CN108333343A - Immune magnetic compound, preparation method including its antigen capture agent, kit, system and application - Google Patents

Abstract

The invention discloses a kind of immune magnetic compound, preparation method including its antigen capture agent, kit, system and applications.Wherein, which includes：Using the magnetic microsphere for the amino functional that amino polymer modified magnetic microballoon obtains；Agglutinin, agglutinin are connect with amino polymer.It applies the technical scheme of the present invention, agglutinin is mediated indeed through the amino polymer for being coated on magnetic microsphere surface and is connected on magnetic microsphere, amino polymer coating magnetic microsphere makes the upper a large amount of amino groups of magnetic ball surface modification, the outer layer copolymer has dendrimer networks shape structure, coupling carrying capacity can fully be improved, steric hindrance is reduced simultaneously, and makes rigid surface of the target conjugate agglutinin far from magnetic microsphere, farthest ensure that the bioactivity of target conjugate.

Description

Immune magnetic compound, preparation method including its antigen capture agent, kit, System and application

Technical field

The present invention relates to biomedicine technical fields, in particular to a kind of immune magnetic compound, its preparation side Method including its antigen capture agent, kit, system and application.

Background technology

Primary carcinoma of liver (primary hepatic carcinoma, PHC) is a kind of common malignant tumour, case fatality rate Third position is occupied in all tumours, is only second to lung cancer and gastric cancer, and the number that China dies of liver cancer every year accounts for world's death toll 45%.In recent years, generally believe PHC be multifactor, multipath, multi-step long term as a result, including external environment Carcinogenic factor (parasite, bacterium, the infection of virus, the intake of aflatoxin, pollution of waterhead and smoking are drunk) and itself Inherent cause.Due to PHC incidence of occult, progress is rapid, treatment is difficult, lethality is high, particularly heavy to the early diagnosis of PHC It wants.

Currently, being mainly alpha-fetoprotein to the common blood serum designated object of the diagnosis of PHC.Alpha-fetoprotein (AFP) is a kind of point The single chain glycoprotein of son amount about 69Kda has been widely used for the generaI investigation of PHC, diagnosis, judges therapeutic effect and prediction recurrence. But detection AFP has that specific deficiency is low with sensitivity to diagnose PHC, can cause false positive and false negative.Some The AFP of the malignant tumour of benign hepatopathy, digestion and reproductive system can also be increased, and some liver cancer AFP may but keep low water It is flat.Early in the seventies, researcher has found that there are different migrations by AFP when carrying out the analysis of agglutinin affinity electrophoresis to AFP Rate, electrophoresis are divided into three bands, are named as AFP-L1, AFP-L2 and AFP-L3 successively, and wherein AFP-L1 comes from benign hepatopathy, AFP- L2 comes from pregnant woman, and AFP-L3 is originated from secretion of hepatoma.Its is quick in PHC Diagnosis and differential diaggnosis for AFP heteroplasmons (AFP-L3) Perception is superior to AFP with specificity, by as liver cancer marker of new generation.U.S. FDA was ratified the index in 2005 and is applied to Liver cancer early warning, and the positive of AFP-L3/AFP diagnosing liver cancers is defined value and is set to 10%.It is than simple that AFP-L3/AFP, which is recognized, The more special PHC indexs of AFP-L3.

Clinically detection AFP-L3 be typically it is different from the binding ability of phytolectin according to different types of AFP and into Row detaches, and then the method for applied immunology carries out quantitative detection.Common method for separating and detecting mainly has affine immunological cross Electrophoretic techniques, affine blotting, LiBASys automatic analysis systems detection method, " two-site sandwich " enzyme linked immunosorbent assay, miniflow Control chip prize law, affinity chromatography and magnetic microsphere prize law etc..Wherein, affine immunological cross electrophoretic techniques was once most often With one of method, colouring method has Coomassie Brilliant Blue, autoradiography, silver Staining Method, enzyme linked immunosorbent assay, the technical method Simply, but have the shortcomings that complicated for operation, disturbing factor is more, poor sensitivity, only have a small amount of application in laboratory at present；Affine print Mark method have many advantages, such as it is reproducible, take it is short, but due to it is complicated for operation, take time and effort, limit its application clinically； LiBASys automatic analysis system detection methods have convenient, fast, simple operation and other advantages, but since its equipment is expensive, sensitivity It is low, also limit the application of the technology clinically；Micro-fluidic chip prize law high sensitivity, but not yet wide popularization and application； Principle of the affinity chromatography based on sugar capture is given birth to by the hot scape in Beijing because method is simple, is not necessarily to special installation, reproducible Object Technology Co., Ltd. develops into microcentrifugation column extracts kit, is that current domestic unique acquisition Bureau of Drugs Supervision approval is suitable for The method that AFP-L3 is isolated and purified, has at home and abroad started extensive use.

In recent years, the extensive use with magnetic microsphere isolation technics in immunodiagnosis field, research staff start to endeavour In the separation for usually carrying out AFP-L3 using magnetic microsphere coupling plant lectin, existing Full-automatic chemiluminescence is recycled to detect The kit of total AFP detects isolated APF-L3, improves the automation journey of AFP-L3 separation detections to a certain extent Degree and detection efficiency.However magnetic microsphere and phytolectin are coupled by this method using carbodlimide method by amido bond Come so that phytolectin is directly in direct contact with the rigid interface of magnetic microsphere, also solidifying to plant while Conjugate ratio is relatively low The activated centre of collection element forms steric hindrance, reduces the efficiency of phytolectin capture AFP-L3.

The technical solution of the separation about alpha-fetoprotein variant in the prior art is exemplified below.

Example 1 selects from patent CN104714026A：A kind of separation detection composition of alpha-fetoprotein variant, system and its Using being related to a kind of separation detection composition of alpha-fetoprotein variant, including separation agent and detection reagent and first tire The separation detecting system of albumen heteroplasmon and application have also related to a kind of separation detection reagent of alpha-fetoprotein variant Box, separation detection composition, system and its application of a kind of alpha-fetoprotein variant provided through the invention, can prompt morning Phase primary carcinoma of liver has high sensitivity, and method is fast and convenient and automates.Wherein, separation agent includes that coupling is solidifying The magnetic-particle and eluent for collecting element, the magnetic-particle for being coupled agglutinin are used for and the AFP-L3 specificity knots in detected sample It closes；Detection reagent includes the magnetic-particle for being coated with alpha-fetoprotein antibody, the anti-alpha-fetoprotein antibody of marker enzyme.

Example 2 selects from patent CN102879567B：Alpha-fetoprotein variant separating kit and its group for diagnosing cancer of liver At reagent and application, it is related to a kind of alpha-fetoprotein variant separating kit for diagnosing cancer of liver and its composition reagent and answers With.In a preferred embodiment, protection liquid that is uniform, stablizing successfully is obtained, and by the optimization to kit, is obtained With stable, high-bond and special agglutinin coating magnetic bead, agglutinin-magnetic bead solution that is uniform and stablizing, most Good reaction solution and eluent.Wherein, magnetic particle is that phytolectin passes through 1- (3- dimethylaminos third with active magnetic bead Base) -3- ethyl carbodiimide method covalent couplings and obtain, phytolectin is LcA, is coated with used in active magnetic bead The feed concentrations of lens lectin are every milligram of magnetic bead 70ug；Kit further includes protection liquid, and protection liquid refers to: By mass volume ratio dissolved with following component in PH7.510mM PBS or pH5.0l00mM citrate buffer solutions:3%BSA, 0.5% Solution obtained by gelatin, 5% casein, 0.03%NaN3,0.05%Tween-20.

The prior art that premise arrives therewith is the same, and there are still following disadvantages in above-mentioned patent：1) agglutinin is in crosslinking agent Effect is lower to be directly coupled at magnetic microsphere surface by amido bond, and the rigid interface of agglutinin and magnetic ball is in direct contact, may Cause activity of lectin to reduce, can not ensure higher Conjugate ratio, be easy to cause certain space hyte, influence subsequent capture Detach the efficiency of reaction；2) it after by magnetic microsphere capture separation, needs to be eluted the AFP-L3 of capture with eluent, step is numerous Trivial, the factor for being easily introduced manual operation error interferes testing result；3) the auxiliary inspection of additional detection kit is needed It surveys, it is difficult to realize automation.

Invention content

The present invention is intended to provide a kind of immune magnetic compound, preparation method including its antigen capture agent, reagent Box, system and application, to solve, in the prior art since agglutinin is directly coupled at magnetic microsphere surface, to lead to agglutinin and magnetic The technical issues of rigid interface of ball is in direct contact, reduces activity of lectin.

To achieve the goals above, according to an aspect of the invention, there is provided a kind of immune magnetic compound.This is immune Magnetic composite includes：The amino functional obtained using amino polymer modified magnetic microballoon；Agglutinin, agglutinin and ammonia Based polyalcohol connects.

Further, agglutinin is oxidation agglutinin；Preferably, oxidation agglutinin is connect with amino on amino polymer； Preferably, agglutinin is the oxidation agglutinin that hydroxyl is oxidized to aldehyde radical, and oxidation agglutinin passes through ammonia on aldehyde radical and amino polymer Base connects.

Further, amino polymer be polyethyleneimine, polyamide, polyacrylamide or poly-o-phenylenediamine, preferably Polyethyleneimine.

Further, immune magnetic compound is the immune magnetic compound for capturing separation AFP-L3, it is preferred that solidifying Integrate element as lentil lectin, small Lens culinaris agglutinin or ConA, more preferably lentil lectin.

Further, magnetic microsphere includes magnetic ball ontology, and magnetic ball ontology carries one or more work by surface modification Sexual function group；Preferably, active group includes at least one of carboxyl, hydroxyl and amino；Preferably, magnetic ball ontology is Fe₂O₃The complex of magnetic nano-particle and high-molecular organic material, or be Fe₃O₄Magnetic nano-particle and organic polymer The complex of material；Preferably, magnetic ball ontology grain size is 0.1 μm~5 μm；Preferably, amino polymer by with active group The magnetic microsphere of amino functional is obtained by the reaction.

According to another aspect of the present invention, a kind of preparation method of any of the above-described kind of immune magnetic compound is provided.It should Preparation method includes the following steps：1) amino polymer modified magnetic microballoon is used, the magnetic microsphere of amino functional is obtained；2) It connect agglutinin to form immune magnetic compound with amino polymer.

Further, before agglutinin is connect with amino polymer, further include the steps that being aoxidized to agglutinin, obtain To oxidation agglutinin；Preferably, oxidation agglutinin is connect with amino on amino polymer；Preferably, by the hydroxyl in agglutinin It is oxidized to aldehyde radical, obtains oxidation agglutinin, oxidation agglutinin is connect by aldehyde radical with amino on amino polymer.

Further, the oxidant used in oxidation step is periodate, perchlorate, and permanganate is preferably high Sodium iodate, sodium perchlorate or potassium permanganate, more preferably sodium metaperiodate；Preferably, agglutinin and oxidant in oxidation step Mass concentration ratio is 1:50-1:500；Preferably, the reaction temperature of oxidation is 20-30 DEG C, and the reaction time is 1-4 hours；It is preferred that , the concentration range of agglutinin is 1mg/mL-5mg/mL.

Further, step 1) specifically includes：11) magnetic microsphere is activated using activator；It 12) will be activated Magnetic microsphere is added to amino polymer aqueous solution and is reacted；13) isolating surface modification has the magnetism of amino polymer micro- Ball is reacted with sealer, obtains the magnetic microsphere of amino functional；Preferably, activator be dicyclohexylcarbodiimide, At least one of 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and N, N'- diisopropylcarbodiimide；It is excellent Choosing, the mass concentration of magnetic microsphere and activator is ranging from：2:1–4:1；Preferably, the reaction temperature of activation is 37 DEG C -40 DEG C, the reaction time is 1h -4h；Preferably, step 12) specifically includes：Activated magnetic microsphere is added to amino polymer Aqueous solution after supersound process, carries out rotation mixing reaction；Preferably, the concentration of amino polymer Ammonia In Aqueous Solution based polyalcohol For 10mg/mL-200mg/mL；Preferably, when activated magnetic microsphere is added to amino polymer aqueous solution and is reacted, magnetic A concentration of 10mg/mL-20mg/mL of property microballoon；Preferably, sealer be ethanol amine, BSA or gelatin, sealer it is a concentration of 0.5-1mol/L。

Further, step 2) specifically includes：The magnetic microsphere of amino functional is resuspended with re-suspension liquid, oxygen is then added Change agglutinin, reducing agent is added after mixing and is stirred to react, immune magnetic compound is obtained after going supernatant to wash；Preferably, it is resuspended Liquid is sodium bicarbonate solution, Tris-HCl buffer solutions or borate buffer solution, a concentration of 0.05mol/L-0.2mol/ of re-suspension liquid L, pH 8-10；Preferably, reducing agent is sodium borohydride, potassium borohydride or Lithium Aluminium Hydride, a concentration of 1mg/ of the reducing agent ML-5mg/mL, more preferably 5mg/mL, it is preferred that the mass concentration ratio of the magnetic microsphere of oxidation agglutinin and amino functional Example is 50:1-1:100；Preferably, the reaction temperature of step 2) is 20 DEG C -30 DEG C, reaction time 2h-6h.

According to a further aspect of the invention, a kind of activity preservative fluid for above-mentioned immune magnetic compound is provided.It should Activity preservative fluid includes：Dilution component, metal-binding sites component, metal-chelator component and biological preservative component；It is excellent Choosing, dilution group is divided into phosphate buffer, borate buffer solution, Tris-HCl buffer solutions or carbonate buffer solution, dilution A concentration of 0.01-0.2mol/L of liquid, pH 7-9；Preferably, metal-binding sites component includes manganese ion and calcium ion, gold Belong to a concentration of 0.5-5mmol/L of binding site component, wherein manganese ion concentration is 0.1-1mmol/L, calcium ion concentration 0.5- 5mmol/L；Preferably, the metal-binding sites group is divided into calcium chloride, calcium nitrate, calcium bromide, manganese chloride, manganese sulfate and nitric acid At least two in manganese；Preferably, metal-chelator group is divided into ethylenediamine tetra-acetic acid, tetrasodium ethylenediamine tetraacetate, ethylenediamine tetraacetic At least one of acetic acid dipotassium and disodium ethylene diamine tetraacetate；Wherein, the working concentration of metal-chelator is 0.05-0.2mg/ mL；Preferably, biological preservative component includes in isothiazolinone, ε-poly-D-lysine, Sodium azide, thimerosal and gentamicin At least one, the working concentration of biological preservative component is 0.5mg/mL-2mg/mL.

Further, activity preservative fluid further includes surface active agent composition；Preferably, surface active agent composition is poly- second two At least one of alcohol, polyoxyethylene 20 sorbitan monolaurate -20 and octyl phenyl polyoxyethylene ether, surfactant The working concentration of component is 0.1mg/mL-0.5mg/mL；Preferably, activity preservative fluid further includes non-protein class site sealer； Preferably, non-protein class site sealer is at least one of gelatin, fish glue from skin and ethanol amine, non-protein class site sealer Working concentration be 0.5g/mL-2g/mL.

According to a further aspect of the invention, a kind of antigen capture for capturing separation and quantitatively detecting AFP-L3 is provided Agent.The antigen capture agent includes any of the above-described kind of immune magnetic compound and activity preservative fluid.

Further, a concentration of in activity preservative fluid with the densimeter immune magnetic compound of the magnetic microsphere 0.5-2mg/mL。

According to a further aspect of the invention, a kind of kit for capturing separation and quantitatively detecting AFP-L3 is provided. The kit includes any of the above-described kind of antigen capture agent.

Further, which further includes：Low concentration calibration product, altitude calibration product, the anti-AFP of luminescent label are anti- Body and buffer solution；Preferably, a concentration of 10.936-20.313IU/mL of low concentration calibration product, a concentration of 350- of altitude calibration product 650IU/mL；Preferably, the working concentration of luminous marker is 5ng/mL-500ng/mL, and the working concentration of AntiAFP antibody is 50ng/mL-5000ng/mL；Preferably, buffer solution be carbonate buffer solution, phosphate buffer, borate buffer solution or Tris-HCl buffer solutions；Preferably, luminous marker is luminol, different luminol and its derivative；Alkaline phosphatase, horseradish mistake Oxide enzyme, fluorescent material, rare earth ion and its chelate aglucon, acridinium ester and its derivative or tris (bipyridine) ruthenium.

According to a further aspect of the invention, a kind of above-mentioned immune magnetic compound is provided, antigen capture agent, kit exist Application in capture separation and quantitatively detection AFP-L3.

Further, above-mentioned immune magnetic compound, antigen capture agent, kit are applied to semi-automatic or are automatically immunized Analyzer.

According to a further aspect of the invention, a kind of system that capture detaches and quantitatively detects AFP-L3 is provided.The system Including mentioned reagent box and semi-automatic or automatic lmunoassays analyzer.

In the prior art, agglutinin is directly coupled at magnetic microsphere surface, causes the rigid interface of agglutinin and magnetic ball straight Contact, reduces activity of lectin, can not ensure higher Conjugate ratio, be easy to cause certain space hyte, influences follow-up Capture separation reaction efficiency.It applies the technical scheme of the present invention, agglutinin is indeed through being coated on magnetic microsphere table The amino polymer mediation in face is connected on magnetic microsphere, and amino polymer coating magnetic microsphere so that magnetic ball surface modification is upper big Amino group is measured, which has dendrimer networks shape structure, can fully improve coupling carrying capacity, while reducing space bit Resistance, and make rigid surface of the target conjugate agglutinin far from magnetic ball, it farthest ensure that the life of target conjugate Object activity.

Specific implementation mode

It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase Mutually combination.Below in conjunction with embodiment, the present invention will be described in detail.

Abbreviation in the present invention is explained as follows：

Alpha-fetoprotein (Alpha-fetoprotein/AFP), alpha-fetoprotein variant (Alpha-fetoprotein Variant/AFP-L3), primary carcinoma of liver (primary hepatocellular carcinoma), lentil lectin (lens Culinaris agglutinin/LCA), magnetic microsphere (Magnetic microspheres/MB), capture separation (Capture And separation), it is CLIA (chemiluminescence immunoassay), ABEI [N- (4- ammonia butyl)-N- ethyls different luminol], poly- Aziridine (polyethyleneimine/PEI), DCC (dicyclohexylcarbodiimide), EDC (1- (3- dimethylamino-propyls)- 3- ethyl-carbodiimide hydrochlorides), CDC (N, N'- diisopropylcarbodiimide), DMF (n,N-Dimethylformamide), PEI are (poly- Aziridine), PA (polyamide), PAM (polyacrylamide), PDI (poly-o-phenylenediamine).

In the prior art, agglutinin is directly coupled at magnetic microsphere surface, causes the rigid interface of agglutinin and magnetic ball straight Contact, reduces activity of lectin, can not ensure higher Conjugate ratio, be easy to cause certain space hyte, influences follow-up Capture separation reaction efficiency.

According to one kind of the invention, typically embodiment there is provided a kind of immune magnetic compounds.The immune magnetic is compound Object includes：Using the magnetic microsphere for the amino functional that amino polymer modified magnetic microballoon obtains, magnetic microsphere surface modification There is amino polymer；Agglutinin, agglutinin are connect with amino polymer.

It applies the technical scheme of the present invention, agglutinin is indeed through the amino polymer for being coated on magnetic microsphere surface Mediation is connected on magnetic microsphere, and amino polymer coating magnetic microsphere makes the upper a large amount of amino groups of magnetic ball surface modification, should Outer layer copolymer has dendrimer networks shape structure, can fully improve coupling carrying capacity, while reducing steric hindrance, and makes mesh Rigid surface of the conjugate agglutinin far from magnetic microsphere is marked, farthest ensure that the bioactivity of target conjugate.When When being detached to the capture of AFP-L3 using the immune magnetic compound, capture separative efficiency will be greatly improved.

In the present invention, the part being connected on magnetic microsphere after amino polymer modified magnetic microballoon is not amino The entirety of polymer, but eliminate the part after reactive group, but in order to indicate agglutinin specifically with amino functional Which part of magnetic microsphere connects, also referred to as amino polymer.

Preferably, agglutinin is oxidation agglutinin；It is furthermore preferred that oxidation agglutinin connects with amino on the amino polymer It connects；It is further preferred that agglutinin hydroxyl is oxidized to the oxidation agglutinin of aldehyde radical, oxidation agglutinin is polymerize by aldehyde radical with amino Amino connects on object.Hydroxyl on agglutinin sugar chain structure is oxidized to aldehyde radical, and the oxidation agglutinin is mediated with amino polymer Magnetic microsphere directly reacted in room temperature, be not necessarily to crosslinking agent facilitation, pass through the Schiff formed between the two group Key covalent bond forms compound.It due to there is no crosslinking agent participation in reaction, carries out at room temperature, system pH is in neutrality on the weak side Alkalinity, reaction is mild, ensure that the activity of agglutinin to greatest extent.

According to a kind of typical embodiment of the present invention, amino polymer is polyethyleneimine, polyamide, polyacrylamide Or poly-o-phenylenediamine, preferably polyethyleneimine.

According to a kind of typical embodiment of the present invention, immune magnetic compound is for capturing the immune of separation AFP-L3 Magnetic composite, it is preferred that agglutinin is lentil lectin, small Lens culinaris agglutinin or ConA, and more preferably hyacinth bean is solidifying Collection element.

According to a kind of typical embodiment of the present invention, magnetic microsphere includes magnetic ball ontology, and magnetic ball ontology is changed by surface Property and carry one or more activity functional groups；Preferably, active group includes at least one in carboxyl, hydroxyl and amino Kind；Preferably, magnetic ball ontology is Fe₂O₃The complex of magnetic nano-particle and high-molecular organic material, or be Fe₃O₄It is magnetic The complex of nano-particle and high-molecular organic material；It is further preferred that magnetic ball ontology grain size is 0.1 μm~5 μm；Amino is poly- Object is closed by the way that the magnetic microsphere of the amino functional is obtained by the reaction with active group.The magnetic microsphere is by will be nano level Fe₂O₃Magnetic particle and high-molecular organic material carry out compound (or nano level Fe₃O₄Magnetic particle and high-molecular organic material Carry out compound), the micron-sized solid phase microballoon with superparamagnetism and huge amount protein adsorption capacity is formed, has and is adding magnetic outside It can be magnetized rapidly under field action, the attribute that remanent magnetism is zero after withdrawing magnetic field.In addition, this magnetic ball is modified by surface makes magnetic Ball surface connects active group, not only reduces non-specific adsorption, and increase the stability of system, does not generate cohesion, shows simultaneously It writes and improves Percentage bound.

According to a kind of typical embodiment of the present invention, a kind of preparation side of any of the above-described kind of immune magnetic compound is provided Method.The preparation method includes the following steps：1) amino polymer modified magnetic microballoon is used, the magnetism for obtaining amino functional is micro- Ball；2) it connect agglutinin to form immune magnetic compound with amino polymer.

Amino polymer coating magnetic microsphere makes a large amount of amino groups in magnetic microsphere surface modification, the outer layer copolymer With dendrimer networks shape structure, coupling carrying capacity can be fully improved, while reducing steric hindrance, and so that target conjugate is solidifying Rigid surface of the collection element far from magnetic microsphere, farthest ensure that the bioactivity of target conjugate.When immune using this When magnetic composite detaches the capture of AFP-L3, capture separative efficiency will be greatly improved.

Preferably, further include being aoxidized to agglutinin before agglutinin is connect with the amino on amino polymer Step obtains oxidation agglutinin；Preferably, oxidation agglutinin is connect with amino on the amino polymer；Preferably, it will be aggregated Hydroxyl in element is oxidized to aldehyde radical, obtains oxidation agglutinin, and oxidation agglutinin passes through amino on aldehyde radical and the amino polymer Connection.Hydroxyl on agglutinin sugar chain structure is oxidized to aldehyde radical, and the magnetism that the oxidation agglutinin and amino polymer mediate is micro- Ball is directly reacted in room temperature, is not necessarily to the facilitation of crosslinking agent, is covalently tied by the Schiff key formed between the two group It closes, forms compound.It due to there is no crosslinking agent participation in reaction, carries out at room temperature, system pH is in neutrality alkalinity on the weak side, reaction Mildly, it ensure that the activity of agglutinin to greatest extent.

According to a kind of typical embodiment of the present invention, the oxidant used in oxidation step is periodate, perchloric acid Salt, permanganate, preferably sodium metaperiodate, sodium perchlorate or potassium permanganate, more preferably sodium metaperiodate.Because sodium metaperiodate is most The just right hydroxyl by agglutinin sugar chain of energy is oxidized to aldehyde radical, and is unlikely to excessive oxidation at carboxyl, because if raw Carboxyl is produced, self-crosslinking is easily led to, easily leads to agglutinin inactivation.In addition, carboxyl reacts the effect for needing crosslinking agent with amino, no Conducive to the maximum activity of maintenance agglutinin.

Preferably, the mass concentration ratio of agglutinin and oxidant is 1 in oxidation step:50-1:500；In this proportional region It is interior, easily ensure the hydroxyl on agglutinin sugar chain being oxidized to aldehyde radical.It is further preferred that the reaction temperature of oxidation is 20-30 DEG C, Reaction time is 1-4 hours；Under the conditions of this reaction temperature and time, be conducive to the maximum activity for maintaining agglutinin.Preferably, Agglutinin concentration range be 1mg/mL-5mg/mL.

According to a kind of typical embodiment of the present invention, step 1) specifically includes：11) use activator to magnetic microsphere into Row activation；12) activated magnetic microsphere amino polymer aqueous solution is added to react；13) surface modification is isolated The magnetic microsphere for having amino polymer, is reacted with sealer, and the magnetic microsphere of amino functional is obtained after going supernatant to wash； Preferably, activator be dicyclohexylcarbodiimide, 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and N, At least one of N'- diisopropylcarbodiimide；Preferably, the mass concentration of magnetic microsphere and activator is：2:1–4:1； Preferably, the reaction temperature of activation is 37 DEG C -40 DEG C, and the reaction time is 1h -4h；Preferably, step 12) specifically includes：It will live The magnetic microsphere changed is added to amino polymer aqueous solution, after supersound process, carries out rotation mixing reaction；Preferably, ultrasonic The time of processing is 5min-15min；Preferably, the temperature of rotation mixing reaction is 25 DEG C -30 DEG C, time 2h-6h；It is preferred that , a concentration of 10mg/mL-200mg/mL of amino polymer Ammonia In Aqueous Solution based polyalcohol；Preferably, activated magnetism is micro- When ball is added to amino polymer aqueous solution and is reacted, a concentration of 10mg/mL-20mg/mL of magnetic microsphere；Preferably, it seals It is ethanol amine, BSA or gelatin, a concentration of 0.5-1mol/L of sealer, reaction time 1h-4h to close agent.

According to a kind of typical embodiment of the present invention, step 2) specifically includes：The magnetic microsphere of amino functional is used Re-suspension liquid is resuspended, and oxidation agglutinin is then added, and reducing agent is added after mixing and is stirred to react, and immune magnetic is obtained after going supernatant to wash Property compound；Preferably, re-suspension liquid is that 0.1mol/L pH9.2 sodium bicarbonate aqueous solutions, Tris-HCl buffer solutions or borate are slow Fliud flushing, a concentration of 0.05mol/L-0.2mol/L of re-suspension liquid, pH 8-10；Preferably, preferred reducing agent be sodium borohydride, Potassium borohydride or Lithium Aluminium Hydride, more preferably sodium borohydride, a concentration of 1mg/mL-5mg/mL of the reducing agent, more preferably 5mg/mL；Preferably, the mass concentration ratio for aoxidizing the magnetic microsphere of agglutinin and amino functional is 1:50-1:100；It is preferred that , the reaction temperature of step 2) is 20 DEG C -30 DEG C, reaction time 2h-6h.

The final purpose of above-mentioned reaction condition setting, is for the reactivity of enhancing agglutinin, it is even to improve agglutinin The Conjugate ratio for joining magnetic ball, to improve the capture rate of agglutinin.

According to a kind of typical embodiment of the present invention, a kind of vital preservation for above-mentioned immune magnetic compound is provided Liquid.The activity preservative fluid includes：Dilution component, metal-binding sites component, metal-chelator component and biological preservative group Point.Since agglutinin is to be combined by the fucosyl residues on agglutinin surface, and there are multiple metal ion knots on agglutinin surface Site is closed, the bivalent metal ion (example of the addition suitable concentration in the magnetic ball suspension (preserving liquid) that coupling synthesis finishes is passed through Such as calcium ion, manganese ion, magnesium ion) and protease inhibitors (metal-chelator component), preservative etc., it can ensure to coagulate Activity and stability of the collection element in liquid environment, further ensure the reactivity of agglutinin magnetic microsphere.

Dilution component includes but not limited to phosphate buffer, borate buffer solution, Tris-HCl buffer solutions or carbonic acid Salt buffer, a concentration of 0.01-0.2mol/L of dilution, pH 7-9；Effect be offset to a certain extent, mitigate it is additional The influence of strong acid or highly basic to solution acid alkalinity, to maintain the pH value of solution to stablize relatively.

Metal-binding sites component includes manganese ion and calcium ion, a concentration of 0.5- of the metal-binding sites component 5mmol/L, wherein manganese ion concentration are 0.1-1mmol/L, calcium ion concentration 0.5-5mmol/L；Preferably, metal-binding sites Component includes but not limited at least two in calcium chloride, calcium nitrate, calcium bromide, manganese chloride, manganese sulfate and manganese nitrate；It is acted on It is mainly combined with the metal recognition site of agglutinin, activates the bioactivity of lentil lectin.

Metal-chelator component includes but not limited to ethylenediamine tetra-acetic acid, tetrasodium ethylenediamine tetraacetate, ethylenediamine tetra-acetic acid At least one of dipotassium and disodium ethylene diamine tetraacetate；Wherein, the working concentration of metal-chelator is 0.05-0.2mg/mL； Effect is the strong combination by chelator molecule and metal ion, inside metal ion inclusion to chelating agent, stablizes gold Belong to ion, prevents active metal ion from oxidation or dissociation occurs.

Biological preservative component includes but not limited to that isothiazolinone, ε-poly-D-lysine, Sodium azide, thimerosal and celebrating are big The working concentration of at least one of mycin, biological preservative component is 0.5mg/mL -2mg/mL.Effect is inhibited to microorganism Enzyme activity or the cytogenetics structure of microorganism is had an impact, to inhibit or kill the microorganisms such as bacterium.

The characteristic for preserving liquid and being directed to agglutinin in the present invention, can preferably maintain the activity of agglutinin, protection immune Magnetic microsphere is not destroyed or reunites in homogeneous system.

Preferably, it further includes surface active agent composition to preserve liquid；Surface active agent composition include but not limited to polyethylene glycol, At least one of polyoxyethylene 20 sorbitan monolaurate -20 and octyl phenyl polyoxyethylene ether, surfactant group The working concentration divided is 0.1mg/mL-0.5mg/mL；Effect is the surface tension reduced between immune magnetic microsphere, is improved immune Dispersibility of the magnetic microsphere in homogeneous system.

Preferably, it further includes non-protein class site sealer to preserve liquid；Non-protein class site sealer includes but not limited to The working concentration of at least one of gelatin, fish glue from skin and ethanol amine, non-protein class site sealer is 0.5g/mL-2g/mL； Effect is to close the active group not reacted with participation on magnetic ball.

According to a kind of typical embodiment of the present invention, provide a kind of for the preservation of above-mentioned immune magnetic complex activity Liquid.The preservation liquid includes following components：(1) dilution group is divided into phosphate buffer, borate buffer solution, Tris-HCl bufferings Liquid or carbonate buffer solution；Wherein, a concentration of 0.01-0.2mol/L of dilution；PH is 7-9；(2) metal-binding sites group Divide, calcium chloride, calcium nitrate, calcium bromide, manganese chloride, at least two in manganese sulfate and manganese nitrate；Its a concentration of 0.5-5mmol/ L, and manganese ion, a concentration of 0.1-1mmol/L, calcium ion concentration 0.5-5mmol/L；(3) metal-chelator group is divided into ethylenediamine Tetraacethyl (EDTA), tetrasodium ethylenediamine tetraacetate (EDTA-4Na), EDTAP dipotassium ethylene diamine tetraacetate (EDTA-2K) and ethylenediamine tetrem At least one of acid disodium (EDTA-2Na)；Wherein, the working concentration of metal-chelator is 0.05-0.2mg/mL；(4) table Face active agent component is polyethylene glycol, in polyoxyethylene 20 sorbitan monolaurate -20 and octyl phenyl polyoxyethylene ether At least one；Wherein, the working concentration of surfactant is 0.1mg/mL-0.5mg/mL；(5) non-protein class site sealer For gelatin, at least one of fish glue from skin and ethanol amine；Wherein, the working concentration of non-protein class site sealer is 0.5g/mL- 2g/mL；(6) biological preservative group is divided into isothiazolinone, ε-poly-D-lysine, Sodium azide, in thimerosal and gentamicin It is at least one；Wherein, the working concentration of biological preservative is 0.5mg/mL -2mg/mL.

According to a kind of typical embodiment of the present invention, provide a kind of for capturing separation and quantitatively detecting the anti-of AFP-L3 Former capturing agent.The antigen capture agent includes any of the above-described kind of immune magnetic compound and activity preservative fluid.Preferably, with magnetic micro- A concentration of 0.5-2mg/mL of the densimeter immune magnetic compound of ball in activity preservative fluid.

According to a kind of typical embodiment of the present invention, a kind of examination for capturing separation and quantitatively detecting AFP-L3 is provided Agent box.The kit includes any of the above-described kind of antigen capture agent.

Preferably, which further includes：Low concentration calibration product, altitude calibration product, the anti-AFP of luminescent label are anti- Body (preferably AFP monoclonal antibody) and buffer solution；Preferably, a concentration of 10.936-20.313IU/ of low concentration calibration product Ml, a concentration of 350-650IU/Ml of altitude calibration product；Preferably, the working concentration of luminous marker is 5ng/mL-500ng/mL, The working concentration of AntiAFP antibody is 50ng/mL-5000ng/mL；Preferably, buffer solution is carbonate buffer solution, phosphate-buffered Liquid, borate buffer solution or Tris-HCl buffer solutions；Preferably, luminous marker is luminol, different luminol and its derivative； Alkaline phosphatase, horseradish peroxidase, fluorescent material, rare earth ion and its chelate aglucon, acridinium ester and its derivative or Tris (bipyridine) ruthenium.

According to a kind of typical embodiment of the present invention, the kit for capturing separation and quantitatively detect AFP-L3 is specific Including following component：

A.AFP-L3 antigen capture agent, a concentration of 0.5-2mg/mL of magnetic ball

B. low concentration calibration product (AFP-L3 antigens), concentration：10.936-20.313IU/Ml

C. high concentration degree calibration object (AFP-L3 antigens), concentration：350-650IU/Ml

D. the AFP monoclonal antibody of luminescent label

Luminous marker includes but not limited to luminol, different luminol and its derivative, specifically there is ABEI (N- (4- ammonia Base butyl)-N- ethyls different luminol), AHEI (N- (6- amino base)-N- ethyls different luminol) and, (the different Shandongs of isothiocyanic acid ITCI Minot), ITCBEI (N- (4- isothiocyanos butyl)-N- ethyls different luminol)

Luminous marker concentration：5ng/mL-500ng/mL,

The concentration of AFP monoclonal antibody：50ng/mL-5000ng/mL；

E.Buffer buffer solutions

The ingredient of Buffer buffer solutions includes but not limited to：Carbonate buffer solution, phosphate buffer, boric acid salt buffer Liquid, Tris-HCl buffer solutions.

According to a kind of typical embodiment of the present invention, a kind of above-mentioned immune magnetic compound, antigen capture agent, examination are provided Application of the agent box in capture separation and in quantitatively detecting AFP-L3.

Capture separation in the prior art and the magnetic microsphere for quantitatively detecting AFP-L3, need manually to grasp after capture separation Make, such manual operation is easily introduced error and interference, can not ensure the reliability of testing result.The immune magnetic of the present invention is compound Object, antigen capture agent, kit can be applied to semi-automatic or automatic lmunoassays analyzer, to realize capture and detection one Change, automation, improves the accuracy of testing result.

According to a kind of typical embodiment of the present invention, a kind of system that capture detaches and quantitatively detects AFP-L3 is provided. The system includes mentioned reagent box and semi-automatic or automatic lmunoassays analyzer.

According to a kind of typical embodiment of the present invention, the magnetic microsphere of agglutinin coupling, detects antibody and shows dilution Track marker is integrated in a kit, using Maglumi full automatic chemiluminescence immunoassay instruments can realize sample-adding, One is captured, detached and be detected on, can ensure high accuracy of the operating process without artificial disturbing factor and testing result, Gao Ling Sensitivity.The coated magnetic microsphere of agglutinin is after capture has detached the AFP-L3 in determinand, by Maglumi full-automatic chemicals After the automatic cleaning of luminescent immunoassay instrument system liquid, other unbonded substances are washed away, luminous marker ABEI labels are added AFP monoclonal antibody, with agglutinin capture AFP-L3 magnetic ball compound formed immune complex, realize the inspection of AFP-L3 It surveys.

According to a kind of typical embodiment of the present invention, a kind of side for enhancing AFP-L3 capture separative efficiencies is provided Method.This approach includes the following steps：

1. the oxidation of agglutinin is transformed：Agglutinin is mixed with oxidation processes agent aqueous solution, is protected from light, and is mixed slowly reaction, is obtained To the oxidation product (lectin-CHO) of agglutinin.

Wherein, the agglutinin includes but not limited to lentil lectin, small Lens culinaris agglutinin, ConA, and preferred Lentil lectin；Oxidation processes agent includes but not limited to sodium metaperiodate, sodium perchlorate, potassium permanganate；Reaction temperature is 20-30 DEG C, the reaction time is 1-4 hours；Agglutinin and the mass concentration ratio of oxidation processes agent are 1:50-1:500.

2. the preparation of polyethyleneimine magnetic microsphere：

A certain amount of magnetic ball is taken, activator is added, re-suspension liquid is then added and is configured to certain density magnetic ball solution, by it It is placed in water-bath and shakes or mechanic whirl-nett reaction, activated nanoscale magnetic bead solution is removed into supernatant, and be added The carbonate buffer solution of pH9.5 is configured to certain density magnetic ball solution, spare.Amino polymer is added in activated magnetic ball Aqueous solution, after supersound process, place it under certain temperature and carry out rotation mixing reaction, Magneto separate removes supernatant, PBS is used in combination Buffer solution washs 3 times, is then reacted with sealer, then washed 3 times with PBS buffer solution, amino functional is obtained after Magneto separate The magnetic microsphere of change, it is spare.

Wherein, activator includes but not limited to DCC (dicyclohexylcarbodiimide), EDC (1- (3- dimethylamino-propyls)- 3- ethyl-carbodiimide hydrochlorides), CDC (N, N'- diisopropylcarbodiimide), the ratio of magnetic ball and activator is：2:1-4: 1；Re-suspension liquid includes but not limited to water, ethyl alcohol, DMF (n,N-Dimethylformamide)；The temperature of priming reaction is：37℃-40℃； The priming reaction time is 1h-4h；The mass ratio of magnetic microsphere and amino polymer is 1:1-10:1；Amino polymer includes but not It is limited to PEI (polyethyleneimine), PA (polyamide), PAM (polyacrylamide), PDI (poly-o-phenylenediamine)；Amino polymer it is dense Degree is 10mg/mL-200mg/mL, a concentration of 10mg/mL-20mg/mL of magnetic microsphere；Ultrasonic time is：5min-15min；Rotation Turn reaction temperature be：25 DEG C -30 DEG C, reaction time 2h-6h；Sealer includes but not limited to ethanol amine, BSA, gelatin, envelope Close a concentration of 0.5-1mol/L of agent, reaction time 1h-4h.

3. the preparation of the magnetic microsphere coupled product of agglutinin-amino functional of oxidation processes：

A certain amount of amino polymer functionalization magnetic ball is taken, is resuspended, is made with 0.1mol/L pH9.2 sodium bicarbonate aqueous solutions It reaches a certain concentration (in terms of nanoscale magnetic bead), and the oxidation product (LECTIN-CHO) of agglutinin is added, and after mixing, 5mg/ is added The NaBH of mL₄Solution is stirred to react, and removes completely reacted magnetic microsphere solution supernatant, clear with washing lotion (0.5%BSA solution) It washes solid three times, obtains the coated magnetic microsphere of agglutinin oxide.

Wherein, the mass concentration ratio of the agglutinin of oxidation processes and amino polymer functionalization magnetic ball is 1:50-1:100, The temperature of reaction is：20℃-30℃；Reaction time is 2h -6h.

According to a kind of typical embodiment of the present invention, a kind of method of immunity of detection AFP-L3 is provided comprising Using the above-mentioned method capture separation AFP-L3 for capturing separative efficiency for enhancing AFP-L3.

The advantageous effect further illustrated the present invention below in conjunction with embodiment.

Part raw material sources involved in following embodiment and comparative example are as follows：

(the sources lentil lectin LCA：Aladdin company L140096)

Sodium metaperiodate (source：Sigma companies)

Polyethyleneimine (source：Sigma companies)

Magnetic microsphere (source：NPD projects biomedical engineering limited liability company produces)

(the sources ABEI：NPD projects biomedical engineering limited liability company produces)

(the sources Anti-AFP antibody：Meridian)

AFP calibration objects (source：Meridian)

Embodiment 1

A kind of method and its detection kit for enhancing AFP-L3 capture separative efficiencies

Prepare 1：Lentil lectin aoxidizes the preparation of modification

The lentil lectin that 2mg is dissolved with the 0.2mol/L sodium bicarbonate aqueous solutions of Fresh, in above-mentioned solution by The 0.5mL 8mmol/L sodium metaperiodate aqueous solutions of Fresh are added dropwise in drop, are protected from light, and reaction 2 hours is mixed slowly under the conditions of 25 DEG C, Obtain the oxidation product (LECTIN-CHO) of lentil lectin.

Prepare 2：The preparation of polyethyleneimine magnetic microsphere

A certain amount of magnetic ball is taken, the DCC of 0.5mg is added by every milligram of magnetic ball, ethanol solution, which is then added, makes the dense of magnetic ball Degree is 20mg/mL, is placed in 38 DEG C of water-baths and shakes or mechanical agitation 2 hours；Activated nanoscale magnetic bead solution removal Then supernatant is added the carbonate buffer solution of pH9.5, its concentration is made to reach 20mg/mL (in terms of nanoscale magnetic bead)；With every milligram Magnetic ball adds 10 milligrams of aq. polyethyleneimines, and ultrasonic 10min rotates mixing and react 4h, discarded after Magneto separate at room temperature Supernatant；It is washed 3 times with the PBS buffer solution of 0.01mol/L pH7.5, each 3min；With the ethanol amine capping 2h of 1mol/L； It is washed 3 times with the PBS buffer solution of 0.01mol/L pH7.5, is discarded supernatant after Magneto separate, the magnetism for obtaining amino functional is micro- Ball, it is spare.

Prepare 3：The preparation of lentil lectin modification-polyethyleneimine magnetic ball conjugate

A certain amount of amino polymer functionalization magnetic ball is taken, is resuspended, is made with 0.1mol/L pH9.2 sodium bicarbonate aqueous solutions Its concentration reaches 20mg/mL (in terms of nanoscale magnetic bead), and the oxidation product of the lentil lectin of 0.5mg is added by every milligram of magnetic ball (LECTIN-CHO), after mixing, the NaBH of 5mg/mL is added₄Solution, 25 DEG C are stirred to react 4 hours, completely reacted magnetic microsphere Solution removes supernatant, cleans solid three times with washing lotion (0.5%BSA solution), obtains the coated magnetic of lentil lectin oxide Property microballoon.

Prepare 4：The preparation of lentil lectin oxide magnetic microballoon activity preservative fluid

(1) dilution component：Borate buffer solution, pH7.2, a concentration of 0.02mol/L；

(2) metal-binding sites component：Calcium chloride, a concentration of 5mmol/L,

Manganese chloride, a concentration of 1mmol/L,

(3) metal-chelator component：Ethylenediamine tetra-acetic acid (EDTA), a concentration of 0.1mg/mL；

(4) surface active agent composition：Polyethylene glycol, a concentration of 0.1mg/mL；

(5) site sealant components：Gelatin, a concentration of 0.5g/mL；

(6) biological preservative component：Methylisothiazolinone, a concentration of 1.5mg/mL.

Prepare 5：The preparation of AFP-L3 antigen capture reagents

The magnetic ball for preparing coating lentil lectin oxide in 3 is suspended in the immune magnetic activity for preparing and preparing in 4 to protect In liquid storage, the capture agent of AFP-L3 antigens is obtained.

Prepare 6：ABEI marks AFP monoclonal antibody

1mg AFP monoclonal antibodies are taken, its volume are adjusted to 1mL with 0.1mol/L carbonate buffer solutions (pH9.5), so After be put into bag filter, be placed in pH9.5 carbonate buffer solutions and dialyse 1 hour.It is anti-to take out the anti-AFP monoclonals dialysed Body, is added 100 μ g ABEI-, half succinamic acid-NHS, and room temperature shakes 1.5 hours.G-25 gel columns are installed, purified water is used After elution is clean, then with the PBS buffer solution of pH value 7.4 it is balanced elution.After G-25 gel column equilibrations elute, it will mark The AFP monoclonal antibody of good ABEI crosses column purification, then collects the protein solution for peak value occur.The albumen gathered is molten Isometric protection liquid containing 5% BSA is added in liquid, and diluted is added to 0.15 μ g/mL.

A kind of kit of capture separation and detection AFP-L3, contains following components：

A. according to the AFP-L3 antigen captures reagent that 5 obtain is prepared, (agglutinin oxidation product and magnetic microsphere are immune The part of magnetic composite, but in order to measure conveniently, respectively with the concentration measurement of agglutinin oxidation product and magnetic microsphere.Subsequently Embodiment and the equally similar metering of comparative example)：

The concentration of agglutinin oxidation product：20 μ g/mL,

Magnetic microsphere concentration：1mg/mL,

Dilution component：Borate buffer solution, pH7.2, a concentration of 0.02mol/L；

Metal-binding sites component：Calcium chloride, a concentration of 5mmol/L,

Manganese chloride, a concentration of 1mmol/L；

Metal-chelator component：Ethylenediamine tetra-acetic acid (EDTA), a concentration of 0.1mg/mL；

Surface active agent composition：Polyethylene glycol, a concentration of 0.1mg/mL；

Site sealant components：Gelatin, a concentration of 0.5g/mL；

Biological preservative component：Methylisothiazolinone, a concentration of 1.5mg/mL

B. low concentration calibration product, concentration：15.625IU/mL；

C. high concentration calibration object, concentration：500.000IU/mL；

D. according to the AFP monoclonal antibody for preparing the ABEI labels that 6 obtain

ABEI concentration：250ng/mL,

The concentration of AFP monoclonal antibody：5000ng/mL；

E.Buffer buffer solutions

The ingredient of Buffer buffer solutions：The borate buffer solution of 0.02mol/L pH7.2,5mmol/L sodium chloride, 1mmol/ L manganese chlorides.

A method of it is captured with above-mentioned kit and detaches and detect 3 AFP-L in serum, step is specific as follows：

1) preparation of 10 standard curves

A concentration of 4.6*10^6IU/mL of AFP-L3 enterprises calibration object, is diluted to 0.02M PBS buffer solution The intermediate product of 1000IU/mL successively gradient dilution are respectively obtained the concentration point of ten point curves by 1000IU/mL, fixed by 2 points Working curve is calculated in mark calibration curve.

2) use of capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes

Capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes are given birth to using this method, in this department development ＆ production Maglumi 2000Plus instruments on detect, detecting step includes：

A. 20 μ L samples to be tested, high-concentration and low-concentration calibration object are added in reaction cup；

B. 20 μ L lentil lectins oxides-magnetic microsphere solution is added；

C. 100 μ L Buffer buffer solutions are added；

D.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

E. the AFP monoclonal antibody of 200 μ L ABEI labels is added；

F.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

G. luminous substrate is added, detects light signal strength；

H. sample to be tested can be calculated automatically according to pattern detection luminous intensity by the revised working curve of calibration object AFP-L3 concentration.

It captures the detection for detaching and detecting AFP-L3 chemical luminescence immune analysis reagent boxes to primary carcinoma of liver sample and answers With

The serum sample for having selected 3 primarys carcinoma of liver through clinical definite is measured by the method for agglutinin electrophoresis It is tried to the actual content of AFP-L3 in its sample, then with the prepared AFP-L3 chemiluminescence immune assays of this embodiment scheme Agent box measures the content of AFP-L3 in its sample, and the prepared AFP-L3 chemistry of this programme is calculated by two measured concentrations The capture separative efficiency of the AFP-L3 of luminescence immunoassay kit.

Embodiment 2

A kind of method and its detection kit for enhancing AFP-L3 capture separative efficiencies

Prepare 1：Lentil lectin aoxidizes the preparation of modification

The lentil lectin that 2mg is dissolved with the 0.2mol/L sodium bicarbonate aqueous solutions of Fresh, in above-mentioned solution by The 0.5mL 16mmol/L sodium metaperiodate aqueous solutions of Fresh are added dropwise in drop, are protected from light, and it is small that reaction 2 is mixed slowly under the conditions of 25 DEG C When, obtain the oxidation product (LECTIN-CHO) of lentil lectin.

Prepare 2：The preparation of polyethyleneimine magnetic microsphere

A certain amount of magnetic ball is taken, the DCC of 0.5mg is added by every milligram of magnetic ball, ethanol solution, which is then added, makes the dense of magnetic ball Degree is 20mg/mL, is placed in 38 DEG C of water-baths and shakes or mechanical agitation 2 hours；Activated nanoscale magnetic bead solution is gone Except supernatant, the carbonate buffer solution of pH9.5 is then added, its concentration is made to reach 20mg/mL (in terms of nanoscale magnetic bead)；With every milli Gram magnetic ball adds 10 milligrams of aq. polyethyleneimines, and ultrasonic 10min rotates mixing and react 4h, abandoned after Magneto separate at room temperature Remove supernatant；It is washed 3 times with the PBS buffer solution of 0.01mol/L pH7.5, each 3min；With the ethanol amine capping of 1mol/L 2h；It is washed 3 times with the PBS buffer solution of 0.01mol/L pH7.5, is discarded supernatant after Magneto separate, obtain the magnetism of amino functional Microballoon, it is spare.

Prepare 3：The preparation of lentil lectin modification-polyethyleneimine magnetic ball conjugate

A certain amount of amino polymer functionalization magnetic ball is taken, is resuspended, is made with 0.1mol/L pH9.2 sodium bicarbonate aqueous solutions Its concentration reaches 20mg/mL (in terms of nanoscale magnetic bead), and the oxidation product of the lentil lectin of 0.5mg is added by every milligram of magnetic ball (LECTIN-CHO), after mixing, the NaBH of 5mg/mL is added₄Aqueous slkali, 25 DEG C are stirred to react 4 hours, and completely reacted magnetism is micro- Ball solution removes supernatant, cleans solid three times with washing lotion (0.5%BSA solution), it is coated to obtain lentil lectin oxide Magnetic microsphere.

Prepare 4：The preparation of lentil lectin oxide magnetic microballoon activity preservative fluid

(1) dilution component：Borate buffer solution, pH7.2, a concentration of 0.02mol/L；

(2) metal-binding sites component：Calcium chloride, a concentration of 5mmol/L,

Manganese chloride, a concentration of 1mmol/L,

(3) metal-chelator component：Ethylenediamine tetra-acetic acid (EDTA), a concentration of 0.1mg/mL；

(4) surface active agent composition：Polyethylene glycol, a concentration of 0.1mg/mL；

(5) site sealant components：Gelatin, a concentration of 0.5g/mL；

(6) biological preservative component：Methylisothiazolinone, a concentration of 1.5mg/mL.

Prepare 5：The preparation of AFP-L3 antigen capture reagents

The magnetic ball for preparing coating lentil lectin oxide in 3 is suspended in the immune magnetic activity for preparing and preparing in 4 to protect In liquid storage, the capture agent of AFP-L3 antigens is obtained.

Prepare 6：ABEI marks AFP monoclonal antibody

1mg AFP monoclonal antibodies are taken, its volume are adjusted to 1mL with 0.1mol/L carbonate buffer solutions (pH9.5), so After be put into bag filter, be placed in pH9.5 carbonate buffer solutions and dialyse 1 hour.It is anti-to take out the anti-AFP monoclonals dialysed Body, is added 100 μ g ABEI-, half succinamic acid-NHS, and room temperature shakes 1.5 hours.G-25 gel columns are installed, purified water is used After elution is clean, then with the PBS buffer solution of pH value 7.4 it is balanced elution.After G-25 gel column equilibrations elute, it will mark The AFP monoclonal antibody of good ABEI crosses column purification, then collects the protein solution for peak value occur.The albumen gathered is molten Isometric protection liquid containing 5% BSA is added in liquid, and diluted is added to 0.15 μ g/mL.

A kind of kit of capture separation and detection AFP-L3, contains following components：

A. the AFP-L3 antigen capture reagents obtained according to preparation 5,

The 20 μ g/mL of concentration of agglutinin oxidation product,

Magnetic microsphere working concentration：1mg/mL,

Dilution component：Borate buffer solution, pH7.2, a concentration of 0.02mol/L；

Metal-binding sites component：Calcium chloride, a concentration of 5mmol/L,

Manganese chloride, a concentration of 1mmol/L；

Metal-chelator component：Ethylenediamine tetra-acetic acid (EDTA), a concentration of 0.1mg/mL；

Surface active agent composition：Polyethylene glycol, a concentration of 0.1mg/mL；

Site sealant components：Gelatin, a concentration of 0.5g/mL；

Biological preservative component：Methylisothiazolinone, a concentration of 1.5mg/mL

B. low concentration calibration product, concentration：15.625IU/mL；

C. high concentration calibration object, concentration：500.000IU/mL；

D. according to the AFP monoclonal antibody for preparing the ABEI labels that 6 obtain

ABEI concentration：250ng/mL,

The concentration of AFP monoclonal antibody：5000ng/mL；

E.Buffer buffer solutions

The ingredient of Buffer buffer solutions：The borate buffer solution of 0.02mol/L pH7.2,5mmol/L sodium chloride, 1mmol/ L manganese chlorides.

A method of it is captured with above-mentioned kit and detaches and detect AFP-L3 in serum, step is specific as follows：

1) preparation of 10 standard curves

A concentration of 4.6*10^6IU/mL of AFP-L3 enterprises calibration object, is diluted to 0.02M PBS buffer solution The intermediate product of 1000IU/mL successively gradient dilution are respectively obtained the concentration point of ten point curves by 1000IU/mL, fixed by 2 points Working curve is calculated in mark calibration curve.

2) use of capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes

Capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes are given birth to using this method, in this department development ＆ production Maglumi 2000Plus instruments on detect, detecting step includes：

A. 20 μ L samples to be tested, high-concentration and low-concentration calibration object are added in reaction cup；

B. 20 μ L lentil lectins oxides-magnetic microsphere solution is added；

C. 100 μ L Buffer buffer solutions are added；

D.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

E. the AFP monoclonal antibody of 200 μ L ABEI labels is added；

F.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

G. luminous substrate is added, detects light signal strength；

H. sample to be tested can be calculated automatically according to pattern detection luminous intensity by the revised working curve of calibration object AFP-L3 concentration.

It captures the detection for detaching and detecting AFP-L3 chemical luminescence immune analysis reagent boxes to primary carcinoma of liver sample and answers With

The serum sample for having selected 3 primarys carcinoma of liver through clinical definite is measured by the method for agglutinin electrophoresis To the actual content of AFP-L3 in its sample, tried with the prepared AFP-L3 chemiluminescence immune assays of this embodiment scheme Agent box measures the content of AFP-L3 in its sample, and the prepared AFP-L3 chemistry of this programme is calculated by two measured concentrations The capture separative efficiency of the AFP-L3 of luminescence immunoassay kit.

Embodiment 3

A kind of method and its detection kit for enhancing AFP-L3 capture separative efficiencies

Prepare 1：Lentil lectin aoxidizes the preparation of modification

The lentil lectin that 2mg is dissolved with the 0.2mol/L sodium bicarbonate aqueous solutions of Fresh, in above-mentioned solution by The 0.5mL 8mmol/L sodium perchlorate aqueous solutions of Fresh are added dropwise in drop, are protected from light, and reaction 2 hours is mixed slowly under the conditions of 25 DEG C, Obtain the oxidation product (LECTIN-CHO) of lentil lectin.

Prepare 2：The preparation of polyethyleneimine magnetic microsphere

A certain amount of magnetic ball is taken, the DCC of 0.5mg is added by every milligram of magnetic ball, ethanol solution, which is then added, makes the dense of magnetic ball Degree is 20mg/mL, is placed in 38 DEG C of water-baths and shakes or mechanical agitation 2 hours；Activated nanoscale magnetic bead solution is gone Except supernatant, the carbonate buffer solution of pH9.5 is then added, its concentration is made to reach 20mg/mL (in terms of nanoscale magnetic bead)；With every milli Gram magnetic ball adds 10 milligrams of aq. polyethyleneimines, and ultrasonic 10min rotates mixing and react 4h, abandoned after Magneto separate at room temperature Remove supernatant；It is washed 3 times with the PBS buffer solution of 0.01mol/L pH7.5, each 3min；With the ethanol amine capping of 1mol/L 2h；It is washed 3 times with the PBS buffer solution of 0.01mol/L pH7.5, is discarded supernatant after Magneto separate, obtain the magnetism of amino functional Microballoon, it is spare.

It prepares:3：The preparation of lentil lectin modification-polyethyleneimine magnetic ball conjugate

A certain amount of amino polymer functionalization magnetic ball is taken, is resuspended, is made with 0.1mol/L pH9.2 sodium bicarbonate aqueous solutions Its concentration reaches 20mg/mL (in terms of nanoscale magnetic bead), and the oxidation product of the lentil lectin of 0.5mg is added by every milligram of magnetic ball (LECTIN-CHO), after mixing, the NaBH of 5mg/mL is added₄Aqueous slkali, 25 DEG C are stirred to react 4 hours, and completely reacted magnetism is micro- Ball solution removes supernatant, cleans solid three times with washing lotion (0.5%BSA solution), it is coated to obtain lentil lectin oxide Magnetic microsphere.

Prepare 4：The preparation of lentil lectin oxide magnetic microballoon activity preservative fluid

(1) dilution component：Borate buffer solution, pH7.2, a concentration of 0.02mol/L；

(2) metal-binding sites component：Calcium chloride, a concentration of 5mmol/L,

Manganese chloride, a concentration of 1mmol/L,

(3) metal-chelator component：Ethylenediamine tetra-acetic acid (EDTA), a concentration of 0.1mg/mL；

(4) surface active agent composition：Polyethylene glycol, a concentration of 0.1mg/mL；

(5) site sealant components：Gelatin, a concentration of 0.5g/mL；

(6) biological preservative component：Methylisothiazolinone, a concentration of 1.5mg/mL.

Prepare 5：The preparation of AFP-L3 antigen capture reagents

The magnetic ball for preparing coating lentil lectin oxide in 3 is suspended in the immune magnetic activity for preparing and preparing in 4 to protect In liquid storage, the capture agent of AFP-L3 antigens is obtained.

Prepare 6：ABEI marks AFP monoclonal antibody

1mg AFP monoclonal antibodies are taken, its volume are adjusted to 1mL with 0.1mol/L carbonate buffer solutions (pH9.5), so After be put into bag filter, be placed in pH9.5 carbonate buffer solutions and dialyse 1 hour.It is anti-to take out the anti-AFP monoclonals dialysed Body, is added 100 μ g ABEI-, half succinamic acid-NHS, and room temperature shakes 1.5 hours.G-25 gel columns are installed, purified water is used After elution is clean, then with the PBS buffer solution of pH value 7.4 it is balanced elution.After G-25 gel column equilibrations elute, it will mark The AFP monoclonal antibody of good ABEI crosses column purification, then collects the protein solution for peak value occur.The albumen gathered is molten Isometric protection liquid containing 5% BSA is added in liquid, and diluted is added to 0.15 μ g/mL.

A kind of kit of capture separation and detection AFP-L3, contains following components：

A. the AFP-L3 antigen capture reagents obtained according to preparation 5,

The 20 μ g/mL of concentration of agglutinin oxidation product,

Magnetic microsphere concentration：1mg/mL,

Dilution component：Borate buffer solution, pH7.2, a concentration of 0.02mol/L；

Metal-binding sites component：Calcium chloride, a concentration of 5mmol/L,

Manganese chloride, a concentration of 1mmol/L；

Metal-chelator component：Ethylenediamine tetra-acetic acid (EDTA), a concentration of 0.1mg/mL；

Surface active agent composition：Polyethylene glycol, a concentration of 0.1mg/mL；

Site sealant components：Gelatin, a concentration of 0.5g/mL；

Biological preservative component：Methylisothiazolinone, a concentration of 1.5mg/mL

B. low concentration calibration product, concentration：15.625IU/mL；

C. high concentration calibration object, concentration：500.000IU/mL；

D. AFP monoclonal antibodies are marked according to the ABEI that preparation 6 obtains

ABEI concentration：250ng/mL,

The concentration of AFP monoclonal antibody：5000ng/mL；

E.Buffer buffer solutions

The ingredient of Buffer buffer solutions：The borate buffer solution of 0.02mol/L pH7.2,5mmol/L sodium chloride, 1mmol/ L manganese chlorides.

A method of it is captured with above-mentioned kit and detaches and detect AFP-L3 in serum, step is specific as follows：

1) preparation of 10 standard curves

A concentration of 4.6*10^6IU/mL of AFP-L3 enterprises calibration object, is diluted to 0.02M PBS buffer solution The intermediate product of 1000IU/mL successively gradient dilution are respectively obtained the concentration point of ten point curves by 1000IU/mL, fixed by 2 points Working curve is calculated in mark calibration curve.

2) use of capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes

Capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes are given birth to using this method, in this department development ＆ production Maglumi 2000Plus instruments on detect, detecting step includes：

A. 20 μ L samples to be tested, high-concentration and low-concentration calibration object are added in reaction cup；

B. 20 μ L lentil lectins oxides-magnetic microsphere solution is added；

C. 100 μ L Buffer buffer solutions are added；

D.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

E. the AFP monoclonal antibody of 200 μ L ABEI labels is added；

F.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

G. luminous substrate is added, detects light signal strength；

H. sample to be tested can be calculated automatically according to pattern detection luminous intensity by the revised working curve of calibration object AFP-L3 concentration.

It captures the detection for detaching and detecting AFP-L3 chemical luminescence immune analysis reagent boxes to primary carcinoma of liver sample and answers With

The serum sample for having selected 3 primarys carcinoma of liver through clinical definite is measured by the method for agglutinin electrophoresis To the actual content of AFP-L3 in its sample, tried with the prepared AFP-L3 chemiluminescence immune assays of this embodiment scheme Agent box measures the content of AFP-L3 in its sample, and the prepared AFP-L3 chemistry of this programme is calculated by two measured concentrations The capture separative efficiency of the AFP-L3 of luminescence immunoassay kit.

Embodiment 4

A kind of method and its detection kit for enhancing AFP-L3 capture separative efficiencies

Prepare 1：Lentil lectin aoxidizes the preparation of modification

The lentil lectin that 2mg is dissolved with the 0.2mol/L sodium bicarbonate aqueous solutions of Fresh, in above-mentioned solution by The 0.5mL 8mmol/L sodium metaperiodate aqueous solutions of Fresh are added dropwise in drop, are protected from light, and reaction 2 hours is mixed slowly under the conditions of 25 DEG C, Obtain the oxidation product (LECTIN-CHO) of lentil lectin.

Prepare 2：Polyamide mediates the preparation of magnetic microsphere

A certain amount of magnetic ball is taken, the DCC of 0.5mg is added by every milligram of magnetic ball, ethanol solution, which is then added, makes the dense of magnetic ball Degree is 20mg/mL, is placed in 38 DEG C of water-baths and shakes or mechanical agitation 2 hours；Activated nanoscale magnetic bead solution is gone Except supernatant, the carbonate buffer solution of pH9.5 is then added, its concentration is made to reach 20mg/mL (in terms of nanoscale magnetic bead)；With every milli Gram magnetic ball adds 10 milligrams of aqueous polyamide solutions, and ultrasonic 10min rotates mixing and react 4h, discarded after Magneto separate at room temperature Clearly；It is washed 3 times with the PBS buffer solution of 0.01mol/L pH7.5, each 3min；With the ethanol amine capping 2h of 1mol/L；With The PBS buffer solution of 0.01mol/L pH7.5 is washed 3 times, is discarded supernatant after Magneto separate, is obtained the magnetic microsphere of amino functional, It is spare.

Prepare 3：Lentil lectin modification-polyamide mediates the preparation of magnetic ball conjugate

A certain amount of amino polymer functionalization magnetic ball is taken, is resuspended, is made with 0.1mol/L pH9.2 sodium bicarbonate aqueous solutions Its concentration reaches 20mg/mL (in terms of nanoscale magnetic bead), and the oxidation product of the lentil lectin of 0.5mg is added by every milligram of magnetic ball (LECTIN-CHO), after mixing, the NaBH of 5mg/mL is added₄Aqueous slkali, 25 DEG C are stirred to react 4 hours, and completely reacted magnetism is micro- Ball solution removes supernatant, cleans solid three times with washing lotion (0.5%BSA solution), it is coated to obtain lentil lectin oxide Magnetic microsphere.

Prepare 4：The preparation of lentil lectin oxide magnetic microballoon activity preservative fluid

(1) dilution component：Borate buffer solution, pH7.2, a concentration of 0.02mol/L；

(2) metal-binding sites component：Calcium chloride, a concentration of 5mmol/L,

Manganese chloride, a concentration of 1mmol/L,

(3) metal-chelator component：Ethylenediamine tetra-acetic acid (EDTA), a concentration of 0.1mg/mL；

(4) surface active agent composition：Polyethylene glycol, a concentration of 0.1mg/mL；

(5) site sealant components：Gelatin, a concentration of 0.5g/mL；

(6) biological preservative component：Methylisothiazolinone, a concentration of 1.5mg/mL.

Prepare 5：The preparation of AFP-L3 antigen capture reagents

The magnetic ball for preparing coating lentil lectin oxide in 3 is suspended in the immune magnetic activity for preparing and preparing in 4 to protect In liquid storage, AFP-L3 antigen capture reagents are obtained.

Prepare 6：ABEI marks AFP monoclonal antibody

1mg AFP monoclonal antibodies are taken, its volume are adjusted to 1mL with 0.1mol/L carbonate buffer solutions (pH9.5), so After be put into bag filter, be placed in pH9.5 carbonate buffer solutions and dialyse 1 hour.It is anti-to take out the anti-AFP monoclonals dialysed Body, is added 100 μ g ABEI-, half succinamic acid-NHS, and room temperature shakes 1.5 hours.G-25 gel columns are installed, purified water is used After elution is clean, then with the PBS buffer solution of pH value 7.4 it is balanced elution.After G-25 gel column equilibrations elute, it will mark The AFP monoclonal antibody of good ABEI crosses column purification, then collects the protein solution for peak value occur.The albumen gathered is molten Isometric protection liquid containing 5% BSA is added in liquid, and diluted is added to 0.15 μ g/mL.

A kind of kit of capture separation and detection AFP-L3, contains following components：

A. the AFP-L3 antigen capture reagents obtained according to preparation 5,

The 20 μ g/mL of concentration of agglutinin oxidation product,

Magnetic microsphere concentration：1mg/mL,

Dilution component：Borate buffer solution, pH7.2, a concentration of 0.02mol/L；

Metal-binding sites component：Calcium chloride, a concentration of 5mmol/L,

Manganese chloride, a concentration of 1mmol/L；

Metal-chelator component：Ethylenediamine tetra-acetic acid (EDTA), a concentration of 0.1mg/mL；

Surface active agent composition：Polyethylene glycol, a concentration of 0.1mg/mL；

Site sealant components：Gelatin, a concentration of 0.5g/mL；

Biological preservative component：Methylisothiazolinone, a concentration of 1.5mg/mL

B. low concentration calibration product, concentration：15.625IU/mL；

C. high concentration calibration object, concentration：500.000IU/mL；

The AFP monoclonal antibody of d.ABEI labels

ABEI working concentrations：250ng/mL,

The concentration of AFP monoclonal antibody：5000ng/mL；

E.Buffer buffer solutions

The ingredient of Buffer buffer solutions：The borate buffer solution of 0.02mol/L pH7.2,5mmol/L sodium chloride, 1mmol/ L manganese chlorides.

A method of it is captured with above-mentioned kit and detaches and detect AFP-L3 in serum, step is specific as follows：

1) preparation of 10 standard curves

A concentration of 4.6*10^6IU/mL of AFP-L3 enterprises calibration object, is diluted to 0.02M PBS buffer solution The intermediate product of 1000IU/mL successively gradient dilution are respectively obtained the concentration point of ten point curves by 1000IU/mL, fixed by 2 points Working curve is calculated in mark calibration curve.

2) use of capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes

Capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes are given birth to using this method, in this department development ＆ production Maglumi 2000Plus instruments on examine

It surveys, detecting step includes：

A. 20 μ L samples to be tested, high-concentration and low-concentration calibration object are added in reaction cup；

B. 20 μ L lentil lectins oxides-magnetic microsphere solution is added；

C. 100 μ L Buffer buffer solutions are added；

D.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

E. the AFP monoclonal antibody of 200 μ L ABEI labels is added；

F.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

G. luminous substrate is added, detects light signal strength；

H. sample to be tested can be calculated automatically according to pattern detection luminous intensity by the revised working curve of calibration object AFP-L3 concentration.

It captures the detection for detaching and detecting AFP-L3 chemical luminescence immune analysis reagent boxes to primary carcinoma of liver sample and answers With

The serum sample for having selected 3 primarys carcinoma of liver through clinical definite is measured by the method for agglutinin electrophoresis To the actual content of AFP-L3 in its sample, tried with the prepared AFP-L3 chemiluminescence immune assays of this embodiment scheme Agent box measures the content of AFP-L3 in its sample, and the prepared AFP-L3 chemistry of this programme is calculated by two measured concentrations The capture separative efficiency of the AFP-L3 of luminescence immunoassay kit.

Embodiment 5

A kind of method and its detection kit for enhancing AFP-L3 capture separative efficiencies

Prepare 1：Lentil lectin aoxidizes the preparation of modification

The lentil lectin that 2mg is dissolved with the 0.2mol/L sodium bicarbonate aqueous solutions of Fresh, in above-mentioned solution by The 0.5mL 8mmol/L sodium metaperiodate aqueous solutions of Fresh are added dropwise in drop, are protected from light, and reaction 2 hours is mixed slowly under the conditions of 25 DEG C, Obtain the oxidation product (LECTIN-CHO) of lentil lectin.

Prepare 2：The preparation of polyethyleneimine magnetic microsphere

A certain amount of magnetic ball is taken, the DCC of 0.5mg is added by every milligram of magnetic ball, ethanol solution, which is then added, makes the dense of magnetic ball Degree is 20mg/mL, is placed in 38 DEG C of water-baths and shakes or mechanical agitation 2 hours；Activated nanoscale magnetic bead solution is gone Except supernatant, the carbonate buffer solution of pH9.5 is then added, its concentration is made to reach 20mg/mL (in terms of nanoscale magnetic bead)；With every milli Gram magnetic ball adds 5 milligrams of aq. polyethyleneimines, and ultrasonic 10min rotates mixing and react 4h, discarded after Magneto separate at room temperature Supernatant；It is washed 3 times with the PBS buffer solution of 0.01mol/L pH7.5, each 3min；With the ethanol amine capping 2h of 1mol/L； It is washed 3 times with the PBS buffer solution of 0.01mol/L pH7.5, is discarded supernatant after Magneto separate, the magnetism for obtaining amino functional is micro- Ball, it is spare.

Prepare 3：The preparation of lentil lectin modification-polyethyleneimine magnetic ball conjugate

A certain amount of amino polymer functionalization magnetic ball is taken, is resuspended, is made with 0.1mol/L pH9.2 sodium bicarbonate aqueous solutions Its concentration reaches 20mg/mL (in terms of nanoscale magnetic bead), and the oxidation product of the lentil lectin of 0.5mg is added by every milligram of magnetic ball (LECTIN-CHO), after mixing, the NaBH of 5mg/mL is added₄Aqueous slkali, 25 DEG C are stirred to react 4 hours, and completely reacted magnetism is micro- Ball solution removes supernatant, cleans solid three times with washing lotion (0.5%BSA solution), it is coated to obtain lentil lectin oxide Magnetic microsphere.

Prepare 4：The preparation of lentil lectin oxide magnetic microballoon activity preservative fluid

(1) dilution component：Borate buffer solution, pH7.2, a concentration of 0.02mol/L；

(2) metal-binding sites component：Calcium chloride, a concentration of 5mmol/L,

Manganese chloride, a concentration of 1mmol/L,

(3) metal-chelator component：Ethylenediamine tetra-acetic acid (EDTA), a concentration of 0.1mg/mL；

(4) surface active agent composition：Polyethylene glycol, a concentration of 0.1mg/mL；

(5) site sealant components：Gelatin, a concentration of 0.5g/mL；

(6) biological preservative component：Methylisothiazolinone, a concentration of 1.5mg/mL.

Prepare 5：The preparation of AFP-L3 antigen capture reagents

The magnetic ball for preparing coating lentil lectin oxide in 3 is suspended in the immune magnetic activity for preparing and preparing in 4 to protect In liquid storage, AFP-L3 antigen capture reagents are obtained.

Prepare 6：Luminescent label AFP monoclonal antibody

1mg AFP monoclonal antibodies are taken, its volume are adjusted to 1mL with 0.1mol/L carbonate buffer solutions (pH9.5), so After be put into bag filter, be placed in pH9.5 carbonate buffer solutions and dialyse 1 hour.It is anti-to take out the anti-AFP monoclonals dialysed Body, is added 100 μ g ABEI-, half succinamic acid-NHS, and room temperature shakes 1.5 hours.G-25 gel columns are installed, purified water is used After elution is clean, then with the PBS buffer solution of pH value 7.4 it is balanced elution.After G-25 gel column equilibrations elute, it will mark The AFP monoclonal antibody of good ABEI crosses column purification, then collects the protein solution for peak value occur.The albumen gathered is molten Isometric protection liquid containing 5% BSA is added in liquid, and diluted is added to 0.15 μ g/mL.

A kind of kit of capture separation and detection AFP-L3, contains following components：

A. the AFP-L3 antigen capture reagents obtained according to preparation 5,

The 20 μ g/mL of concentration of agglutinin oxidation product,

Magnetic microsphere concentration：1mg/mL,

Dilution component：Borate buffer solution, pH7.2, a concentration of 0.02mol/L；

Metal-binding sites component：Calcium chloride, a concentration of 5mmol/L,

Manganese chloride, working concentration 1mmol/L；

Metal-chelator component：Ethylenediamine tetra-acetic acid (EDTA), a concentration of 0.1mg/mL；

Surface active agent composition：Polyethylene glycol, a concentration of 0.1mg/mL；

Site sealant components：Gelatin, a concentration of 0.5g/mL；

Biological preservative component：Methylisothiazolinone, a concentration of 1.5mg/mL

B. low concentration calibration product, concentration：15.625IU/mL；

C. high concentration calibration object, concentration：500.000IU/mL；

The AFP monoclonal antibody of d.ABEI labels

ABEI concentration：250ng/mL,

The concentration of AFP monoclonal antibody：5000ng/mL；

E.Buffer buffer solutions

The ingredient of Buffer buffer solutions：The borate buffer solution of 0.02mol/L pH7.2,5mmol/L sodium chloride, 1mmol/ L manganese chlorides.

A kind of to capture the foundation for detaching and detecting AFP-L3 methods in serum with above-mentioned kit, step is specific as follows：

1) preparation of 10 standard curves

A concentration of 4.6*10^6IU/mL of AFP-L3 enterprises calibration object, is diluted to 0.02M PBS buffer solution The intermediate product of 1000IU/mL successively gradient dilution are respectively obtained the concentration point of ten point curves by 1000IU/mL, fixed by 2 points Working curve is calculated in mark calibration curve.

2) use of capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes

Capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes are given birth to using this method, in this department development ＆ production Maglumi 2000Plus instruments on examine

It surveys, detecting step includes：

A. 20 μ L samples to be tested, high-concentration and low-concentration calibration object are added in reaction cup；

B. 20 μ L lentil lectins oxides-magnetic microsphere solution is added；

C. 100 μ L Buffer buffer solutions are added；

D.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

E. the AFP monoclonal antibody of 200 μ L ABEI labels is added；

F.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

G. luminous substrate is added, detects light signal strength；

H. sample to be tested can be calculated automatically according to pattern detection luminous intensity by the revised working curve of calibration object AFP-L3 concentration.

It captures the detection for detaching and detecting AFP-L3 chemical luminescence immune analysis reagent boxes to primary carcinoma of liver sample and answers With

The serum sample for having selected 3 primarys carcinoma of liver through clinical definite is measured by the method for agglutinin electrophoresis To the actual content of AFP-L3 in its sample, tried with the prepared AFP-L3 chemiluminescence immune assays of this embodiment scheme Agent box measures the content of AFP-L3 in its sample, and the prepared AFP-L3 chemistry of this programme is calculated by two measured concentrations The capture separative efficiency of the AFP-L3 of luminescence immunoassay kit.

Embodiment 6

A kind of method and its detection kit for enhancing AFP-L3 capture separative efficiencies

Prepare 1：Lentil lectin aoxidizes the preparation of modification

The lentil lectin that 2mg is dissolved with the 0.2mol/L sodium bicarbonate aqueous solutions of Fresh, in above-mentioned solution by The 0.5mL 8mmol/L sodium metaperiodate aqueous solutions of Fresh are added dropwise in drop, are protected from light, and reaction 2 hours is mixed slowly under the conditions of 25 DEG C, Obtain the oxidation product (LECTIN-CHO) of lentil lectin.

Prepare 2：The preparation of polyethyleneimine magnetic microsphere

A certain amount of magnetic ball is taken, the DCC of 0.5mg is added by every milligram of magnetic ball, ethanol solution, which is then added, makes the dense of magnetic ball Degree is 20mg/mL, is placed in 38 DEG C of water-baths and shakes or mechanical agitation 2 hours；Activated nanoscale magnetic bead solution is gone Except supernatant, the carbonate buffer solution of pH9.5 is then added, its concentration is made to reach 20mg/mL (in terms of nanoscale magnetic bead)；With every milli Gram magnetic ball adds 10 milligrams of aq. polyethyleneimines, and ultrasonic 10min rotates mixing and react 4h, abandoned after Magneto separate at room temperature Remove supernatant；It is washed 3 times with the PBS buffer solution of 0.01mol/L pH7.5, each 3min；With the ethanol amine capping of 1mol/L 2h；It is washed 3 times with the PBS buffer solution of 0.01mol/L pH7.5, is discarded supernatant after Magneto separate, obtain the magnetism of amino functional Microballoon, it is spare.

Prepare 3：The preparation of lentil lectin modification-polyethyleneimine magnetic ball conjugate

A certain amount of amino polymer functionalization magnetic ball is taken, is resuspended, is made with 0.1mol/L pH9.2 sodium bicarbonate aqueous solutions Its concentration reaches 20mg/mL (in terms of nanoscale magnetic bead), and the oxidation product of the lentil lectin of 0.5mg is added by every milligram of magnetic ball (LECTIN-CHO), after mixing, the NaBH of 5mg/mL is added₄Aqueous slkali, 25 DEG C are stirred to react 4 hours, and completely reacted magnetism is micro- Ball solution removes supernatant, cleans solid three times with washing lotion (0.5%BSA solution), it is coated to obtain lentil lectin oxide Magnetic microsphere.

Prepare 4：The preparation of lentil lectin oxide magnetic microballoon activity preservative fluid

(1) dilution component：Borate buffer solution, pH7.2, a concentration of 0.02mol/L；

(2) metal-binding sites component：Calcium chloride, a concentration of 5mmol/L,

Manganese chloride, a concentration of 1mmol/L,

(3) metal-chelator component：Ethylenediamine tetra-acetic acid (EDTA), a concentration of 0.1mg/mL；

(4) biological preservative component：Methylisothiazolinone, a concentration of 1.5mg/mL.

Prepare 5：The preparation of AFP-L3 antigen capture reagents

The magnetic ball for preparing coating lentil lectin oxide in 3 is suspended in the immune magnetic activity for preparing and preparing in 4 to protect In liquid storage, AFP-L3 antigen capture reagents are obtained.

Prepare 6：Luminescent label AFP monoclonal antibody

1mg AFP monoclonal antibodies are taken, its volume are adjusted to 1mL with 0.1mol/L carbonate buffer solutions (pH9.5), so After be put into bag filter, be placed in pH9.5 carbonate buffer solutions and dialyse 1 hour.It is anti-to take out the anti-AFP monoclonals dialysed Body, is added 100 μ g ABEI-, half succinamic acid-NHS, and room temperature shakes 1.5 hours.G-25 gel columns are installed, purified water is used After elution is clean, then with the PBS buffer solution of pH value 7.4 it is balanced elution.After G-25 gel column equilibrations elute, it will mark The AFP monoclonal antibody of good ABEI crosses column purification, then collects the protein solution for peak value occur.The albumen gathered is molten Isometric protection liquid containing 5% BSA is added in liquid, and diluted is added to 0.15 μ g/mL.

A kind of kit of capture separation and detection AFP-L3, contains following components：

A. the AFP-L3 antigen capture reagents obtained according to preparation 5,

The 20 μ g/mL of concentration of agglutinin oxidation product,

Magnetic microsphere concentration：1mg/mL,

Dilution component：Borate buffer solution, pH7.2, a concentration of 0.02mol/L；

Metal-binding sites component：Calcium chloride, a concentration of 5mmol/L,

Manganese chloride, working concentration 1mmol/L；

Metal-chelator component：Ethylenediamine tetra-acetic acid (EDTA), a concentration of 0.1mg/mL；

Biological preservative component：Methylisothiazolinone, a concentration of 1.5mg/mL

B. low concentration calibration product, concentration：15.625IU/mL；

C. high concentration calibration object, concentration：500.000IU/mL；

The AFP monoclonal antibody of d.ABEI labels

ABEI concentration：250ng/mL,

The concentration of AFP monoclonal antibody：5000ng/mL；

E.Buffer buffer solutions

The ingredient of Buffer buffer solutions：The borate buffer solution of 0.02mol/L pH7.2,5mmol/L sodium chloride, 1mmol/ L manganese chlorides.

A kind of to capture the foundation for detaching and detecting AFP-L3 methods in serum with above-mentioned kit, step is specific as follows：

1) preparation of 10 standard curves

A concentration of 4.6*10^6IU/mL of AFP-L3 enterprises calibration object, is diluted to 0.02M PBS buffer solution The intermediate product of 1000IU/mL successively gradient dilution are respectively obtained the concentration point of ten point curves by 1000IU/mL, fixed by 2 points Working curve is calculated in mark calibration curve.

2) use of capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes

Capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes are given birth to using this method, in this department development ＆ production Maglumi 2000Plus instruments on detect, detecting step includes：

A. 20 μ L samples to be tested, high-concentration and low-concentration calibration object are added in reaction cup；

B. 20 μ L lentil lectins oxides-magnetic microsphere solution is added；

C. 100 μ L Buffer buffer solutions are added；

D.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

E. the AFP monoclonal antibody of 200 μ L ABEI labels is added；

F.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

G. luminous substrate is added, detects light signal strength；

H. sample to be tested can be calculated automatically according to pattern detection luminous intensity by the revised working curve of calibration object AFP-L3 concentration.

It captures the detection for detaching and detecting AFP-L3 chemical luminescence immune analysis reagent boxes to primary carcinoma of liver sample and answers With

The serum sample for having selected 3 primarys carcinoma of liver through clinical definite is measured by the method for agglutinin electrophoresis To the actual content of AFP-L3 in its sample, tried with the prepared AFP-L3 chemiluminescence immune assays of this embodiment scheme Agent box measures the content of AFP-L3 in its sample, and the prepared AFP-L3 chemistry of this programme is calculated by two measured concentrations The capture separative efficiency of the AFP-L3 of luminescence immunoassay kit.

Embodiment 7

A kind of method and its detection kit for enhancing AFP-L3 capture separative efficiencies

Prepare 1：ConA aoxidizes the preparation of modification

The ConA that 2mg is dissolved with the 0.2mol/L sodium bicarbonate aqueous solutions of Fresh, in above-mentioned solution by The 0.5mL 8mmol/L sodium metaperiodate aqueous solutions of Fresh are added dropwise in drop, are protected from light, and reaction 2 hours is mixed slowly under the conditions of 25 DEG C, Obtain the oxidation product of ConA.

Prepare 2：The preparation of polyethyleneimine magnetic microsphere

A certain amount of magnetic ball is taken, the DCC of 0.5mg is added by every milligram of magnetic ball, ethanol solution, which is then added, makes the dense of magnetic ball Degree is 20mg/mL, is placed in 38 DEG C of water-baths and shakes or mechanical agitation 2 hours；Activated nanoscale magnetic bead solution is gone Except supernatant, the carbonate buffer solution of pH9.5 is then added, its concentration is made to reach 20mg/mL (in terms of nanoscale magnetic bead)；With every milli Gram magnetic ball adds 10 milligrams of aq. polyethyleneimines, and ultrasonic 10min rotates mixing and react 4h, abandoned after Magneto separate at room temperature Remove supernatant；It is washed 3 times with the PBS buffer solution of 0.01mol/L pH7.5, each 3min；With the ethanol amine capping of 1mol/L 2h；It is washed 3 times with the PBS buffer solution of 0.01mol/L pH7.5, is discarded supernatant after Magneto separate, obtain the magnetism of amino functional Microballoon, it is spare.

Prepare 3：The preparation of ConA modification-polyethyleneimine magnetic ball conjugate

A certain amount of amino polymer functionalization magnetic ball is taken, is resuspended, is made with 0.1mol/L pH9.2 sodium bicarbonate aqueous solutions Its concentration reaches 20mg/mL (in terms of nanoscale magnetic bead), and the oxidation product of the ConA of 0.5mg is added by every milligram of magnetic ball (LECTIN-CHO), after mixing, the NaBH of 5mg/mL is added₄Aqueous slkali, 25 DEG C are stirred to react 4 hours, and completely reacted magnetism is micro- Ball solution removes supernatant, cleans solid three times with washing lotion (0.5%BSA solution), it is coated to obtain ConA oxide Magnetic microsphere.

Prepare 4：The preparation of ConA oxide magnetic microballoon activity preservative fluid

(1) dilution component：Phosphate buffer, pH7.2, a concentration of 0.02mol/L；

(2) metal-binding sites component：Calcium nitrate, a concentration of 5mmol/L,

Manganese nitrate, a concentration of 1mmol/L,

(3) metal-chelator component：EDTAP dipotassium ethylene diamine tetraacetate, a concentration of 0.1mg/mL；

(4) surface active agent composition：Polyethylene glycol, a concentration of 0.1mg/mL；

(5) site sealant components：Gelatin, a concentration of 0.5g/mL；

(6) biological preservative component：Sodium azide, a concentration of 1.5mg/mL.

Prepare 5：The preparation of AFP-L3 antigen capture reagents

The magnetic ball for preparing coating ConA oxide in 3 is suspended in the immune magnetic activity for preparing and preparing in 4 to protect In liquid storage, AFP-L3 antigen capture reagents are obtained.

Prepare 6：Luminescent label AFP monoclonal antibody

1mg AFP monoclonal antibodies are taken, its volume are adjusted to 1mL with 0.1mol/L carbonate buffer solutions (pH9.5), so After be put into bag filter, be placed in pH9.5 carbonate buffer solutions and dialyse 1 hour.It is anti-to take out the anti-AFP monoclonals dialysed Body, is added 100 μ g ABEI-, half succinamic acid-NHS, and room temperature shakes 1.5 hours.G-25 gel columns are installed, purified water is used After elution is clean, then with the PBS buffer solution of pH value 7.4 it is balanced elution.After G-25 gel column equilibrations elute, it will mark The AFP monoclonal antibody of good ABEI crosses column purification, then collects the protein solution for peak value occur.The albumen gathered is molten Isometric protection liquid containing 5% BSA is added in liquid, and diluted is added to 0.15 μ g/mL.

A kind of kit of capture separation and detection AFP-L3, contains following components：

A. the AFP-L3 antigen capture reagents obtained according to preparation 5,

The 20 μ g/mL of concentration of agglutinin oxidation product,

Magnetic microsphere concentration：1mg/mL,

Dilution component：Phosphate buffer, pH7.2, a concentration of 0.02mol/L；

Metal-binding sites component：Calcium nitrate, a concentration of 5mmol/L,

Manganese nitrate, working concentration 1mmol/L；

Metal-chelator component：EDTAP dipotassium ethylene diamine tetraacetate, a concentration of 0.1mg/mL；

Surface active agent composition：Polyethylene glycol, a concentration of 0.1mg/mL；

Site sealant components：Gelatin, a concentration of 0.5g/mL；

Biological preservative component：Sodium azide, a concentration of 1.5mg/mL

B. low concentration calibration product, concentration：15.625IU/mL；

C. high concentration calibration object, concentration：500.000IU/mL；

The AFP monoclonal antibody of d.ABEI labels

ABEI concentration：250ng/mL,

The concentration of AFP monoclonal antibody：5000ng/mL；

E.Buffer buffer solutions

The ingredient of Buffer buffer solutions：The borate buffer solution of 0.02mol/L pH7.2,5mmol/L sodium chloride, 1mmol/ L manganese chlorides.

A method of it is captured with above-mentioned kit and detaches and detect AFP-L3 in serum, step is specific as follows：

1) preparation of 10 standard curves

A concentration of 4.6*10^6IU/mL of AFP-L3 enterprises calibration object, is diluted to 0.02M PBS buffer solution The intermediate product of 1000IU/mL successively gradient dilution are respectively obtained the concentration point of ten point curves by 1000IU/mL, fixed by 2 points Working curve is calculated in mark calibration curve.

2) use of capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes

Capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes are given birth to using this method, in this department development ＆ production Maglumi 2000Plus instruments on examine

It surveys, detecting step includes：

A. 20 μ L samples to be tested, high-concentration and low-concentration calibration object are added in reaction cup；

B. 20 μ L ConAs oxides-magnetic microsphere solution is added；

C. 100 μ L Buffer buffer solutions are added；

D.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

E. the AFP monoclonal antibody of 200 μ L ABEI labels is added；

F.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

G. luminous substrate is added, detects light signal strength；

H. sample to be tested can be calculated automatically according to pattern detection luminous intensity by the revised working curve of calibration object AFP-L3 concentration.

It captures the detection for detaching and detecting AFP-L3 chemical luminescence immune analysis reagent boxes to primary carcinoma of liver sample and answers With

The serum sample for having selected 3 primarys carcinoma of liver through clinical definite is measured by the method for agglutinin electrophoresis To the actual content of AFP-L3 in its sample, tried with the prepared AFP-L3 chemiluminescence immune assays of this embodiment scheme Agent box measures the content of AFP-L3 in its sample, and the prepared AFP-L3 chemistry of this programme is calculated by two measured concentrations The capture separative efficiency of the AFP-L3 of luminescence immunoassay kit.

Comparative example 1

Prepare 1：Lentil lectin is coated with the preparation of magnetic microsphere

A certain amount of magnetic microsphere is taken, is resuspended with 0.1mol/L pH9.2 sodium bicarbonate aqueous solutions, its concentration is made to reach The lentil lectin of 0.5mg is added by every milligram of magnetic ball, after mixing, is added 5mg/mL's by 20mg/mL (in terms of nanoscale magnetic bead) NaBH₄Aqueous slkali, 25 DEG C are stirred to react 4 hours, and completely reacted magnetic microsphere solution removes supernatant, with washing lotion (0.5%BSA Solution) clean solid three times, obtain the coated magnetic microsphere of lentil lectin.

Prepare 2：ABEI marks AFP monoclonal antibody

1mg AFP monoclonal antibodies are taken, its volume are adjusted to 1mL with 0.1mol/L carbonate buffer solutions (pH9.5), so After be put into bag filter, be placed in pH9.5 carbonate buffer solutions and dialyse 1 hour.It is anti-to take out the anti-AFP monoclonals dialysed Body, is added 100 μ g ABEI-, half succinamic acid-NHS, and room temperature shakes 1.5 hours.G-25 gel columns are installed, purified water is used After elution is clean, then with the PBS buffer solution of pH value 7.4 it is balanced elution.After G-25 gel column equilibrations elute, it will mark The AFP monoclonal antibody of good ABEI crosses column purification, then collects the protein solution for peak value occur.The albumen gathered is molten Isometric protection liquid containing 5% BSA is added in liquid, and diluted is added to 0.15 μ g/mL.

A kind of kit of capture separation and detection AFP-L3, contains following components：

A. the AFP-L3 antigen capture reagents obtained according to preparation 1,

The 20 μ g/mL of concentration of agglutinin,

Magnetic microsphere concentration：1mg/mL,

B. low concentration calibration product, concentration：15.625IU/mL；

C. high concentration calibration object, concentration：500.000IU/mL；

D. according to the AFP monoclonal antibody for preparing the ABEI labels that 2 obtain

ABEI working concentrations：250ng/mL,

The working concentration of AFP monoclonal antibody：5000ng/mL；

E.Buffer buffer solutions

The ingredient of Buffer buffer solutions：The borate buffer solution of 0.02mol/L pH7.2,5mmol/L sodium chloride, 1mmol/ L manganese chlorides.

A method of it is captured with above-mentioned kit and detaches and detect AFP-L3 in serum, step is specific as follows：

1) preparation of 10 standard curves

A concentration of 4.6*10^6IU/mL of AFP-L3 enterprises calibration object, is diluted to 0.02M PBS buffer solution The intermediate product of 1000IU/mL successively gradient dilution are respectively obtained the concentration point of ten point curves by 1000IU/mL, fixed by 2 points Working curve is calculated in mark calibration curve.

2) use of capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes

Capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes are given birth to using this method, in this department development ＆ production Maglumi 2000Plus instruments on detect, detecting step includes：

A. 20 μ L samples to be tested, high-concentration and low-concentration calibration object are added in reaction cup；

B. 20 μ L lentil lectins-magnetic microsphere solution is added；

C. 100 μ L Buffer buffer solutions are added；

D.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

E. the AFP monoclonal antibody of 200 μ L ABEI labels is added；

F.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

G. luminous substrate is added, detects light signal strength；

H. sample to be tested can be calculated automatically according to pattern detection luminous intensity by the revised working curve of calibration object AFP-L3 concentration.

It captures the detection for detaching and detecting AFP-L3 chemical luminescence immune analysis reagent boxes to primary carcinoma of liver sample and answers With

The serum sample for having selected 3 primarys carcinoma of liver through clinical definite is measured by the method for agglutinin electrophoresis It is tried to the actual content of AFP-L3 in its sample, then with the prepared AFP-L3 chemiluminescence immune assays of this embodiment scheme Agent box measures the content of AFP-L3 in its sample, and the prepared AFP-L3 chemistry of this programme is calculated by two measured concentrations The capture separative efficiency of the AFP-L3 of luminescence immunoassay kit.

Comparative example 2

The preparation of polyethyleneimine magnetic microsphere

A certain amount of magnetic ball is taken, the DCC of 0.5mg is added by every milligram of magnetic ball, ethanol solution, which is then added, makes the dense of magnetic ball Degree is 20mg/mL, is placed in 38 DEG C of water-baths and shakes or mechanical agitation 2 hours；Activated nanoscale magnetic bead solution is gone Except supernatant, the carbonate buffer solution of pH9.5 is then added, its concentration is made to reach 20mg/mL (in terms of nanoscale magnetic bead)；With every milli Gram magnetic ball adds 10 milligrams of aq. polyethyleneimines, and ultrasonic 10min rotates mixing and react 4h, abandoned after Magneto separate at room temperature Remove supernatant；It is washed 3 times with the PBS buffer solution of 0.01mol/L pH7.5, each 3min；With the ethanol amine capping of 1mol/L 2h；It is washed 3 times with the PBS buffer solution of 0.01mol/L pH7.5, is discarded supernatant after Magneto separate, obtain the magnetism of amino functional Microballoon, it is spare.

Prepare 2：Lentil lectin is coated with the preparation of polyethyleneimine magnetic microsphere

A certain amount of magnetic ball is taken, is resuspended with 0.1mol/L pH9.2 sodium bicarbonate aqueous solutions, its concentration is made to reach 20mg/mL (in terms of nanoscale magnetic bead) lentil lectin of 0.5mg is added by every milligram of magnetic ball, after mixing, the NaBH of 5mg/mL is added₄It is molten Liquid, 25 DEG C are stirred to react 4 hours, and completely reacted magnetic microsphere solution removes supernatant, is cleaned with washing lotion (0.5%BSA solution) Solid three times obtains the coated magnetic microsphere of lentil lectin.

Prepare 3：The preparation of activity preservative fluid

(1) dilution component：Borate buffer solution, pH7.2, a concentration of 0.02mol/L；

(2) metal-binding sites component：Calcium chloride, a concentration of 5mmol/L,

Manganese chloride, a concentration of 1mmol/L,

(3) metal-chelator component：Ethylenediamine tetra-acetic acid (EDTA), a concentration of 0.1mg/mL；

(4) surface active agent composition：Polyethylene glycol, a concentration of 0.1mg/mL；

(5) site sealant components：Gelatin, a concentration of 0.5g/mL；

(6) biological preservative component：Methylisothiazolinone, a concentration of 1.5mg/mL.

Prepare 4：The preparation of AFP-L3 antigen capture reagents

Coating lentil lectin coating magnetic microsphere in 2 will be prepared and be suspended in the immune magnetic activity guarantor for preparing and being prepared in 3 In liquid storage, the capture agent of AFP-L3 antigens is obtained.

Prepare 5：ABEI marks AFP monoclonal antibody

1mg AFP monoclonal antibodies are taken, its volume are adjusted to 1mL with 0.1mol/L carbonate buffer solutions (pH9.5), so After be put into bag filter, be placed in pH9.5 carbonate buffer solutions and dialyse 1 hour.It is anti-to take out the anti-AFP monoclonals dialysed Body, is added 100 μ g ABEI-, half succinamic acid-NHS, and room temperature shakes 1.5 hours.G-25 gel columns are installed, purified water is used After elution is clean, then with the PBS buffer solution of pH value 7.4 it is balanced elution.After G-25 gel column equilibrations elute, it will mark The AFP monoclonal antibody of good ABEI crosses column purification, then collects the protein solution for peak value occur.The albumen gathered is molten Isometric protection liquid containing 5% BSA is added in liquid, and diluted is added to 0.15 μ g/mL.

A kind of kit of capture separation and detection AFP-L3, contains following components：

A. the AFP-L3 antigen capture reagents obtained according to preparation 4,

The 20 μ g/mL of concentration of agglutinin,

Magnetic microsphere concentration：1mg/mL,

Dilution component：Borate buffer solution, pH7.2, working concentration 0.02mol/L；

Metal-binding sites component：Calcium chloride, working concentration 5mmol/L,

Manganese chloride, working concentration 1mmol/L；

Metal-chelator component：Ethylenediamine tetra-acetic acid (EDTA), working concentration 0.1mg/mL；

Surface active agent composition：Polyethylene glycol, working concentration 0.1mg/mL；

Site sealant components：Gelatin, working concentration 0.5g/mL；

Biological preservative component：Methylisothiazolinone, working concentration 1.5mg/mL

B. low concentration calibration product, concentration：15.625IU/mL；

C. high concentration calibration object, concentration：500.000IU/mL；

D. according to the AFP monoclonal antibody for preparing the ABEI labels that 5 obtain

ABEI working concentrations：250ng/mL,

The working concentration of AFP monoclonal antibody：5000ng/mL；

E.Buffer buffer solutions

The ingredient of Buffer buffer solutions：The borate buffer solution of 0.02mol/L pH7.2,5mmol/L sodium chloride, 1mmol/ L manganese chlorides.

A kind of to capture the foundation for detaching and detecting AFP-L3 methods in serum with above-mentioned kit, step is specific as follows：

1) preparation of 10 standard curves

A concentration of 4.6*10^6IU/mL of AFP-L3 enterprises calibration object, is diluted to 0.02M PBS buffer solution The intermediate product of 1000IU/mL successively gradient dilution are respectively obtained the concentration point of ten point curves by 1000IU/mL, fixed by 2 points Working curve is calculated in mark calibration curve.

2) use of capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes

Capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes are given birth to using this method, in this department development ＆ production Maglumi 2000Plus instruments on detect, detecting step includes：

A. 20 μ L samples to be tested, high-concentration and low-concentration calibration object are added in reaction cup；

B. 20 μ L lentil lectins-magnetic microsphere solution is added；

C. 100 μ L Buffer buffer solutions are added；

D.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

E. the AFP monoclonal antibody of 200 μ L ABEI labels is added；

F.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

G. luminous substrate is added, detects light signal strength；

H. sample to be tested can be calculated automatically according to pattern detection luminous intensity by the revised working curve of calibration object AFP-L3 concentration.

It captures the detection for detaching and detecting AFP-L3 chemical luminescence immune analysis reagent boxes to primary carcinoma of liver sample and answers With

The serum sample for having selected 3 primarys carcinoma of liver through clinical definite is measured by the method for agglutinin electrophoresis It is tried to the actual content of AFP-L3 in its sample, then with the prepared AFP-L3 chemiluminescence immune assays of this embodiment scheme Agent box measures the content of AFP-L3 in its sample, and the prepared AFP-L3 chemistry of this programme is calculated by two measured concentrations The capture separative efficiency of the AFP-L3 of luminescence immunoassay kit.

Comparative example 3

A kind of method and its detection kit for enhancing AFP-L3 capture separative efficiencies

Prepare 1：Lentil lectin aoxidizes the preparation of modification

The lentil lectin that 2mg is dissolved with the 0.2mol/L sodium bicarbonate aqueous solutions of Fresh, in above-mentioned solution by The 0.5mL 8mmol/L sodium metaperiodate aqueous solutions of Fresh are added dropwise in drop, are protected from light, and reaction 2 hours is mixed slowly under the conditions of 25 DEG C, Obtain the oxidation product (LECTIN-CHO) of lentil lectin.

Prepare 2：Lentil lectin modification is coated with the preparation of magnetic ball conjugate

A certain amount of magnetic microsphere is taken, is resuspended with 0.1mol/L pH9.2 sodium bicarbonate aqueous solutions, its concentration is made to reach Oxidation product (the LECTIN- of the lentil lectin of 0.5mg is added by every milligram of magnetic ball by 20mg/mL (in terms of nanoscale magnetic bead) CHO), after mixing, the NaBH of 5mg/mL is added₄Aqueous slkali, 25 DEG C are stirred to react 4 hours, and completely reacted magnetic microsphere solution is gone Except supernatant, cleans solid three times with washing lotion (0.5%BSA solution), obtain the coated magnetic microsphere of lentil lectin oxide.

Prepare 3：The preparation of lentil lectin oxide magnetic microballoon activity preservative fluid

(1) dilution component：Borate buffer solution, pH7.2, a concentration of 0.02mol/L；

(2) metal-binding sites component：Calcium chloride, a concentration of 5mmol/L,

Manganese chloride, a concentration of 1mmol/L,

(3) metal-chelator component：Ethylenediamine tetra-acetic acid (EDTA), a concentration of 0.1mg/mL；

(4) surface active agent composition：Polyethylene glycol, a concentration of 0.1mg/mL；

(5) site sealant components：Gelatin, a concentration of 0.5g/mL；

(6) biological preservative component：Methylisothiazolinone, a concentration of 1.5mg/mL.

Prepare 4：The preparation of AFP-L3 antigen capture reagents

The magnetic ball for preparing coating lentil lectin oxide in 2 is suspended in the immune magnetic activity for preparing and preparing in 3 to protect In liquid storage, the capture agent of AFP-L3 antigens is obtained.

Prepare 5：ABEI marks AFP monoclonal antibody

1mg AFP monoclonal antibodies are taken, its volume are adjusted to 1mL with 0.1mol/L carbonate buffer solutions (pH9.5), so After be put into bag filter, be placed in pH9.5 carbonate buffer solutions and dialyse 1 hour.It is anti-to take out the anti-AFP monoclonals dialysed Body, is added 100 μ g ABEI-, half succinamic acid-NHS, and room temperature shakes 1.5 hours.G-25 gel columns are installed, purified water is used After elution is clean, then with the PBS buffer solution of pH value 7.4 it is balanced elution.After G-25 gel column equilibrations elute, it will mark The AFP monoclonal antibody of good ABEI crosses column purification, then collects the protein solution for peak value occur.The albumen gathered is molten Isometric protection liquid containing 5% BSA is added in liquid, and diluted is added to 0.15 μ g/mL.

A kind of kit of capture separation and detection AFP-L3, contains following components：

A. the AFP-L3 antigen capture reagents obtained according to preparation 4,

The 20 μ g/mL of concentration of agglutinin oxidation product,

Magnetic microsphere concentration：1mg/mL,

Dilution component：Borate buffer solution, pH7.2, a concentration of 0.02mol/L；

Metal-binding sites component：Calcium chloride, a concentration of 5mmol/L,

Manganese chloride, a concentration of 1mmol/L；

Metal-chelator component：Ethylenediamine tetra-acetic acid (EDTA), a concentration of 0.1mg/mL；

Surface active agent composition：Polyethylene glycol, a concentration of 0.1mg/mL；

Site sealant components：Gelatin, a concentration of 0.5g/mL；

Biological preservative component：Methylisothiazolinone, a concentration of 1.5mg/mL

B. low concentration calibration product, concentration：15.625IU/mL；

C. high concentration calibration object, concentration：500.000IU/mL；

D. according to the AFP monoclonal antibody for preparing the ABEI labels that 5 obtain

ABEI concentration：250ng/mL,

The concentration of AFP monoclonal antibody：5000ng/mL；

E.Buffer buffer solutions

The ingredient of Buffer buffer solutions：The borate buffer solution of 0.02mol/L pH7.2,5mmol/L sodium chloride, 1mmol/ L manganese chlorides.

A kind of to capture the foundation for detaching and detecting AFP-L3 methods in serum with above-mentioned kit, step is specific as follows：

1) preparation of 10 standard curves

A concentration of 4.6*10^6IU/mL of AFP-L3 enterprises calibration object, is diluted to 0.02M PBS buffer solution The intermediate product of 1000IU/mL successively gradient dilution are respectively obtained the concentration point of ten point curves by 1000IU/mL, fixed by 2 points Working curve is calculated in mark calibration curve.

2) capture separation and the use of detection AFP-L3 chemical luminescence immune analysis reagent boxes utilize this method life capture point From with detection AFP-L3 chemical luminescence immune analysis reagent boxes, examined on the Maglumi 2000Plus instruments of this department development ＆ production It surveys, detecting step includes：

A. 20 μ L samples to be tested, high-concentration and low-concentration calibration object are added in reaction cup；

B. 20 μ L lentil lectins oxides-magnetic microsphere solution is added；

C. 100 μ L Buffer buffer solutions are added；

D.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

E. the AFP monoclonal antibody of 200 μ L ABEI labels is added；

F.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

G. luminous substrate is added, detects light signal strength；

H. sample to be tested can be calculated automatically according to pattern detection luminous intensity by the revised working curve of calibration object AFP-L3 concentration.

It captures the detection for detaching and detecting AFP-L3 chemical luminescence immune analysis reagent boxes to primary carcinoma of liver sample and answers With

The serum sample for having selected 3 primarys carcinoma of liver through clinical definite is measured by the method for agglutinin electrophoresis It is tried to the actual content of AFP-L3 in its sample, then with the prepared AFP-L3 chemiluminescence immune assays of this embodiment scheme Agent box measures the content of AFP-L3 in its sample, and the prepared AFP-L3 chemistry of this programme is calculated by two measured concentrations The capture separative efficiency of the AFP-L3 of luminescence immunoassay kit.

Comparative example 4

Prepare 1：Lentil lectin aoxidizes the preparation of modification

The lentil lectin that 2mg is dissolved with the 0.2mol/L sodium bicarbonate aqueous solutions of Fresh, in above-mentioned solution by The 0.5mL 8mmol/L sodium metaperiodate aqueous solutions of Fresh are added dropwise in drop, are protected from light, and reaction 2 hours is mixed slowly under the conditions of 25 DEG C, Obtain the oxidation product (LECTIN-CHO) of lentil lectin.

Prepare 2：The preparation of polyethyleneimine magnetic microsphere

A certain amount of magnetic ball is taken, the DCC of 0.5mg is added by every milligram of magnetic ball, ethanol solution, which is then added, makes the dense of magnetic ball Degree is 20mg/mL, is placed in 38 DEG C of water-baths and shakes or mechanical agitation 2 hours；Activated nanoscale magnetic bead solution is gone Except supernatant, the carbonate buffer solution of pH9.5 is then added, its concentration is made to reach 20mg/mL (in terms of nanoscale magnetic bead)；With every milli Gram magnetic ball adds 10 milligrams of aq. polyethyleneimines, and ultrasonic 10min rotates mixing and react 4h, abandoned after Magneto separate at room temperature Remove supernatant；It is washed 3 times with the PBS buffer solution of 0.01mol/L pH7.5, each 3min；With the ethanol amine capping of 1mol/L 2h；It is washed 3 times with the PBS buffer solution of 0.01mol/L pH7.5, is discarded supernatant after Magneto separate, obtain the magnetism of amino functional Microballoon, it is spare.

Prepare 3：The preparation of lentil lectin modification-polyethyleneimine magnetic ball conjugate

A certain amount of amino polymer functionalization magnetic ball is taken, is resuspended, is made with 0.1mol/L pH9.2 sodium bicarbonate aqueous solutions Its concentration reaches 20mg/mL (in terms of nanoscale magnetic bead), and the oxidation product of the lentil lectin of 0.5mg is added by every milligram of magnetic ball (LECTIN-CHO), after mixing, the NaBH of 5mg/mL is added₄Aqueous slkali, 25 DEG C are stirred to react 4 hours, and completely reacted magnetism is micro- Ball solution removes supernatant, cleans solid three times with washing lotion (0.5%BSA solution), it is coated to obtain lentil lectin oxide Magnetic microsphere.

Prepare 4：The preparation of lentil lectin oxide magnetic microballoon activity preservative fluid

(1) dilution component：Borate buffer solution, pH7.2, a concentration of 0.02mol/L；

(2) surface active agent composition：Polyethylene glycol, a concentration of 0.1mg/mL；

(3) site sealant components：Gelatin, a concentration of 0.5g/mL；

(4) biological preservative component：Methylisothiazolinone, a concentration of 1.5mg/mL.

Prepare 5：The preparation of AFP-L3 antigen capture reagents

The magnetic ball for preparing coating lentil lectin oxide in 3 is suspended in the immune magnetic activity for preparing and preparing in 4 to protect In liquid storage, the capture agent of AFP-L3 antigens is obtained.

Prepare 6：ABEI marks AFP monoclonal antibody

1mg AFP monoclonal antibodies are taken, its volume are adjusted to 1mL with 0.1mol/L carbonate buffer solutions (pH9.5), so After be put into bag filter, be placed in pH9.5 carbonate buffer solutions and dialyse 1 hour.It is anti-to take out the anti-AFP monoclonals dialysed Body, is added 100 μ g ABEI-, half succinamic acid-NHS, and room temperature shakes 1.5 hours.G-25 gel columns are installed, purified water is used After elution is clean, then with the PBS buffer solution of pH value 7.4 it is balanced elution.After G-25 gel column equilibrations elute, it will mark The AFP monoclonal antibody of good ABEI crosses column purification, then collects the protein solution for peak value occur.The albumen gathered is molten Isometric protection liquid containing 5% BSA is added in liquid, and diluted is added to 0.15 μ g/mL.

A kind of kit of capture separation and detection AFP-L3, contains following components：

A. the AFP-L3 antigen capture reagents obtained according to preparation 5,

The 20 μ g/mL of working concentration of agglutinin oxidation product,

Magnetic microsphere working concentration：1mg/mL,

Dilution component：Borate buffer solution, pH7.2, working concentration 0.02mol/L；

Surface active agent composition：Polyethylene glycol, working concentration 0.1mg/mL；

Site sealant components：Gelatin, working concentration 0.5g/mL；

Biological preservative component：Methylisothiazolinone, working concentration 1.5mg/mL

B. low concentration calibration product, concentration：15.625IU/mL；

C. high concentration calibration object, concentration：500.000IU/mL；

D. according to the AFP monoclonal antibody for preparing the ABEI labels that 6 obtain

ABEI working concentrations：250ng/mL,

The working concentration of AFP monoclonal antibody：5000ng/mL；

E.Buffer buffer solutions

The ingredient of Buffer buffer solutions：The borate buffer solution of 0.02mol/L pH7.2,5mmol/L sodium chloride, 1mmol/ L manganese chlorides.

A method of it is captured with above-mentioned kit and detaches and detect AFP-L3 in serum, step is specific as follows：

1) preparation of 10 standard curves

A concentration of 4.6*10^6IU/mL of AFP-L3 enterprises calibration object, is diluted to 0.02M PBS buffer solution The intermediate product of 1000IU/mL successively gradient dilution are respectively obtained the concentration point of ten point curves by 1000IU/mL, fixed by 2 points Working curve is calculated in mark calibration curve.

2) use of capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes

Capture separation and detection AFP-L3 chemical luminescence immune analysis reagent boxes are given birth to using this method, in this department development ＆ production Maglumi 2000Plus instruments on detect, detecting step includes：

A. 20 μ L samples to be tested, high-concentration and low-concentration calibration object are added in reaction cup；

B. 20 μ L lentil lectins oxides-magnetic microsphere solution is added；

C. 100 μ L Buffer buffer solutions are added；

D.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

E. the AFP monoclonal antibody of 200 μ L ABEI labels is added；

F.37 DEG C incubation 15min, is placed under magnetic environment and cleans 3 times；

G. luminous substrate is added, detects light signal strength；

H. sample to be tested can be calculated automatically according to pattern detection luminous intensity by the revised working curve of calibration object AFP-L3 concentration.

It captures the detection for detaching and detecting AFP-L3 chemical luminescence immune analysis reagent boxes to primary carcinoma of liver sample and answers With

The serum sample for having selected 3 primarys carcinoma of liver through clinical definite is measured by the method for agglutinin electrophoresis It is tried to the actual content of AFP-L3 in its sample, then with the prepared AFP-L3 chemiluminescence immune assays of this embodiment scheme Agent box measures the content of AFP-L3 in its sample, and the prepared AFP-L3 chemistry of this programme is calculated by two measured concentrations The capture separative efficiency of the AFP-L3 of luminescence immunoassay kit.

Experimental data and interpretation of result

From above-mentioned experimental data it is found that detecting AFP-L3, the capture to AFP-L3 using the kit that embodiment provides Separative efficiency is significantly better than that the capture separative efficiency of comparative example A FP-L3, illustrates that technology using the present invention passes through agglutinin After oxidant effect, the aldehyde groups of generation can directly be reacted with amino group, be not necessarily to the assisted reaction of activator and crosslinking agent, It ensure that the activity of agglutinin；By magnetic microsphere after amino polymer functionalization, magnetic microsphere surface forms branch Network-like structure reduces the steric hindrance between magnetic ball and agglutinin, and the carrying capacity of amino group is made to greatly improve, to improve Capture separative efficiency of the agglutinin to AFP-L3；Using agglutinin magnetic microsphere active protection liquid provided by the invention, for The characteristic of agglutinin can preferably maintain the activity of agglutinin, immune magnetic microsphere is protected not to be destroyed in homogeneous system Or reunite, to a certain extent to playing facilitation in the activity of agglutinin and capture separative efficiency, therefore, using this hair Bright technical solution can enhance the reactivity of agglutinin, improve the efficiency of agglutinin coupling magnetic ball, improve the capture of AFP-L3 Efficiency.

By the comparison of the measurement result of embodiment 1 and embodiment 2 it is found that when handling the oxidant concentration excess of agglutinin, The oxidation results of agglutinin can be caused to form carboxyl rather than aldehyde radical, to further influence and amino polymer functionalization magnetic ball Coupling efficiency.

It, may be right by the comparison of the measurement result of embodiment 1 and embodiment 3 it is found that replacing the oxidant of processing agglutinin Oxidation effectiveness impacts, and the oxidisability of sodium perchlorate is better than sodium metaperiodate, can lead to the amount of activated inactivation of agglutinin.

By the comparison of the measurement result of embodiment 1 and embodiment 4 it is found that polyethyleneimine is modified as amino polymer to magnetic Property microsphere surface, the carrying capacity and coupling efficiency for capableing of coupled antigen are higher than polyamide.

By the measurement result comparison of embodiment 1 and embodiment 5 it is found that the inventory of amino polymer is very few, ammonia can be influenced Based polyalcohol is coupled magnetic ball and the coupling efficiency of agglutinin coupling amino polymer magnetic ball and reduces, to influence capture separation effect Rate.

By the comparison of the measurement result of embodiment 1 and embodiment 6 it is found that under the protection of active protection liquid, agglutinin coupling The activity of amino polymer magnetic ball can be ensured well.

By the measurement result comparison of embodiment 1 and embodiment 7 it is found that lentil lectin specific recognition AFP-L3 antigens Specificity is better than ConA.

If being compared by the measurement result of embodiment 1 and comparative example 1-4 it is found that not handled agglutinin and being directly coupled Magnetic ball can affect to the activity or capture rate of agglutinin itself, without even using the magnetic ball of amino functional Join agglutinin, the decline for being coupled carrying capacity also directly affects the capture separative efficiency of agglutinin；The activity preservative fluid of magnetic ball is certain To playing facilitation in the activity of agglutinin and capture separative efficiency in degree.

The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, any made by repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.

Claims (19)

1. a kind of immune magnetic compound, which is characterized in that including：

Using the magnetic microsphere for the amino functional that amino polymer modified magnetic microballoon obtains；Agglutinin, the agglutinin with The amino polymer connection.

2. immune magnetic compound according to claim 1, which is characterized in that the agglutinin is oxidation agglutinin；

Preferably, the oxidation agglutinin is connect with amino on the amino polymer；

Preferably, the agglutinin is the oxidation agglutinin that hydroxyl is oxidized to aldehyde radical, and the oxidation agglutinin passes through aldehyde radical and institute Amino on amino polymer is stated to connect.

3. immune magnetic compound according to claim 1, which is characterized in that the amino polymer is polyethyleneimine Amine, polyamide, polyacrylamide or poly-o-phenylenediamine, preferably polyethyleneimine.

4. immune magnetic compound according to any one of claim 1 to 3, which is characterized in that the immune magnetic is multiple It is the immune magnetic compound for capturing separation AFP-L3 to close object, it is preferred that the agglutinin is lentil lectin, small Lens culinaris Agglutinin or ConA, more preferably lentil lectin.

5. immune magnetic compound according to any one of claim 1 to 3, which is characterized in that the magnetic microsphere packet Magnetic ball ontology is included, the magnetic ball ontology carries one or more activity functional groups by surface modification；

Preferably, the active group includes at least one of carboxyl, hydroxyl and amino；

Preferably, the magnetic ball ontology is Fe₂O₃The complex of magnetic nano-particle and high-molecular organic material, or be Fe₃O₄ The complex of magnetic nano-particle and high-molecular organic material；

Preferably, the magnetic ball ontology grain size is 0.1 μm~5 μm；

Preferably, the amino polymer is by the way that the magnetic microsphere of the amino functional is obtained by the reaction with the active group.

6. a kind of preparation method of immune magnetic compound as described in any one of claim 1 to 5, which is characterized in that packet Include following steps：

1) amino polymer modified magnetic microballoon is used, the magnetic microsphere of amino functional is obtained；

2) it connect agglutinin to form the immune magnetic compound with the amino polymer.

7. preparation method according to claim 6, which is characterized in that connect with the amino polymer in the agglutinin Before, further include the steps that being aoxidized to the agglutinin, obtain oxidation agglutinin；

Preferably, the oxidation agglutinin is connect with amino on the amino polymer；

Preferably, the hydroxyl in the agglutinin is oxidized to aldehyde radical, obtains oxidation agglutinin, the oxidation agglutinin passes through aldehyde Base is connect with amino on the amino polymer.

8. preparation method according to claim 7, which is characterized in that the oxidant used in the oxidation step is high iodine Hydrochlorate, perchlorate or permanganate, it is preferred that the oxidant be sodium metaperiodate, sodium perchlorate or potassium permanganate, more preferably For sodium metaperiodate；

Preferably, the mass concentration ratio of agglutinin described in the oxidation step and the oxidant is 1:50-1:500；

Preferably, the reaction temperature of the oxidation is 20-30 DEG C, and the reaction time is 1-4 hours；

Preferably, a concentration of 1mg/mL-5mg/mL of the agglutinin.

9. preparation method according to claim 6, which is characterized in that the step 1) specifically includes：

11) magnetic microsphere is activated using activator；

12) activated magnetic microsphere amino polymer aqueous solution is added to react；

13) isolating surface modification has the magnetic microsphere of amino polymer, is reacted with sealer, obtains amino functional Magnetic microsphere；

Preferably, the activator is dicyclohexylcarbodiimide, 1- (3- dimethylamino-propyls) -3- ethyl carbodiimide hydrochlorides Salt and N, at least one of N'- diisopropylcarbodiimide；

Preferably, the mass concentration ratio of the magnetic microsphere and the activator is：2:1–4:1；

Preferably, the reaction temperature of the activation is 37 DEG C -40 DEG C, and the reaction time is 1h -4h；

Preferably, the step 12) specifically includes：Activated magnetic microsphere is added to amino polymer aqueous solution, ultrasound After processing, rotation mixing reaction is carried out；

Preferably, a concentration of 10mg/mL-200mg/mL of the amino polymer Ammonia In Aqueous Solution based polyalcohol；

Preferably, when the activated magnetic microsphere is added to amino polymer aqueous solution and is reacted, the magnetic microsphere A concentration of 10mg/mL-20mg/mL；

Preferably, the sealer is ethanol amine, BSA or gelatin, a concentration of 0.5-1mol/L of the sealer.

10. preparation method according to claim 7, which is characterized in that the step 2) specifically includes：By the amino work( The magnetic microsphere of energyization is resuspended with re-suspension liquid, and the oxidation agglutinin is then added, and reducing agent is added after mixing and is stirred to react, removes The immune magnetic compound is obtained after going supernatant to wash；

Preferably, the re-suspension liquid is sodium bicarbonate aqueous solution, Tris-HCl buffer solutions or borate buffer solution, the re-suspension liquid A concentration of 0.05mol/L-0.2mol/L, pH 8-10；

Preferably, the reducing agent is sodium borohydride, potassium borohydride or Lithium Aluminium Hydride, more preferably sodium borohydride, the reduction A concentration of 1mg/mL-5mg/mL of agent, more preferably 5mg/mL；

Preferably, the mass concentration ratio of the magnetic microsphere of the oxidation agglutinin and the amino functional is 1:50-1: 100；

Preferably, the reaction temperature of the step 2) is 20 DEG C -30 DEG C, reaction time 2h-6h.

11. a kind of activity preservative fluid of immune magnetic compound as described in any one of claim 1 to 5, which is characterized in that Including：Dilution component, metal-binding sites component, metal-chelator component and biological preservative component；

Preferably, it is slow to be divided into phosphate buffer, borate buffer solution, Tris-HCl buffer solutions or carbonate for the dilution group Fliud flushing, a concentration of 0.01-0.2mol/L of the dilution, pH 7-9；

Preferably, the metal-binding sites component includes manganese ion and calcium ion, the concentration of the metal-binding sites component For 0.5-5mmol/L, wherein manganese ion concentration is 0.1-1mmol/L, calcium ion concentration 0.5-5mmol/L；

Preferably, the metal-binding sites group is divided into calcium chloride, calcium nitrate, calcium bromide, manganese chloride, manganese sulfate and manganese nitrate At least two；

Preferably, the metal-chelator group is divided into ethylenediamine tetra-acetic acid, tetrasodium ethylenediamine tetraacetate, EDTAP dipotassium ethylene diamine tetraacetate At least one of with disodium ethylene diamine tetraacetate；Wherein, the working concentration of the metal-chelator is 0.05-0.2mg/mL；

Preferably, the biological preservative component includes that isothiazolinone, ε-poly-D-lysine, Sodium azide, thimerosal and celebrating are big The working concentration of at least one of mycin, the biological preservative component is 0.5mg/mL-2mg/mL.

12. activity preservative fluid according to claim 11, which is characterized in that

The activity preservative fluid further includes surface active agent composition；

Preferably, the surface active agent composition is polyethylene glycol, polyoxyethylene 20 sorbitan monolaurate -20 and octyl The working concentration of at least one of phenyl polyoxyethylene ether, the surface active agent composition is 0.1mg/mL-0.5mg/mL；

Preferably, the activity preservative fluid further includes non-protein class site sealer；

Preferably, non-protein class site sealer is at least one of gelatin, fish glue from skin and ethanol amine, the non-protein The working concentration of class site sealer is 0.5g/mL-2g/mL.

13. a kind of antigen capture agent for capturing separation and quantitatively detecting AFP-L3, which is characterized in that including such as claim Immune magnetic compound described in any one of 1 to 5 and the activity preservative fluid as described in claim 11 or 12.

14. antigen capture agent according to claim 13, which is characterized in that exempt from described in the densimeter of the magnetic microsphere A concentration of 0.5-2mg/mL of the epidemic disease magnetic composite in the activity preservative fluid.

15. a kind of for capture separation and the quantitatively kit of detection AFP-L3, which is characterized in that including such as claim 13 or Antigen capture agent described in 14.

16. kit according to claim 15, which is characterized in that further comprise：Low concentration calibration product, altitude calibration The AntiAFP antibody and buffer solution of product, luminescent label；

Preferably, a concentration of 10.936IU/mL-20.313IU/mL of low concentration calibration product, the altitude calibration product are a concentration of 350IU/mL-650IU/mL；

Preferably, the working concentration of luminous marker is 5ng/mL-500ng/mL, and the working concentration of the AntiAFP antibody is 50ng/mL-5000ng/mL；

Preferably, the buffer solution is carbonate buffer solution, phosphate buffer, borate buffer solution or Tris-HCl bufferings Liquid；

Preferably, the luminous marker is luminol, different luminol and its derivative；Alkaline phosphatase, horseradish peroxidase Enzyme, fluorescent material, rare earth ion and its chelate aglucon, acridinium ester and its derivative or tris (bipyridine) ruthenium.

17. a kind of immune magnetic compound as described in any one of claim 1 to 5, according to claim 13 or 14 The application of antigen capture agent, the kit as described in claim 15 or 16 in capture separation and in quantitatively detecting AFP-L3.

18. application according to claim 17, which is characterized in that the immune magnetic compound, the antigen are caught It obtains agent or kit is applied to semi-automatic or automatic lmunoassays analyzer.

19. a kind of capture separation and the system for quantitatively detecting AFP-L3, which is characterized in that including as described in claim 15 or 16 Kit and semi-automatic or automatic lmunoassays analyzer.