CN108469526A - Double-stranded DNA antigen, preparation method, the reagent comprising it, kit and application - Google Patents

Double-stranded DNA antigen, preparation method, the reagent comprising it, kit and application Download PDF

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CN108469526A
CN108469526A CN201810127235.5A CN201810127235A CN108469526A CN 108469526 A CN108469526 A CN 108469526A CN 201810127235 A CN201810127235 A CN 201810127235A CN 108469526 A CN108469526 A CN 108469526A
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饶微
袁锦云
张云
孙志成
易昊禹
陈顺利
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Shenzhen New Industries Biomedical Engineering Co Ltd
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Abstract

The present invention provides a kind of double-stranded DNA antigen, preparation method, the reagent comprising it, kit and applications.The preparation method includes extraction double-stranded DNA；Using protease and RNA enzyme removal double-stranded DNA in histone and RNA ingredients obtain the first purifying DNA；The segment that first purifying DNA is cut into 500~2000bp obtains shearing DNA；Single-stranded and the modification that methylates DNA molecular in shearing DNA is eliminated using s1 nuclease, exonuclease I and demethylase and obtains the second purifying DNA；And purifying recycling is carried out to the second purifying DNA and obtains double-stranded DNA antigen.The DNA molecular of single stranded DNA and modified using various enzymes to being generated in albumen that may be present, RNA, operating process in the double-stranded DNA that is extracted carries out degradation digestion, so that the purity of gained double-stranded DNA antigen is relatively higher, thus keep testing result specificity high more acurrate.

Description

Double-stranded DNA antigen, preparation method, the reagent comprising it, kit and application
Technical field
The present invention relates to field of immunodetection, in particular to a kind of double-stranded DNA antigen, preparation method, include it Reagent, kit and application.
Background technology
Autoimmunity disease is due to immunologic function disorder, and body generates the pathologic immune responsing reaction for autoantigen And cause a kind of disease of organ or system injury.Currently, the pathogenesis for autoimmunity disease is not fully understood.Generally Thinking autoimmunity disease, to be inheritance susceptible individual such as infect in environmental factor, ultraviolet light, tumour and drug many factors are common The lower generation of effect.The appearance of the autoantibody and sensitization lymphocyte of high titre is the essential characteristic of autoimmunity disease, Middle antinuclear antibodies (antinuclear antibody, ANA) is also known as anti-antigen nucleic acid antibody, is one group by itself eukaryocyte Various composition deoxyribonucleoprotein (DNP), DNA, extractable nuclear antigen (ENA) and RNA etc. itself resist as target antigen The general name of body, the target antigen that antinuclear antibodies is directed to is mainly intracellular nucleic acid and nucleoprotein ingredient, in the transcription of cell cycle It plays an important role in translation process, to specific detection the clarifying a diagnosis with important reference value, especially to disease of ANA It is to systemic loupus erythematosus (systemic lupus erythematosus, SLE), Combination knot hoof tissue disease (mixed Connective tissue disease, MCTD), Sjogren syndrome (Sjogren syndrome, SS), systemic sclerosis (systemic sclerosis, SSc), polymyositis/dermatomyositis (polymyositis/dermatomyositis) and primary The diagnosis of the diseases such as biliary cirrhosis (Primary biliary cirrhosis, PBC) has higher specificity.
Wherein systemic loupus erythematosus (systemic lupus erythematosus, SLE) is a kind of chronic inflammation Autoimmune disease, it is main to invade connective tissue and influence multiple tracts.Worldwide, in Europe, SLE is annual Newly-increased incidence be about 25/,100,000, and north America region incidence higher.SLE easily hairs often recur in young woman, the course of disease It is alternately present with alleviation.Patient's body can generate the antinuclear antibodies and other autoantibodies for nucleic acid, nucleoprotein and histone, These autoantibodies are combined to form immune complex with corresponding antigens, can be deposited on cardiovascular connective tissue, glomerular basement membrane, On serous coat, synovium of joint and a variety of internal organs thin vessels walls, immune complex attracts neutrophil leucocyte leaching in Local activation complement Profit, causes the chronic inflammatory injury of local organization.Therefore SLE patient often suffers from the damage of multisystem, multiple organ.According to the device of damage Official is different, and patient may occur in which that fever, fash, arthralgia, renal damage, cardiovascular pathological changes, scrositis, anaemia, mental symptom etc. are more Kind clinical manifestation.Rheumatism association of the U.S. and 11 ARA standards (ARA, the U.S.'s rheumatism diagnosed about SLE is revised within 1997 Association).11 standards are respectively：1. butterfly erythema；2. other positions of body occur red, scales of skin that peel off shape, limitation, swell Skin rash；3. phenomenon of photosensitivity；4. oral mucosa pain or ulcer；5. arthralgia or joint exudation；6. scrositis；7. kidney disease Disease；8. the nervous system disease；9. hematoligical symptom goes out as what is reduced with reticulosis, Neuroleptic Leukocytopenia, lymphocyte Hemolytic hemolytic anemia；10. immunological method detects Anti-hCG action, Sm antibody, anticardiolipin antibodies；11. indirect immunofluorescence is examined Go out ANA.If occurring 4 in 11 standards, 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.Arana in 1967 is found that a kind of autoantibody that can be combined with n DNA, referred to as n DNA in SLE patients serums Antibody or dsDNA antibody.With going deep into for research, it has been found that Anti-hCG action almost exists only in SLE patients serums, It is the specific antibody of SLE, and there are correlations for the activity of the titre of antibody and SLE.
SLE patient's body anti-DNA antibodies are divided into as Anti-hCG action and anti-ssDNA antibody, and Anti-hCG action is mainly known Other site be located at DNA double helical structure on ribonucleic acid skeleton structure, be mainly seen in SLE, positive rate be 40%~ 90%, specificity seldom occurs, general positive rate is below more than 90% in other autoimmune diseases and normal person 10%, and antibody titer is also low, and also such patient is commonly considered as SLE overlap syndromes.The main identification of anti-ssDNA antibody Site is the purine being exposed after double-stranded DNA uncoiling and pyrimidine bases multimeric structure, and the measurement knot of anti-ssDNA antibody Fruit lacks disease specific, in addition to SLE patient has compared with high detection rate (50%-60%), other rheumatism such as Combination connective group Disease, drug-induced lupus, chorionitis, dermatomyositis, Sjogren syndrome are knitted, rheumatoid arthritis etc. also has 10%-70% Recall rate.Therefore, meaning of the Anti-hCG action in terms of autoimmune disease especially SLE clinical diagnosises is apparently higher than anti- SsDNA antibody, and the detection of Anti-hCG action is very easy to be interfered by anti-ssDNA Antibody Results in clinical application, thus it is anti- DsDNA antibody is highly sensitive, high specific detection is most important for clarifying a diagnosis for auxiliary SLE diseases, and for anti- The detection of dsDNA antibody, it is necessary first to solve the problems, such as it is that highly purified and complete double chain DNA molecule is prepared.
Double-stranded DNA is the double-spiral structure made of two long chain-coilings of antiparallel deoxynucleotide；The outside of DNA The basic framework alternately connected and composed by deoxyribose and phosphoric acid, inside is that base connects the base-pair to be formed, alkali by hydrogen bond Pairing between base follows base pair complementarity principle (A-T, C-G).In normal cell, the hydrogen bond between base-pair is opened, Need the effect of unwindase.The ratio for the base-pair that G and C is formed in DNA is higher, and structure is more stable, untwists into single-stranded institute completely The temperature needed is higher.
Under normal circumstances, DNA double helical structure is very stable, and there are mainly two types of active forces, and DNA double helical structure to be made to tie up It is fixed to keep steady, and a kind of active force is the hydrogen bond formed between A-T, C-G complementary base, and second of active force is the base in DNA molecular Accumulation force, layer upon layer between base form a hydrophobic core inside DNA, and almost without trip in hydrophobic core heart district From hydrone, this forms hydrogen bond between being more advantageous to complementary base.And double chain DNA molecule is in vitro, easily by environment Factor influences, such as various physics (high temperature, ultraviolet irradiation), chemical (pH changes, methanol, ethyl alcohol, urea and formamide), machinery Etc. the faint variation of factors, damage or fracture will be brought it about, it is random that DNA molecular is loosened by the double-spiral structure stablized , the single-stranded linear structure of out-of-flatness, when denaturation maintains the hydrogen bond fracture of duplex stability, and the accumulation force between base is by broken It is bad, so that internally positioned purine pyrimidine base is exposed.Base has the function of absorbing ultraviolet light, outside the base of single stranded DNA Dew so that system after denaturation is referred to as hyperchromicity to the influx and translocation of ultraviolet light at wavelength 260nm, the process, this also at To detect the most important means of DNA denaturation at present.The denaturation temperature Tm of DNA refers to the double-stranded DNA denaturation when system has 50% When corresponding temperature.Thus, when the system residing for the double chain DNA molecule keeps the Tm of double chain DNA molecule higher, double chain DNA molecule Structure is more stable.And dsDNA molecules be coated with processing procedure and it is follow-up preserve transport and use during there may be double Ssdna molecule is degraded and destruction formed it is single-stranded, therefore, optimization antigen-magnetic microsphere coating buffer solution and magnetic microsphere conjugate Preserve liquid maintains the duplex structure of DNA molecular most important for improving the Tm of system residing for double chain DNA molecule and stablizing.
At present in terms of detection method, the method that detects Anti-hCG action is mainly radiommunoassay, it is glimmering to be immunized indirectly Light method, colloidal gold method, Western blot and enzyme linked immunosorbent assay, wherein radiommunoassay tend to be naughty because there is radiocontamination It eliminates, indirect immunofluorescence result interpretation needs fluorescence microscope, and operator is needed to have certain morphological base, by Artificial subjective factor is affected, and difficult quantitative, and colloidal gold method is only suitable for qualitative detection, and sensitivity is poor, is easily disturbed, and exempts from Epidemic disease blotting is possible to antigen loss or denaturation occur in transfer process, so the combination of antigen-antibody is influenced, in addition, IBT bands do not develop the color or unintelligible, and result is caused to be not easy to observe, and enzyme linked immunosorbent assay can be used for quantifying detection, but due to DsDNA is more difficult to be coated on solid phase carrier, thus detection range is relatively narrow, and detection sensitivity and accuracy are unable to get guarantee.
Therefore, poor specificity, sensitivity and accuracy are detected existing for the context of detection for Anti-hCG action at present Relatively low defect there is no effective solution scheme.
Invention content
The main purpose of the present invention is to provide a kind of double-stranded DNA antigen, preparation method, the reagent comprising it, reagents Box and application, to solve the problems, such as that double-stranded DNA antigen in the prior art is low there are purity and causes testing result impacted.
To achieve the goals above, according to an aspect of the invention, there is provided a kind of preparation side of double-stranded DNA antigen Method, the preparation method include：Extract double-stranded DNA；Using protease and RNA enzyme removal double-stranded DNA in histone and RNA at Point, obtain the first purifying DNA；The segment that first purifying DNA is cut into 500~2000bp, obtains shearing DNA；Using nucleic acid Enzyme S1, exonuclease I and demethylase eliminate single-stranded and the modification that methylates the DNA molecular in shearing DNA, obtain second Purify DNA；And purifying recycling is carried out to the second purifying DNA, obtain double-stranded DNA antigen.
Further, double-stranded DNA is extracted from calf thymus tissue, salmon essence, Hela cells, human blood or saliva；It is excellent First purifying DNA is cut into 500 by selection of land in such a way that No. 4.5 hypodermic needles pair first purify DNA progress pressure-vaccums The segment of~2000bp obtains shearing DNA；Preferably, the frequency of pressure-vaccum is 10~30 times/min, the number of pressure-vaccum is 90~ 120 times；Further include the steps that being concentrated to the first purifying DNA, into one it is highly preferred that before to the first purifying DNA shearings First purifying DNA is preferably concentrated into a concentration of 1.8~3mg/mL by step.
According to the second aspect of the invention, a kind of double-stranded DNA antigen is provided, which uses above-mentioned A kind of preparation method is prepared.
According to the third aspect of the present invention, a kind of reagent of detection Anti-hCG action is provided, reagent includes double-strand DNA antigens, double-stranded DNA antigen are prepared using any of the above-described kind of preparation method.
Further, reagent further includes the solid phase carrier for loading double-stranded DNA antigen, and preferably solid phase carrier is magnetic micro- Ball or microwell plate.
Further, solid phase carrier is magnetic microsphere, and reagent further includes for being coated with double-stranded DNA antigen to magnetic microsphere Coating buffer solution, preferably be coated with buffer solution include：Tris-HCl buffer solutions, cationic metal salt and enzyme inhibitor, coating The pH of buffer solution is 7.5~8.5；It is highly preferred that coating buffer solution includes：The Tris-HCl buffer solutions of 10~50mM, 0.1~ The NaCl and/or KCl of 0.3M；And the EDTA of 0.5~2mM.
Further, reagent further includes the storage buffer solution that pH is 7.5~8.5, and storage buffer solution includes：Basis buffering Liquid, basis buffer include Tris-HCl buffer solutions, cationic metal salt and preservative, and preferably basis buffer includes：10~ The Tris-HCl buffer solutions of 50mM, the NaCl and/or KCl of 0.1~0.5M and the preservative of 0.05~0.2wt%, more preferably Ground, preservative are Proclin300 or thimerosal；Stable storage liquid, stable storage liquid include DNA molecular protective agent, enzyme inhibition Any one or more of agent, surfactant, DNA double chain structural stabilizing agent and sealer in；Preferably, DNA molecular is protected Shield agent is cycloheptaamylose, cyclohexaamylose or cyclooctaamylose；In more preferable stable storage liquid, DNA molecular is protectant to be contained Amount is 1~3wt%；Preferably, surfactant is anion surfactant or nonionic surfactant, it is more preferably cloudy from Sub- surfactant is SDS or SLS；Nonionic surfactant is PEG6000, polysorbate80 or polysorbate20； It is further preferred that in stable storage liquid, the content of anion surfactant is 0.02~0.05wt%, and non-ionic surface is lived Property agent content be 0.05~0.1wt%；Preferably, DNA double chain structural stabilizing agent is containing Al3+、Co3+Or Ce3+Compound； In more preferable stable storage liquid, a concentration of 5~10mM of DNA double chain structural stabilizing agent；Preferably, enzyme inhibitor is 0.5~2mM EDTA；Preferably, sealer is BSA, casein or the gelatin of 0.5~2wt%.
Further, reagent further includes detection buffer solution, detects in buffer solution and contains s1 nuclease, more preferably detection buffering A concentration of 0.5~2 μ g/L of liquid amplifying nucleic acid enzyme S1, it is further preferred that detection buffer solution further includes：10~50mM phosphate is slow Fliud flushing, 0.5~2wt%BSA, 0.1~0.3M sodium chloride and 0.05~0.2wt%Proclin300.
According to the fourth aspect of the present invention, a kind of kit of detection anti-dsDNA antibody is provided, kit includes Any of the above-described kind of reagent.
Further, kit further includes any one or more in luminous marker, calibration product and quality-control product；It is preferred that Luminescent marking is the ABEI that anti-human igg, IgA or IgM are marked, and calibration product and quality-control product are each independently the anti-double-strand of humanized The antibody of DNA.
Further, the double-stranded DNA antigen in kit exists in the form of antigen-solid phase carrier conjugate, and solid phase carries Body is magnetic microsphere.
Further, kit further includes the magnetic microsphere pipe for holding antigen-solid phase carrier conjugate, magnetic microsphere Pipe use is protected from light material and is made.
According to the fifth aspect of the present invention, a kind of detection method of anti-dsDNA antibody is provided, detection method includes Pass through the dense of the anti-dsDNA antibody in Chemiluminescence immunoassay detection sample to be tested using the target antigen of anti-dsDNA antibody The step of spending, the target antigen of anti-dsDNA antibody are the double-stranded DNA antigen that any of the above-described kind of preparation method is prepared.
Further, it is detected in sample to be tested by Chemiluminescence immunoassay using the target antigen of anti-dsDNA antibody In the step of concentration of anti-dsDNA antibody, reaction temperature is 25~30 DEG C.
According to the sixth aspect of the invention, the double-stranded DNA antigen that any of the above-described kind of preparation method is prepared is provided Or any of the above-described kind of reagent for detecting Anti-hCG action or any of the above-described kind of kit are examined in anti-dsDNA antibody screening clinic Application in survey.
Further, which includes the clinical assistant diagnosis to systemic loupus erythematosus.
According to the sixth aspect of the invention, application of any of the above-described kind of kit in antinuclear antibodies detection is provided, The application includes：Kit is applied to semi-automatic immunity analysis instrument or automatic lmunoassays analyzer.
It is few to consider single stranded DNA generally to remove based on RNA and histone in terms of double-stranded DNA antigen extraction purification Influence, but the preparation method of the above-mentioned double-stranded DNA antigen of the application by using various enzymes to may in the double-stranded DNA that is extracted The DNA molecular of the single stranded DNA and modified that are generated in existing albumen, RNA, operating process carries out degradation digestion so that institute The purity for obtaining double-stranded DNA antigen is relatively higher, has specificity high when for detecting the double-stranded-DNA antibody in sample, detects As a result more accurate advantage.
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.
Term is explained:
ANA：Antinuclear antibodies.
Target antigen：The substance (including nucleic acid or nucleoprotein) that can be reacted with corresponding antibody specificity.
Anti-hCG action：Anti-dsDNA antibody.
Anti- ssDNA antibody：Anti-ssDNA antibody.
Systemic loupus erythematosus：(systemic lupus erythematosus,SLE).
ABEI：N- (4- aminobutyls)-N- ethyl different luminols.
SDS：Lauryl sodium sulfate.
SLS：Sodium laureth sulfate.
EDC：1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides.
DCC：Dicyclohexylcarbodiimide.
As background technology is previously mentioned, there is specificity in the antigen in the prior art for anti-dsDNA antibody detection Defect that is poor and being difficult to the double-stranded-DNA antibody in accurately detection sample, it is a kind of typical in the application in order to improve this situation In embodiment, a kind of preparation method of double-stranded DNA antigen is provided, which includes：Extract double-stranded DNA；Using egg White enzyme and RNA enzyme removal obtain the first purifying DNA to the histone and RNA ingredients in double-stranded DNA；By the first purifying DNA shearings At the segment of 500~2000bp, shearing DNA is obtained；It is eliminated and is sheared using s1 nuclease, exonuclease I and demethylase Single-stranded or the modification that methylates DNA molecular in DNA, obtains the second purifying DNA；And the second purifying DNA is purified back It receives, obtains double-stranded DNA antigen.
The preparation method of the above-mentioned double-stranded DNA antigen of the application, by using various enzymes to can in the double-stranded DNA that is extracted The DNA molecular of the single stranded DNA and the modified that methylates that are generated in albumen, RNA, operating process existing for energy carries out degradation and disappears Change so that the purity of gained double-stranded DNA antigen is relatively higher, has when for detecting the double-stranded-DNA antibody in sample special The relatively high advantage of property.
In the preparation method of above-mentioned double-stranded DNA, DNA fragmentation difference is relatively large when the first step purifies, and is carried using phenol chloroform It is pure preferable.DNA fragmentation is in a certain size substantially when second step purifies, can first Purified in electrophoresis, then cut glue and cross column affinity purification, Not only gained double-stranded DNA antigen purity is high after purification, but also can determine that clip size.Specific purity detecting can pass through A280/A260To carry out indirect detection.
The source of above extracted double-stranded DNA include but are not limited to calf thymus tissue, salmon essence, Hela cells, Human blood or saliva.Since the purpose of the application is the content for detecting humanized's Anti-hCG action, antibody identifies target antigen Double chain DNA molecule predominantly in human body, thus, select people or anti-as target with the higher kind DNA molecular of people's DNA homology Originally detecting Anti-hCG action in human serum has better antigen-antibody affinity and detection sensitivity.But from raw material can Acquired angle considers, is because of ox and people's gene more preferably from the dsDNA molecules of calf thymus tissue extraction as target antigen Group size is substantially suitable, and DNA molecular sequence and space structure similarity are high, thus, it is possible to ensure antigen-antibody reaction have compared with High affinity and good detection sensitivity, avoid the occurrence of missing inspection situation.
The purpose repeatedly purified in above-mentioned preparation method is higher in order to obtain the purity without single stranded DNA as far as possible Double chain DNA molecule, and double chain DNA molecule is made to be maintained at above-mentioned suitable length.Since antibody identifies the site size one of DNA molecular As be 6~12bp, more than 60bp DNA molecular could with antibody firm connection, it is contemplated that dsDNA antigens packet when subsequent detection Situation is preserved by, steric hindrance and later stage, by prepared double chain DNA molecule control in 1kb or so, with improve coating efficiency and Repeatability, while promoting to disperse, and preferably maintain the duplex structure of DNA molecular.
In above-mentioned preparation method, it is special that the concrete mode of the first purifying DNA segments for cutting into 500~2000bp is had no It limits, as long as can shear DNA to the segment of above-mentioned size under the premise of not destroying duplex structure as far as possible.At this Apply in a kind of preferred embodiment, using No. 4.5 syringe needles pair first purify DNA carry out it is pure by first by the way of pressure-vaccum Change the segment that DNA cuts into 500~2000bp, obtains shearing DNA.The pore size of No. 4.5 syringe needles is in pressure-vaccum process In it is smaller to the mechanical force shear property model of DNA, can ensure segment of the DNA longitudinal shears at 500~2000bp, while ensureing cross To the stabilization of Hyarogen-bonding between base.Thus No. 4.5 syringe needles can reduce the destruction to double-stranded DNA.Certainly, Syringe needle aperture can also be optimized on the basis of the syringe of above-mentioned pore size, to select more suitable aperture Syringe carry out above-mentioned pressure-vaccum shear history.
During above-mentioned pressure-vaccum, the syringe needle of different size has different an internal diameter and outer diameter, usually with outer diameter come The outer diameter of name, used in this application No. 4.5 syringe needles is 0.45mm.The frequency or number of specific pressure-vaccum be not special It is different to limit, be subject to the DNA fragmentation actually obtained size within the above range.In a kind of preferred embodiment, pressure-vaccum Frequency is 10~30 times/min, and the number of pressure-vaccum is 90~120 times.According to the above-mentioned number of pressure-vaccum frequency pressure-vaccum so that purpose The size of DNA fragmentation is more concentrated relatively.
In above-mentioned shearing step, according to the concentration of the DNA extracted, it can will be waited for by concentration or diluted mode The DNA of shearing is controlled in suitable concentration range.Using above-mentioned mechanical force to DNA carry out shearing interrupt when, concentration high point effect Fruit can be more preferable.Therefore, before being sheared to the first purifying DNA, the step of can also be concentrated.By using 1 butanol Concentration of DNA solution is repeated several times in (n-butyl alcohol or 1-butanol) or 2 butanol (2-butanol), in this way, can incite somebody to action The volume concentration of DNA sample to be precipitated to can use absolute ethyl alcohol precipitate volume.It is dense in a kind of more specific embodiment The operating procedure of contracting DNA is as follows：1) it is added in 2 isometric butanol to DNA solution, mixes well；2) it centrifuges 1 minute, 1600g Remove upper organic phase；3) step 1) and 2) is repeated, until required volume；4) it with water saturated ether extraction, removes in solution 2 butanol；5) vacuum extraction removes ether.
Further include being purified to first before to the first purifying DNA shearings in a kind of preferred embodiment of the application First purifying DNA is further preferably concentrated into a concentration of 1.8~3mg/mL, most preferably 2mg/ by the step of DNA is concentrated mL.DNA to be sheared is concentrated into above-mentioned concentration range, with the high advantage of shear efficiency.
In second of typical embodiment of the application, a kind of double-stranded DNA antigen is provided, which adopts It is prepared with above-mentioned any preparation method.Double-stranded DNA antigen prepared by above-mentioned preparation method has purity high and length is fitted Suitable advantage.
In the application in the third typical embodiment, a kind of reagent of detection Anti-hCG action, the reagent are provided Including double-stranded DNA antigen, double-stranded DNA antigen is prepared using any of the above-described kind of preparation method.Due to above-mentioned preparation method system Double-stranded DNA antigen made of standby has the advantages that purity height and suitable length, thus uses the reagent containing the double-stranded DNA antigen When detecting Anti-hCG action, also have the effect of that detection specificity is high and accuracy is high under the premise of ensureing sensitivity.
In order to keep detection more convenient, the reagent of above-mentioned detection Anti-hCG action further includes for loading double-stranded DNA antigen Solid phase carrier.By loading to double-stranded DNA antigen on solid phase carrier, with joint efficiency is high, steric hindrance is small, conjugate knot The advantages such as structure stabilization.Above-mentioned solid phase carrier can be existing solid phase carrier, in this application including but not limited to magnetic microsphere Or microwell plate.
In order to further increase above-mentioned detection reagent double center chain DNA antigens duplex structure stability and reduce by its The degradation or unwinding of double-stranded DNA during solid phase carrier are loaded into single stranded DNA, in a kind of preferred embodiment of the application, Above-mentioned solid phase carrier is magnetic microsphere, and mentioned reagent further includes that the coating for being coated with double-stranded DNA antigen to magnetic microsphere is delayed Fliud flushing, being preferably coated with buffer solution includes：Tris-HCl buffer solutions, cationic metal salt and enzyme inhibitor, are coated with buffer solution PH is 7.5~8.5；It is highly preferred that coating buffer solution includes：The Tris-HCl buffer solutions of 10~50mM, the NaCl of 0.1~0.3M And/or KCl；And the EDTA of 0.5~2mM.
In above-mentioned preferred embodiment, the pH for being coated with buffer solution is controlled 7.5~8.5, to prevent DNA molecular or RNA Dissociation.The effect of Tris-HCl buffer solutions is maintenance system in certain pH range, protects the base integrality of double-stranded DNA, is prevented Only open loop or fracture.The enzyme inhibitors such as EDTA are energy and Mg2+、Ca2+、Mn2+、Fe2+The chelating that equal bivalent metal ions combine Agent prevents DNA molecular from degrading.Since the cation of the salt form of cationic metal salt such as NaCl and KCl in solution may act as Screener, the repulsion between two strands of chains to reduce double-stranded DNA enhance the stability of duplex structure, improve DNA molecular and become Warm-natured degree Tm values.
Above-mentioned coating buffer solution can prevent or reduce during double-stranded DNA antigen is coated with magnetic microsphere to double-strand The destruction of DNA molecular.In addition, double-stranded DNA antigen-magnetic microsphere the conjugate formed after coating is in long-term holding and transportational process In still remain generate open loop or fracture generate single stranded DNA risk, to cause false positive results.It is deposited to be further reduced Or transportational process in above-mentioned damage, in a kind of preferred embodiment of the application, mentioned reagent further include pH be 7.5~ 8.5 storage buffer solution, the storage buffer solution include：Basis buffer and stable storage liquid, basis buffer include Tris- HCl buffer solutions, cationic metal salt and preservative, preferably basis buffer include：The Tris-HCl buffer solutions of 10~50mM, The NaCl and/or KCl of 0.1~0.5M；And the preservative of 0.05~0.2wt%, it is preferable that preservative be Proclin300 or Thimerosal；Stable storage liquid includes DNA molecular protective agent, surfactant, DNA double chain structural stabilizing agent, sealer and enzyme Any one or more in inhibitor；Preferably, cycloheptaamylose, cyclohexaamylose or cyclooctaamylose；More preferably storage In stabilizing solution, the protectant content of DNA molecular is 1~3wt%；Preferably, surfactant includes anion surfactant And nonionic surfactant, more preferable anion surfactant are SDS or SLS；Nonionic surfactant is PEG6000, polysorbate80 or polysorbate20；It is further preferred that in stable storage liquid, anion surfactant Content be 0.02~0.05wt%, the content of nonionic surfactant is 0.05~0.1wt%；Preferably, DNA double chains Structure stabilizer contains Al3+、Co3+Or Ce3+Compound；In more preferable stable storage liquid, the content of DNA double chain structural stabilizing agent is 5 ~10mM；Preferably, sealer is BSA, casein or the gelatin of 0.5~2wt%.Preferably, enzyme inhibitor is 0.5~2mM EDTA.
In above-mentioned storage buffer solution, the ingredient of basis buffer is similar with coating buffer components, and effect is also similar, herein It repeats no more.It further includes the stable storage liquid that stabilization is played to DNA molecular structure and double-spiral structure to store buffer solution, is appointed The substance what in the prior art can play DNA molecular structure and double-spiral structure stabilization can be steady as the storage Determine the component of liquid.
In above preferred embodiment, DNA molecular protective agent cycloheptaamylose can be arranged in around DNA molecular and mitigate pair The direct effect of DNA has obvious protective effect to have the function that protection to DNA molecular.It is in stable storage liquid In content adjustment can be optimized according to effect.It is 1~3wt% in the application preferably its content, it is right in the content range The protective effect of double chain DNA molecule is more preferable.
In above preferred embodiment, surfactant includes anion surfactant and nonionic surfactant, In, anion surfactant can also promote magnetic microsphere to disperse, and magnetic microsphere is avoided to reunite, and reduce DNA molecular and magnetism Absorption between microballoon pipe.Anion surfactant enters proteinase activity center and complex reaction occurs, and so that enzyme is inactivated, prevents DNA molecular is degraded.And nonionic surfactant can promote magnetic microsphere to disperse, and magnetic microsphere is avoided to reunite, and reduce DNA Absorption between molecule and magnetic microsphere pipe.Its specific content can also optimize adjustment according to final stablizing effect. The content of preferred anion surfactant is 0.02~0.05wt% in the stable storage liquid of the application, and non-ionic surface is lived Property agent content be 0.05~0.1wt%, the coated magnetic microsphere dispersibility of double-stranded DNA antigen is made in the content range more It is good, and then the absorption between DNA molecular and magnetic microsphere pipe is reduced, keep the stability and dispersibility of double chain DNA molecule.
In above preferred embodiment, in DNA double chain structural stabilizing agent, contain Al3+、Co3+Or Ce3+Compound (such as chlorination Aluminium, cobalt chloride or cerium chloride) in trivalent metal ion and double chain DNA molecule in phosphate oxygen affinity it is very high, the two energy Rapid reaction, it is strong bonded, very stable complex compound is formed, as a result, two chains of DNA is made to be crosslinked, double helix is had mercy on Closer, duplex structure is more stable, to improve DNA molecular denaturation temperature Tm values.Its content in stable storage liquid also may be used To optimize adjustment according to stablizing effect.And the application preferably controls its content within the scope of 5~10mM, having makes double-strand The better effect of DNA molecular stability.
In above preferred embodiment, sealer such as 0.5%BSA are for closing possible not coated position in magnetic microsphere Point.And the effect of preservative Proclin300 or thimerosal is to inhibit the growth and breeding of microorganism, to extend the holding time.
In a kind of preferred embodiment, mentioned reagent further includes detection buffer solution, detects in buffer solution and contains nuclease S1 more preferably detects a concentration of 0.5~2 μ g/L of buffer solution amplifying nucleic acid enzyme S1, it is further preferred that detection buffer solution also wraps It includes：10~50mM phosphate buffers, 0.5~2wt%BSA, 0.1~0.3M sodium chloride and 0.05~0.2wt% Proclin300。
Highly purified double chain DNA molecule can form some chemical bonds in coating magnetic microsphere process, inevitably right Double chain DNA molecule causes centainly to destroy.In addition, double-stranded DNA antigen-magnetic microsphere conjugate after coating in long-term preservation and The risk for generating single stranded DNA is still remained in transportational process, it is also possible to lead to false positive results.Therefore, it also needs single stranded DNA point Sub further removal, most straightforward approach are that s1 nuclease is added in magnetic microsphere suspension, but magnetic microsphere suspension In there are enzyme inhibitors, the two to coexist.On the other hand, the single-stranded significant portion that double chain DNA molecule is formed can natural renaturation Restore duplex structure, if there are single stranded DNA degrading enzyme in magnetic microsphere suspension, generation and the degradation of single stranded DNA continue into Row, may finally cause most DNA molecular above magnetic microsphere to be degraded, and to influence to detect potency, cannot provide Correct result.
Above preferred embodiment passes through the s1 nuclease of the addition debita spissitudo in the detection buffer composition of kit, energy Enough two problems avoided well above.The detection of anti-dsDNA antibody generally uses two step indirect methods, is that coating is double first The magnetic microsphere of ssdna molecule warm bath together with sample to be tested (analyte-containing) and detection buffer solution, if existing in sample anti- The antibody of double-stranded DNA can then form the compound of magnetic microsphere-dsDNA- Anti-hCG actions, because of magnetic microsphere and sample application Measure equal very little, thus this step must add larger amount of detection buffer solution provide allow magnetic microsphere and sample mixing and fully The environment of reaction.It is larger because detecting buffer solution additive amount in reaction process, so the activity of s1 nuclease is suspended by magnetic microsphere In liquid enzyme inhibitor influence can it is smaller, meanwhile, because s1 nuclease be with coating double chain DNA molecule magnetic microsphere and wait for test sample Reaction cup is added simultaneously in this, and enzymic catalytic reaction speed is exceedingly fast, and can eliminate single strand dna wherein that may be present rapidly, The problem of s1 nuclease is with magnetic microsphere long-term co-existence sustaining degradation DNA molecular is in turn avoided simultaneously.
In the 4th kind of typical embodiment of the application, a kind of kit of detection anti-dsDNA antibody is provided, it should Kit includes any of the above-described kind of reagent.Mentioned reagent includes double-stranded DNA antigen, and double-stranded DNA antigen is using any of the above-described Kind preparation method is prepared.The kit for the double-stranded DNA antigen being prepared containing the application above method has detection special Anisotropic advantage high, accuracy is high.
More convenient in order to make detection kit use, in a kind of preferred embodiment of the application, kit further includes hair Signal object, calibration product and quality-control product in any one or more；It is preferred that luminescent marking is anti-human igg, IgA or IgM labels ABEI, calibration product and quality-control product are each independently the antibody of the anti-double-chain DNA of humanized.
In a kind of preferred embodiment of the application, double-stranded DNA antigen is deposited in the form of the conjugate for being coated with solid phase carrier Having in, coating to the double-stranded DNA antigen on solid phase carrier can quantify that detection and joint efficiency are high, steric hindrance is small, even Join the advantages such as object stable structure.
In a kind of preferred embodiment of the application, solid phase carrier is magnetic microsphere, and kit further includes for holding idol Join the magnetic microsphere pipe of object, magnetic microsphere pipe use is protected from light material and is made.In external environment, ultraviolet light irradiation can make DNA Adjacent thymidine forms thymine dimer in molecule, and thymine dimer is usually happened at two in same DNA chain Between a adjacent thymidine, can also be happened at two it is single-stranded between, this dimer is very stable.If it occurs Between two adjacent thymines of same chain, the key of two chains of double helix will be made to weaken, double-stranded DNA structure is made locally to become Shape；If it is happened between two chains, separating for double-strand will be hindered due to its crosslinking, to influence point of magnetic microsphere Property is dissipated, experimental result is influenced.Thus double chain DNA molecule can be avoided to form thymine dimer using material is protected from light, to Influence the accuracy of testing result.
In the 5th kind of typical embodiment of the application, a kind of detection method of anti-dsDNA antibody, the inspection are provided Survey method detects the anti-double-strand in sample to be tested including the use of the target antigen of anti-dsDNA antibody by Chemiluminescence immunoassay The step of concentration of DNA antibody, the wherein target antigen of anti-dsDNA antibody are pair that any of the above-described kind of preparation method is prepared Chain DNA antigen.The detection method has specificity high and the high advantage of accuracy.
In a kind of preferred embodiment, detected by Chemiluminescence immunoassay using the target antigen of anti-dsDNA antibody In the step of concentration of anti-dsDNA antibody in sample to be tested, reaction temperature is 25~30 DEG C.Inventor has found, at 37 DEG C When lower reaction, motion collision aggravates between molecule, it is possible that local solution chain structure, the base pyrimidine polymer quilt exposed Anti- ssDNA antibody identification, increases false positive risk.And by reaction temperature control within the scope of relatively low 25~30 DEG C, energy It is enough to reduce probability of the local unwinding at single stranded DNA, and then the probability identified by single-stranded DNA antibody can be also reduced, to reduce inspection Survey the false positive of result.
In the 6th kind of typical embodiment of the application, the double-strand that any of the above-described kind of preparation method is prepared is provided The reagent or any of the above-described kind of kit for detecting anti-dsDNA antibody of DNA antigens or any of the above-described kind of detection Anti-hCG action Application in anti-dsDNA antibody clinical detection.The application of the application has specificity high and the high advantage of accuracy.
In a kind of preferred embodiment of the application, which includes the clinical assistant diagnosis to systemic loupus erythematosus. The application can improve clinical detection specificity and accuracy, reduce false positive results, auxiliary to the clinic of systemic loupus erythematosus Help diagnostic result reliability higher.
In the 7th kind of typical embodiment of the application, the examination of any of the above-described kind of detection anti-dsDNA antibody is provided Agent box antinuclear antibodies detection in application, using including：Kit is applied to semi-automatic immunity analysis instrument or is automatically exempted from Epidemic disease analyzer.Herein by kit be applied to semi-automatic or automatic lmunoassays analyzer in refer to will be corresponding in mentioned reagent box Reagent carries out handling to be placed in semi-automatic immunity analysis instrument or automatic lmunoassays analyzer analyzing to sample, to obtain Testing result.The application can realize the automatic detection of kit detection and high-throughput detection, improve clinical detection specificity And accuracy, false positive results are reduced, detection flux and efficiency are improved.
In a kind of preferred embodiment of the application, above application includes the following steps：
1) plus in 5-20 μ L anti-dsDNA antibodies calibration objects or quality-control product sample to detection pipe to be measured；
2) plus 10-30 μ L are coated with the magnetic microsphere of anti-dsDNA antibody target antigen and 100-300 μ L detect buffer solution extremely In detection pipe, after mixing, 30 ± 0.5 DEG C of Warm educate 5-20min, carry out Magneto separate, remove supernatant；
3) it is added in 200-400 μ L cleaning solutions to detection pipe, mixing, carries out Magneto separate, remove supernatant；
4) step 3) is repeated twice；
5) plus in 100-300 μ L anti-human igg conjugates to step 4) detection pipe, after mixing, 37 ± 0.5 DEG C of Warm educate 5- 20min carries out Magneto separate, removes supernatant；
6) it is added in 200-400 μ L cleaning solutions to detection pipe, mixing, carries out Magneto separate, remove supernatant；
7) step 6) is repeated twice；
8) it is added in 100-400 μ L chemiluminescent substrates to step 7) detection pipe, mixing, detects luminous intensity, instrument root Concentration value is calculated automatically according to luminous intensity.
Further illustrate the advantageous effect of the application below in conjunction with specific embodiments.It should be noted that following In embodiment and comparative example, unless otherwise specified, agents useful for same and consumptive material are commercial product, such as DNA extraction kit, tool The step of preliminary extraction of body DNA, can illustrate to be operated according to kit.% therein indicates mass percentage, MM indicates mmol/L.
Detect sample：Detection sample standard deviation is detected by clinical diagnosis and other commercial reagents screenings and individual event detection reagent It makes a definite diagnosis.
Sample is collected：Subject phlebotomizes in morning on an empty stomach：3mL.Serum is detached, -20 DEG C of refrigerators is placed in and preserves.
Clinical Sensitivity assesses sample：Selecting system lupus erythematosus (Systemic lupus erythematosus, SLE) patient 385.
Clinical specificity assesses sample：Mixed connective tissue disease (Mixed connective tissue diseases, MCTD) patient 142, systematicness scleroderma Systemic sclerosis, PSS) patient 78, Patients with Sjogren Syndrome (Sjogren's syndrome, SSc) 114, polymyositis/dermatomyositis (Poly/Dermatomyositis, DM) patient 45, Primary biliary cirrhosis (Primary biliary liver cirrhosis, PBC) patient 66, rheumatoid joint Scorching (Rheumatoid arthritis, RA) patient 135,150, health examination sample, amount to 730.
Comparative example 1：
1, prepared by antigen：
Using the DNA extraction kit of Sigma from calf thymus tissue extraction double-stranded DNA, Proteinase K and RNase A are added Histone and RNA ingredients are removed, the double-stranded DNA antigen of purifying is prepared.
2, magnetic microsphere coating and suspension：
10-30mg is taken to contain-NH2Magnetic microsphere, washed 3-5 times with phosphate buffer, be added 1mL be coated with buffer solution, then 0.1-0.2mL DCC and 0.05-0.2mg target antigens, room temperature concussion coating 3h is added.
The ingredient and its concentration of magnetic microsphere coating buffer solution are as follows, remaining is water：
Acetate buffer 20mM
PH value pH is 3.6
Magnetic microsphere stores the ingredient and its concentration of buffer solution, remaining is water：
3, reagent constituents：
1) magnetic microsphere (double chain DNA molecule for being coated with purifying)
The coated magnetic microsphere solution of dsDNA molecules of purifying, the wherein a concentration of 20mg/mL of magnetic microsphere are coated in A concentration of 15 μ g/mL of dsDNA on magnetic microsphere；
2) the mouse anti-human igg monoclonal antibody solution of ABEI labels, a concentration of 50ug/mL of wherein ABEI, the mouse marked by ABEI A concentration of 1mg/mL of anti-human igg monoclonal antibody；
3) buffer solution (being free of s1 nuclease) is detected
4) calibration product (containing humanized's anti-dsDNA antibody)
Low spot calibrates product：20IU/mL
High point calibrates product：400IU/mL
5) quality-control product (containing humanized's anti-dsDNA antibody)
Quality-control product 1：50IU/mL
Quality-control product 2：200IU/mL
Testing principle：
It is to be coated with magnetic microsphere and the sample to be tested (analyte-containing) of double chain DNA molecule and detect warm together with buffer solution first Bath, if there are anti-dsDNA antibodies in sample, then can form the compound of magnetic microsphere-dsDNA- Anti-hCG actions, clearly It washes three times, removes other IgG ingredients being not associated in sample, add luminous marker, anti-human igg is formed with first time warm bath Anti-dsDNA antibody response in compound forms the compound of magnetic microsphere-dsDNA- Anti-hCG actions-anti-human IgG antibodies-ABEI Object, in the case where exciting substrate-function, ABEI luminous intensities are proportional to the concentration of Anti-hCG action in sample to be tested.
4, detection method：
The detection method specifically comprises the following steps：
1) plus in 5-20 μ L anti-dsDNA antibodies calibration objects or quality-control product or sample to detection pipe to be measured；
2) plus 10-30 μ L are coated with the magnetic microsphere of anti-dsDNA antibody target antigen and 100-300 μ L detect buffer solution extremely In detection pipe, after mixing, 37 ± 0.5 DEG C of Warm educate 5-20min, carry out Magneto separate, remove supernatant；
3) 200-400 μ L cleaning solutions (60mM Tris-HCl, 0.007% polysorbas20,180mM sodium chloride, pH 7.2 is added ~8.8) in detection pipe, mixing carries out Magneto separate, removes supernatant；
4) step 3) is repeated twice；
5) plus in the mouse anti-human igg monoclonal antibody to step 4) detection pipe of 100-300 μ LABEI labels, after mixing, 37 ± 0.5 DEG C Warm educates 5-20min, carries out Magneto separate, removes supernatant；
6) it is added in 200-400 μ L cleaning solutions to detection pipe, mixing, carries out Magneto separate, remove supernatant；
7) step 6) is repeated twice；
8) it is added in 100-400 μ L chemiluminescent substrates to step 7) detection pipe, mixing, detects luminous intensity, instrument root Concentration value is calculated automatically according to luminous intensity
5, result counts：
Table 1：Sensitivity：
Table 2：Specificity：
6, interpretation of result：
Anti-hCG action primary recognition site is the ribonucleic acid skeleton structure being located on DNA double helical structure, is mainly seen In SLE, positive rate is 40%~90%, and specificity seldom goes out more than 90% in other autoimmune diseases and normal person Existing, general positive rate is below 10%.And the primary recognition site of anti-ssDNA antibody is exposed after double-stranded DNA uncoiling Purine and pyrimidine bases multimeric structure, and the measurement result of anti-ssDNA antibody lack disease specific, except SLE patient has Outside compared with high detection rate (50%-60%), other rheumatism such as mixed connective tissue disease, drug-induced lupus, chorionitis, skin Myositis, Sjogren syndrome, rheumatoid arthritis etc. also have the recall rate of 10%-70%.
It is existing single-stranded as can be seen that double-stranded DNA antigen is in extraction process from the testing result of above-mentioned comparative example DNA molecular is not eliminated, and DNA molecular is not cut to suitable fragments yet.During coating, it is not added in coating buffer solution anti- The only enzyme inhibitor of double chain DNA molecule degradation, pH and ion concentration are also improper, are unfavorable for double chain DNA molecule stable structure.It deposits It stores up in buffer solution, is also not added with the enzyme inhibitor for preventing double chain DNA molecule from degrading, wherein Mg2+The effect of nuclease is also activated, It is unfavorable for the preservation of DNA molecular, in addition, being not added with DNA molecular protective agent and duplex structure stabilizer, is generated in long-term preservation single Chain risk is higher.In addition, during subsequent detection, detects in buffer composition and s1 nuclease is not also added, thus, it is magnetic micro- There are the single strand dnas of larger proportion in the conjugate that ball is formed with double chain DNA molecule, during the reaction on magnetic microsphere Single strand dna by sample anti-ssDNA antibody identify, to cause false positive results higher.Therefore, in mixed type A large amount of positive findings are also detected in the non-SLE diseases such as connective tissue disease, rheumatoid arthritis, with clinical research and practical report It is not inconsistent, causes kit whole specific relatively low (70.14%).
Embodiment 1：
1, prepared by antigen：
Using the DNA extraction kit of Sigma from calf thymus tissue extraction double-stranded DNA, Proteinase K and RNase A are added Histone and RNA ingredients are removed, then phenol chloroform is used to purify, obtains the first purifying DNA；Using 2 butanol pair first purify DNA into Row concentration, obtains a concentration of 2mg/mL double-stranded DNAs.Then use No. 4.5 hypodermic needles according to the frequency of 20 times/min Suction beats solution about 100 times, obtains the DNA fragmentation of 500bp or so, then adds s1 nuclease and the elimination of DpnI demethylases carries It takes and the single-stranded structure formed during shear treatment and is methylated the DNA molecular of modified, then to carry out running gel pure Change and cut glue and cross column purification, highly purified double-stranded DNA antigen is prepared in recycling.
2, magnetic microsphere coating and suspension：
10-30mg is taken to contain-NH2Naked magnetic microsphere, washed 3-5 times with phosphate buffer, be added 1mL be coated with buffer solution, Add 0.1-0.2mL EDC and 0.05-0.2mg target antigens, room temperature concussion coating 3h.
Magnetic microsphere is coated with the ingredient and its concentration of buffer solution, remaining is water：
Acetate buffer 20mM
PH value pH is 3.6
Magnetic microsphere stores the ingredient and its concentration of buffer solution, remaining is water：
3, reagent constituents：
Magnetic microsphere (double chain DNA molecule for being coated with purifying)
3) buffer solution (being free of s1 nuclease) is detected
4) calibration product (containing humanized's anti-dsDNA antibody)
Low spot calibrates product：20IU/mL
High point calibrates product：400IU/mL
5) quality-control product (containing humanized's anti-dsDNA antibody)
Quality-control product 1：50IU/mL
Quality-control product 2：200IU/mL
Testing principle (with comparative example 1)
4, detection method：
The detection method specifically comprises the following steps：：
1) plus in 5-20 μ L anti-dsDNA antibodies calibration objects or quality-control product or sample to detection pipe to be measured；
2) plus 10-30 μ L are coated with the magnetic microsphere of anti-dsDNA antibody target antigen and 100-300 μ L detect buffer solution extremely In the detection pipe, after mixing, 37 ± 0.5 DEG C of Warm educate 5-20min, carry out Magneto separate, remove supernatant；
3) it is added in 200-400 μ L cleaning solutions to detection pipe, mixing, carries out Magneto separate, remove supernatant；
4) step 3) is repeated twice；
5) plus in 100-300 μ L anti-human igg conjugates to step 4) detection pipe, after mixing, 37 ± 0.5 DEG C of Warm educate 5- 20min carries out Magneto separate, removes supernatant；
6) it is added in 200-400 μ L cleaning solutions to detection pipe, mixing, carries out Magneto separate, remove supernatant；
7) step 6) is repeated twice；
8) it is added in 100-400 μ L chemiluminescent substrates to step 7) detection pipe, mixing, detects luminous intensity, instrument root Concentration value is calculated automatically according to luminous intensity
5, result counts：
Table 3：Sensitivity：
Table 4：Specificity：
6, interpretation of result：
By the table 3 of embodiment 1 and table 4 it is found that the kit clinic inspection of embodiment 1 compared with the Tables 1 and 2 of comparative example 1 It surveys specificity and is increased to 76.16% from 70.14%.Although the embodiment shows that some may be will produce during coating single-stranded Core is not also added there is also the risk for generating single stranded DNA and when detecting in buffer composition during structure, subsequent preservation When sour enzyme S1, it is also possible to lead to some false positive results, but be prepared that purity is relatively high and the double-stranded DNA of suitable size Molecule is testing result accurately and reliably precondition.
Embodiment 2：
1, prepared by antigen：
Salmon essence double-stranded DNA is extracted using the DNA extraction kit of Sigma, adds Proteinase K and RNase A removal group egg White and RNA ingredients, are obtained the first purifying DNA, are then concentrated using 1 butanol, obtain the concentration of DNA of 1.8mg/mL, then Solution is beaten according to the frequency suction of 10 times/min about 90 times, obtain the DNA pieces of 2000bp or so using No. 4.5 hypodermic needles Then section is formed during addition s1 nuclease, exonuclease I and demethylase DpnI eliminations extraction and shear treatment Single-stranded structure and the DNA molecular for being methylated modified, then carry out running gel purifying and cut glue crossing column purification, it is prepared by recycling Obtain highly purified double-stranded DNA antigen.
2, magnetic microsphere coating and suspension：
10-30mg is taken to contain-NH2Naked magnetic microsphere, washed 3-5 times with phosphate buffer, be added 1mL be coated with buffer solution, Add 0.1-0.2mL DCC and 0.05-0.2mg target antigens, room temperature concussion coating 3h.
Magnetic microsphere is coated with the ingredient and its concentration of buffer solution, remaining is water：
Magnetic microsphere stores the ingredient and its concentration of buffer solution, remaining is water：
3, reagent constituents：
1) magnetic microsphere (double chain DNA molecule for being coated with purifying)
2) the mouse anti-human igg monoclonal antibody solution of ABEI labels, a concentration of 50ug/mL of wherein ABEI, the mouse marked by ABEI A concentration of 1mg/mL of anti-human igg monoclonal antibody；
3) buffer solution (being free of s1 nuclease) is detected
4) calibration product (containing humanized's anti-dsDNA antibody)
Low spot calibrates product：20IU/mL
High point calibrates product：400IU/mL
5) quality-control product (containing humanized's anti-dsDNA antibody)
Quality-control product 1：50IU/mL
Quality-control product 2：200IU/mL
Testing principle (with comparative example 1)
4, detection method
With embodiment 1, incubation temperature is changed to 30 DEG C.
5, result counts：
Table 5：Sensitivity：
Table 6：Specificity：
6, interpretation of result：
It can be seen that compared with comparative example 1 from the data of table 5 and table 6, the kit of embodiment 2 is in terms of clinical detection Specificity from 70.14% be increased to 89.86%, clinical detection specificity is greatly improved.Show the double-stranded DNA of purifying Molecular concentration meets testing requirements, and it is few in coating processing and later stage to preserve the single stranded DNA that is formed in the process, therefore, except The positive is seldom detected in the other diseases sample of SLE, and there is higher clinical detection specificity.
Embodiment 3：
1, prepared by antigen：
Using the DNA extraction kit of Sigma from calf thymus tissue extraction double-stranded DNA, Proteinase K and RNase A are added Histone and RNA ingredients are removed, then phenol chloroform is used to purify, obtains the first purifying DNA；Using 2 butanol pair first purify DNA into Row concentration, obtains a concentration of 3mg/mL double-stranded DNAs.Then use No. 4.5 hypodermic needles according to the frequency of 30 times/min Suction beats solution about 100 times, to cut off DNA molecular to the DNA fragmentation of 1000bp or so, then adds s1 nuclease, exonuclease Enzyme I and demethylase DpnI eliminates the single-stranded structure formed during extraction and shear treatment and is methylated modified DNA molecular, then carry out running gel purifying and cut glue crossing column purification, the double-stranded DNA antigen of purifying is prepared in recycling.
2, magnetic microsphere coating and suspension：
10-30mg is taken to contain-NH2Naked magnetic microsphere, washed 3-5 times with phosphate buffer, be added 1mL be coated with buffer solution, Add 0.1-0.2mL DCC and 0.05-0.2mg target antigens, room temperature concussion coating 3h.
Magnetic microsphere is coated with the ingredient and its concentration of buffer solution, remaining is water：
Magnetic microsphere stores the ingredient and its concentration of buffer solution, remaining is water：
3, reagent constituents：
1) magnetic microsphere (double chain DNA molecule for being coated with purifying)
2) the mouse anti-human igg monoclonal antibody solution of ABEI labels, a concentration of 50ug/mL of wherein ABEI, the mouse marked by ABEI A concentration of 1mg/mL of anti-human igg monoclonal antibody；
3) buffer solution (containing s1 nuclease) is detected
4) calibration product (containing humanized's anti-dsDNA antibody)
Low spot calibrates product：20IU/mL
High point calibrates product：400IU/mL
5) quality-control product (containing humanized's anti-dsDNA antibody)
Quality-control product 1：50IU/mL
Quality-control product 2：200IU/mL
Testing principle (with comparative example 1)
4, detection method
The detection method is the same as embodiment 2
5, result counts：
Table 7：Sensitivity：
Table 8：Specificity：
6, interpretation of result：
It can be seen that compared with comparative example 1 from the data of table 7 and table 8, the clinical detection of the kit of embodiment 3 is special Property is increased to 95.62% from 70.14%.Compared with Example 2, kit clinical detection specificity carries embodiment 3 from 89.86% For height to 95.62%, clinical detection is so specific that further increase.Both show the double chain DNA molecule concentration (magnetic microsphere of purifying： DsDNA proportional regions 1mg:5~20 μ g), purity and size meet testing requirements, in addition, also indicating that at magnetic microsphere coating A small amount of single stranded DNA is still unavoidably produced during reason process and storage and transport, but by adding in detecting buffer solution Add suitable s1 nuclease, the single stranded DNA of generation is made to be degraded, to further reduced false positive, therefore, utilizes the implementation The kit of example seldom detects the positive in the other diseases sample except SLE, has high clinical detection specificity.
Embodiment 4：
1, prepared by antigen：
Using the DNA extraction kit of Sigma from calf thymus tissue extraction double-stranded DNA, Proteinase K and RNase A are added Histone and RNA ingredients are removed, then phenol chloroform is used to purify, obtains the first purifying DNA；Using 2 butanol pair first purify DNA into Row concentration, obtains a concentration of 2mg/mL double-stranded DNAs.It is beaten according to the frequency suction of 20 times/min using No. 4.5 hypodermic needles Solution about 120 times adds s1 nuclease, exonuclease I and goes first to cut off DNA molecular to the DNA fragmentation of 1000bp or so Base enzyme eliminates the single-stranded structure formed and the DNA molecular being modified during extraction and shear treatment, then carries out electrophoresis It gel-purified and cuts glue and crosses column purification, the double-stranded DNA antigen of purifying is prepared in recycling.
2, magnetic microsphere coating and suspension：
10-30mg is taken to contain-NH2Naked magnetic microsphere, washed 3-5 times with phosphate buffer, be added 1mL be coated with buffer solution, Add 0.1-0.2mL DCC and 0.05-0.2mg target antigens, room temperature concussion coating 3h.
Magnetic microsphere is coated with the ingredient and its concentration of buffer solution, remaining is water：
Magnetic microsphere stores the ingredient and its concentration of buffer solution, remaining is water：
3, reagent constituents：
1) magnetic microsphere (double chain DNA molecule for being coated with purifying)
2) luminous marker：ABEI label mouse anti-human igg monoclonal antibody solution, a concentration of 50ug/mL of wherein ABEI, by A concentration of 1mg/mL of the mouse anti-human igg monoclonal antibody of ABEI labels；
3) buffer solution (containing s1 nuclease) is detected
4) calibration product (containing humanized's anti-dsDNA antibody)
Low spot calibrates product：20IU/mL
High point calibrates product：400IU/mL
5) quality-control product (containing humanized's anti-dsDNA antibody)
Quality-control product 1：50IU/mL
Quality-control product 2：200IU/mL
Testing principle (with comparative example 1)
4, detection method
(in addition to reaction temperature is 25 ± 0.5 DEG C, remaining step is with embodiment 2)
5, result counts：
Table 9：Sensitivity：
Table 10：Specificity：
6, interpretation of result：
Compared with Example 3, clinical detection sensitivity almost indifference, clinical detection specificity is from 95.62% for embodiment 4 It is increased to 96.03%, clinical detection is so specific that further increase, and illustrate reduces reaction temperature during the reaction also contributes to Improve anti-dsDNA antibody clinical detection specificity.
It can be seen that in terms of sensitivity from above-mentioned comparative example 1 and embodiment 1-4, comparative example 1 detects the positive rate of SLE It is 66.23%, the SLE positive rates that embodiment 1 detects are 61.04%, and the SLE positive rates that embodiment 2 detects are 56.62%, real The SLE positive rates for applying the detection of example 3 are 54.55%, and the SLE positive rates that embodiment 4 detects are 55.06%.As it can be seen that as detection is special Anisotropic significantly improves, and detection sensitivity, which has no, to be substantially reduced.
Since positive rate of the anti-dsDNA antibody in SLE is 40%~90%, other also 10%~60% SLE Anti-dsDNA antibody is not present in patient's body.Under normal circumstances, it is anti-usually to occur anti-double-chain DNA simultaneously in SLE patients serums Body and anti-ssDNA antibody also have part SLE patient's body individualism anti-ssDNA antibodies that anti-double-chain DNA may be not present anti- Body.Therefore, it is analyzed from the comparison of above-mentioned comparative example 1 and the detection sensitivity of Examples 1 to 4 it is found that comparative example more than 1 detected SLE positive samples be most likely due to the presence of single strand dna and caused by false positive results.
In addition, considering from the clinical application angle of anti-dsDNA antibody, generally there are more in SLE Serum It is anti-generally to carry out antinuclear antibodies, anti-double-chain DNA to the patient of the doubtful systemic loupus erythematosus of clinical symptoms for kind autoantibody A variety of autoantibodies such as body, anti-Sm antibody are detected, and are integrated in combination with the various clinical signs of patient and other testing results Consider, thus, the non-apparent reduction of anti-dsDNA antibody detection sensitivity, on SLE make a definite diagnosis influence it is smaller.But anti-double-strand The reduction of DNA antibody specificity frequently can lead to mistaken diagnosis, because systemic autoimmune diseases are before there is typical clinical symptom It is all more similar, and it is the specific antibody of SLE that anti-dsDNA antibody, which is reported, seldom appears in other autoimmune diseases In.Thus, for example, Patients with SLE and rheumatoid arthritis patients in early stage it is possible that heating paresthesia, And be possible in the rheumatoid arthritis patients of early stage because of disease advanced symptoms unobvious, simultaneously because anti-double-chain DNA There are single chain components in antibody kit, to obtain anti-dsDNA antibody it is positive as a result, being easy mistaken diagnosis in conjunction with symptom and being The error diagnosis of SLE causes mistaken diagnosis and wrong medicine to treat, may cause serious consequence.Therefore, anti-dsDNA antibody faces Bed detection specificity is particularly important.
Therefore, compared with comparative example 1, the detection sensitivity of above-described embodiment 1~4 of the application is reduced to from 66.23% 55.06% range of decrease influences the application of the kit smaller.Under the premise of herein, the clinical detection specificity of Examples 1 to 4 from 70.14% is increased to 96.03%, and specificity is greatly improved, substantially increase anti-dsDNA antibody clinical detection specificity and Accuracy.
In addition, inventor is according further to operating method similar to Example 4, and according to the preparation condition in table 10 and table 11 It is carried out example 5 to 15 respectively, and the testing result of embodiment 5 to 15 is counted, statistical result is shown in Table 12 to table 15. Wherein, in the preparation process of 5 to 15 double center chain DNA of embodiment, s1 nuclease, exonuclease I and DpnI demethyls are added Change enzyme to be handled.
Table 10：
Table 11：
Table 12：
Table 13：
Table 14：
Table 15：
The effect data of preparation condition and table 12 to table 15 from above-mentioned table 10 and 11 can be seen that embodiment 15 with it is right Ratio is compared, and specificity improves 10% or so, and compared with Example 1, specificity improves 4% or so to embodiment 15, illustrates double S1 nuclease, exonuclease I and DpnI demethylases is added in the preparation process of chain DNA to be conducive to improve anti-double-chain DNA The clinical detection specificity of antibody, meanwhile, s1 nuclease is added in detecting buffer solution to be also contributed to improve anti-dsDNA antibody Clinical detection specificity.It can be obtained from comparison of the embodiment 2 with embodiment 3, nuclease added in detecting buffer solution S1 improves 5% or so to specificity.Embodiment 3 compared with Example 10, illustrates that 10% left side can be improved in DNA double chain structural stabilizing agent Right detection specificity.Embodiment 3 detects specificity and improves 4% or so compared with Example 7, illustrates that DNA molecular protection is added The clinical detection specificity of anti-dsDNA antibody can be improved in agent.It is compared with embodiment 3 it is found that DNA molecular is protected by embodiment 11 Agent and DNA double chain structural stabilizing agent can cooperate with the detection for improving 12% specific.By embodiment 12-15 and embodiment 3 and mutually Between specific size be compared it is found that there is synergistic effect between storing the stability component of buffer solution, can cooperate with and carry High detection specificity.
To sum up, the above embodiments of the present invention realize following technique effect：The application is highly purified by being prepared With the smooth double chain DNA molecule of suitable length, optimization antigen-magnetic microsphere coating buffer solution and magnetic microsphere conjugate preserve Liquid, the Tm for improving system residing for double chain DNA molecule, to stablize maintain DNA molecular duplex structure, in addition, by The s1 nuclease of debita spissitudo is added in the detection buffer composition of kit, if magnetic microsphere in coating processing, preserves and fortune A small amount of single stranded DNA is still unavoidably produced during defeated, it can be during the reaction by detecting the nuclease in buffer solution S1 degrades single stranded DNA, reduces the risk for false positive results occur, improves the clinical detection specificity of anti-dsDNA antibody.Together When reduce reaction process temperature, to improve anti-dsDNA antibody clinical detection specificity, finally, magnetic microsphere pipe use It is protected from light material, further decreases the risk for generating single stranded DNA.Further it is provided that the case where one kind can ensure detection sensitivity Under, improve the Anti-hCG action assay kit of specificity.
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 (17)

1. a kind of preparation method of double-stranded DNA antigen, which is characterized in that the preparation method includes：
Extract double-stranded DNA；
Histone and RNA ingredients in the double-stranded DNA are removed using protease and RNA enzyme, obtain the first purifying DNA；
The segment that DNA cuts into 500~2000bp is purified by described first, obtains shearing DNA；
The single-stranded and modification that methylates in the shearing DNA is eliminated using s1 nuclease, exonuclease I and demethylase DNA molecular obtains the second purifying DNA；
Purifying recycling is carried out to the second purifying DNA, obtains the double-stranded DNA antigen.
2. preparation method according to claim 1, which is characterized in that from calf thymus tissue, salmon essence, Hela cells, The double-stranded DNA is extracted in human blood or saliva；
Preferably, pure by described first in such a way that No. 4.5 hypodermic needles carry out the first purifying DNA pressure-vaccum Change the segment that DNA cuts into 500~2000bp, obtains the shearing DNA；
Preferably, the frequency of the pressure-vaccum is 10~30 times/min, and the number of the pressure-vaccum is 90~120 times；
Further include the step concentrated to the first purifying DNA it is highly preferred that before to the first purifying DNA shearings Suddenly, further preferably DNA is purified by described first be concentrated into a concentration of 1.8~3mg/mL.
3. a kind of double-stranded DNA antigen, which is characterized in that the double-stranded DNA antigen uses preparation side as claimed in claim 1 or 2 Method is prepared.
4. a kind of reagent of detection anti-dsDNA antibody, the reagent includes double-stranded DNA antigen, which is characterized in that the double-strand DNA antigens are prepared using preparation method as claimed in claim 1 or 2.
5. reagent according to claim 4, which is characterized in that the reagent further includes anti-for loading the double-stranded DNA Former solid phase carrier, the preferably described solid phase carrier are magnetic microsphere or microwell plate.
6. reagent according to claim 5, which is characterized in that the solid phase carrier is magnetic microsphere, and the reagent also wraps It includes for being coated with the double-stranded DNA antigen to the coating buffer solution of the magnetic microsphere, the preferably described coating buffer solution includes：
The pH of Tris-HCl buffer solutions, cationic metal salt and enzyme inhibitor, the coating buffer solution is 7.5~8.5；
It is highly preferred that the coating buffer solution includes：The Tris-HCl buffer solutions of 10~50mM, the NaCl of 0.1~0.3M and/or The EDTA of KCl and 0.5~2mM.
7. the reagent according to any one of claim 4 to 6, which is characterized in that the reagent further include pH be 7.5~ 8.5 storage buffer solution, the storage buffer solution include：
Basis buffer, the basis buffer include Tris-HCl buffer solutions, cationic metal salt and preservative, preferably described Basis buffer includes：The Tris-HCl buffer solutions of 10~50mM, the NaCl and/or KCl of 0.1~0.5M and 0.05~ The preservative of 0.2wt%, it is highly preferred that the preservative is Proclin300 or thimerosal；
Stable storage liquid, the stable storage liquid include DNA molecular protective agent, enzyme inhibitor, surfactant, DNA double link Any one or more of structure stabilizer and sealer in；
Preferably, the DNA molecular protective agent is cycloheptaamylose, cyclohexaamylose or cyclooctaamylose；It is more preferably described to deposit It stores up in stabilizing solution, the protectant content of DNA molecular is 1~3wt%；
Preferably, the surfactant is anion surfactant or nonionic surfactant, more preferably it is described it is cloudy from Sub- surfactant is SDS or SLS；The nonionic surfactant is PEG6000, polysorbate80 or polysorbate 20；It is further preferred that in the stable storage liquid, the content of the anion surfactant is 0.02~0.05wt%, The content of the nonionic surfactant is 0.05~0.1wt%；
Preferably, the DNA double chain structural stabilizing agent is containing Al3+、Co3+Or Ce3+Compound；The more preferable stable storage In liquid, a concentration of 5~10mM of the DNA double chain structural stabilizing agent；
Preferably, the enzyme inhibitor is the EDTA of 0.5~2mM；
Preferably, the sealer is BSA, casein or the gelatin of 0.5~2wt%.
8. reagent according to claim 7, which is characterized in that the reagent further includes detection buffer solution, and the detection is slow Contain s1 nuclease in fliud flushing, a concentration of 0.5~2 μ g/L of s1 nuclease described in the more preferably described detection buffer solution, into one Preferably, the detection buffer solution further includes step：10~50mM phosphate buffers, 0.5~2wt%BSA, 0.1~0.3M chlorine Change sodium and 0.05~0.2wt%Proclin300.
9. a kind of kit of detection anti-dsDNA antibody, which is characterized in that the kit includes appointing in claim 4 to 8 Reagent described in one.
10. kit according to claim 9, which is characterized in that the kit further includes luminous marker, calibration product And any one or more in quality-control product；It is preferred that the luminescent marking is the ABEI that anti-human igg, IgA or IgM are marked, it is described Calibration product and the quality-control product are each independently the antibody of the anti-double-chain DNA of humanized.
11. kit according to claim 9, which is characterized in that the double-stranded DNA antigen in the kit is with antigen- The form of solid phase carrier conjugate exists, and the solid phase carrier is magnetic microsphere.
12. kit according to claim 11, which is characterized in that the kit further includes described anti-for holding The magnetic microsphere pipe of original-solid phase carrier conjugate, the magnetic microsphere pipe use are protected from light material and are made.
13. a kind of detection method of anti-dsDNA antibody, the detection method including the use of anti-dsDNA antibody target antigen The step of concentration of the anti-dsDNA antibody in sample to be tested being detected by Chemiluminescence immunoassay, which is characterized in that described anti- The target antigen of double-stranded-DNA antibody is the double-stranded DNA antigen that preparation method as claimed in claim 1 or 2 is prepared.
14. detection method according to claim 13, which is characterized in that the target antigen using anti-dsDNA antibody By Chemiluminescence immunoassay detect sample to be tested in anti-dsDNA antibody concentration the step of in, reaction temperature be 25~ 30℃。
15. any one of double-stranded DNA antigen that preparation method as claimed in claim 1 or 2 is prepared or claim 4 to 8 The reagent of the described detection anti-dsDNA antibody or by the detection anti-dsDNA antibody described in any one of claim 9 to 12 Application of the kit in anti-dsDNA antibody clinical detection.
16. application according to claim 15, which is characterized in that the application includes the clinic to systemic loupus erythematosus Auxiliary diagnosis.
17. application of the kit in antinuclear antibodies detection described in any one of claim 9 to 12, which is characterized in that institute It states to apply and includes：The kit is applied to semi-automatic immunity analysis instrument or automatic lmunoassays analyzer.
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