CN108333346A - DsDNA immune magnetic microspheres system, preparation method and application and preservation liquid - Google Patents

DsDNA immune magnetic microspheres system, preparation method and application and preservation liquid Download PDF

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CN108333346A
CN108333346A CN201710040242.7A CN201710040242A CN108333346A CN 108333346 A CN108333346 A CN 108333346A CN 201710040242 A CN201710040242 A CN 201710040242A CN 108333346 A CN108333346 A CN 108333346A
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dsdna
magnetic
magnetic microsphere
immune magnetic
immune
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CN108333346B (en
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饶微
刘坤
袁锦云
刘来壮
李婷华
卢东林
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Shenzhen New Industries Biomedical Engineering Co Ltd
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01—MEASURING; TESTING
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
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    • G01N2446/00—Magnetic particle immunoreagent carriers
    • G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
    • G01N2446/90—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids characterised by small molecule linker used to couple immunoreagents to magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/104—Lupus erythematosus [SLE]

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Abstract

The invention discloses a kind of dsDNA immune magnetic microspheres system and preparation method thereof and preserve liquid, Anti-hCG action detection reagent and kit and system.DsDNA immune magnetic microspheres system of the present invention includes magnetic microsphere ontology and amido polymer group and dsDNA antigens, and magnetic microsphere ontology described in the amido polymer base group modification, the dsDNA antigens are coupled with the amido polymer group.Preserve the preservation that liquid is used for dsDNA immune magnetic microsphere systems.Anti-hCG action detection reagent and kit and system include dsDNA immune magnetic microsphere systems.DsDNA immune magnetic microspheres architecture of the present invention is stablized, and the package amount of dsDNA antigens is high, and space steric effect is low, high with the joint efficiency and binding capacity of antibody, improves antibody test sensitivity.

Description

DsDNA immune magnetic microspheres system, preparation method and application and preservation liquid
Technical field
The invention belongs to medical reagent technical fields, and in particular to a kind of Anti-hCG action detection reagent and its preparation side Method, Anti-hCG action detection kit, Anti-hCG action detecting system and their application.
Background technology
Systemic loupus erythematosus (systemic lupus erythematosus/SLE) is that one kind involving multiple organ, polyphyly The thin vessels of system and the systemic autoimmune disease of connective tissue.In Europe, newly-increased incidence annual SLE is about 25/ 100000, and north America region incidence higher.Often recurrence is alternately present SLE easily hairs with alleviation in young woman, the course of disease.Patient Can generate the antinuclear antibodies and other autoantibodies for nucleic acid, nucleoprotein and histone in vivo, these autoantibodies with it is corresponding Antigen binding forms immune complex, can be deposited on cardiovascular connective tissue, glomerular basement membrane, serous coat, synovium of joint and more On kind internal organs thin vessels wall, immune complex attracts neutrophil infiltration, causes the slow of local organization in Local activation complement Property inflammatory damage.Therefore SLE patient often suffers from the damage of multisystem, multiple organ.Different according to the organ of damage, patient may occur in which hair Heat, fash, arthralgia, renal damage, the various clinical symptoms such as cardiovascular pathological changes, scrositis, anaemia, mental symptom.U.S.'s rheumatism Association and the 11 ARA standards (ARA, American Rheumatism Association) diagnosed about SLE are revised within 1997.11 standard difference For：1. butterfly erythema；2. there is red, scales of skin that peel off shape, limitation, protuberance skin rash in other positions of body；3. phenomenon of photosensitivity；4. Oral mucosa pain or ulcer；5. arthralgia or joint exudation；6. scrositis；7. kidney trouble；8. the nervous system disease；9. The hemorrhagic anemia of hematoligical symptom such as companion's reticulosis, Neuroleptic Leukocytopenia, lymphocyte reduction；10. immunology side Method detects Anti-hCG action, Sm antibody, anticardiolipin antibodies；11. indirect immunofluorescence detects ANA.If going out in 11 standards 4 existing, the possibility for diagnosing SLE is more than 80%.
Anti-hCG action is characteristic mark's antibody of SLE patient, the important diagnostic standard which is SLE it One.The active level of Anti-hCG action potency and disease has correlation, the dynamic of antibody titer to be measured as monitoring treatment and provide Effective laboratory techniques.Anti-hCG action plays an important role in the pathogenesis of SLE, and lupus nephritis concurrent SLE is By the antibody-mediated immune complex, Anti-hCG action can form a variety of cryoprecipitates and cause vasculitis, SLE ephritis and typical case Butterfly erythema it is related with the antibody.
The reaction site of Anti-hCG action and nucleus is predominantly located on DNA (external zones) deoxyribonucleotide frame, The specificity of Anti-hCG action diagnosis SLE is up to 95% or more.The common method master of Anti-hCG action is clinically detected at present There are indirect immunofluorescence, enzyme linked immunosorbent assay, radio immunoassay and chemoluminescence method.Wherein, the most specific Method with sensibility is to carry out indirect immunofluorescene assay as antigen using pferdepest trypanosome or crithidia luciliae.Due to such The kinetoplast of polypide is made of pure cyclic DNA, is in addition to this substantially free of other cell nuclear antigens, can be risen with kinetoplast The autoantibody of reaction only has Anti-hCG action, thus has high degree of specificity.But this method cannot achieve quantitative detection and behaviour Make very complicated, needs to be equipped with expensive fluorescence microscope, can not be promoted in basic hospital.Radio immunoassay is due to tracer There are radioactivity for object, there is pollution risk to environment and operating personnel.Enzyme linked immunosorbent assay needs manual operation, takes consumption Power, and accuracy and precision are poor, it is difficult to obtain reliable result.
On the basis of above-mentioned existing detection method existing defects, chemiluminometry has high sensitivity, linearly Range is wide, and specificity is good, and instrument and equipment simple operation and other advantages have been successfully applied to life science, Food Science, clinical medicine And the fields such as environment measuring, become a kind of effective micro and Analytical Methods of Trace in modern analysis field.However, chemistry hair Light immunoassay is homogeneous reaction system, and dsDNA antigen coat magnetic balls are simultaneously suspended in homogeneous system, dsDNA antigens easily by Unstable situation is caused to the factors such as crosslinking process and system environment, the judgement of test result is seriously affected, to limit Make application of the project on chemiluminescence immune analysis method.
Such as presently disclosed one kind is sent out using gold-magnetic particles as carrier with indirect elisa method, immunofluorescence technique or chemistry The method that light method detects anti-dsDNA antibody.This approach includes the following steps：(1) gold-magnetic particles and double-stranded DNA are mixed, Double-stranded DNA is fixed on gold-magnetic particles surface or with gold-magnetic particles from people's whole blood, saliva, sperm or plasmid under certain condition Extraction DNA is simultaneously fixed on gold-magnetic particles surface；(2) use indirect elisa method, immunofluorescence technique or chemoluminescence method detection to be measured Anti-dsDNA antibody present in sample.Wherein double-stranded DNA is fixed on golden magnetic by the prior art using the method for physical absorption Microparticle surfaces.But it being found during Clinical practice, it is physical absorption that this method double center chain DNA, which fixes gold-magnetic particles table mode, It is combined with the electrostatic force between dsDNA antigens by magnetic microsphere surface, however, dsDNA molecules are easy in homogeneous system It is influenced and is destroyed by many uncertain factors such as enzyme, therefore pass through the steady of the magnetic microsphere of physical absorption fixation dsDNA Qualitative to be difficult to ensure, the product after fixing is easy to fall off in storage and transportational process, and shadow is caused to reagent sensitivity and stability It rings.
In presently disclosed another related art, it is related to using ELISA Plate as solid phase carrier, with chemiluminescence The method that method detects anti-dsDNA antibody.This approach includes the following steps：(1) artificial synthesized double-stranded DNA is fixed on solid phase The surface of carrier；(2) anti-dsDNA antibody present in chemoluminescence method detection sample to be tested is used.But this method is still To be fixed on dsDNA antigens on ELISA Plate by way of physical absorption, i.e., by magnetic microsphere surface and dsDNA antigens it Between electrostatic force be combined, but due to ELISA Plate fix dsDNA antigens carrying capacity it is limited, immunological response carry out process In, fixed dsDNA antigens and the target antibody reaction contact area in sample are limited on ELISA Plate, easily cause steric hindrance, Therefore reaction bonded efficiency (joint efficiency of antigen coat carrier, the efficiency of envelope antigen and test antibodies reaction bonded) can be by To limitation.
It is existing in the related technology, due to the functional group limited amount of magnetic ball surface, can effectively combine dsDNA antigens Quantity is similarly limited, simultaneously because dsDNA antigens and the rigid interface of magnetic ball are in direct contact, can cause one to the activity of antigen Fixed influence, and be easy to cover active site, cause effectively combine test antibodies in detection process, therefore reaction bonded Efficiency (efficiency of envelope antigen and test antibodies reaction bonded) can be restricted.
Invention content
It is an object of the invention to overcome the above-mentioned deficiency of the prior art, a kind of dsDNA immune magnetic microspheres system is provided And preparation method thereof, with overcome it is existing dsDNA antigens are combined by electrostatic force on magnetic microsphere using physical absorption, Caused by dsDNA combine unstable and there is technical issues that steric hindrance etc..
Another object of the present invention is to provide a kind of dsDNA immune magnetic microspheres to preserve liquid, to solve the application dsDNA Immune magnetic microsphere preserves unstable technical problem using existing preservation liquid.
Another object of the present invention is to provide a kind of Anti-hCG action detection reagent, kit and detecting system, with solution Certainly existing related preparations are for detecting the not high technical problem of Anti-hCG action accuracy.
In order to achieve the above-mentioned object of the invention, an aspect of of the present present invention provides a kind of dsDNA immune magnetic microspheres system. The dsDNA immune magnetic microspheres system includes magnetic microsphere ontology, further includes amido polymer group and dsDNA antigens, And magnetic microsphere ontology described in the amido polymer base group modification, the dsDNA antigens and the amido polymer base Dough is keyed.
Another aspect of the present invention provides a kind of preparation method of dsDNA immune magnetic microspheres system.The preparation side Method includes the following steps：
The magnetic microsphere of activated processing is reacted with amino polymer, then carries out reaction product and sealer Processing obtains the magnetic microsphere of amido polymer base group modification；
The magnetic microsphere of the amido polymer base group modification and dsDNA antigens are carried out under activator effect anti- It answers.
Another aspect of the present invention provides a kind of dsDNA immune magnetic microspheres preservation liquid.The dsDNA immune magnetics It includes following component that microballoon, which preserves liquid,：
Another aspect of the present invention provides a kind of Anti-hCG action detection reagent.The Anti-hCG action detection examination Agent includes the preservation liquid of dsDNA immune magnetic microspheres and the dsDNA immune magnetic microspheres, the dsDNA immune magnetic microspheres It is scattered in the preservation liquid；And the dsDNA immune magnetic microspheres are for dsDNA immune magnetic microspheres of the present invention or by the present invention DsDNA immune magnetic microspheres prepared by preparation method, the preservation liquid are that dsDNA immune magnetic microspheres of the present invention preserve liquid.
Another aspect of the present invention provides a kind of Anti-hCG action detection kit.The Anti-hCG action detection Kit includes Anti-hCG action detection reagent of the present invention.
Another aspect of the present invention provides a kind of Anti-hCG action detecting system.The Anti-hCG action detection system System includes：
Anti-hCG action detection reagent of the present invention or Anti-hCG action detection kit of the present invention；
Magneto separate module coordinates with the dsDNA immune magnetic microspheres, for detaching magnetic microsphere from liquid；
Detection module, the magnetic microsphere detached with the Magneto separate module and luminous substrate cooperation, for completing confrontation The detection of dsDNA antibody；
Data processing module, the content for calculating the Anti-hCG action.
Another aspect of the present invention provides dsDNA immune magnetic microspheres system of the present invention, Anti-hCG action of the present invention Detection reagent, Anti-hCG action detection kit of the present invention, Anti-hCG action detecting system of the present invention are any to be examined in separation Survey the application in Anti-hCG action.
Compared with prior art, dsDNA immune magnetic microspheres system of the present invention is coupled by amido polymer group It is anti-to effectively increase dsDNA in this way, the chemistry on the one hand realizing dsDNA antigens and magnetic microsphere is keyed for dsDNA antigens The stability of primordial covering；On the other hand, since the characteristic of amido polymer group and dsDNA antigens pass through chemical bond such as coupling It is connected to amido polymer group this structure, effectively increases the package amount of dsDNA antigens, and reduces steric hindrance effect It answers, enhances dsDNA immune magnetic microspheres the system of the present invention joint efficiency of binding purpose antibody and combination in sample to be tested Amount, to improve detection sensitivity.
DsDNA immune magnetic microspheres system preparation method of the present invention first uses amido polymer modified magnetic microballoon, so It is coupled dsDNA antigens afterwards so that dsDNA antigens are coupled on amido polymer group so that the dsDNA immune magnetics of preparation Microballoon system not only stable structure, and space steric effect effectively reduces, and ensure that the activity of antigen.In addition, system of the present invention Preparation Method process conditions are easily-controllable, and the performance that product dsDNA immune magnetic microsphere systems have been effectively ensured is stablized.
DsDNA immune magnetic microspheres of the present invention preserve liquid energy and reach effective protection dsDNA immune magnetic microspheres system of the present invention It is not degraded or destroys in homogeneous system, to improve the stability of dsDNA immune magnetic microspheres system of the present invention.
Anti-hCG action detection reagent, Anti-hCG action detection kit and Anti-hCG action detecting system of the present invention And its application due to be by dsDNA immune magnetic microspheres system of the present invention and preserve liquid based on, this has been effectively ensured Corresponding reagent is steady in Anti-hCG action detection reagent, Anti-hCG action detection kit and Anti-hCG action detecting system Qualitative and raising and purpose antibody joint efficiency, to improve detection sensitivity degree and accuracy.
Description of the drawings
Fig. 1 is dsDNA immune magnetic microsphere architectural schematics of the embodiment of the present invention；
Fig. 2 is dsDNA immune magnetic microsphere system preparation method flow charts of the embodiment of the present invention；
Fig. 3 is Anti-hCG action detection method flow chart of the embodiment of the present invention.
Specific implementation mode
In order to make technical problems, technical solutions and advantageous effects to be solved by the present invention be more clearly understood, below in conjunction with Embodiment and attached drawing, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used To explain the present invention, it is not intended to limit the present invention.
Abbreviation is explained：
Anti-dsDNA antibody (Anti dsDNA), deoxyribonucleotide DNA (Deoxyribose Nucleic Acid), systemic loupus erythematosus SLE (systemic lupus erythematosus), DCC (dicyclohexylcarbodiimide), EDC (1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides), CDC (N, N'- diisopropylcarbodiimide), DMF (N,N-dimethylformamide), PEI (polyethyleneimine), PA (polyamide), PAM (polyacrylamide), PDI (poly- neighbour's benzene two Amine), Isoluminol (different luminol), ABEI (N- (4- aminobutyls)-N- ethyls different luminol), ((6- amino is by N- by AHEI Base)-N- ethyls different luminol), ITCI (isothiocyanic acid different luminol), (N- (4- isothiocyanos butyl)-N- ethyls are different by ITCBEI Luminol).
On the one hand, an embodiment of the present invention provides a kind of stable structure, and the dsDNA immune magnetics that space steric effect is low Microballoon system.The structure of the dsDNA immune magnetic microspheres system is as shown in Figure 1 comprising such as lower part：
Magnetic microsphere ontology (being hereafter referred to as abbreviation magnetic microsphere) 1, amido polymer group 2, dsDNA antigens 3, and contain Amino polymer group 2 is connect with magnetic microsphere 1, plays the role of modified magnetic microballoon 1, while amido polymer group is logical Chemistry key connection dsDNA antigens 3 are crossed, 3 structure of magnetic microsphere 1- amido polymer group 2-dsDNA antigens is formed.Wherein, magnetic Property microballoon 1 and dsDNA antigens 3 be not directly coupled, but by the function served as bridge of amido polymer group 2 realize between in succession It is connected in one.
Wherein, above-mentioned magnetic microsphere 1 is as amido polymer group 2, the carrier of dsDNA antigens 3.In an embodiment In, the mass ratio of magnetic microsphere 1 and amido polymer group 2 contained by above-mentioned dsDNA immune magnetic microspheres system is 1:(1- 10).The amount for controlling the amido polymer group 2 of magnetic microsphere 1 surface connection, with regard to the amount of indirect control dsDNA antigens 3, from And improve the amount of dsDNA immune magnetic microsphere systems capture Anti-hCG action.Therefore, in another embodiment, above-mentioned dsDNA Amido polymer group 2 and 3 mass ratio of dsDNA antigens are (50-2000) in immune magnetic microsphere system：1.
On the basis of the various embodiments described above, above-mentioned magnetic microsphere 1 can select magnetic microsphere commonly used in the art, specifically Can select nano-magnetic microsphere.As long as all magnetism in this field that technical solution of the embodiment of the present invention can be suitable for Microballoon is in range disclosed by the invention.
In one embodiment, which is the magnetic microsphere that surface is activated processing.At magnetic microsphere surface active Reason can carry out activation process according to magnetic microsphere activation method in hereafter dsDNA immune magnetic microspheres system preparation method.
Above-mentioned amido polymer group 2 effectively connection magnetic microsphere 1 and dsDNA antigens 3, realize dsDNA antigens 3 and magnetic Property microballoon 1 between chemistry key connection, to effectively increase 3 coated stability of dsDNA antigens, that is to say improve it is above-mentioned The stable structure of dsDNA immune magnetic microsphere systems；And dsDNA antigens 3 and magnetism are realized by amido polymer group 2 Chemistry key connection between microballoon 1, and it is based on 2 self character of amido polymer group, effectively prevent space steric effect Generation or effectively reduce space steric effect, improve the package amount of dsDNA antigens 3, and ensure that the work of antigen Property.
In one embodiment, the amido polymer group 2 and dsDNA antigens 3 are coupling connections, such as in specific embodiment In, amido polymer group 2 is to connect the dsDNA antigens 3 by phosphorylated amino ester bond, to realize that dsDNA antigens 3 wrap The stability of quilt.
In another embodiment, it is by polyethyleneimine, polyamide, polypropylene to provide the amido polymer group 2 Any one of amide, poly-o-phenylenediamine amino polymer provides.The amido polymer group 2 that the compound provides can have Effect coupling dsDNA antigens 3, and improve that dsDNA antigens 3 are coated to stabilize and increase 3 coated amount of dsDNA antigens, it reduces simultaneously Or avoid space steric effect.
Above-mentioned dsDNA antigens 3 can be Anti-hCG action specific bond with characteristic mark's antibody specific of SLE patient.
Therefore, above-mentioned dsDNA immune magnetic microspheres system realizes dsDNA antigens 3 and the chemical bond of magnetic microsphere 1 connects It connects, effectively increases 3 coated stability of dsDNA antigens；Characteristic and dsDNA additionally, due to amido polymer group 2 is anti- Original 3 is connected to amido polymer group 2 this structure by chemical bond, effectively increases the coating carrying capacity of dsDNA antigens 3, And space steric effect is reduced, enhance above-mentioned dsDNA immune magnetic microspheres system binding purpose antibody in sample to be tested Joint efficiency and binding capacity, to improve detection sensitivity.
Correspondingly, the embodiment of the present invention additionally provides a kind of system about dsDNA immune magnetic microspheres system described above Preparation Method.The preparation method flow includes the following steps as shown in Fig. 2, refer to Fig. 1 simultaneously：
S01. the magnetic microsphere of amido polymer base group modification is prepared：By the magnetic microsphere 1 and amino of activated processing Polymer is reacted, and then handles reaction product and sealer, obtains the magnetism of 2 modification of amido polymer group Microballoon 1；
S02. by the magnetic microsphere of amido polymer base group modification and dsDNA antigen-reactives：Described it will polymerize containing amino The magnetic microsphere 1 that object group 2 is modified is reacted with dsDNA antigens 3 under activator existence condition.
Specifically, in above-mentioned steps S01, the activator for activation process magnetic microsphere 1 includes but not limited to N, N'- bis- Diisopropylcarbodiimide (CDC), 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides (EDC), dicyclohexylcarbodiimide Any one of (DCC), certainly, if adding proportion is appropriate, can be those types activator in two or more mixing Object that is to say according to above-mentioned steps, while the use of two or more activator be also range disclosed by the embodiments of the present invention.Activation The method of magnetic microsphere 1 can with but not only according to activation method in Examples below 1.The magnetic microsphere 1 such as dsDNA above exempts from Magnetic microsphere 1 in epidemic disease magnetic microsphere system, in order to save length, details are not described herein.
In one embodiment, step S01 in the activation process of magnetic microsphere 1, magnetic microsphere 1 and activator Workable proportions are (2-4):1(w/w)；Re-suspension liquid for magnetic microsphere 1 to be resuspended includes but not limited to water, ethyl alcohol, N, N- diformazans Base formamide (DMF)；The temperature of activation process water-bath can be 37 DEG C -40 DEG C；Reaction time is 1h-4h.By should Activation process condition optimizing is controlled in those ranges, to improve 1 activation effect of magnetic microsphere.
In step S01, after the magnetic microsphere 1 of activated processing is reacted with amino polymer, the amino polymer It is reacted with magnetic microsphere 1 being activated the group formed in processing procedure so that amino polymer is with amino polymer group It is connected to by chemical bond on the surface of magnetic microsphere 1.Specifically, the chemical reaction that magnetic microsphere 1 is carried out with amino polymer As shown in following chemical reactions：
In the magnetic microsphere 1 of the activated processing and amino polymer reaction system, the content of the amino polymer The content of relative magnetism microballoon 1 is enough, so that 1 surface of magnetic microsphere combines enough amido polymer groups 2.Such as one In embodiment, the mass ratio of the magnetic microsphere and the amino polymer is 1：(1-10) controls the reacting dose of the two at this Both proportional region, on the one hand so that combining enough amido polymer groups on 1 surface of magnetic microsphere, on the other hand improve Reaction efficiency, and amino polymer waste is reduced, it is cost-effective.In another embodiment, amino polymer can be, but not limited to Select any one of polyethyleneimine (PEI), polyamide (PA), polyacrylamide (PAM), poly-o-phenylenediamine (PDI) or two Kind or more mixture.Those amino polymers contain abundant amino group, to improve the package amount of coupling dsDNA antigens 3. In addition, the working concentration control for the magnetic microsphere 1 that is activated that treated but not just for 10mg/mL -20mg/mL, the two reaction Temperature be preferred 25 DEG C -30 DEG C of control, the reaction time should be sufficient, such as 2h-6h.
It, preferably will reaction before being reacted magnetic microsphere 1 with sealer with the reaction product of amino polymer Product carries out Magneto separate, washing, to improve the function and effect of sealer.
It is handled using sealer and the reaction product, is the site group that will not reacted with amino polymer on magnetic ball It is combined, prevents the site group not reacted with amino polymer from being interfered to subsequent reactions.Certainly, sealer is anti-with this Answer the combination of product may be physical bond be also likely to be chemical bonding,
In the reaction product and the reaction system of sealer, the content relative magnetism microballoon 1 and amino of the sealer The content of polymeric reaction products is enough, so that reaction product is fully closed agent reaction.As in one embodiment, reaction is produced The quality control of object and the sealer is (1-50) in the mass ratio of magnetic microsphere 1 and the sealer:1.Another embodiment At least one of in, which selects but be not limited to casein, BSA, gelatin.The working concentration of the sealer controls but not Only 10mg/mL, the reaction time should be sufficient, such as 1h-4h.
In above-mentioned steps S02, under the action of activator, the amido polymer base group modification that is obtained in step S01 Magnetic microsphere and dsDNA antigens generate coupling reaction and generate phosphorylated amino ester bond, to realize dsDNA antigens by containing amino Polymeric groups are connected on magnetic microsphere 1.Specifically, the magnetic microsphere dsDNA antigens of amido polymer base group modification it Between chemical equation it is as follows：
In the activation process of above-mentioned steps S02, magnetic of the activator relative to the amido polymer base group modification Property microballoon amount it is enough, if the mass ratio of magnetic microsphere and the activator of the amido polymer base group modification is (2-4)：1. In another embodiment, the temperature of the reaction of activation process is 37 DEG C -40 DEG C；Reaction time should be sufficient, such as be 1h-4h. In another embodiment, the activator selects N, N'- diisopropylcarbodiimide, 1- (3- dimethylamino-propyls) -3- ethyls Any one of carbodiimide, dicyclohexylcarbodiimide or two or more mixtures.In addition, the activation in this step S02 Agent and the activator in step S01 can it is identical can not also be identical.Magnetism for amido polymer base group modification to be resuspended The re-suspension liquid of microballoon includes but not limited at least one of water, ethyl alcohol, N,N-dimethylformamide (DMF).
In the magnetic microsphere of amido polymer base group modification and the reaction system of dsDNA antigens, dsDNA antigens contain Amount be it is enough, such as in one embodiment, the magnetic microsphere of the amido polymer base group modification and the dsDNA antigens Mass ratio is (50-200)：1.And the reaction of the two can be carried out directly at room temperature, such as be reacted at 25 DEG C, the reaction time Should be sufficient, such as 1h-4h.
After abundant reaction, isolated target product is carried out to reactant.Magneto separate is such as used, reaction product is obtained, It is dsDNA immune magnetic microsphere systems as shown in Figure 1.Magneto separate should be to reaction product carrying out washing treatment.
In dsDNA immune magnetic microsphere systems prepared by the preparation method of above-mentioned dsDNA immune magnetic microspheres system, contain ammonia Based polyalcohol group 2 is connect with magnetic microsphere 1, plays the role of modified magnetic microballoon 1, while amido polymer group passes through Chemistry key connection dsDNA antigens 3 so that 1 structure of dsDNA antigen 3- amido polymer group 2- magnetic microspheres of preparation DsDNA immune magnetic microsphere architectures are stablized, and space steric effect effectively reduces, and ensure that the activity of antigen, improve The activity of dsDNA antigens 3.In addition, shown preparation method process conditions are easily-controllable, product dsDNA immune magnetics have been effectively ensured The performance of microballoon system is stablized.
On the other hand, an embodiment of the present invention provides a kind of dsDNA immune magnetic microspheres to preserve liquid.The dsDNA is immune It includes following component that magnetic microsphere, which preserves liquid,：
Specifically, above-mentioned dsDNA immune magnetic microspheres preserve the dilution contained by liquid and select but be not limited only to phosphate At least one of buffer solution, borate buffer solution, Tris-HCl buffer solutions, carbonate buffer solution.It not only acts as solvent load The effect of body, additionally it is possible to which the effect for effectively playing buffer plays counteracting, mitigates additional strong acid or highly basic to solution acid alkalinity It influences, to keep the pH value of solution to stablize relatively.As in one embodiment, pass through the effect of the thinner composition so that on It is 7-9 to state dsDNA immune magnetic microspheres and preserve the pH value of liquid.
Above-mentioned dsDNA immune magnetic microspheres preserve the dnase inhibitor contained by liquid and select but be not limited only to pancreas deoxidation core At least one of sour enzyme (DNase I), DNA topoisomerase enzyme inhibitors, Distacin.Select those dnase inhibitor energy Enough activity for effectively inhibiting deoxyribonuclease, prevent the dsDNA antigen quilts contained by dsDNA immune magnetic microspheres system above Deoxyribonuclease is degraded, to ensure that the bioactivity and stability of dsDNA immune magnetic microspheres system above.
Above-mentioned dsDNA immune magnetic microspheres preserve the metal-chelator contained by liquid and select but be not limited to ethylenediamine tetra-acetic acid (EDTA), tetrasodium ethylenediamine tetraacetate (EDTA-4Na), EDTAP dipotassium ethylene diamine tetraacetate (EDTA-2K), disodium ethylene diamine tetraacetate At least one of (EDTA-2Na).The chelating agent can effectively chelate Mg2+、Ca2+Equal metal ions, inhibit deoxyribose core Degradation of the sour enzyme to dsDNA；Therefore, which plays synergistic effect with dnase inhibitor, inhibits deoxyribose Degradation of the nuclease to dsDNA ensure that the bioactivity and stability of dsDNA immune magnetic microspheres system above.
Above-mentioned dsDNA immune magnetic microspheres preserve the polymer protective agent contained by liquid select but be not limited to trehalose, sucrose, At least one of fructose, lactose.The polymer protective agent of the selection is capable of providing more hydroxyl, can with it is described above The absorption of dsDNA immune magnetic microsphere systems, which combines, forms hydrogen bond, and so that dsDNA immune magnetic microsphere systems surface is formed one layer has The hydrated sheath of protective effect, to improve the stability of its preservation.
Above-mentioned dsDNA immune magnetic microspheres preserve the surfactant contained by liquid and select but be not limited to polyethylene glycol, polyoxy At least one of ethylene sorbitan mono-laurate -20, octyl phenyl polyoxyethylene ether.The surfactant of the selection The surface tension between dsDNA immune magnetic microsphere systems described above can be effectively reduced, dsDNA described above is improved Dispersibility of the immune magnetic microsphere system in homogeneous system.
Above-mentioned dsDNA immune magnetic microspheres preserve the site sealer contained by liquid and select but be not limited to bovine serum albumin(BSA) (BSA), at least one of casein, gelatin.The site sealer of the selection can be closed effectively and not tied with dsDNA on magnetic ball The active group of conjunction avoids the contained dsDNA antigens of these active groups interference and test antibodies specific bond, to improve DsDNA immune magnetic microsphere systems above are used to detect the accuracy of Anti-hCG action to be measured, while improving dsDNA above and exempting from The stability of epidemic disease magnetic microsphere architecture.
Above-mentioned dsDNA immune magnetic microspheres preserve the biological preservative contained by liquid and select but be not limited to isothiazolinone, ε- At least one of poly-D-lysine, Sodium azide, thimerosal, gentamicin.The biological preservative of selection can inhibit microorganism Enzyme activity or the cytogenetics structure of microorganism is had an impact, to inhibit or kill such as bacterium microorganism, with raising DsDNA immune magnetic microspheres system above is interfered from microorganism, improves the stability of its preservation, especially bioactivity is steady It is qualitative.
In addition, the preparation method that above-mentioned dsDNA immune magnetic microspheres preserve liquid can mix each component preparation Uniformly.
Therefore, it is to be directed to dsDNA immune magnetic microsphere systems described above that above-mentioned dsDNA immune magnetic microspheres, which preserve liquid, Characteristic and research and develop, therefore, be used for the preservation of dsDNA immune magnetic microsphere systems described above so that described above DsDNA immune magnetic microspheres system can stable dispersion wherein, and improve dsDNA immune magnetic microsphere systems described above Structural stability, bioactivity and dispersibility.Certainly, if not considering the optimum efficiency preserved, above-mentioned dsDNA immune magnetics Microballoon preserves the preservation that liquid can be also used for other immune magnetic microspheres.
On the basis of above-mentioned dsDNA immune magnetic microspheres system and preparation method thereof and its preservation liquid, the present invention is implemented Example provides a kind of Anti-hCG action detection reagent.The Anti-hCG action detection reagent includes that dsDNA described above exempts from Epidemic disease magnetic microsphere system and dsDNA immune magnetic microspheres described above preserve liquid.Wherein, the dsDNA immune magnetic microspheres System is scattered in the dsDNA immune magnetic microspheres and preserves in liquid.In this way, based on above to dsDNA immune magnetic microsphere systems The elaboration of liquid is preserved with dsDNA immune magnetic microspheres, the present embodiment Anti-hCG action detection reagent can effectively improve institute above Structural stability, bioactivity and the dispersibility for the dsDNA immune magnetic microsphere systems stated.
In one embodiment, in Anti-hCG action detection reagent, contained dsDNA immune magnetic microsphere systems are controlled Working concentration be 0.5mg/mL-2.0mg/mL, to improve its dispersion stabilization, and the above-mentioned dsDNA of performance that can be best is immune Magnetic microsphere preserves the effect of liquid, improves the structural stability and bioactivity of dsDNA immune magnetic microsphere systems.
Another aspect, an embodiment of the present invention provides a kind of Anti-hCG action detection kits.The Anti-hCG action The reagent that detection kit includes has：Anti-hCG action detection reagent described above.
In one embodiment, as described above, in the Anti-hCG action detection reagent contained by kit, dsDNA above is immune The working concentration of magnetic microsphere system is 0.5mg/mL-2.0mg/mL.
It is used in order to facilitate the Anti-hCG action detection kit, improves the efficiency of Anti-hCG action detection and accurate Property.In one embodiment, which further includes the anti-human igg of calibration sample, trace labelling substance markers At least one of antibody, buffer solution.
Wherein, the calibration sample contained by mentioned reagent box can be Anti dsDNA enterprises calibration object, such as include a concentration of The low concentration calibration product of 8.750IU/mL-16.250IU/mL and a concentration of 350.000IU/mL-520.000IU/mL high concentrations school Quasi- product.
The trace labelling object of the anti-human IgG antibodies of trace labelling substance markers contained by mentioned reagent box includes but not limited to gold In rigid alkane, luminol and its derivative, different luminol and its derivative, acridinium ester, alkaline phosphatase and horseradish peroxidase At least one, preferably N- (4- ammonia butyl)-N- ethyl different luminols.In a particular embodiment, trace labelling object working concentration： 5ng/mL-500ng/mL, the working concentration of anti-human IgG antibodies：50ng/mL-5000ng/mL.In one embodiment, the anti-human igg Antibody can be any one of mouse anti-human igg, rabbit anti-human igg, goat anti-human igg etc..
It is slow that carbonate buffer solution, phosphate buffer, borate are selected but be not limited to buffer solution contained by mentioned reagent box At least one of fliud flushing, Tris-HCl buffer solutions.
In addition, in order to further coordinate the use of mentioned reagent box, on the basis of the reagent that mentioned reagent box is configured, also The reagents such as luminous substrate can be configured, in order to avoid need provisional configuration when checking.The luminous substrate can be directed to trace labelling The conventional luminous substrate of object, in a particular embodiment, which can be, but not limited to sodium hydroxide and hydrogen peroxide.
Therefore, based on Anti-hCG action of embodiment of the present invention detection reagent described above and Anti-hCG action detection examination Agent box, Anti-hCG action method for separating and detecting is as shown in figure 3, include the following steps：
S03. dsDNA immune magnetic microsphere systems are utilized to capture Anti-hCG action：By sample to be tested and sheet described above Inventive embodiments Anti-hCG action detection reagent carries out incubation processing, carries out Magneto separate afterwards, and obtaining capture has sample to be tested anti- The dsDNA immune magnetic microsphere systems of dsDNA antibody；
S04. there are the dsDNA immune magnetics of Anti-hCG action micro- the anti-human IgG antibodies of trace labelling substance markers and capture Sphere system carries out immune response：Capture has the dsDNA immune magnetic microspheres system and the tracer of sample to be tested Anti-hCG action The anti-human IgG antibodies of label substance markers carry out incubation processing, carry out Magneto separate afterwards, and magnetic is immunized in the dsDNA for obtaining trace labelling object Property microballoon system；
S05. luminous signal is detected, Anti-hCG action concentration is calculated：To the dsDNA immune magnetics of the trace labelling object Luminous substrate is added in microballoon system, obtains luminous signal, and detects the luminous signal intensity and calculates the sample to be tested In the anti-concentration of anti-dsDNA.
Wherein, in above-mentioned steps S04-S05 each reagent as described in Anti-hCG action detection kit above.
Therefore, Anti-hCG action detection method Anti-hCG action detection reagent of the embodiment of the present invention described herein above Based on Anti-hCG action detection kit, the accurate and quantitative detection to Anti-hCG action in sample, and its spirit are realized Sensitivity is high.In addition, the Anti-hCG action detection method includes using Anti-hCG action detection reagent above or Anti-hCG action Detection kit is detected calibration object, to be compareed with sample to be tested testing result, quantitatively obtains sample to be tested moderate resistance The content of dsDNA antibody.
Correspondingly, above-mentioned Anti-hCG action detection reagent and Anti-hCG action detection kit and anti-dsDNA are based on Antibody detection method, an embodiment of the present invention provides a kind of Anti-hCG action detecting systems.The Anti-hCG action detection system System includes：
Anti-hCG action of embodiment of the present invention detection reagent described above or the embodiment of the present invention described above are anti- DsDNA antibody assay kits, Magneto separate module, detection module and data processing module.
Wherein, Magneto separate module, with Anti-hCG action of embodiment of the present invention detection reagent described above or described above Anti-hCG action detection kit of the embodiment of the present invention contained by dsDNA immune magnetic microspheres system and trace labelling object mark The anti-human IgG antibodies of note coordinate, and for detaching magnetic microsphere from liquid, complete the separation to Anti-hCG action；Particularly DsDNA immune magnetic microspheres system detaches magnetic microsphere using Magneto separate module, so with after sample to be tested hybrid reaction The magnetic microsphere of separation is mixed with the anti-human IgG antibodies of trace labelling substance markers again afterwards and is reacted, uses Magneto separate again Module detaches magnetic microsphere.
Detection module, the magnetic microsphere detached with Magneto separate module and luminous substrate cooperation, it is anti-to anti-dsDNA for completing The detection of body；Specifically, the detection module is to obtain the magnetic microsphere that Magneto separate module detaches under the action of luminous substrate Optical signal.
Data processing module, the content for calculating Anti-hCG action can show the content data of Anti-hCG action. Data processing specifically is carried out to optical signal in detection module, calculates the content of Anti-hCG action.Therefore, in an embodiment Data processing module can also include optical signal reading device.
In addition, above-mentioned Anti-hCG action detecting system can also include sampling module, for sampling to above-mentioned Magneto separate mould In block.
It can be seen from the above, the detection examination of Anti-hCG action of embodiment of the present invention detection agent described above, Anti-hCG action Agent box and Anti-hCG action detecting system due to based on being the above text inventive embodiments dsDNA immune magnetic microsphere systems, And it is first captured and is detached the Anti-hCG action in sample to be tested, formed immune complex, then by immune complex again with The anti-human IgG antibodies of trace labelling substance markers react again forms immune complex, after with luminous substrate excite trace labelling object It shines.In this way, since dsDNA immune magnetic microspheres system of the present invention is with structure as described above stabilization, steric hindrance Small, dsDNA antigen actives are high, therefore, can improve inspection with the abundant specific bond of Anti-hCG action in sample to be tested Survey sensitivity and the accuracy of result.
In conjunction with specific example, to dsDNA immune magnetic microsphere systems of the embodiment of the present invention and preparation method thereof and resist DsDNA antibody tests reagent, Anti-hCG action detection kit, Anti-hCG action detection method carry out further specifically It is bright.Material source used in following each embodiments is as follows：
Magnetic microsphere：NPD projects biomedical engineering limited liability company produces
Polyethyleneimine：SIGMA companies,
Polyamide：SIGMA companies,
ABEI：NPD projects biomedical engineering limited liability company produces
Double-stranded DNA antigen：Meridian companies,
Double-stranded-DNA antibody：NPD projects biomedical engineering limited liability company produces
Double-stranded-DNA antibody WHO international standard substances：NIBSC, WHO Wo/80
DsDNA immune magnetic microsphere systems and preparation method thereof embodiment
Embodiment 11
Present embodiments provide a kind of dsDNA immune magnetic microspheres system and preparation method thereof.Wherein, magnetic is immunized in dsDNA Property microballoon architecture it is as described in Figure 1 comprising nano-magnetic microsphere 1, polyethyleneimine amine groups 2 and dsDNA antigens are formed Nano-magnetic microsphere 1- polyethyleneimine amine groups 2-dsDNA antigens 3.
DsDNA immune magnetic microsphere system preparation methods include the following steps：
S11. the preparation of amino polymer functionalized magnetic microsphere：
1) a certain amount of magnetic ball is taken, the DCC of 0.5mg is added by every milligram of magnetic ball, DMF 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；
2) activated nanoscale magnetic bead solution removes supernatant, and the carbonate buffer solution of pH 9.5 is then added, keeps its dense Degree reaches 20mg/mL (in terms of nanoscale magnetic bead)；10 milligrams of aq. polyethyleneimines are added with every milligram of magnetic ball, ultrasonic 10min, Rotation mixing reacts 4h at room temperature, is discarded supernatant after Magneto separate；It is washed 3 times with the PBS buffer solution of 0.01mol/L pH 7.5, Each 3min；With the BSA cappings 2h of 10mg/mL；3 times are washed with the PBS buffer solution of 0.01mol/L pH7.5, Magneto separate After discard supernatant, obtain the magnetic microsphere of amino functional, it is spare；
The functionalized magnetic ball of S12.dsDNA cross linked aminos：
The magnetic microsphere for taking the amino polymer functionalization of a certain amount of step S11 preparations, is then added the carbonic acid of pH9.5 Salt buffer makes its concentration reach 20mg/mL (in terms of nanoscale magnetic bead)；It is anti-with the amount addition dsDNA of every milligram of 10 μ g of magnetic ball again Original, while the CDC of 0.5mg is added by every milligram of magnetic ball is subsequently placed in 38 DEG C of water-baths and shakes or mechanical agitation 2 is small When.Magnetic Isolation cleans solid three times with washing lotion (0.5%BSA solution), obtains the magnetic ball for being coated with dsDNA antigens, be DsDNA immune magnetic microsphere systems.
Embodiment 12
Present embodiments provide a kind of dsDNA immune magnetic microspheres system and preparation method thereof.Wherein, magnetic is immunized in dsDNA Property microballoon architecture is identical as embodiment 11.
DsDNA immune magnetic microsphere system preparation methods include the following steps：
S21. the preparation of amino polymer functionalized magnetic microsphere：With reference to the step S11 of embodiment 11, the difference is that poly- second The dosage of alkene imines is changed to 5mg.
The functionalized magnetic ball of S22.dsDNA cross linked aminos：Such as the step S12 of embodiment 11.
Embodiment 13
Present embodiments provide a kind of dsDNA immune magnetic microspheres system and preparation method thereof.Wherein, magnetic is immunized in dsDNA Property microballoon architecture it is identical as embodiment 11, wherein polyethyleneimine amine groups polyamide group substitutes, it is other with implement Example 11 is identical.
DsDNA immune magnetic microsphere system preparation methods include the following steps：
S31. the preparation of amino polymer functionalized magnetic microsphere：With reference to the step S11 of embodiment 11, the difference is that polyamides Amine substitutes polyethyleneimine.
The functionalized magnetic ball of S32.dsDNA cross linked aminos：Such as the step S12 of embodiment 11.
DsDNA immune magnetic microspheres preserve liquid embodiment
Embodiment 21
The present embodiment 21 provides a kind of dsDNA immune magnetic microspheres preservation liquid, and it includes following components：
1) dilution：Tris-HCl buffer solutions, working concentration 0.02mol/L；
2) dnase inhibitor：Pancreas picodna enzyme (DNase I), working concentration 0.01mg/mL；
3) metal-chelator：Ethylenediamine tetra-acetic acid (EDTA), working concentration 0.1mg/mL；
4) polymer protective agent：Trehalose, working concentration 1g/mL；
5) surfactant：Polyethylene glycol, working concentration 0.1mg/mL；
6) site sealer：Bovine serum albumin(BSA) (BSA), working concentration 1mg/mL；
7) biological preservative：Isothiazolinone, working concentration 1.5mg/mL.
Embodiment 22
The present embodiment 22 provides a kind of dsDNA immune magnetic microspheres preservation liquid, and it includes following components：
1) dilution：Phosphate buffer, working concentration 0.02mol/L；
2) dnase inhibitor：Distacin, working concentration 0.01mg/mL；
3) metal-chelator：EDTAP dipotassium ethylene diamine tetraacetate (EDTA-2K), working concentration 0.1mg/mL；
4) polymer protective agent：Sucrose, working concentration 1g/mL；
5) surface active agent composition：Polyethylene glycol, working concentration 0.1mg/mL；
6) site sealant components：Bovine serum albumin(BSA) (BSA), working concentration 1g/mL；
7) biological preservative component, Sodium azide, working concentration 1.5mg/mL.
Anti-hCG action detection reagent embodiment
Embodiment 31
The present embodiment 31 provides a kind of Anti-hCG action detection reagent.The dsDNA antibody tests reagent includes above-mentioned The dsDNA immune magnetic microspheres that the dsDNA immune magnetic microspheres system and above-described embodiment 21 that embodiment 11 provides provide preserve Liquid；Wherein, dsDNA immune magnetic microspheres system is dispersed in dsDNA immune magnetic microspheres and preserves in liquid, and magnetic is immunized in dsDNA Property microsphere ties up to a concentration of 1mg/mL in Anti-hCG action detection reagent.
Embodiment 32
The present embodiment 32 provides a kind of Anti-hCG action detection reagent.The dsDNA antibody tests reagent includes above-mentioned The dsDNA immune magnetic microspheres system and state the dsDNA immune magnetic microspheres preservation liquid that embodiment 21 provides that embodiment 12 provides； Wherein, dsDNA immune magnetic microspheres system is dispersed in dsDNA immune magnetic microspheres and preserves in liquid, and dsDNA immune magnetics are micro- Sphere ties up to a concentration of 1mg/mL in Anti-hCG action detection reagent.
Embodiment 33
The present embodiment 33 provides a kind of Anti-hCG action detection reagent.The dsDNA antibody tests reagent includes above-mentioned The dsDNA immune magnetic microspheres that the dsDNA immune magnetic microspheres system and above-described embodiment 21 that embodiment 13 provides provide preserve Liquid；Wherein, dsDNA immune magnetic microspheres system is dispersed in dsDNA immune magnetic microspheres and preserves in liquid, and magnetic is immunized in dsDNA Property microsphere ties up to a concentration of 1mg/mL in Anti-hCG action detection reagent.
Embodiment 34
The present embodiment 34 provides a kind of Anti-hCG action detection reagent.The dsDNA antibody tests reagent includes above-mentioned The dsDNA immune magnetic microspheres that the dsDNA immune magnetic microspheres system and above-described embodiment 22 that embodiment 11 provides provide preserve Liquid；Wherein, dsDNA immune magnetic microspheres system is dispersed in dsDNA immune magnetic microspheres and preserves in liquid, and magnetic is immunized in dsDNA Property microsphere ties up to a concentration of 1mg/mL in Anti-hCG action detection reagent.
Anti-hCG action detection kit embodiment
Embodiment 41
The present embodiment 41 provides a kind of Anti-hCG action detection kit.The dsDNA antibody assay kits include Following preparation：
A. the Anti-hCG action detection reagent that embodiment 31 provides：The 10 μ g/mL of working concentration of dsDNA antigens, it is magnetic micro- The working concentration 1mg/mL of ball working concentration 1mg/mL, dsDNA immune magnetic microsphere system；
B. low concentration calibration product, concentration 12.500IU/mL；
C. high concentration calibration object, concentration 400.000IU/mL；
The mouse anti-human IgG antibodies of d.ABEI labels：ABEI working concentration 250ng/mL, the work of mouse anti-human IgG antibodies are dense Spend 5000ng/mL；Wherein, the mouse anti-human IgG antibodies of ABEI labels are prepared as follows：1mg mouse anti-human IgG antibodies are taken, Its volume is adjusted to 1mL with 0.1mol/L carbonate buffer solutions (pH9.5), is then placed in bag filter, is placed in pH9.5 carbonic acid It dialyses 1 hour in salt buffer.The mouse anti human IgG antibody dialysed is taken out, 100 μ g ABEI-, half succinamic acids-are added NHS, room temperature shake 1.5 hours；Install G-25 gel columns, with purifying water elution it is clean after, then with the PBS of pH value 7.4 buffering Liquid is balanced elution；After G-25 gel column equilibrations elute, the mouse anti-human IgG antibodies for having marked ABEI are crossed into column purification, Then the protein solution for peak value occur is collected；Isometric protection liquid containing 5% BSA is added in the protein solution that will be gathered, Diluted is added to 0.15 μ g/mL.
E.Buffer buffer solutions：Tris-HCl buffer solutions.
Embodiment 42
The present embodiment 42 provides a kind of Anti-hCG action detection kit.The dsDNA antibody assay kits include The following preparation of component：
A. the Anti-hCG action detection reagent that embodiment 32 provides：The 10 μ g/mL of working concentration of dsDNA antigens, it is magnetic micro- The working concentration 1mg/mL of ball working concentration 1mg/mL, dsDNA immune magnetic microsphere system；
B. low concentration calibration product：Such as embodiment 41；
C. high concentration calibration object：Such as embodiment 41；
The anti-human IgG antibodies of d.ABEI labels：Such as embodiment 41；
E.Buffer buffer solutions：Tris-HCl buffer solutions.
Embodiment 43
The present embodiment 43 provides a kind of Anti-hCG action detection kit.The dsDNA antibody assay kits include Following preparation：
A. the Anti-hCG action detection reagent that embodiment 33 provides：The 10 μ g/mL of working concentration of dsDNA antigens, it is magnetic micro- The working concentration 1mg/mL of ball working concentration 1mg/mL, dsDNA immune magnetic microsphere system；
B. low concentration calibration product：Such as embodiment 41；
C. high concentration calibration object：Such as embodiment 41；
The anti-human IgG antibodies of d.ABEI labels：Such as embodiment 41；
E.Buffer buffer solutions：Tris-HCl buffer solutions.
Embodiment 44
The present embodiment 44 provides a kind of Anti-hCG action detection kit.The dsDNA antibody assay kits include Following preparation：
A. the Anti-hCG action detection reagent that embodiment 34 provides：The 10 μ g/mL of working concentration of dsDNA antigens, it is magnetic micro- The working concentration 1mg/mL of ball working concentration 1mg/mL, dsDNA immune magnetic microsphere system；
B. low concentration calibration product：Such as embodiment 41；
C. high concentration calibration object：Such as embodiment 41；
The anti-human IgG antibodies of d.ABEI labels：Such as embodiment 41；
E.Buffer buffer solutions：Such as embodiment 41.
Comparative example 1
This comparative example 1 provides a kind of Anti-hCG action detection reagent and kit.
The dsDNA antibody tests reagent includes the dsDNA immune magnetic microspheres system and guarantor that above-described embodiment 11 provides Liquid storage；Wherein, dsDNA immune magnetic microspheres system, which is dispersed in, preserves in liquid, and dsDNA immune magnetic microspheres system is anti- A concentration of 1mg/mL in dsDNA antibody test reagents, preservation liquid are Tris-HCl buffer solutions.
Anti-hCG action detection kit includes following preparation：
A. this comparative example 1 provides a kind of Anti-hCG action detection reagent：The 10 μ g/mL of working concentration of dsDNA antigens, Magnetic microsphere working concentration 1mg/mL；
B. low concentration calibration product：Such as embodiment 41；
C. high concentration calibration object：Such as embodiment 41；
The anti-human IgG antibodies of d.ABEI labels：Such as embodiment 41；
E.Buffer buffer solutions：Such as embodiment 41.
Comparative example 2
This comparative example 2 provides a kind of Anti-hCG action detection reagent and kit.
The dsDNA antibody tests reagent includes that existing dsDNA immune magnetic microspheres and above-described embodiment 21 provide DsDNA immune magnetic microspheres preserve liquid；Wherein, existing dsDNA immune magnetic microspheres are dispersed in dsDNA immune magnetic microspheres guarantor In liquid storage, and a concentration of 1mg/mL of the existing dsDNA immune magnetic microspheres in Anti-hCG action detection reagent.
Wherein, existing dsDNA immune magnetic microspheres are prepared as follows acquisition：
1) a certain amount of magnetic ball is taken, the DCC of 0.5mg is added by every milligram of magnetic ball, DMF 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；
2) activated nanoscale magnetic bead solution removes supernatant, and the carbonate buffer solution of pH9.5 is then added, makes its concentration Reach 20mg/mL (in terms of nanoscale magnetic bead)；DsDNA antigens are added with the amount of every milligram of 10 μ g of magnetic ball again, are subsequently placed in room temperature shake Shake reaction 2 hours.Magnetic Isolation cleans solid three times with washing lotion (0.5%BSA solution), obtains the magnetic for being coated with dsDNA antigens Ball.
The Anti-hCG action detection kit that this comparison 2 provides includes following preparation：
A. this comparative example 2 provides a kind of Anti-hCG action detection reagent：The 10 μ g/mL of working concentration of dsDNA antigens, Magnetic microsphere working concentration 1mg/mL；
B. low concentration calibration product：Such as embodiment 41；
C. high concentration calibration object：Such as embodiment 41；
The anti-human IgG antibodies of d.ABEI labels：Such as embodiment 41；
E.Buffer buffer solutions：Such as embodiment 41.
Comparative example 3
This comparative example 3 provides a kind of Anti-hCG action detection reagent and kit.
The dsDNA antibody tests reagent includes existing dsDNA immune magnetic microspheres and preservation liquid.Existing dsDNA is immune Magnetic microsphere, which is dispersed in, to be preserved in liquid, and existing dsDNA immune magnetic microspheres are dense in Anti-hCG action detection reagent Degree is 1mg/mL.
Wherein, existing dsDNA immune magnetic microspheres are according to existing dsDNA immune magnetic microspheres preparation method in comparative example 2 It obtains, preservation liquid is carbonate buffer solution.
The Anti-hCG action detection kit that this comparison 3 provides includes following preparation：
A. this comparative example 3 provides a kind of Anti-hCG action detection reagent：The 10 μ g/mL of working concentration of dsDNA antigens, Magnetic microsphere working concentration 1mg/mL；
B. low concentration calibration product：Such as embodiment 41；
C. high concentration calibration object：Such as embodiment 41；
The anti-human IgG antibodies of d.ABEI labels：Such as embodiment 41；
E.Buffer buffer solutions：Such as embodiment 41.
Experimental method, data and interpretation of result
The anti-dsDNA provided with above-described embodiment 41-44 Anti-hCG action detection kits provided and comparative example 1-3 Based on antibody assay kit, according to the Anti-hCG action detecting system specifically above described in embodiment part, according to Following detection method is immune using the Full-automatic chemiluminescence of Shenzhen New Industries Biomedical Engineering Co., Ltd.'s production Analyzer Maglumi 2000Plus carry out Performance Evaluation, and separation detection is carried out to Anti-hCG action in sample.Using Anti- Above-mentioned detection reagent while being placed in 37 DEG C of perseverances by sensitivity reference material in dsDNA enterprises reference material into the assessment of line sensitivity Accelerated stability test is carried out in constant temperature and humidity case.
1. the Anti-hCG action that Anti-hCG action detection kit and comparative example 1-3 that embodiment 41-44 is provided provide Anti-hCG action detection method based on detection kit is as follows：
1) preparation of 10 standard curves
Anti dsDNA enterprises calibration object is diluted to ten concentration points by a certain percentage with 0.02M PBS buffer solution is dilute, And working curve is calculated by 2 scaling correction curves.
2) use of Anti dsDNA chemical luminescence immune analysis reagent boxes
Anti dsDNA detection kits are produced using this method, in the Maglumi 2000Plus instrument of this department development ＆ production It is detected on device, detecting step includes：
A. 20 μ L samples to be tested, high-concentration and low-concentration calibration object are added in reaction cup；
B. the detection reagent of 20 μ L Anti-hCG actions is added；
C. 100 μ L Buffer buffer solutions are added；
D.37 DEG C incubation 10min, is placed under magnetic environment and cleans 3 times；
E. the anti-human IgG antibodies of 200 μ L ABEI labels are added；
F.37 DEG C incubation 10min, 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 Anti dsDNA concentration.
3) testing result and analysis
Testing result is as shown in the following table 1,2 according to the method described above.Wherein, sensitivity technique result is as described in Table 1, surely It is qualitative as shown in table 2.
As can be known from Table 1, the sensitivity in 1 scheme of 41-44 of the embodiment of the present invention and comparative example is obviously better than comparative example 3 Sensitivity, illustrate the characteristic due to amino polymer, improve the coating carrying capacity of double-stranded DNA antigen, while reducing space Steric effect enhances the combination effect of the present embodiment dsDNA immune magnetic microsphere systems binding purpose antibody in sample to be tested Rate, to improve detection sensitivity；And from embodiment 41 with the result of embodiment 42 it is found that magnetic microsphere polymerize with amino The conjugation ratios of object can influence the modification capacity of amino on magnetic ball, and the coupling efficiency of double-stranded DNA antigen is caused to be affected, to The joint efficiency of the present embodiment dsDNA immune magnetic microsphere systems binding purpose antibody in sample to be tested, can make sensitivity At certain influence；From the result of embodiment 41 and embodiment 43 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；And from embodiment 41-44 and comparative example 1-3 result Comprehensive Correlations show that immune magnetic microsphere stablizes protection liquid and equally has facilitation to the detection sensitivity of reagent.
The different embodiment detection sensitivity comparisons of table 1
As known from Table 2,41-44 of the embodiment of the present invention, reagent of the reagent stability than comparative example 1,2 of comparative example 2 are steady It is qualitative good, it is that the reagent stability especially in embodiment 41 is apparently higher than comparative example 3 as a result, 7 days falls 5% with It is interior, illustrate that dsDNA immune magnetic microspheres of the embodiment of the present invention preserve the enzyme inhibitor contained by liquid, metal ion chelation agent, poly Object protective agent, the active principles such as surfactant can preferably stablize protection dsDNA immune magnetic microspheres system in homogeneous body It is not degraded or destroys in system, to improve the stability of dsDNA immune magnetic microsphere systems.And from embodiment 41-44 with The comparing result synthesis of comparative example 1-3 shows steady to its using the structure of dsDNA immune magnetic microsphere systems of the embodiment of the present invention Qualitative to play certain facilitation, reason is to make antigen after magnetic ball is coupled with double-stranded DNA antigen by amino polymer Magnetic ball interface far from rigidity, reduces space steric effect, and ensure that the activity of antigen to a certain extent, to Protective effect is played to antigen.
The different embodiment high temperature of table 2 accelerate thermal stability comparison
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention All any modification, equivalent and improvement etc., should all be included in the protection scope of the present invention made by within refreshing and principle.

Claims (16)

1. a kind of dsDNA immune magnetic microspheres system, including magnetic microsphere ontology, which is characterized in that further include polymerizeing containing amino Object group and dsDNA antigens, and magnetic microsphere ontology described in the amido polymer base group modification, the dsDNA antigens with The amido polymer group chemical key connection.
2. dsDNA immune magnetic microspheres system according to claim 1, it is characterised in that：The amido polymer base Group is coupled the dsDNA antigens.
3. dsDNA immune magnetic microspheres system according to claim 1 or 2, it is characterised in that：The amido polymer Group is by least any one offer in polyethyleneimine, polyamide, polyacrylamide, poly-o-phenylenediamine；And/or
The mass ratio of the magnetic microsphere ontology and amido polymer group is 1：(1-10)；The amido polymer group It is (50-2000) with the dsDNA antigenic qualities ratio：1.
4. a kind of preparation method of dsDNA immune magnetic microspheres system, includes the following steps：
The magnetic microsphere of activated processing is reacted with amino polymer, then by reaction product at sealer Reason obtains the magnetic microsphere of amido polymer base group modification；
The magnetic microsphere of the amido polymer base group modification and dsDNA antigens are reacted under activator effect.
5. preparation method according to claim 4, it is characterised in that：In the magnetic microsphere and amino polymer reactant In system,
The mass ratio of the magnetic microsphere and the amino polymer is 1：(1-10)；And/or
The mass ratio of the magnetic microsphere and the sealer is (1-50):1；And/or
A concentration of 10mg/mL-20mg/mL of the magnetic microsphere；And/or
The amino polymer is at least one of polyethyleneimine, polyamide, polyacrylamide, poly-o-phenylenediamine；And/or
The sealer is at least one of casein, BSA, gelatin.
6. preparation method according to claim 4 or 5, it is characterised in that：In the magnetic microsphere and the dsDNA antigens In reaction system,
The mass ratio of the magnetic microsphere and the dsDNA antigens is (50-200)：1；And/or
The mass ratio of the magnetic microsphere and the activator is (2-4):1；And/or
The activator selects N, N'- diisopropylcarbodiimide, 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides, two Any one of carbodicyclo hexylimide.
7. a kind of dsDNA immune magnetic microspheres preserve liquid, which is characterized in that include following component：
8. dsDNA immune magnetic microspheres according to claim 7 preserve liquid, it is characterised in that：The dnase inhibitor is At least one of pancreas picodna enzyme, DNA topoisomerase enzyme inhibitors, Distacin；And/or
The polymer protective agent is at least one of trehalose, sucrose, fructose, lactose；And/or
The site sealer is at least one of bovine serum albumin(BSA), casein, gelatin；And/or
The biological preservative is at least one in isothiazolinone, ε-poly-D-lysine, Sodium azide, thimerosal, gentamicin Kind；And/or
The dilution be phosphate buffer, borate buffer solution, Tris-HCl buffer solutions, in carbonate buffer solution at least It is a kind of；And/or
The metal-chelator is ethylenediamine tetra-acetic acid, tetrasodium ethylenediamine tetraacetate, EDTAP dipotassium ethylene diamine tetraacetate, ethylenediamine tetrem At least one of acid disodium；And/or
The surfactant is polyethylene glycol, polyoxyethylene 20 sorbitan monolaurate -20, octyl phenyl polyoxyethylene At least one of ether.
9. a kind of Anti-hCG action detection reagent, including dsDNA immune magnetic microspheres and the dsDNA immune magnetic microspheres Liquid is preserved, the dsDNA immune magnetic microspheres are scattered in the preservation liquid；And the dsDNA immune magnetic microspheres are right It is required that any dsDNA immune magnetic microspheres systems of 1-3 or being prepared by any preparation methods of claim 4-6 DsDNA immune magnetic microsphere systems, the liquid that preserves is that any dsDNA immune magnetic microspheres of claim 7-8 preserve Liquid.
10. detection reagent according to claim 9, it is characterised in that：In the detection reagent, the dsDNA is immune The working concentration of magnetic microsphere is 0.5mg/mL-2.0mg/mL.
11. a kind of Anti-hCG action detection kit, including any detection reagents of claim 9-10.
12. kit according to claim 11, it is characterised in that：Further include calibration sample, trace labelling substance markers At least one of anti-human IgG antibodies, buffer solution.
13. kit according to claim 12, it is characterised in that：The trace labelling object is selected from adamantane, luminol And its at least one of derivative, different luminol and its derivative, acridinium ester, alkaline phosphatase and horseradish peroxidase.
14. kit according to claim 13, it is characterised in that：The trace labelling object is N- (4- ammonia butyl)-N- Ethyl different luminol.
15. a kind of Anti-hCG action detecting system, which is characterized in that including：
Claim 9-10 any one of them Anti-hCG action detection reagents or any reagents of claim 11-14 Box；
Magneto separate module coordinates with the dsDNA immune magnetic microspheres, for detaching magnetic microsphere from liquid；
Detection module, the magnetic microsphere detached with the Magneto separate module and luminous substrate cooperation, it is anti-to anti-dsDNA for completing The detection of body；
Data processing module, the content for calculating the Anti-hCG action.
16. a kind of any dsDNA immune magnetic microspheres system of claim 1-3 or claim 9-10 any one of them Anti-dsDNA described in Anti-hCG action detection reagent or claim the 11-14 any kit or claim 15 is anti- Application of the physical examination examining system in separation detection Anti-hCG action.
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CN109521194A (en) * 2018-11-30 2019-03-26 暨南大学 DNA immunization adsorbent is preparing the application in anti-ds-DNA antibody detection reagent
CN109521193A (en) * 2018-11-05 2019-03-26 暨南大学 DNA immunization adsorbent is preparing the application in Anti-hCG action detection reagent
CN110609134A (en) * 2019-10-14 2019-12-24 珠海丽珠试剂股份有限公司 Magnetic particle preservation solution, preparation method and use method thereof, immunoreaction reagent and application thereof
CN110850076A (en) * 2019-11-28 2020-02-28 南京迪安医学检验所有限公司 Anti-double-chain DNA antibody IgG chemiluminescence immunoassay kit and preparation method and application thereof
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CN109521193A (en) * 2018-11-05 2019-03-26 暨南大学 DNA immunization adsorbent is preparing the application in Anti-hCG action detection reagent
CN109521194A (en) * 2018-11-30 2019-03-26 暨南大学 DNA immunization adsorbent is preparing the application in anti-ds-DNA antibody detection reagent
CN109521194B (en) * 2018-11-30 2023-03-31 暨南大学 Application of DNA immunoadsorbent in preparation of anti-ds-DNA antibody detection reagent
CN109355287A (en) * 2018-12-14 2019-02-19 长春市志昂生物科技有限公司 A method of exempting from pre-treatment automation and extracts large volume whole blood DNA
CN110609134A (en) * 2019-10-14 2019-12-24 珠海丽珠试剂股份有限公司 Magnetic particle preservation solution, preparation method and use method thereof, immunoreaction reagent and application thereof
CN110609134B (en) * 2019-10-14 2022-10-04 珠海丽珠试剂股份有限公司 Magnetic particle preservation solution, preparation method and use method thereof, immunoreaction reagent and application thereof
CN110850076A (en) * 2019-11-28 2020-02-28 南京迪安医学检验所有限公司 Anti-double-chain DNA antibody IgG chemiluminescence immunoassay kit and preparation method and application thereof
CN112881680A (en) * 2021-01-19 2021-06-01 深圳市卓润生物科技有限公司 Method for preparing dsDNA-linked magnetic particles
CN113866157A (en) * 2021-08-19 2021-12-31 宁波瑞源生物科技有限公司 Application of polyethyleneimine compound in luminescent method in-vitro diagnostic reagent
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