material, chemiluminescent substance, Coloring matter, radioactive substance etc..In order to improve detection sensitivity, simplify operating procedure, reduce Radiocontamination, is preferably used fluorescein labelling.Fluorescent dye for labelling is also not particularly limited, Material as the label that can use and use in known Immunofluorescence test system, tool Style can be enumerated: Fluorescein isothiocyanate (fluoresceinisothiocyanate, FITC), and four Ethyl rhodamine (rhoda mine, RIB200), Tetramethylrhodamine isothiocyanate (tetramethylrhoda mineisothiocyanate, TRITC), lanthanide series (europium Eu₃, terbium Tb₃, cerium Ce₃Deng) chelate, phycoerythrin (phycoerythrin, PE) and other fluorescence Matter (beta galactosidase, alkaline acid enzyme, horseradish peroxidase) etc..The present invention can make With any one as fluorescent dye, but it is not limited to these.

When using biotin as label, it is possible to use combine enzyme, fluorescent material, chemistry The streptavidin of stimulative substance, coloring matter or radioactive substance etc. or hapten antibody etc. are examined Survey.

The detection of signal suitably can select according to the kind of label.Such as signal if colour developing, Then can use tintometer or extinction photometer, if fluorescence then can use fluorescence spectrophotometry Meter, if luminescence then can use photon, if lonizing radiation then can use radiation Line detector.For containing standard sample known to the concentration of AFP-L3 with various concentration, press Method according to the present invention detects AFP-L3, by the semaphore and standard sample of labelling The dependency relation drawing of the concentration of AFP-L3, draws standard curve, to the unknown of AFP-L3 concentration Sample carries out detection operation equally, detects the semaphore from labelling, detected value substitutes into this standard Curve, thus can be carried out quantitatively AFP-L3 in sample.

The sample that the method for the present invention is suitable for is the sample separated in subject, preferably blood Sample, particularly preferred blood plasma or serum.According to the detection method of the present invention, be no matter blood plasma or Serum, all can stably detect AFP-L3 content.As required can suitable dilute sample, with really Protect and detect in the range of working concentration.

Antibody described in this AFP-L3 magnetic particle immunofluorescent detection method is not to be coated onboard, and It is to be coated magnetic particle with antibody, coated magnetic particle is used as reagent, be more conducive to quantitatively behaviour Make, conveniently stablize and sensitivity is higher, detection interference factor can be dropped in antibody antigen reacts Minimum.

The present invention also provides for AFP-L3 magnetic particle immunofluorescence detection agent, and this reagent comprises: magnetic Microgranule, AFP-L3 antigen, anti-AFP-L3 antibody, fluorescein, coupling buffer, washing buffer Liquid, eluent, confining liquid, preserve liquid.

Above-mentioned AFP-L3 magnetic particle immunofluorescence detection agent can be appropriately combined, as AFP-L3 Magnetic particle immunofluorescence detection agent box provides.AFP-L3 magnetic particle immunofluorescence detection agent is also Can be appropriately combined with other reagent class etc., such as, in addition to above-mentioned detectable, the present invention's Detection kit 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 is known.

Sensitive quick AFP-L3 magnetic particle immunofluorescence detection agent box provided by the present invention, Provide a kind of easy to be quick for the detection of AFP-L3 and the early diagnosis of disease, high sensitivity, High accuracy, safety non-toxic, callable approach.Detection kit includes described AFP-L3 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 AFP-L3 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 AFP-L3 As a example by situation as target detection thing, the AFP-L3 magnetic particle immunity illustrating the present invention is glimmering Light detection method.The present invention is not limited by these embodiments etc..Those skilled in the art can be by Content disclosed by this specification understands other advantages and effect of the present invention easily.The present invention is also Can be carried out by the most different detailed description of the invention or apply, every in this specification Details can also carry out various based on different viewpoints and application under the spirit without departing from the present invention Modify or change.

In the present invention, when numerical ranges are given, it should be appreciated that unless the present invention is otherwise noted, Between two end points and two end points of each numerical range, any one numerical value all can be selected for.Remove Non-other definition, all technology used in the present invention and scientific terminology and the art technology people The same meaning that member is generally understood that.In addition to the concrete grammar used in embodiment, 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 The prior art that method described in use and the embodiment of the present invention, equipment, material are similar or equivalent Any method, equipment and material realize the present invention.

The preparation of embodiment 1.AFP-L3 magnetic particle separating immune fluorescence analysis detectable

1, the preparation of AFP-L3 antigen

(1) structure of pCold II-AFP-L3 expression vector

Design primer with reference to the gene order of people AFP-L3 in GenBank and synthesize AFP-L3 base Cause, after PCR amplification, 1% agarose gel electrophoresis detection AFP-L3 amplified production, cuts purpose Band, uses glue to reclaim test kit and reclaims purpose fragment.It is used restricted enzyme Nde I He The prokaryotic expression carrier pCold II of Hind III double digestion connects, and converts to E. coli. DH5 α expands, then proceeds to E.coli.BL21 (DE3) competent cell, be inoculated in LB solid In culture medium, containing ampicillin (Amp) 100mg/L, extract plasmid, carry out Nde I and Hind III enzyme action is identified and order-checking.Recombiant plasmid pCold II-AFP-L3 is through 5'Nde I and 3'Hind III Carry out double digestion, two specific bands, position and pCold II and the size of purpose fragment occur Unanimously.Sequencing result display target sequence is consistent with NCBI corresponding sequence, it was demonstrated that expression vector PCold II-AFP-L3 successfully constructs.

(2) expression of AFP-L3 fusion protein and purification

The picking colony inoculation containing recombiant plasmid pCold II-AFP-L3-BL21 is trained in 10mL LB Supporting in base (Amp, 100mg/L), 220r/min 37 DEG C shakes bacterium 12h, in the ratio of 1:100 It is inoculated in the LB culture medium (Amp, 100mg/L) of 1000mL, cultivates under the same terms 3h, treats that bacterium solution reaches A₆₀₀Time add IPTG to final concentration of 0.5mmol/L, 15 DEG C of shaking trainings Support 16h, after inducing culture terminates, collection thalline to 50mL sterile centrifugation tube, 5000r/min 4 DEG C Centrifugal 10min, abandons supernatant, is resuspended in 20mL lysis buffer (20mmol/L Tris-HCl Comprise 1mmol/L protease inhibitor cocktail, pH 8.0) in, break through 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, mistake Ni-NTA chromatographic column is purified；The column volume lysis buffer of 10 times is (containing 8mol/L carbamide+20 Mmol/L imidazoles) washing；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 the albumen of eluting with containing finite concentration gradient carbamide (6,5,4,2,1mol/L) carry out Renaturation, finally with PBS, detecting lyophilizing protein concentrate concentration after having dialysed is 1.5 mg/mL。

2, the preparation of AFP-L3 antibody

(1) preparation of hybridoma cell strain

By S p2/0 myeloma cell good for growth conditions with immune mouse spleen cell with 1:10's Ratio mixes, and adds 50% Polyethylene Glycol and merges, and fusion process is carried out according to a conventional method.With The supernatant having fused cell hole, as detection antigen, is carried out indirectly by AFP-L3 expressing protein ELISA detects, and one resists for cells and supernatant, and two resist the goat for horseradish peroxidase-labeled Anti-mouse IgG (1:2000 dilution), the screening positive cell clone strain of TMB color developing detection.Will sieve The positive cell clone strain limiting dilution assay of choosing carries out monoclonal screening, and indirect elisa method is examined Surveying, until positive rate reaches 100%, the hybridoma filtering out stably excreting anti-AFP-L3 antibody is thin Born of the same parents' strain is enlarged cultivating, and frozen in liquid nitrogen.

(2) preparation of monoclonal antibody

Select BALb/c mice, every lumbar injection sterilized liquid paraffin 0.5mL, warp after 1 week Intraperitoneal inoculation hybridoma 0.5mL (1 × 10⁶Cell/only).After 10～14d, mouse web portion is bright Ascites is collected in aobvious swelling, and 12000r/min is centrifuged 10min, removes the fat on upper strata, liquid paraffin And precipitation, draw faint yellow ascites, ascites is obtained pure through Protein A agarose affinity chromatography post Change antibody, carry out in PBS afterwards 4 DEG C dialysis 12h, the next day use BCA method detection antibody Concentration, ELISA method detection antibody titer, it is subsequently adding 50% glycerol mixing, after subpackage-80 DEG C in a small amount Save backup.

AFP-L3 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 Entering 0.5～1.0mL lavation buffer solution, lavation buffer solution is: pH is the 20mM phosphorus of 7.2～7.6 Acid sodium, 150mM sodium chloride, mixing, then Magnetic Isolation on magnetic frame, abandons supernatant. Add 1mL lavation buffer solution suspension magnetic particle, mixing, Magnetic Isolation on magnetic frame, abandon Clear liquid.Repeat previous step, then magnetic particle is suspended in 50 μ L coupling buffers, coupling Buffer is: pH is 25mM～the 50mM ammonium hydrogen carbonate of 8.0～8.2, stand-by.

(2) coupling of targeted biological specimen

During by immune with preparation to targeted biological specimen and magnetic particle coupling magnetic particle, monoclonal used The concentration of antibody or antigen coated liquid is to affect the direct factor of late detection, and its concentration is direct Affect accuracy and the range of linearity of testing result.If the concentration being coated liquid used by is too low, in the later stage Immunoreation in the reaction efficiency of magnetic particle poor, antigen antibody reaction is incomplete, makes 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 use with Lower method carries out condition optimizing screening:

Taking 10～15 μ L needs the AFP-L3 antibody of coupling or the antigen in filling 2mL particle diameter to be In the EP pipe of 100～1000nm magnetic particles, and adding 500 μ L coupling buffers, coupling buffers Liquid is: pH is 25mM～the 50mM ammonium hydrogen carbonate of 8.0～8.2, mixing.EP pipe is placed in rotation Turn and hatch on mixed instrument.Then EP pipe is placed in Magnetic Isolation on magnetic frame, retain magnetic particle and Supernatant, the protein content of anti-AFP-L3 antibody standard solution before and after employing BCA method detection coupling. Described magnetic particle is preferably with anti-AFP-L3 antibody coupling condition:

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

Described magnetic particle is preferably with anti-AFP-L3 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%, After mixing, EP pipe is placed on rotary mixer incubated at room 2～6h or 4 DEG C hatch 12～20h, Then EP pipe is placed in Magnetic Isolation on magnetic frame, abandons supernatant.Repeat previous step once.

(4) preservation of magnetic particle

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

2, magnetic particle and the detection of anti-AFP-L3 antibody coupling efficiency

After anti-AFP-L3 antibody and magnetic particle coupling, anti-before and after using the detection coupling of BCA method The protein content of AFP-L3 antibody standard solution, calculates magnetic particle coupling efficiency.Result shows, After adding magnetic particle, solution protein concentration significantly reduces, and the group on magnetic particle surface can be with anti- AFP-L3 antibody generation coupling so that magnetic particle has biological activity, i.e. have capture antibody or The ability of antigen becomes immunity magnetic particle.Magnetic particle with anti-AFP-L3 antibody coupling efficiency is 78.43% ± 4.25.

3, the drafting of AFP-L3 standard curve

Take the anti-AFP-L3 antibody 100 μ L of FITC labelling in EP pipe, be separately added into AFP-L3 The each 100 μ L of standard substance, with 0.01mol/L PBS be diluted to 7 Concentraton gradient (0ng/mL, 1.25ng/mL, 2.50ng/mL, 5.00ng/mL, 10.00ng/mL, 20.00ng/mL, 40.00 Ng/mL), in room temperature rotate hatch 2～6h or 4 DEG C hatch more than 12h, make AFP-L3 Antigen-antibody fully reacts.Return to zero by blank well, use spectrofluorophotometer examination criteria product glimmering Light value, draws standard curve according to AFP-L3 standard concentration and fluorescent value thereof, sees Fig. 1.

4, the detection of AFP-L3 content

Concrete operation step is as follows:

(1) take 10 parts of human serum sample 200 μ L, add the magnetic of 2mL coupling AFP-L3 antibody Particles A, in room temperature rotate hatch 2～6h or 4 DEG C hatch 12～20h, make AFP-L3 antigen Antibody fully reacts.

(2) it is placed in Magnetic Isolation on magnetic frame, abandons supernatant, clean magnetic particle with lavation buffer solution Complex, lavation buffer solution is: pH is the 20mM sodium phosphate of 7.2～7.6,150mM chlorination Sodium, 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, mixed Even, resuspended magnetic particle, incubated at room 5min, it is placed in Magnetic Isolation on magnetic frame, retains supernatant Liquid.

(4) add the anti-AFP-L3 antibody of 2mL FITC labelling, rotate in room temperature and hatch 2～6h Or hatch 12～20h for 4 DEG C.Add the magnetic particle of 20mL coupling AFP-L3 antigen, in room temperature Rotation hatch 2～6h or 4 DEG C hatch 12～20h.

(5) it is placed in Magnetic Isolation on magnetic frame, retains supernatant, use spectrofluorophotometer inspection Survey sample fluorescence value, and reference standard curve calculates AFP-L3 content in sample.

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

Immunity magnetic particle has higher surface quality ratio, can participate in conjunction with more antibody or antigen Immunoreation；

Use two set magnetic particle coupled antibody and antigens respectively, compared with conventional double antibody sandwich method, this Method has only to the anti-AFP-L3 antibody of a kind of correspondence can complete reaction, and cost is relatively low；

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

5, the stability of AFP-L3 detection method compares

Above-mentioned testing result being compareed with ELISA method, result shows that the two has good phase Guan Xing: the range of linearity using magnetic particle separating immune fluorimetry to detect AFP-L3 is 0.1～500.0ng/mL, detection is limited to 0.1ng/mL, and the equation of linear regression of standard curve is y =-1.6679+1.0742x, R²=0.9981, (wherein x is AFP-L3 concentration, and y is absorbance Value), its Monitoring lower-cut is lower 50 times than ELISA method.Blood serum sample AFP-L3 content detection is tied Fruit 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 AFP-L3 antibody or antigen, eluent is: PH is the 0.1M glycine of 2.5, and fully reaction is placed on Magnetic Isolation on magnetic frame, uses immediately Lavation buffer solution cleans magnetic particle, and lavation buffer solution is: pH is the 20mM phosphoric acid of 7.2～7.6 Sodium, 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, mixing, save backup in 4 DEG C.By anti-AFP-L3 antibody with After the magnetic particle coupling reclaimed, before and after using the detection coupling of BCA method, anti-AFP-L3 antibody standard is molten The protein content of liquid, calculating the magnetic particle response rate is 79.53% ± 9.25.

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 that operator use the consumptive materials such as DCP magnetic particle immunofluorescence detection agent, magnetic frame And instrument, have been manually done whole testing process.The magnetic frame structure comparison letter manually used in sorting Single, mainly it is made up of the permanent magnet of support and generation magnetic field, plays support test tube and additional magnetic is provided The effect of field.The hatching of magnetic particle, external magnetic field add with remove, the cleaning of adsorbate with wash Each committed step such as de-is all completed by manual operation.Manually sorting need not the equipment of complexity, form Flexibly, cost relatively low, be suitable for test use on a small quantity.

When needs frequently carry out operating and sample size is bigger, use that to be automatically separated detection method the most square The most efficient.Automatic separation process mainly utilizes full-automatic magnetic particle sorter, and operator will wait to sort Sample joins in sorter, sorter be automatically performed separation process.Full-automatic magnetic sorter Sorting flux relatively big, and typically have the process of separation of multiple optimization to be available for calling, simple to operate, Sorting reliability is high.The most representative commercialization Full-automatic magnetic sorter has U.S. sky Ni (MACS), BD, R&D, StemCell RoboSep and Dynal Bead Retriever etc. Brand.

The detection method of magnetic particle separating immune fluorescence analysis DCP content of the present invention, can be used for complete Active immunity magnetic bead sorting 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, anti-DCP antibody, coupling buffer, the lavation buffer solution of FITC labelling, wash De-liquid, confining liquid, preservation liquid；

(2) cell sorting policy selection " positive sorting strategy ", mark mode selects " directly Labelling "；

(3) take 25 μ L DCP standard substance in S1-7 pipe, in 2 blank tubes, add 1 ML lavation buffer solution, is separately added into 25 μ L magnetic particle A and 50 μ L fluorescence marks in centrifuge tube Remember anti-DCP antibody, response procedures condition, upper machine testing are set.By testing result and manually inspection Survey method compares, and result shows that the two has good dependency.

The composition of embodiment 2.AFP-L3 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 AFP-L3, by using this test kit, makes simple to operate, time saving and energy saving, it is to avoid existing Loaded down with trivial details with now join, 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: coupling AFP-L3 The magnetic particle A of antibody, magnetic particle B, the AFP-L3 antigen of coupling AFP-L3 antigen, anti-AFP-L3 Antibody, fluorescein, coupling buffer, lavation buffer solution, eluent, confining liquid, preserve liquid, Magnetic frame.

The another kind of test kit of the present invention, including: the magnetic particle A of coupling AFP-L3 antibody, even The magnetic particle B of connection AFP-L3 antigen, and optional following components: AFP-L3 antigen, anti- AFP-L3 antibody, fluorescein, coupling buffer, lavation buffer solution, eluent, confining liquid, Preserve liquid, magnetic frame.

Wherein said optionally for any of which component can not be selected, it is also possible to selection 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 bag In mounted box, operate according to the method described in description during use.

In test kit, coupling buffer is selected from: bicarbonate buffer, carbonate buffer solution, sulfur The combination of one or more of phthalate buffer, 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, dense Degree is 2～200mM, and pH is 7.0～8.0；

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

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

The detection kit of the present invention, including: the reagent in box body, box body, described reagent is AFP-L3 magnetic particle immunofluorescence detection agent, described tray interior is provided with some reagent troughs, institute Stating and place the EP pipe filling magnetic particle in reagent trough, the amount of seminal plasma fructose detection kit can be a person-portion, Can also be many person-portions.

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

Use two set magnetic particle coupled antibody and antigens respectively, compared with conventional double antibody sandwich method, this Method has only to the anti-immunological marker thing antibody of a kind of correspondence can complete reaction, and cost is relatively low；

In sum, AFP-L3 magnetic particle immunofluorescence detection agent box provided by the present invention tool Have good accuracy and specificity and highly sensitive, effectively overcome 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 this Invention.Any person skilled in the art all can be right under the spirit and the scope of the present invention Above-described embodiment is modified or changes.Therefore, such as art has usual knowledge All equivalences that person is completed under without departing from disclosed spirit and technological thought are modified Or change, must be contained by the claim of the present invention.

Claims

1. one kind is detected the method for AFP-L3 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-AFP-L3 antibody, and the preparation of the magnetic particle B of surface coupling AFP-L3 antigen；

Step 2, the preparation of fluorescently-labeled anti-AFP-L3 antibody；

Step 3, the detection of AFP-L3 content: blood plasma or blood serum sample and magnetic particle A reaction, react complete by AFP-L3 eluting, add fluorescently-labeled anti-AFP-L3 antibody incubation, add magnetic particle B to continue to hatch, then Magnetic Isolation, detects sample fluorescence value with spectrofluorophotometer, calculates AFP-L3 content in sample.

Method the most according to claim 1, it is characterized 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-AFP-L3 antibody or AFP-L3 antigen coupling: anti-AFP-L3 antibody or AFP-L3 antigen are mixed with magnetic particle suspension and hatches coupling, close with confining liquid after coupling, obtain the magnetic particle A and the magnetic particle B of coupling AFP-L3 antigen of coupling AFP-L3 antibody.

Method the most according to claim 1, it is characterised in that wherein, the described antibody specific binding with AFP-L3 is monoclonal antibody or polyclonal antibody.

The coupling condition of method the most according to claim 1, it is characterised in that wherein, magnetic particle and AFP-L3 antigen is selected from following condition:

The coupling condition of method the most according to claim 1, it is characterised in that wherein, magnetic particle and anti-AFP-L3 antibody is selected from 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, and mixing, then Magnetic Isolation on magnetic frame, 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 suspension that the step (1) of volume 0.01～1 times processes in anti-AFP-L3 antibody or AFP-L3 antigen, adds coupling buffer, hatches, until magnetic particle coupling is complete；Magnetic Isolation, retains magnetic particle；After magnetic particle coupling terminates, add confining liquid mixing, hatch, Magnetic Isolation, obtain the magnetic particle A and the magnetic particle B of coupling AFP-L3 antigen of coupling AFP-L3 antibody；

The preparation of the fluorescently-labeled anti-AFP-L3 antibody of step (3): anti-AFP-L3 antibody is dissolved in carbonate buffer solution, fluorescein is dissolved in DMSO, fluorescein is dropwise slowly added in anti-AFP-L3 antibody-solutions, 4 DEG C of lucifuge stirrings 12～20h, remove free fluorescein with G-25 polydextran gel；

Step (4) takes testing sample, adds sample volume 1～the magnetic particle A of 100 times, in room temperature rotate hatch 2～6h or 4 DEG C hatch 12～20h, make AFP-L3 antigen-antibody fully react；It is placed in Magnetic Isolation on magnetic frame, abandons supernatant, clean magnetic particle complex with lavation buffer solution, be placed in Magnetic Isolation on magnetic frame, abandon supernatant；Add the eluent with antibody equivalent, mixing, resuspended magnetic particle, incubated at room 5min, be placed in Magnetic Isolation on magnetic frame, retain supernatant；Add sample volume 1～the fluorescent labeling AFP-L3 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, retains supernatant, use spectrofluorophotometer detection sample fluorescence value, and reference standard curve calculates AFP-L3 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 Magnetic Isolation on magnetic frame, abandons supernatant；Repeat previous step, then magnetic particle is being suspended in the coupling buffer of its equivalent, obtaining magnetic particle suspension；

(2) addition magnetic particle suspension vol 0.2～the anti-AFP-L3 antibody needing coupling of 0.3 times or AFP-L3 antigen are in the EP pipe filling magnetic particle suspension, and add the coupling buffer of magnetic particle suspension vol 10 times, mixing；EP pipe is placed 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 Isolation on magnetic frame, retains magnetic particle；Surface coupling anti-AFP-L3 antibody for magnetic particle A, surface coupling AFP-L3 antigen for magnetic particle B, supernatant 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 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；Repeat previous step once；

(4) take test plasma or blood serum sample, add sample volume 1～the magnetic particle A of 100 times, in room temperature rotate hatch 2～6h or 4 DEG C hatch 12～20h, make AFP-L3 antigen-antibody fully react；It is placed in Magnetic Isolation on magnetic frame, abandons supernatant, clean magnetic particle complex with lavation buffer solution, be placed in Magnetic Isolation on magnetic frame, abandon supernatant；Add the eluent with antibody equivalent, mixing, resuspended magnetic particle, incubated at room 5min, be placed in Magnetic Isolation on magnetic frame, retain supernatant；Add sample volume 1～the fluorescent labeling AFP-L3 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, retains supernatant, use spectrofluorophotometer detection sample fluorescence value, and reference standard curve calculates AFP-L3 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 combination of one or more of sulfate buffer, and 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, and concentration is 2～200mM, and pH is 7.0～8.0；

Eluent is selected from: glycine, Tris Buffer, EDTA solution, IPTG solution, Pidolidone solution, the combination of one or more of MES Buffer, and 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, and pH is 8～10.

9. use a test kit of claim 1 method detection AFP-L3, including:

Magnetic particle, and optional following components: the magnetic particle A of coupling AFP-L3 antibody, magnetic particle B, the AFP-L3 antigen of coupling AFP-L3 antigen, anti-AFP-L3 antibody, fluorescein, coupling buffer, lavation buffer solution, eluent, confining liquid, preserve liquid, magnetic frame.

10. use a test kit of claim 1 method detection AFP-L3, including:

The magnetic particle A of coupling AFP-L3 antibody,

The magnetic particle B of coupling AFP-L3 antigen,

And optional following components: AFP-L3 antigen, anti-AFP-L3 antibody, fluorescein, coupling buffer, lavation buffer solution, eluent, confining liquid, preserve liquid, magnetic frame.