CN101165491A - Method for detecting double-chain DNA resistant antibody using gold magnetic particle as carrier - Google Patents
Method for detecting double-chain DNA resistant antibody using gold magnetic particle as carrier Download PDFInfo
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Abstract
The method comprises: 1) the GoldMag nano-particles are mixed with the double strand DNA; under certain condition, the double strand DNA is fixed on the surface of GoldMag nano-particles, or the GoldMag nano-particles are used to extract the DNA from the human blood, saliva and semen and to fix the extracted DNA on the surface of GoldMag nano-particles; 2) using indirect ELISA method, immumofluorescence method or chemiluminescent method to detect the anti double strand DNA antibody existed in the sampler under test.
Description
Technical field
The present invention relates to a kind of method that detects the anti-dsDNA antibody in the sample, relate in particular to a kind of method that adopts enzyme linked immunosorbent assay to detect the anti-dsDNA antibody in the sample.
Background technology
Systemic loupus erythematosus (SLE) is comparatively common clinically a kind of autoimmune disease, and patient's outstanding behaviours is to have multiple autoantibody, wherein the most important thing is anti-dsDNA antibody.This is a kind of autoantibody that can combine with n DNA, almost only can detect this antibody in SLE patient.The growth and decline of anti-dsDNA antibody titre are relevant with SLE patient's change of illness state, and along with the control of disease activity, the titre of anti-dsDNA antibody can descend or disappear.
Methods such as early stage indirect hemagglutination, complement fixation test (CFT) and counter immunoelectrophoresis are eliminated because of sensitivity and poor specificity, and using more now, detection method roughly has following several: immunofluorescence technique (IFT), Western blot (Immunoblot assay), radioimmunology (RIA), spot immune gold percolation (DIGFA), colloidal gold chromatography and enzyme linked immunosorbent assay (ELISA).
Immunofluorescence technique (IFA) is with dilute serum and experiment matrix are carried out incubation, make specific antibody combine with corresponding antigens in the experiment matrix, and then with fluorescently-labeled second antibody incubation, last specificity fluorescent pattern of under fluorescent microscope, observing corresponding site.The IFA method can only be as the reference of clinical diagnosis, can not be as the important evidence of making a definite diagnosis.This in addition method also needs quality fluorescent microscope preferably, and is higher to operating personnel, and complicated operation length consuming time and have other interfering materials is prone to false positive.
Western blot (Immunoblot assay) is the technology that polyacrylamide gel electrophoresis protein isolate and polypeptide are combined with immunoreactive high specific, but its preparation method is loaded down with trivial details, cost is higher relatively.
Radioimmunology (RIA) is to combine with anti-dsDNA antibody in the test sample with isotope-labeled double-stranded DNA, easily produces radioactive contamination, and has other interfering materials easily to produce false positive; Detecting needs special instrument and equipment, and detection time is long; Can not realize that individual event detects.
Colloidal gold immunity chromatography is with envelope antigen, colloid gold label antibody immobilization, combines with sample sorbing material etc. and is prepared as the immunochromatography diagnosis test paper.This method can only be carried out qualitative detection.
Enzyme linked immunosorbent assay (ELISA) is a kind of nonradioactive labeling's immune analysis method that development sets up that combines of the efficient catalytic effect with the high degree of specificity of antigen-antibody reaction and enzyme.This method is that the DNA of purifying is antigen coated on solid phase carrier, with certain dilution test serum reaction, resist (anti-human IgGs with two of HRP mark then, IgA, the potpourri of IgM or anti-human IgG .A.M) reaction, HRP can adopt two waveband (450nm, 630nm) to measure its absorbance on microplate reader in colour developing liquid generation chromogenic reaction.Because it is more stable that ELISA has the character of highly sensitive, high specificity, enzyme immunoreagent, lot of advantages such as easy quick, "dead" pollution of method of operating and applied range are immunological experiments that is most widely used at present.Because being used for the high-quality ELISA kit of anti-dsDNA antibody detection, the maturation of antigen production technology purifying or reorganization, commercialization day by day increase.Owing to highly sensitive, the high specificity of this detection method, can quantize and get rid of artificial subjective judgement, expense is cheap, is the breadboard first-selected detection method of external most of autoimmunity diagnostic detection at present.But abroad the kit of some companies' productions costs an arm and a leg, and can't promote and a large amount of the use.The kit that the at present domestic application indirect elisa method of also not producing voluntarily detects anti-dsDNA antibody appears on the market.
At present, the method for existing detection anti-dsDNA antibody has the following disadvantages:
1, detect complex steps, detection time is long, as RIA method and IFA method.
2, detectable needs refrigeration, as RIA method, IFA method, Western blot and spot immune gold percolation.
3, detection needs special instrument and equipment, needs the fluorescence Electronic Speculum to detect as the IFA method, and the RIA method needs γ-calculating instrument or γ-scintillation counter detection.
There is radioactive contamination when 4, detecting, as the RIA method.
5, can only carry out qualitative and can not carry out detection by quantitative, as colloidal gold immunity chromatography, RIA method, IFA method and spot immune gold filter method.
6, there is very strong subjectivity in detection method itself, all needs artificial interpreting blueprints as IFA method, colloidal gold immunity chromatography, has very strong subjective judgement factor.
7, present domestic most of laboratories also are in the qualitative stage to the traditional E LISA detection method of anti-dsDNA antibody.Though the traditional E LISA detection method of anti-dsDNA antibody is fast and convenient, and is comparatively accurate, also there is not enough aspect as clinical diagnosis, as the poor sensitivity that detects.Because the carrier of traditional E LISA method is a polystyrene board, reaction is only carried out at micropore surface, thereby has limited the detection sensitivity of this method.So domestic main employing colloid gold label and quick film spot percolation detect anti-dsDNA antibody qualitatively at present.
The preparation method of packaging magnetic composite particle and core/shell type super-paramagnetic composite particle and structure composition disclose respectively at Chinese patent ZL 03153486.4 and ZL 03124061.5, but it comprises that mainly molecule is fixed and the content of coherent detection in using, and does not relate to the content of anti-dsDNA antibody context of detection.
Summary of the invention
The object of the invention provides a kind of method that detects anti-dsDNA antibody, and it has solved the shortcoming of the poor sensitivity of existing detection anti-dsDNA antibody.
Technical solution of the present invention is:
A kind of is the method for carrier sense anti-dsDNA antibody with the gold-magnetic particles, may further comprise the steps:
1] DNA fixing: get gold-magnetic particles and add in the centrifuge tube on the gold-magnetic particles surface, after magnetic resolution is abandoned supernatant, wash gold-magnetic particles with coupling buffer, magnetic resolution is abandoned the natural double-stranded DNA that adds purifying behind the supernatant or plasmid DNA and coupling buffer in gold-magnetic particles, under 20～40 ℃ of conditions, fully react 20～40min, form gold-magnetic particles-DNA compound; Perhaps whole blood, saliva, seminal fluid or plasmid are made solution system, therefrom extract DNA and make it be fixed on the gold-magnetic particles surface with gold-magnetic particles;
2] sealing of gold-magnetic particles: after the gold-magnetic particles magnetic resolution abandoned supernatant, add sealer in centrifuge tube, abundant reaction 1～2h under 20～40 ℃ of conditions takes out, and 4 ℃ are spent the night;
3] cleaning of gold-magnetic particles: with the gold-magnetic particles after the cleaning buffer solution cleaning sealing;
4] preservation of gold-magnetic particles: the gold-magnetic particles that cleaning is finished adds the preservation damping fluid, and 4 ℃ of preservations are standby;
5] detection of anti-dsDNA antibody: the DNA with the washed gold magnetic particles surface is an antigen, and utilization indirect elisa method, immunofluorescence technique or chemoluminescence method detect anti-dsDNA antibody.
The gold-magnetic particles quality of above-mentioned adding is 0.5～5mg; The DNA quality of above-mentioned adding is 5～25 μ g.
Above-mentioned coupling buffer is 0.5 *～10 * the PBS of pH=5.0～8.0, the perhaps CBS of the pH=9.0 of 5mM～10M～11.0, the perhaps Tris-HCl damping fluid of the pH=7.0 of 5mM～10M～9.0, the perhaps acetate buffer of the pH=3.6 of 5mM～10M～5.6, the perhaps citrate buffer of the pH=3.0 of 5mM～10M～7.0, the perhaps TBS damping fluid of the pH=7.0 of 5mM～10M～9.0, perhaps be the salt of polyglycol (PEG) 6000～10000 addings 0.5～5.0M between 10～25, described salt can be sodium chloride, lithium chloride, barium chloride, potassium chloride, lime chloride, magnesium chloride, cesium chloride etc.; Described sealer is the mixed solution of irrelevant albumen and above-mentioned coupling buffer, and described irrelevant albumen is one or more in 1%～10% bovine serum albumin(BSA), 1%～10% lysine, 1%～10% skimmed milk power, 1%～10% monoethanolamine or 1%～10% animal blood serum; Described cleaning buffer solution is coupling buffer or the coupling buffer that contains 0.02%～0.2% tween; Described preservation damping fluid is coupling buffer or adds antiseptic and protectant coupling buffer; described antiseptic is that 0.01%～0.1% nitrine is received or 0.01%～0.1% thimerosal, and described protective agent is 0.05%～1% BSA, 0.05%～1% animal blood serum or 0.05%～1% protease inhibitors.
The mixed solution of the PEG 8000 of above-mentioned coupling buffer preferred 10% and the sodium chloride of 2.5M; The mixed solution of the PBS damping fluid of the pH=7.0 of the preferred 0.1M of sealer, 2% BSA, 5% calf serum; The mixed solution of the PBS damping fluid of the pH=7.0 of the preferred 0.1M of cleaning buffer solution, 0.5% Tween-20; Preserve the PBS damping fluid of the pH=7.0 of the preferred 0.1M of damping fluid.
The detection of above-mentioned anti-dsDNA antibody can be used indirect elisa method, may further comprise the steps:
1] numbering: test tube is placed on the magnetic separator, establish 2 holes, sample tube to be checked hole respectively, 2 holes, positive control test tube hole, 3 holes, negative control test tube hole, blank 1 hole;
2] add antigen: every hole adds the gold-magnetic particles of the fixed dna of 30 μ L;
3] application of sample: add testing sample 50 μ L respectively in sample tube to be checked hole, positive control hole, negative control hole respectively add yin, yang contrast 50～100 μ L and shake up;
4] incubation: cover all test tube holes, put into biochemical incubator and react 15min at least;
5] washing: magnetic resolution 2min abandons supernatant, adds 500 μ L 0.01M PBST in every test tube, fully shakes up, and magnetic resolution 2min abandons supernatant; Repeating this step all washes off until the material that does not participate in antigen-antibody reaction;
6] enzyme-added: as in positive control test tube hole, negative control test tube hole and sample tube to be checked hole, respectively to add the anti-human IgG of 50～100 μ L enzyme targets respectively;
7] antibody response: cover all test tube holes, put into biochemical incubator and react 15min at least;
8] washing: magnetic resolution 2min abandons supernatant, adds 500 μ L 0.01M PBST in every test tube, fully shakes up, and magnetic resolution 2min abandons supernatant; Repeating this step all washes off until the anti-human IgG that does not participate in antibody response;
9] chromogenic reaction: each test tube hole respectively adds colour developing liquid A, each 50～100 μ L of B, and 5～15min develops the color in the rearmounted biochemical incubator of jog mixing;
10] result judges: each test tube hole respectively adds stop buffer 100～200 μ L, the jog mixing, after the magnetic resolution 2 minutes, from each test tube hole, respectively get 100 μ L supernatants and be transferred in the enzyme mark bar, on microplate reader, adopt 450nm and 630nm two waveband to measure each test tube hole OD value.
The detection of above-mentioned anti-dsDNA antibody also can be adopted immunofluorescence technique, may further comprise the steps:
1] numbering: test tube is placed on the magnetic separator, establish 2 holes, sample tube to be checked hole respectively, 2 holes, positive control test tube hole, 3 holes, negative control test tube hole, blank 1 hole;
2] add antigen: every hole adds the gold-magnetic particles of the fixed dna of 30 μ L;
3] application of sample: add testing sample 50 μ L respectively in sample tube to be checked hole, positive control hole, negative control hole respectively add yin, yang contrast 50～100 μ L and shake up;
4] incubation: cover all test tube holes, put into biochemical incubator and react 15min at least;
5] washing: magnetic resolution 2min abandons supernatant, adds 500 μ L 0.01M PBST in every test tube, fully shakes up, and magnetic resolution 2min abandons supernatant; Repeating this step all washes off until the material that does not participate in antigen-antibody reaction;
6] add fluorescently-labeled anti-human IgG: the anti-human IgG that in positive control test tube hole, negative control test tube hole and sample tube to be checked hole, respectively adds 50～100 μ L FITC marks respectively;
7] antibody response: cover all test tube holes, put into biochemical incubator and react 15min at least;
8] washing: magnetic resolution 2min abandons supernatant, adds 500 μ L 0.01M PBST in every test tube, fully shakes up, and magnetic resolution 2min abandons supernatant; Repeating this step all washes off until the anti-human IgG that does not participate in antibody response;
9] result judges: place microscopically to observe result of determination the specific immune fluorescent composition that obtains.
The detection of above-mentioned anti-dsDNA antibody also can be adopted chemoluminescence method, may further comprise the steps:
1] numbering: test tube is placed on the magnetic separator, establish 2 holes, sample tube to be checked hole respectively, 2 holes, positive control test tube hole, 3 holes, negative control test tube hole, blank 1 hole;
2] add antigen: every hole adds the gold-magnetic particles of the fixed dna of 30 μ L;
3] application of sample: in sample tube to be checked hole, add testing sample 50 μ L, positive control hole, negative control hole respectively and respectively add yin, yang contrast 50～100 μ L and shake up;
4] incubation: cover all test tube holes, put into biochemical incubator and react 15min at least;
5] washing: magnetic resolution 2min abandons supernatant, adds 500 μ L 0.01M PBST in every test tube, fully shakes up, and magnetic resolution 2min abandons supernatant; Repeating this step all washes off until the material that does not participate in antigen-antibody reaction;
6] enzyme-added: the anti-human IgG that in positive control test tube hole, negative control test tube hole and sample tube to be checked hole, respectively adds 50～100 μ L HRP marks respectively;
7] antibody response: cover all test tube holes, put into biochemical incubator and react 15min at least;
8] washing: magnetic resolution 2min abandons supernatant, adds 500 μ L 0.01M PBST in every test tube, fully shakes up, and magnetic resolution 2min abandons supernatant; Repeating this step all washes off until the anti-human IgG that does not participate in antibody response;
9] chromogenic reaction: from vitro change enzyme mark hole over to, each enzyme mark hole respectively adds luminescent substance 100～200 μ L with gold-magnetic particles;
10] result judges: enzyme is marked the signal value that the hole places chemiluminescence detector test sample product, positive control, negative control and blank well.
Compare with existing anti-dsDNA antibody detection method, the advantage that the present invention has is:
1, bag is by the efficient height.The present invention is in gold-magnetic particles pan coating n DNA or plasmid DNA or use gold-magnetic particles to extract DNA as antigen from people's whole blood, saliva, seminal fluid or plasmid.Because of gold-magnetic particles has bigger specific surface area, therefore have bigger fixed capacity, the gold-magnetic particles of unit mass can wrap by relatively large DNA.Such as the bag quilt of antagonist, the gold-magnetic particles of 1mg is the above antibody of 0.3mg fixedly.Because this kind gold-magnetic particles is powerful to the immobilization capacity of biomolecule, the DNA antigen in can high efficiency adsorbent solution.
2, sensitivity of Jian Ceing and specificity height.The present invention is with the carrier matrix of gold-magnetic particles as the detection anti-dsDNA antibody.Gold-magnetic particles has superparamagnetism concurrently, the magnetic grain under the effect in magnetic field, be easy to separate very easily combine with the gold, silver surface with the sulfhydrylation bioactivator and easy adorned advantage to make that this magnetic grain has extremely strong to the response of externally-applied magnetic field and the bigger fixed capacity to bioactive molecule, and gold-magnetic particles is suspended in the liquid phase in the whole testing process, and its bigger specific surface area makes and can detect anti-dsDNA antibody to greatest extent in experimentation.And the carrier of traditional E LISA method is a polystyrene board, and reaction is only carried out at micropore surface, thereby has reduced the detection sensitivity of this method.The whole testing process of the inventive method is carried out in liquid phase, and the DNA antigen on the whole sphere of gold-magnetic particles all participates in reaction, therefore compares with classic method, and the sensitivity and the specificity of this method all improve a lot.
3, can quantize and get rid of artificial subjective judgement.According to the comparison of the testing result and the critical value of microplate reader, judge the negative or positive of testing result and got rid of the artificial subjective judgement according to the visual inspection test findings such as present domestic hospital colloidal gold immunity chromatography commonly used, indirect immunofluorescence, Western blot according to concrete data.
4, the character of enzyme immunoreagent is more stable.Immunofluorescence technique (IFA) is with dilute serum and experiment matrix are carried out incubation, make specific antibody combine with corresponding antigens in the experiment matrix, and then with fluorescently-labeled second antibody incubation, last specificity fluorescent pattern of under fluorescent microscope, observing corresponding site.Wherein testing matrix is the Hep-2 cell than what use always now, can't long preservation.Radioimmunology (RIA) is to combine with anti-dsDNA antibody in the test sample with isotope-labeled double-stranded DNA, because of the isotopic tool half life period, so this reagent can't long preservation.
5, little to the influence of detected sample.The pH value of the various damping fluids that the inventive method is used is compared with the alkaline buffer that traditional ELISA detection method is used near neutral, and the influence of sample is reduced.
Description of drawings
Fig. 1 is the contrast UV figure of solution before and after the gold-magnetic particles adsorption of DNA; Wherein dotted line is represented bag by preceding DNA ultraviolet determination result, and solid line represents that bag is by back DNA ultraviolet determination result.
Fig. 2 is that gold-magnetic particles is that the detection of carrier utilization indirect elisa method contains the blood sample of anti-dsDNA antibody and the comparison of negative control and blank OD value.
Embodiment
1] double-stranded DNA is fixed on the sealing and the preservation of gold-magnetic particles surface and gold-magnetic particles: the gold-magnetic particles of getting 1mg is in the 2mL centrifuge tube, and magnetic resolution is abandoned supernatant.Reach the pH or the salinity of coupling buffer with coupling buffer (mixed solution of 20% PEG 8000 and the sodium chloride of 2.5M) washing gold-magnetic particles until the preservation environment of gold-magnetic particles, each coupling buffer washing consumption is 400 μ L.Magnetic resolution is abandoned the double-stranded DNA that adds 14 μ g behind the supernatant and the above-mentioned coupling buffer of 200 μ L in gold-magnetic particles, and 37 ℃, 180rpm reacts 30min.After magnetic resolution is abandoned supernatant, the sealer (mixed solution of the PBS damping fluid of the pH=7.4 of 0.01M and 10% calf serum) that adds 1mL is in centrifuge tube, 37 ℃, 180rpm reacts 1h, take out, the gold-magnetic particles of sealing is cleaned in 4 ℃ of backs of spending the night with the cleaning buffer solution (mixed solution of the PBS damping fluid of the pH=7.4 of 0.01M, 0.05% Tween-20) of 1mL.After cleaning finished, the preservation damping fluid (the PBS damping fluid of the pH=7.4 of 0.01M) that adds 1mL was preserved the gold-magnetic particles that bag is finished, and 4 ℃ of preservations are standby.
2] detection of anti-dsDNA antibody: get the gold-magnetic particles that 30 μ L have wrapped quilt respectively and put into the test tube hole, shake up; Get the sample to be checked of 50 μ L respectively and put into sample tube to be checked hole, shake up; In positive control test tube hole, add 50 μ L positive control serums, shake up; In negative control test tube hole, add 50 μ L negative control seras, shake up; Blank well wouldn't add any liquid; 37 ℃, take out behind the 180rpm reaction 30min, magnetic resolution 2min, abandon supernatant after, add 500 μ L 0.01M PBST in every test tube, fully shake up, magnetic resolution 2min abandons supernatant.After repeating this step 4 time, in positive control test tube hole, negative control test tube hole and sample tube to be checked hole, respectively add the anti-human IgG of 50 μ L enzyme targets respectively; 37 ℃, take out behind the 180rpm reaction 30min, magnetic resolution 2min abandons supernatant, adds 500 μ L 0.01MPBST in every test tube hole, fully shakes up, and magnetic resolution 2min abandons supernatant; Repeating this step 4 time all washes off until the anti-human IgG that does not participate in antibody response; Add 50 μ L colour developing liquid A and 50 μ L colour developing liquid B in every test tube hole, 37 ℃ of colour developing 5min in the rearmounted biochemical incubator of jog mixing, every test tube hole adds 2M H
2SO
4100 μ L, the jog mixing, magnetic resolution was respectively got 100 μ L supernatants and is transferred in the enzyme mark bar after 2 minutes from test tube, adopt two waveband (450nm, 630nm) to measure on microplate reader and respectively manage the OD value.After detection finished, utilization elution buffer (1 * the TE damping fluid of pH=7.2) wash-out was coated on the DNA on gold-magnetic particles surface, and added at gold-magnetic particles and to preserve preservation in the damping fluid (the PBS damping fluid of the pH=7.4 of 0.01M).
As seen from Figure 1, use gold-magnetic particles can be from samples such as whole blood, saliva, seminal fluid, tissue in suitable solution system purify DNA and be adsorbed on the gold-magnetic particles surface when using as envelope antigen, the existence of solution contrast UV figure clearly before and after the gold-magnetic particles adsorption of DNA, illustrate gold-magnetic particles can be from samples such as whole blood, saliva, seminal fluid, tissue a large amount of purify DNAs.As seen from Figure 2, there is tangible difference between the testing result of the positive blood sample of application the present invention mensuration and negative blood sample and blank.
The purified natural double-stranded DNA of gold-magnetic particles pan coating or plasmid DNA antigen is for preparation voluntarily or available from commercial product, and used two anti-are commercial prod in the experimentation.
The principle of the invention:
1. anti-dsDNA antibody is a kind of autoantibody that can combine with n DNA, almost only in SLE patient, can detect this antibody (Ruffatti A, Calligaro A, Ross D et al.Anti-doublestranded DNA antibodies in the healthy elderly:prevalence andcharacteristics.J Clin Immunol.1990; 10:300-303; Kadlubowski M, JacksonM, Yap PL and NeilG.Lack of specificity for antibodies to double strandedDNA found in four commercial kits.J Clin Path.1991; 44:248-250).Relevant (the Borg EJ of the growth and decline of anti-dsDNA antibody titre with SLE patient's change of illness state, Horst G, HumEJ et al.Measurement of increases in anti-double stranded DNA antibodylevel as a predictor of disease exacerbation in systemic lupuserythematosus:a long term, prospective study. Arth Rheum. 1990; 33:634-643), of the present invention is the method for carrier sense anti-dsDNA antibody with the gold-magnetic particles, be behind natural double-stranded DNA on the gold-magnetic particles pan coating or plasmid DNA antigen, to use ELISA method, chemoluminescence method or immunofluorescence technique to detect test serum, perhaps use gold-magnetic particles from people's whole blood, saliva, seminal fluid or plasmid, to extract DNA, in system, add tested serum then and directly detect.
Chinese invention patent ZL 03153486.4 and ZL 03124061.5 disclose the Fe3O4/Au super-paramagnetic composite particle of packaging magnetic composite particle and core/shell structure respectively.Following these two kinds of magnetic particles are called gold-magnetic particles.Gold-magnetic particles is made of core with superparamagnetism and outer layer segment; the core forms the positive ferrite magnetic particle with superparamagnetism by Fe3O4, γ-Fe2O3, Fe, Co, Ni or Fe and other metallic element and constitutes; housing parts is made of nanoscale noble metal granule or simple substance gold, silver shell, and mean diameter is 0.05～100 μ m.Because metal simple-substance such as gold, silver is Substance Properties such as adhesion protein, nucleic acid efficiently, thus can natural double-stranded DNA or plasmid DNA be coated on gold-magnetic particles surperficial and use gold-magnetic particles can be from blood, seminal fluid, saliva, microorganism and animal tissue etc. the high efficiency extraction double-stranded DNA.This moment, DNA was coated on the antigen that the gold-magnetic particles surface can be used as indirect elisa method, immunofluorescence technique or chemoluminescence method detection anti-dsDNA antibody.
2, add blood sample to be checked, by 37 ℃ of incubations, if contain anti-dsDNA antibody (is anti-) in the sample, the specificity combination can take place between the antigen-antibody, antibody in the blood sample also has been connected the gold-magnetic particles surface by antigen, and other albumen and impurity that antigen and antibody specific reaction do not take place can be removed by washing process.
3, add enzyme (HRP) mark or fluorescently-labeled anti-human IgG (two is anti-), by 37 ℃ of incubations, thereby anti-and two anti-specificitys that take place are in conjunction with making the two anti-gold-magnetic particles surfaces that also have been connected, and remove by washing process and have neither part nor lot in the two anti-of reaction.
4, behind adding colour developing liquid or the luminescent substance, superoxide (the used superoxide of this experiment is a tetramethyl benzidine) in the HRP catalysis colour developing liquid or luminescent substance catalysis HRP react and produce coloured product, the highest absorption peak in 450nm place survey indirect elisa method or the signal value in the detection chemoluminescence method on chemiluminescence detector.OD value or the signal value surveyed are big more, show that two of the HRP mark that is connected with on the gold-magnetic particles are anti-many more, thereby have shown contain in the blood sample one anti-many more, have just shown that also conditions of patients is serious more.Examine under a microscope result of determination in the immunofluorescence technique.
5, select the natural double-stranded DNA of purifying or the principle of plasmid DNA for use: think at present double-stranded DNA the bag quilt with take place to reset relevant with the space conformation of the combination of anti-ds-DNA antibody and double-stranded DNA, especially concern very greatly with double chain DNA molecule whether complete, moreover the big more adhesion of double chain DNA molecule amount is strong more.Have report greater than the DNA of 60pb could with the dna antibody strong bonded, and irrelevant with the kind source.
Gold-magnetic particles is meant that the aggregation with magnetic nano-particle or magnetic nano-particle is a nuclear, magnetic composite particle in noble metal shell formation such as nuclear surface coating simple substance gold, silver, refer to that also the aggregation with magnetic nano-particle or magnetic nano-particle is a nuclear, at the magnetic composite particle of noble metal formation such as nuclear surface-assembled nanometer gold, silver.Described magnetic nano-particle comprises Fe
3O
4, γ-Fe
2O
3Deng the oxide particle of iron, simple substance Fe, Co, Ni particle, or the positive ferrite particle that forms by Fe and other metallic element; The aggregation of magnetic nano-particle is meant the Fe that modified back forms
3O
4, γ-Fe
2O
3Deng the oxide particle aggregation of iron, simple substance Fe, Co, Ni particle agglomeration that modified back forms, or the positive ferrite particle agglomeration of the Fe of modified back formation and the formation of other metallic element.
Claims (7)
1. one kind is the method for carrier sense anti-dsDNA antibody with the gold-magnetic particles, it is characterized in that: said method comprising the steps of:
1] DNA fixing: get gold-magnetic particles and add in the centrifuge tube on the gold-magnetic particles surface, after magnetic resolution is abandoned supernatant, wash gold-magnetic particles with coupling buffer, magnetic resolution is abandoned the natural double-stranded DNA that adds purifying behind the supernatant or plasmid DNA and coupling buffer in gold-magnetic particles, under 20～40 ℃ of conditions, fully react 20～40min, form gold-magnetic particles-DNA compound; Perhaps whole blood, saliva, seminal fluid or plasmid are made solution system, therefrom extract DNA and make it be fixed on the gold-magnetic particles surface with gold-magnetic particles;
2] sealing of gold-magnetic particles: after the gold-magnetic particles magnetic resolution abandoned supernatant, add sealer in centrifuge tube, abundant reaction 1～2h under 20～40 ℃ of conditions takes out, and 4 ℃ are spent the night;
3] cleaning of gold-magnetic particles: with the gold-magnetic particles after the cleaning buffer solution cleaning sealing;
4] preservation of gold-magnetic particles: the gold-magnetic particles that cleaning is finished adds the preservation damping fluid, and 4 ℃ of preservations are standby;
5] detection of anti-dsDNA antibody: the DNA with the washed gold magnetic particles surface is an antigen, and utilization indirect elisa method, immunofluorescence technique or chemoluminescence method detect anti-dsDNA antibody.
2. according to claim 1 a kind of be the method for carrier sense anti-dsDNA antibody with the gold-magnetic particles, it is characterized in that: the gold-magnetic particles quality of described adding is 0.5～5mg; The DNA quality of described adding is 5～25 μ g.
3. according to claim 1 and 2 a kind of be the method for carrier sense anti-dsDNA antibody with the gold-magnetic particles, it is characterized in that: described coupling buffer is 0.5 *～10 * the PBS of pH=5.0～8.0, the perhaps CBS of the pH=9.0 of 5mM～10M～11.0, the perhaps Tris-HCl damping fluid of the pH=7.0 of 5mM～10M～9.0, the perhaps acetate buffer of the pH=3.6 of 5mM～10M～5.6, the perhaps citrate buffer of the pH=3.0 of 5mM～10M～7.0, the perhaps TBS damping fluid of the pH=7.0 of 5mM～10M～9.0, perhaps be the salt of polyglycol (PEG) 6000～10000 addings 0.5～5.0M between 10～25, described salt can be sodium chloride, lithium chloride, barium chloride, potassium chloride, lime chloride, magnesium chloride, cesium chloride etc.; Described sealer is the mixed solution of irrelevant albumen and above-mentioned coupling buffer, and described irrelevant albumen is one or more in 1%～10% bovine serum albumin(BSA), 1%～10% lysine, 1%～10% skimmed milk power, 1%～10% monoethanolamine or 1%～10% animal blood serum; Described cleaning buffer solution is coupling buffer or the coupling buffer that contains 0.02%～0.2% tween; Described preservation damping fluid is coupling buffer or adds antiseptic and protectant coupling buffer; described antiseptic is that 0.01%～0.1% nitrine is received or 0.01%～0.1% thimerosal, and described protective agent is 0.05%～1% BSA, 0.05%～1% animal blood serum or 0.05%～1% protease inhibitors.
4. according to claim 3 a kind of be the method for carrier sense anti-dsDNA antibody with the gold-magnetic particles, it is characterized in that: described coupling buffer is the mixed solution of the sodium chloride of 10% PEG8000 and 2.5M; Described sealer is the PBS damping fluid, 2% BSA of the pH=7.0 of 0.1M, the mixed solution of 5% calf serum; Described cleaning buffer solution is the PBS damping fluid of the pH=7.0 of 0.1M, the mixed solution of 0.5% Tween-20; The PBS damping fluid of the pH=7.0 that described preservation damping fluid is 0.1M.
5. according to claim 1 a kind of be the method for carrier sense anti-dsDNA antibody with the gold-magnetic particles, it is characterized in that: the detection of described anti-dsDNA antibody may further comprise the steps:
1] numbering: test tube is placed on the magnetic separator, establish 2 holes, sample tube to be checked hole respectively, 2 holes, positive control test tube hole, 3 holes, negative control test tube hole, blank 1 hole;
2] add antigen: every hole adds the gold-magnetic particles of the fixed dna of 30 μ L;
3] application of sample: add testing sample 50 μ L respectively in sample tube to be checked hole, positive control hole, negative control hole respectively add yin, yang contrast 50～100 μ L and shake up;
4] incubation: cover all test tube holes, put into biochemical incubator and react 15min at least;
5] washing: magnetic resolution 2min abandons supernatant, adds 500 μ L 0.01M PBST in every test tube, fully shakes up, and magnetic resolution 2min abandons supernatant; Repeating this step all washes off until the material that does not participate in antigen-antibody reaction;
6] enzyme-added: as in positive control test tube hole, negative control test tube hole and sample tube to be checked hole, respectively to add the anti-human IgG of 50～100 μ L enzyme targets respectively;
7] antibody response: cover all test tube holes, put into biochemical incubator and react 15min at least;
8] washing: magnetic resolution 2min abandons supernatant, adds 500 μ L 0.01M PBST in every test tube, fully shakes up, and magnetic resolution 2min abandons supernatant; Repeating this step all washes off until the anti-human IgG that does not participate in antibody response;
9] chromogenic reaction: each test tube hole respectively adds colour developing liquid A, each 50～100 μ L of B, and 5～15min develops the color in the rearmounted biochemical incubator of jog mixing;
10] result judges: each test tube hole respectively adds stop buffer 100～200 μ L, the jog mixing, after the magnetic resolution 2 minutes, from each test tube hole, respectively get 100 μ L supernatants and be transferred in the enzyme mark bar, on microplate reader, adopt 450nm and 630nm two waveband to measure each test tube hole OD value.
6. according to claim 1 a kind of be the method for carrier sense anti-dsDNA antibody with the gold-magnetic particles, it is characterized in that: the detection of described anti-dsDNA antibody may further comprise the steps:
1] numbering: test tube is placed on the magnetic separator, establish 2 holes, sample tube to be checked hole respectively, 2 holes, positive control test tube hole, 3 holes, negative control test tube hole, blank 1 hole;
2] add antigen: every hole adds the gold-magnetic particles of the fixed dna of 30 μ L;
3] application of sample: add testing sample 50 μ L respectively in sample tube to be checked hole, positive control hole, negative control hole respectively add yin, yang contrast 50～100 μ L and shake up;
4] incubation: cover all test tube holes, put into biochemical incubator and react 15min at least;
5] washing: magnetic resolution 2min abandons supernatant, adds 500 μ L 0.01M PBST in every test tube, fully shakes up, and magnetic resolution 2min abandons supernatant; Repeating this step all washes off until the material that does not participate in antigen-antibody reaction;
6] add fluorescently-labeled anti-human IgG: the anti-human IgG that in positive control test tube hole, negative control test tube hole and sample tube to be checked hole, respectively adds 50～100 μ L FITC marks respectively;
7] antibody response: cover all test tube holes, put into biochemical incubator and react 15min at least;
8] washing: magnetic resolution 2min abandons supernatant, adds 500 μ L 0.01M PBST in every test tube, fully shakes up, and magnetic resolution 2min abandons supernatant; Repeating this step all washes off until the anti-human IgG that does not participate in antibody response;
9] result judges: place microscopically to observe result of determination the specific immune fluorescent composition that obtains.
7. according to claim 1 a kind of be the method for carrier sense anti-dsDNA antibody with the gold-magnetic particles, it is characterized in that: the detection of described anti-dsDNA antibody may further comprise the steps:
1] numbering: test tube is placed on the magnetic separator, establish 2 holes, sample tube to be checked hole respectively, 2 holes, positive control test tube hole, 3 holes, negative control test tube hole, blank 1 hole;
2] add antigen: every hole adds the gold-magnetic particles of the fixed dna of 30 μ L;
3] application of sample: in sample tube to be checked hole, add testing sample 50 μ L, positive control hole, negative control hole respectively and respectively add yin, yang contrast 50～100 μ L and shake up;
4] incubation: cover all test tube holes, put into biochemical incubator and react 15min at least;
5] washing: magnetic resolution 2min abandons supernatant, adds 500 μ L 0.01M PBST in every test tube, fully shakes up, and magnetic resolution 2min abandons supernatant; Repeating this step all washes off until the material that does not participate in antigen-antibody reaction;
6] enzyme-added: the anti-human IgG that in positive control test tube hole, negative control test tube hole and sample tube to be checked hole, respectively adds 50～100 μ L HRP marks respectively;
7] antibody response: cover all test tube holes, put into biochemical incubator and react 15min at least;
8] washing: magnetic resolution 2min abandons supernatant, adds 500 μ L 0.01M PBST in every test tube, fully shakes up, and magnetic resolution 2min abandons supernatant; Repeating this step all washes off until the anti-human IgG that does not participate in antibody response;
9] chromogenic reaction: from vitro change enzyme mark hole over to, each enzyme mark hole respectively adds luminescent substance 100～200 μ L with gold-magnetic particles;
10] result judges: enzyme is marked the signal value that the hole places chemiluminescence detector test sample product, positive control, negative control and blank well.
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