CN101776682A - Preparation method for double-stranded DNA antigen and kit for testing anti-double-stranded-DNA antibody of human serum - Google Patents
Preparation method for double-stranded DNA antigen and kit for testing anti-double-stranded-DNA antibody of human serum Download PDFInfo
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Abstract
The invention relates to a preparation method for a double-stranded DNA antigen and a kit for testing an anti-double-stranded-DNA antibody of human serum. Particularly, the invention provides a preparation method for a double-stranded DNA antigen used for diagnosing systemic lupus erythematosus and a preparation method for a kit used for in vitro test of an anti-double-stranded-DNA antibody of human serum. The antigen prepared in the method has the advantages of low cost and stable and reliable antigen properties, and also has high sensitivity and specificity for in vitro test of the anti-double-stranded-DNA antibody in the serum of the patient with the systemic lupus erythematosus.
Description
Technical field
The present invention relates to that a kind of in the immunological technique field detects the preparation of double-stranded DNA antigen and as the preparation method of antigen, be used for auxiliary diagnosis SLE and in the application of other disease at the kit that detects anti-double-stranded-DNA antibody of human serum.
Background technology
(systemic lupus erythematosus is a kind of autoimmunity disease of invading the whole body connective tissue SLE) to systemic loupus erythematosus, is more common in young women, and complicated clinical manifestation is various.Dna antibody roughly is divided into natural double-stranded DNA (double stranded DNA, dsDNA) and sex change single stranded DNA (singlestranded DNA, ssDNA) antibody, there is anti-dsDNA antibody in 40% SLE patients serum, 70% exists anti-ssDNA antibody, but anti-ssDNA antibody deficiency specificity is meaningless to diagnosing.And anti-dsDNA antibody has very high specificity, is called as " labelled antibody " of SLE, is one of important sign antibody among the SLE patients serum, and SLE has higher specificity to diagnosis.
The method of the detection anti-dsDNA antibody that present laboratory is commonly used mainly contains enzyme-linked immunosorbent test (enzyme linked immunosorbent assay, ELISA), indirect immunofluorescence (IIF, to lack film worm or pferdepest trypanosome is antigen), put the method for exempting from (Farr method), spot immune gold percolation (DotImmunogoldfiltration Assay, DIGFA) etc.Farr method repeatability is better, but can only survey high-affinity antibody, can not distinguish the type (as IgG or IgM type antibody) of antibody, and need particular instrument equipment, has isotopic contamination.Indirect immunofluorescence can be made semiquantitative determination, but the fluorescence microscope result must be arranged and certain subjective artificial error in judgement is arranged, and the standardization difficulty also is not suitable for the detection of high flux sample.The DIGFA method detects easy, quick, visual result, but its specificity is lower, can not make detection by quantitative, the difficult control of differences between batches.The ELISA method detects and can be used for high flux sample mensuration, method is simple, simple operation, easily standardization, sensitivity is also higher, is extensively approved at present, it has "dead" isotopic contamination, not influenced in conjunction with albumen by non-DNA, can measure dissimilar antibody, high and low affinity antibodies such as can measure simultaneously at advantage.
Along with deep day by day to the research of the bag quilt of double-stranded DNA and double-stranded DNA antigen and anti-dsDNA antibody binding mechanism, think at present double-stranded DNA the bag quilt with take place to reset relevant with the space conformation of the combination of anti-dsDNA and double-stranded DNA, especially the combination of double-stranded DNA antigen and anti-dsDNA antibody and double chain DNA molecule whether complete concerns very big, moreover the big more adhesion of double chain DNA molecule amount is strong more, have report greater than the DNA more than the 60bp could with firm the combining of anti-DNA antibody, and irrelevant with kind source.Plasmid DNA is to be free on to have the double chain DNA molecule that duplicates voluntarily outside the bacterial chromosome.Recombinant plasmid dna stable uniform, and convenient sources.We adopt the purification process of optimization to prepare double chain DNA molecule; guaranteed the purity of antigen; the susceptibility and the specificity that detect have been improved; because the double-stranded DNA of natural purification has the pollution of autoantigen materials such as nucleoprotein composition or nucleosome usually, cause anti-dsDNA antibody test specificity to reduce.
Therefore, this area still needs to prepare the preparation method of double-stranded DNA antigen and the kit of detection anti-double-stranded-DNA antibody of human serum.
Summary of the invention
One of purpose of the present invention provides a kind of systemic loupus erythematosus and detects preparation method with double-stranded DNA antigen, and described method comprises,
(1) use the damping fluid P1 contain Tris, edta salt and RNase A to handle plasmid DNA, centrifugal removing adds this damping fluid P1 again behind the supernatant and suspends;
(2) use alkaline buffer P2 treatment step (1) the gained suspension that contains SDS and alkali metal ion; With
(3) use contains the product of the damping fluid P3 treatment step (2) of potassium acetate, centrifugal acquisition supernatant, and supernatant filters the back and handles with isopropyl alcohol, centrifugal acquisition precipitation.
In a preferred embodiment, in described step (3) afterwards, use the precipitation with alcohol mycoprotein respectively, use PEG8000 deposit D NA, carry out phenol-chloroform extracting DNA then.
In a preferred embodiment, in the mixed liquor of gained, the ratio of damping fluid P1, P2 and P3 is roughly 1: 1: 1 behind the adding damping fluid P3.
In a further advantageous embodiment, in the mixed liquor of gained, the ratio of damping fluid P1, P2 and P3 is roughly every milligram of plasmid 4～6ml: 4～6ml: 4～6ml behind the adding damping fluid P3.
In a preferred embodiment, described plasmid DNA is contained in the recombination bacillus coli.
In a preferred embodiment, in step (3), the amount of isopropyl alcohol is 0.5～1 times of supernatant volume.In preferred embodiment, the amount of isopropyl alcohol is 0.7～0.8 times of supernatant volume.
In a preferred embodiment, the pH of damping fluid P3 is 5～6, is preferably 5.2～5.8, more preferably 5.3～5.7.In one embodiment, the pH of damping fluid P3 is 5.5.
In a preferred embodiment, in the weight of thalline or plasmid, the consumption of Tris is 0.1%～1.0% of thalline or a plasmid weight, and is preferred 0.2～0.8%, more preferably 0.3～0.6%.
In a preferred embodiment, edta salt can be sodium salt, sylvite of EDTA etc., and its concentration is 0.1 weight %～0.8 weight %, is preferably 0.1～0.6 weight %, more preferably 0.2～0.4 weight %.
In a preferred embodiment, the amount of RNase A can be 0.005% weight～0.05 weight %, perhaps is 0.01%～0.03 weight.
In a preferred embodiment, the pH of damping fluid P1 is generally 7～9 in 7～10 scope, be preferably 7.5～8.5, and more preferably about 8.0.
In a preferred embodiment, the concentration of SDS is 0.1 weight %～2 weight %, is preferably 0.5～1.5 weight %, more preferably 0.6～1.0 weight %.
In a preferred embodiment, alkali metal ion can be for example sodion, potassium ion etc., and its concentration is generally 0.1 weight %～2 weight %, is preferably 0.5～1.5 weight %, more preferably 0.6～1.0 weight %.
In a preferred embodiment, described damping fluid P2 contains SDS and NaOH or potassium hydroxide.
In a preferred embodiment, the concentration of KAC is generally 5～15 weight %, for example can be 6～12 weight %, 7～10 weight %, 8～10 weight % etc.
In an embodiment, the inventive method comprises following successively the processing: damping fluid P1 processing, damping fluid P2 processing, damping fluid P3 processing, isopropyl alcohol processing, Ethanol Treatment, the TE processing that contains RNase A, phenol-chloroform extracting, chloroform extracting, Ethanol Treatment, NaCl and PEG8000 handle, Ethanol Treatment, and last TE dissolving, preserve the TE dissolved matter standby then.These handle used reagent and condition as described in this instructions other parts.
Two of purpose of the present invention provides a kind of systemic loupus erythematosus detection double-stranded DNA antigen that adopts the inventive method to make.
In a preferred embodiment, described method comprises:
(1) use the damping fluid P1 contain Tris, edta salt and RNase A to handle plasmid DNA, centrifugal removing adds this damping fluid P1 again behind the supernatant and suspends;
(2) use alkaline buffer P2 treatment step (1) the gained suspension that contains SDS and alkali metal ion; With
(3) use contains the product of the damping fluid P3 treatment step (2) of potassium acetate, centrifugal acquisition supernatant, and supernatant filters the back and handles with isopropyl alcohol, centrifugal acquisition precipitation.
In a preferred embodiment, in described step (3) afterwards, use the precipitation with alcohol mycoprotein respectively, use PEG8000 deposit D NA, carry out phenol-chloroform extracting DNA then.
In a preferred embodiment, in the mixed liquor of gained, the ratio of damping fluid P1, P2 and P3 is roughly 1: 1: 1 behind the adding damping fluid P3.
In a further advantageous embodiment, in the mixed liquor of gained, the ratio of damping fluid P1, P2 and P3 is roughly every milligram of plasmid 4～6ml: 4～6ml: 4～6ml behind the adding damping fluid P3.
In a preferred embodiment, described plasmid DNA is contained in the recombination bacillus coli.
In a preferred embodiment, in step (3), the amount of isopropyl alcohol is 0.5～1 times of supernatant volume.In preferred embodiment, the amount of isopropyl alcohol is 0.7～0.8 times of supernatant volume.
In a preferred embodiment, the pH of damping fluid P3 is 5～6, is preferably 5.2～5.8, more preferably 5.3～5.7.In one embodiment, the pH of damping fluid P3 is 5.5.
In a preferred embodiment, described damping fluid P2 contains SDS and NaOH or potassium hydroxide.
In a preferred embodiment, in the weight of thalline or plasmid, the consumption of Tris is 0.1%～1.0% of thalline or a plasmid weight, and is preferred 0.2～0.8%, more preferably 0.3～0.6%.
In a preferred embodiment, edta salt can be sodium salt, sylvite of EDTA etc., and its concentration is 0.1 weight %～0.8 weight %, is preferably 0.1～0.6 weight %, more preferably 0.2～0.4 weight %.
In a preferred embodiment, the amount of RNase A can be 0.005% weight～0.05 weight %, perhaps is 0.01%～0.03 weight.
In a preferred embodiment, the pH of damping fluid P1 is generally 7～9 in 7～10 scope, be preferably 7.5～8.5, and more preferably about 8.0.
In a preferred embodiment, the concentration of SDS is 0.1 weight %～2 weight %, is preferably 0.5～1.5 weight %, more preferably 0.6～1.0 weight %.
In a preferred embodiment, alkali metal ion can be for example sodion, potassium ion etc., and its concentration is generally 0.1 weight %～2 weight %, is preferably 0.5～1.5 weight %, more preferably 0.6～1.0 weight %.
In a preferred embodiment, described damping fluid P2 contains SDS and NaOH or potassium hydroxide.
In a preferred embodiment, the concentration of KAC is generally 5～15 weight %, for example can be 6～12 weight %, 7～10 weight %, 8～10 weight % etc.
Three of purpose of the present invention provides a kind of kit that detects anti-double-stranded-DNA antibody of human serum, and this kit comprises systemic loupus erythematosus detection double-stranded DNA antigen as herein described.
In a preferred embodiment, described systemic loupus erythematosus detects and adopts method as herein described to make with double-stranded DNA antigen.
In a preferred embodiment, described method comprises:
(1) use the damping fluid P1 contain Tris, edta salt and RNase A to handle plasmid DNA, centrifugal removing adds this damping fluid P1 again behind the supernatant and suspends;
(2) use alkaline buffer P2 treatment step (1) the gained suspension that contains SDS and alkali metal ion; With
(3) use contains the product of the damping fluid P3 treatment step (2) of potassium acetate, centrifugal acquisition supernatant, and supernatant filters the back and handles with isopropyl alcohol, centrifugal acquisition precipitation.
In a preferred embodiment, in described step (3) afterwards, use the precipitation with alcohol mycoprotein respectively, use PEG8000 deposit D NA, carry out phenol-chloroform extracting DNA then.
In a preferred embodiment, in the mixed liquor of gained, the ratio of damping fluid P1, P2 and P3 is roughly 1: 1: 1 behind the adding damping fluid P3.
In a further advantageous embodiment, in the mixed liquor of gained, the ratio of damping fluid P1, P2 and P3 is roughly every milligram of plasmid 4～6ml: 4～6ml: 4～6ml behind the adding damping fluid P3.
In a preferred embodiment, described plasmid DNA is contained in the recombination bacillus coli.
In a preferred embodiment, in step (3), the amount of isopropyl alcohol is 0.5～1 times of supernatant volume.In preferred embodiment, the amount of isopropyl alcohol is 0.7～0.8 times of supernatant volume.
In a preferred embodiment, described damping fluid P2 contains SDS and NaOH or potassium hydroxide.
In a preferred embodiment, the pH of damping fluid P3 is 5～6, is preferably 5.2～5.8, more preferably 5.3～5.7.In one embodiment, the pH of damping fluid P3 is 5.5.
In a preferred embodiment, in the weight of thalline or plasmid, the consumption of Tris is 0.1%～1.0% of thalline or a plasmid weight, and is preferred 0.2～0.8%, more preferably 0.3～0.6%.
In a preferred embodiment, edta salt can be sodium salt, sylvite of EDTA etc., and its concentration is 0.1 weight %～0.8 weight %, is preferably 0.1～0.6 weight %, more preferably 0.2～0.4 weight %.
In a preferred embodiment, the amount of RNase A can be 0.005% weight～0.05 weight %, perhaps is 0.01%～0.03 weight.
In a preferred embodiment, the pH of damping fluid P1 is generally 7～9 in 7～10 scope, be preferably 7.5～8.5, and more preferably about 8.0.
In a preferred embodiment, the concentration of SDS is 0.1 weight %～2 weight %, is preferably 0.5～1.5 weight %, more preferably 0.6～1.0 weight %.
In a preferred embodiment, alkali metal ion can be for example sodion, potassium ion etc., and its concentration is generally 0.1 weight %～2 weight %, is preferably 0.5～1.5 weight %, more preferably 0.6～1.0 weight %.
In a preferred embodiment, described damping fluid P2 contains SDS and NaOH or potassium hydroxide.
In a preferred embodiment, the concentration of KAC is generally 5～15 weight %, for example can be 6～12 weight %, 7～10 weight %, 8～10 weight % etc.
In a preferred implementation, described double-stranded DNA antigen is through biotin labeling.
In a preferred implementation, also described kit comprises that also two of horseradish peroxidase or alkali phosphatase enzyme mark resists, and ELISA Plate; Wherein, described ELISA Plate contains through Streptavidin SA and biotin system and has adsorbed the solid phase carrier of described double-stranded DNA antigen absorption.
In one embodiment, described kit also comprises calibration object, sample diluting liquid, concentrated cleaning solution, colour developing liquid A, shows liquid B and/or stop buffer.
In a preferred implementation, colour developing liquid A is hydrogen peroxide or urea peroxide, and colour developing liquid B is o-phenylenediamine or tetramethyl benzidine; Concentrated cleaning solution is the phosphate buffer that contains 0.8～1.2% tween; Sample diluting liquid is the phosphate buffer of 0.8～1.2% tween and 0.1～0.3%BSA; Stop buffer is 1M～3M sulfuric acid or hydrochloric acid solution.
In a preferred embodiment, calibration object is 3 bottles of double-stranded-DNA antibody series standard solution that concentration is respectively 10IU, 100IU, 800IU.
In a preferred embodiment, described marker enzyme can be horseradish peroxidase or alkaline phosphatase.
In a preferred embodiment, two anti-anti-human IgG monoclonal antibody or anti-human IgG polyclonal antibody or SPA (staphylococcal protein A) or the Protein G of can be.
In a preferred embodiment, marker enzyme is a horseradish peroxidase, and two anti-are SPA, and horseradish peroxidase can by glutaraldehyde method or to cross the salt compounded of iodine acid system crosslinked on SPA.
Four of the object of the invention provides a kind of method for preparing the kit that detects anti-double-stranded-DNA antibody of human serum, the systemic loupus erythematosus of the present invention that comprises described kit detects two anti-and ELISA Plate with double-stranded DNA antigen, horseradish peroxidase or alkali phosphatase enzyme mark, it is characterized in that described method comprises:
(1) prepares described systemic loupus erythematosus detection by method of the present invention and use double-stranded DNA antigen, so that described double-stranded DNA antigen to be provided;
(2) provide two of horseradish peroxidase or alkali phosphatase enzyme mark to resist; With
(3) through Streptavidin SA and biotin system with described double-stranded DNA antigen bag by on the solid phase carrier of ELISA Plate;
(4) use above-mentioned (1)-(a 3) described composition to dispose described kit.
In one embodiment, the described systemic loupus erythematosus detection of the described preparation of step (1) is the whole bag of tricks and the preferred implementation thereof described in first purpose of the present invention with the method for double-stranded DNA antigen.
In one embodiment, described kit also comprises calibration object, sample diluting liquid, concentrated cleaning solution, colour developing liquid A, shows that liquid B and/or stop buffer, described method also are included in the described calibration object of configuration in the described kit, sample diluting liquid, concentrated cleaning solution, colour developing liquid A, show liquid B and/or stop buffer.
In a preferred embodiment, colour developing liquid A is hydrogen peroxide or urea peroxide, and colour developing liquid B is o-phenylenediamine or tetramethyl benzidine; Concentrated cleaning solution is the phosphate buffer that contains the 0.8-1.2% tween; Sample diluting liquid is the phosphate buffer of 0.8-1.2% tween and 0.1～0.3%BSA; Stop buffer is 1M-3M sulfuric acid or hydrochloric acid solution.
In a preferred embodiment, calibration object is 3 bottles of double-stranded-DNA antibody series standard solution that concentration is respectively 10IU, 100IU, 800IU.
In a preferred embodiment, described marker enzyme can be horseradish peroxidase or alkaline phosphatase.
In a preferred embodiment, two anti-anti-human IgG monoclonal antibody or anti-human IgG polyclonal antibody or SPA (staphylococcal protein A) or the Protein G of can be.
In a preferred embodiment, marker enzyme is a horseradish peroxidase, and two anti-are SPA, and horseradish peroxidase can by glutaraldehyde method or to cross the salt compounded of iodine acid system crosslinked on SPA.
The present invention provides the kit of the detection anti-double-stranded-DNA antibody of human serum that the method that adopts the present invention to prepare kit makes a purpose.
Therefore, in one embodiment, this kit contains described systemic loupus erythematosus and detects the two anti-and ELISA Plate of using double-stranded DNA antigen, horseradish peroxidase or alkali phosphatase enzyme mark.
In other embodiments, described kit also contains calibration object, colour developing liquid, stop buffer, concentrated cleaning solution and/or sample diluting liquid.Described calibration object, colour developing liquid, stop buffer, concentrated cleaning solution and sample diluting liquid are as hereinbefore defined.
In the used percentage by weight of the present invention, except as otherwise noted, otherwise its calculating benchmark is the weight of corresponding damping fluid.
Description of drawings
Fig. 1 shows that the double-stranded DNA agarose gel electrophoresis detects the result of its purity.After getting 5 μ l stostes adding TE damping fluid, 20 μ l mixings, get 5 μ l and carry out 0.8% agarose gel electrophoresis (the 100ml gel solution adds 5 μ l Goldview), get 5 μ l DNA Marker simultaneously as reference, with 120V voltage electrophoresis 30 minutes, show that as accompanying drawing sample lane do not have microRNA, dna degradation band and bacterial genomes DNA pollute.Show that double-stranded DNA purity is higher.
Fig. 2 shows the agarose gel electrophoresis figure of the double-stranded DNA that the traditional alkaline lysis of employing makes.Arrow among the figure shows that RNA does not eliminate.
Embodiment
The method of many proposition plasmid DNA is disclosed in the prior art.For example Li Gang just waits (" foundation and the improvement of high-purity plasmid DNA large-scale production flow process ", modern food science and technology, 2008, the 4 volumes, 433-438 page or leaf) to disclose a kind of method that adopts the thick upgrading grain of alkaline lysis.But these existing method complex process, not high, the poor stability of the antigen purity that obtains that extracts still can't satisfy the detection applications requirement.
Therefore, the purpose of this invention is to provide a kind of systemic loupus erythematosus and detect the preparation method who uses double-stranded DNA antigen, comprising:
(1) use the damping fluid P1 contain Tris, edta salt and RNase A to handle plasmid DNA, centrifugal removing adds this damping fluid P1 again behind the supernatant and suspends;
(2) use alkaline buffer P2 treatment step (1) the gained suspension that contains SDS (lauryl sodium sulfate) and alkali metal ion; With
(3) use contains the product of the damping fluid P3 treatment step (2) of potassium acetate, centrifugal acquisition supernatant, and supernatant filters the back and handles with isopropyl alcohol, centrifugal acquisition precipitation.
Among the present invention, containing systemic loupus erythematosus, to detect recombinant plasmid dna with double-stranded DNA antigen can be the known various recombinant plasmid dnas that systemic loupus erythematosuses detect the usefulness double-stranded DNA antigen that contain, for example, protokaryon cloning and expression plasmid such as pBV220, pQE3x series plasmid, pET series plasmid, pGET-4T-X series and pcDNA3.1, etc. eukaryon expression plasmid etc. all can be used as the antigen source of detecting double-stranded-DNA antibody.These plasmids can be from obtaining from various commercial sources, and for example plasmid pET-GST purchases the diligent precious Bioisystech Co., Ltd that rises in Shenzhen.
Also can adopt the recombinant technique of this area routine,, and then from transformed host cells, extract this recombinant plasmid dna of acquisition with the plasmid vector transformed host cell (as Escherichia coli) that contains double-stranded DNA antigen of the present invention.
In the inventive method, at first use damping fluid P1 to handle recombinant plasmid dna.Damping fluid P1 contains the known various Tris in this area, edta salt and RNase A.Can use deionized water allotment damping fluid P1.In thalline or plasmid weight, the concentration of Tris is 0.1 weight %～1.0 weight %, preferred 0.2～0.8 weight %, more preferably 0.3～0.6 weight %.
Edta salt can be sodium salt, sylvite of EDTA etc., and its concentration is 0.1 weight %～0.8 weight %, is preferably 0.1～0.6 weight %, more preferably 0.2～0.4 weight %.RNase A can buy from various commercially available approach, for example buy from TIANGEN Biotech (Beijing) Co., Ltd., its amount is conventional consumption, can decide according to the amount of the plasmid DNA of institute's cracking, for example can be 0.005% weight～0.05 weight %, perhaps be 0.01%～0.03 weight.
Usually, the pH of damping fluid P1 is generally 7～9 in 7～10 scope, be preferably 7.5～8.5, and more preferably about 8.0.
Usually, every milligram of recombinant plasmid dna can use the damping fluid P1 of 3～8ml to handle, and for example every milligram of plasmid DNA can use the damping fluid P1 of 4～6ml to handle.After suspending with damping fluid P1 vibration, centrifugal, and then with the suspension of vibrating again of this P1 damping fluid.After using the P1 damping fluid to handle, the endotoxin of plasmid DNA is removed comparatively up hill and dale.
Can add damping fluid P2 afterwards.Damping fluid P2 contains SDS and alkali-metal alkali, available deionized water allotment.The concentration of SDS is 0.1 weight %～2 weight %, is preferably 0.5～1.5 weight %, more preferably 0.6～1.0 weight %.Alkali-metal alkali can be for example NaOH, potassium hydroxide etc., and its concentration is generally 0.1 weight %～2 weight %, is preferably 0.5～1.5 weight %, more preferably 0.6～1.0 weight %.
The addition of P2 is identical with the addition of P1.Usually, it is limpid until solution several times to turn upside down behind the adding P2, places a few minutes then.
Add damping fluid P3 afterwards.Damping fluid P3 can cool off in advance.Damping fluid P3 contains KAC, available deionized water allotment.The concentration of KAC is generally 5～15 weight %, for example can be 6～12 weight %, 7～10 weight %, 8～10 weight % etc.The pH value of damping fluid P3 is preferably 5～6, for example can be about 5.2～5.8.In one embodiment, the pH value of P3 is in 5.3～5.7 scope.After P3 handled, plasmid DNA renaturation better became the closed loop state, formed double-stranded DNA.
After adding P3, can turn upside down several times, make its mixing.Centrifuge tube can be put on ice then and place a few minutes.Centrifugal then, behind the acquisition supernatant, centrifugal again.Centrifugal gained supernatant filters with filter membrane (for example filter membrane of 0.22 μ m), adds isopropanol precipitating nucleic acid.And then centrifugal, the nucleic acid that obtains precipitating.Filter membrane is handled and small amount of impurities and albumen can be removed, and reaches to be further purified purpose.
The amount of isopropyl alcohol can be 0.5～1 times of supernatant volume, can use the isopropyl alcohol of 0.7 times of volume usually.The processing of isopropyl alcohol makes that the plasmid DNA precipitation is more abundant.
In a preferred embodiment, add in (3) behind the damping fluid P3 in the resulting mixed liquor, in milliliter, the ratio of damping fluid P1, P2, P3 is roughly 1: 1: 1.In a preferred embodiment, add in the mixed liquor of damping fluid P3 gained, damping fluid P1, P2 and P3 ratio are roughly 4～6ml: 4～6ml: 4～6ml (with respect to every milligram of thalline or DNA plasmid for the treatment of cracking).Compare with the alkaline lysis of classics, ratio of the present invention makes cellular lysate more abundant, and protein and genomic DNA precipitation are more thorough, and is difficult for producing the plasmid DNA of degraded.
In a preferred embodiment, the inventive method also comprises following extra step except that above-mentioned steps (1)～(3), and is for example aforementioned through isopropanol precipitating, the centrifugal and nucleic acid that obtains with precipitation with alcohol.Dry up precipitation after centrifugal, with the TE dissolution precipitation that contains RNase A.The consumption of TE solution is looked actual conditions can determine that easily for example its consumption is that 1～3ml does not wait by those skilled in the art.The consumption of RNase A enzyme is also decided on the amount of the nucleic acid of actual precipitation among the TE, can be 10～30 μ g/ml TE solution usually.After the dissolving, with this potpourri of phenol-chloroform extracting, and then with isopyknic chloroform extracting.
Afterwards, also can be again with two volumes precipitation with alcohol nucleic acid, mixing is placed after a few minutes centrifugal.Dry up post precipitation, with the aqua sterilisa dissolving, add an amount of NaCl and PEG8000,4 ℃ of placements are spent the night behind the mixing.Centrifugal, supernatant discarded adds ethanol washing precipitation DNA.The centrifugal ethanol that discards is used the ethanol washing precipitation again, discards ethanol, dries up.With sterilization TE dissolving DNA.Can obtain double-stranded DNA antigen of the present invention.The concentration of NaCl can be 4～6M, and its consumption is decided on actual conditions, is generally 120～200 μ l.The concentration of PEG8000 can be 10～18% (V/W), and its consumption is also decided on actual conditions, is generally 500～1200 μ l.These additional steps can further remove foreigh protein removing and nuclease and RNA, improve the purity of double-stranded DNA antigen.
In addition, the adding of RNA enzyme also can be removed RNA better.
It will be understood by those skilled in the art that the technological means that uses in the above-mentioned additional step, comprise concentration of ethanol, consumption, centrifugal rotation speed and time, temperature or the like, all is that those skilled in the art are known.Therefore, those skilled in the art also will understand, and above-mentioned extra concrete steps can be used or omit in concrete enforcement according to the situation of reality, and perhaps its condition can change, but can not have influence on the final effect of technical solution of the present invention.
Second aspect present invention relates to the systemic loupus erythematosus detection double-stranded DNA antigen that adopts the inventive method to make.Via optimize improving and obtain, its purity height, specificity are good, and stability is also high on the basis of classical alkaline lysis for double-stranded DNA antigen of the present invention.
In a preferred embodiment, described systemic loupus erythematosus detection comprises with the method for making of double-stranded DNA antigen:
(1) use the damping fluid P1 contain Tris, edta salt and RNase A to handle plasmid DNA, centrifugal removing adds this damping fluid P1 again behind the supernatant and suspends;
(2) use alkaline buffer P2 treatment step (1) the gained suspension that contains SDS and alkali metal ion; With
(3) use contains the product of the damping fluid P3 treatment step (2) of potassium acetate, centrifugal acquisition supernatant, and supernatant filters the back and handles with isopropyl alcohol, centrifugal acquisition precipitation.
Centrifugal gained precipitation can further adopt existing method purifying in the step (3).
Randomly, double-stranded DNA antigen of the present invention can be used biotin labeling.The method of mark comprises, for example, gets this double-stranded DNA antigen and directly mixes with long arm photobiotin, carries out the illumination mark with the signal lamp.With biotin labeled method is that this area is known.
Third aspect present invention provides a kind of kit that detects anti-double-stranded-DNA antibody of human serum, and this kit comprises systemic loupus erythematosus detection double-stranded DNA antigen of the present invention.
In a preferred embodiment, described systemic loupus erythematosus detects with double-stranded DNA antigen and adopts the described method preparation of first aspect present invention.
In a preferred embodiment, described double-stranded DNA is through biotin labeling.
In a preferred embodiment, kit of the present invention also comprises two anti-, ELISA Plate of horseradish peroxidase or alkali phosphatase enzyme mark.
In a preferred embodiment, described marker enzyme can be horseradish peroxidase or alkaline phosphatase, is preferably horseradish peroxidase, and horseradish peroxidase can pass through glutaraldehyde method or cross the salt compounded of iodine acid system crosslinked on two antibody.
In a preferred embodiment, two anti-anti-human IgG monoclonal antibody or anti-human IgG polyclonal antibody or SPA (staphylococcal protein A) or the Protein G of can be.Described anti-human IgG monoclonal antibody or anti-human IgG polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source and cavy source antibody.Be preferably SPA, because its high specificity is highly sensitive.
In a preferred embodiment, marker enzyme is a horseradish peroxidase, and two anti-are SPA, and horseradish peroxidase can by glutaraldehyde method or to cross the salt compounded of iodine acid system crosslinked on SPA.
In a preferred embodiment, ELISA Plate through Streptavidin SA and biotin system with described double-stranded DNA antigen bag by on solid phase carrier.Particularly, by the biotin labeling double-stranded DNA antigen, bag is incorporated on the solid phase carrier by being tested double-stranded DNA in Streptavidin SA pre-processed board then, thereby obtains through Streptavidin SA and biotin system bag by the ELISA Plate of double-stranded DNA antigen of the present invention.
The material that can be used as the fixing carrier of double-stranded DNA plasmid is a lot, as polystyrene, cellulose, polyacrylamide, tygon, poly-propylamine, cross-link dextran, glass, silicon rubber, Ago-Gel etc.The form of this carrier can be test tube, micro-reaction plate shrinkage pool, globule, sequin etc.
It can be phosphate buffer, Tris damping fluid, borate buffer, carbonic acid buffer that the bag that uses in the coated elisa plate is cushioned liquid, is preferably 0.01～0.5M phosphate buffer.
In an embodiment, with the sterilization deionized water SA is diluted to 10～40 μ g/ml earlier, every hole adds 100 μ l, 37 ℃ of incubation 1h, and liquid inclines, cross once with cleansing solution, pat dry, be cushioned liquid with bag then biotin-double-stranded DNA of the present invention is diluted to 5～7 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h, 4 ℃ are spent the night, and the coating buffer that inclines is with cleansing solution washing 3 times, each 1min, pat dry, in every hole, add 150～200 μ l confining liquids then, 37 ℃ of incubation 1～2h, the liquid in the hole that inclines can obtain the ELISA Plate of described bag quilt after the drying.
For more convenient on-site supervision and great amount of samples examination, kit of the present invention also comprises double-stranded-DNA antibody standard solution (calibration object), colour developing liquid, stop buffer, concentrated cleaning solution and/or sample diluting liquid.
In a preferred embodiment, contain calibration object in the kit, it is respectively 3 bottles of double-stranded-DNA antibody series standard solution of 10IU, 100IU, 800IU for concentration.
In a preferred embodiment, contain colour developing liquid in the kit, it comprises colour developing liquid A and colour developing liquid B.Colour developing liquid A can be hydrogen peroxide or urea peroxide, and colour developing liquid B can be o-phenylenediamine or tetramethyl benzidine.Can adopt prior art preparation colour developing liquid.
In a preferred embodiment, contain concentrated cleaning solution in the kit, it is conventional cleansing solution, for example is selected from the phosphate buffer, Tris damping fluid, borate buffer, the carbonic acid buffer that contain 0.8～1.2% tween, is preferably the phosphate buffer of 0.8～1.2% tween.
In a preferred embodiment, contain sample diluting liquid in the kit, it is conventional cleansing solution, for example is selected from phosphate buffer, Tris damping fluid, borate buffer, carbonic acid buffer, is preferably 0.8～1.2% and 0.1～0.3%BSA (bovine serum albumin(BSA)) phosphate buffer.In an embodiment, sample diluting liquid is the phosphate buffer of 0.8～1.2% tween and 0.1～0.3%BSA.
In a preferred embodiment, stop buffer is 1M～3M sulfuric acid or hydrochloric acid solution.
In a preferred embodiment, kit of the present invention also comprises instructions, and this instructions comprises that the guidance technology personnel adopt the operation steps of the contained composition detection systemic loupus erythematosus of this kit etc.In one embodiment of the invention, described detection comprises: add blood serum sample, add ELIAS secondary antibody again, testing sample double center chain dna antibody combines with the double-stranded DNA antigen generation specificity of solid phase carrier bag quilt, the colour developing back stops, the working sample light absorption value, and this value and blood serum sample double center chain dna antibody content are proportionate, according to the double-stranded-DNA antibody typical curve that draws of the serial concentration of accurate product and the mean light absorbency of standard product, the typical curve concentration of coming calculation sample according to this then.The method of using double-stranded-DNA antibody series standard solution that testing result is judged comprises, is Y-axis with each the standard solution mean light absorbency value that is obtained, and concentration value is an X-axis, drawing standard curve map, the concentration of reading sample from typical curve.In addition, the accurate product 100IU of regulation is a critical value, if sample concentration greater than 100IU, then is judged to be the double-stranded-DNA antibody positive, if sample concentration less than 100IU, then is judged to be the double-stranded-DNA antibody feminine gender.
Then can be in conjunction with clinical, for judging that the patient may suffer from the system lupus erythematosus disease foundation is provided.
Fourth aspect present invention provides a kind of method for preparing the kit that detects anti-double-stranded-DNA antibody of human serum, the systemic loupus erythematosus of the present invention that comprises described kit detects two anti-and ELISA Plate with double-stranded DNA antigen, horseradish peroxidase or alkali phosphatase enzyme mark, and this method comprises:
(1) prepares described systemic loupus erythematosus detection by method of the present invention and use double-stranded DNA antigen, so that described double-stranded DNA antigen to be provided;
(2) provide two of horseradish peroxidase or alkali phosphatase enzyme mark to resist; With
(3) through Streptavidin SA and biotin system with described double-stranded DNA antigen bag by on the solid phase carrier of ELISA Plate;
(4) use above-mentioned (1)～(a 3) described composition to dispose described kit.
The described systemic loupus erythematosus of described preparation detects the whole bag of tricks that the usefulness double-stranded DNA antigen can be as indicated above.
In a preferred embodiment, this method also comprises provides double-stranded-DNA antibody standard solution (calibration object), colour developing liquid, stop buffer, concentrated cleaning solution and sample diluting liquid, and it is configured in the kit.Described calibration object, colour developing liquid, stop buffer, concentrated cleaning solution and sample diluting liquid are as hereinbefore defined.
In a further advantageous embodiment, return the instructions that described kit configuration guide technician adopts the operation steps etc. of the contained composition detection systemic loupus erythematosus of this kit.
Term used herein " configuration " means to be packed described composition, put into into or be placed on kit.
Therefore, fifth aspect present invention also comprises the kit of the detection anti-double-stranded-DNA antibody of human serum that employing the inventive method makes.
Therefore, in one embodiment, this kit contains described systemic loupus erythematosus and detects the two anti-and ELISA Plate of using double-stranded DNA antigen, horseradish peroxidase or alkali phosphatase enzyme mark.Described systemic loupus erythematosus detects with double-stranded DNA antigen such as this instructions mentioned above, or adopts the described method of the application to prepare, and described horseradish peroxidase or alkaline phosphatase, two anti-and ELISA Plate are as hereinbefore defined.
In other embodiments, described kit also contains calibration object, colour developing liquid, stop buffer, concentrated cleaning solution and/or sample diluting liquid.Described calibration object, colour developing liquid, stop buffer, concentrated cleaning solution and sample diluting liquid are as hereinbefore defined.
In a preferred implementation, described kit also contains colour developing liquid, stop buffer, concentrated cleaning solution and/or sample diluting liquid.Described demonstration liquid comprises colour developing liquid A and colour developing liquid B, and it defines as mentioned above.
In another preferred implementation, described kit also comprises calibration object, colour developing liquid, stop buffer, concentrated cleaning solution and sample diluting liquid, and its definition is as indicated above.
Others of the present invention also comprise the various damping fluids that are used for kit of the present invention, for example damping fluid P1, P2, P3 etc. and the TE solution that contains RNase A of the present invention, and these solution, and antigen of the present invention, the application in the kit that preparation detection anti-double-stranded-DNA antibody of human serum is used.The present invention also comprises antigen of the present invention and the application of kit in detecting the robot system lupus erythematosus.
The enzyme linked immunological kit that the present invention detects double-stranded-DNA antibody mainly adopts the qualitative or quantitative serum sample of indirect ELISA method.The main agents of this kit all provides with the working fluid form, and is easy to use, has characteristics such as high specific, high sensitivity, pinpoint accuracy, pin-point accuracy, can play a significant role in detection.
Because the electronegativity of double chain DNA molecule, make it be difficult for direct coated on plastic plate, this product uses biotin Avidin system bag by double-stranded DNA in development process, not only increase absorption property and play the effect that effective epitope exposes amplification simultaneously, anti-human IgG antibody with horseradish peroxidase-labeled is the enzyme labeling thing, and the kit of developing by ELISA method detection serum anti-dsDNA antibody has the characteristics of high susceptibility and high specific.
In this application, when using " containing ", " comprising " and similar terms, its also comprise " by ... form ", " by ... constitute " meaning.
Above the various preferred implementations to the application describe in detail.It should be understood that the present invention be not limited to above-mentioned and hereinafter shown in embodiment.Under the situation that does not depart from the scope of the invention and spirit, can make various modifications and changes to the present invention.In addition, though above be to describe preferred feature in each embodiment in the mode of each embodiment, but those skilled in the art are known, each concrete technical characterictic in these embodiments (for example each numerical range) at random can be combined, the technical scheme of gained also within the scope of the invention.
Except as otherwise noted, the reagent that is used among the embodiment all can obtain from commercially available approach.
Embodiment
Embodiment 1: the preparation of double-stranded DNA antigen
On the basis of classical alkaline lysis, carried out optimizing and improved, obtained highly purified plasmid DNA from the plasmid DNA purifying of commercially available acquisition, the plasmid size is 2.5Kb, have stable dna double chain structure and stronger immunogenicity, with its envelope antigen as the enzyme linked immunological kit of double-stranded-DNA antibody, prepared kit has reached higher detection susceptibility and specificity.The plasmid Stability Analysis of Structures homogeneous of this method preparation, and convenient sources can satisfy the technological requirement of the enzyme linked immunological kit of double-stranded-DNA antibody.
(1) concrete operations are as follows:
1) get 2mg plasmid pET-GST (purchasing the diligent precious Bioisystech Co., Ltd that rises) and suspend with 10mlBuffer P1 vibration in Shenzhen, 8000g, 4 ℃, 2min removes supernatant, adds 10ml Buffer P1 vibration again and suspends.
2) add 10ml Buffer P2 and turn upside down 4～6 times until limpid, room temperature is placed 5min.
3) the 10ml Buffer P3 of adding precooling turns upside down mixing 4～6 times.Place 10min on ice.15000g, 4 ℃, centrifugal 30min.Careful supernatant, 15000g, 4 ℃, the centrifugal 15min of shifting out.
4) get the isopropyl alcohol that supernatant adds 0.7 times of volume after with the membrane filtration of 0.22 μ m, mixing, room temperature is placed 10min.12000g, centrifugal 15min.
5) with 70% the ethanol precipitation that suspends, 12000g, centrifugal 10min.Supernatant discarded in the air dries up precipitation.
6) with the TE that contains RNase A (pH8.0) dissolution precipitation (final concentration of RNase A is 20 μ g/ml) of 1ml, 37 ℃, 30min.
7) with plasmid-RNase A potpourri once with isopyknic phenol-chloroform (1: 1) extracting.
8) with isopyknic chloroform extracting once.
9) with two volumes precipitation with alcohol nucleic acid, mixing, room temperature is placed 5min.12000g，4℃，10min。
10) supernatant discarded is after precipitation dried up.With 1ml aqua sterilisa dissolving, add the PEG8000 of the 4M NaCl of 160 μ l and 800 μ l 13% (V/W), mixing, 4 ℃ of placements are spent the night.
11) 15000g, 4 ℃, centrifugal 15min.Supernatant discarded, the 70% ethanol washing precipitation DNA of adding 1ml.
12) centrifugally discard ethanol, with 70% the ethanol washing precipitation of 1ml, discard ethanol, dry up.
13) with 200ul sterilization TE dissolving DNA.
14)-80 ℃ refrigerator is preserved.
Except that specifying, all purification process is all carried out in 15～25 ℃ of environment.
(2) extract purifying double-stranded DNA antigen agents useful for same and compound method thereof
1)Buffer?A(100ml)：
The Tris:12.1 gram;
Deionized water: 100ml.
2)Buffer?B(100ml)：
SDS：?????????20g；
Deionized water: 100ml.
3)Buffer?P1(pH?8.0、100ml)：
Buffer?A：????5ml；
Na
2EDTA.2H
2O：0.372g；
RNase?A：?????10mg；
Deionized water: 100ml.
4)Buffer?P2(100ml)：
Buffer?B：????5ml；
NaOH：????????0.8g；
Deionized water: 100ml.
5)Buffer?P3(pH?5.5、100ml)：
KAC (potassium acetate): 9.82g;
Deionized water: 100ml.
(3) double-stranded DNA (dsDNA) antigenic quality authentication method
1) method for measurement of concentration and criterion
Get purifying gained double-stranded DNA 5 μ l and add 495 μ l TE damping fluid (10Mm Tris-HCL, 1mMEDTA, pH8.0) mixing, with TE damping fluid (pH8.0) is blank, measure its absorbance respectively, the sample after the dilution calculated original content at the absorbance A260 that wavelength 260nm records at wavelength 260nm and 280nm place:
Double-stranded DNA concentration (mg/ml)=A260 * 50 * extension rate/1000; Being considered as of original content value＞1mg/ml is qualified.
2) method for detecting purity and criterion
Sample after will diluting is as stated above surveyed absorbance at wavelength 260nm and 280nm, calculates A260/A280 ratio, and ratio is judged to be purity in 1.80 to 1.95 scopes qualified.Simultaneously the band of observing behind its electrophoresis with agarose gel electrophoresis method is verified its purity.The double-stranded DNA agarose gel electrophoresis detects its purity method:
After getting 5 μ l stostes adding TE damping fluid, 20 μ l mixings, get 5 μ l and carry out 0.8% agarose gel electrophoresis (the 100ml gel solution adds 5 μ l Goldview (match Parkson bioengineering company limited)), get 5 μ l DNA Marker simultaneously as reference, with 120V voltage electrophoresis 30 minutes, show that as accompanying drawing sample lane do not have microRNA, dna degradation band and bacterial genomes DNA pollute.It is qualified meeting above-mentioned purified double-stranded DNA.See accompanying drawing 1 with reference to electrophoretogram.
Hereinafter describe and do not improve alkaline lysis experimental procedure (can with reference to " molecular cloning laboratory manual ", the third edition).
(1) main agents preparation
Lysate I:25mmol/L Tris-HCl pH8.0,50mmol/L glucose, 10mmol/L EDTApH8.0.
Lysate II:0.2N NaOH, 1%SDS.
Lysate III:3mol/L KAC (pH4.8).
TE solution: 10mmol/L Tris-HCl, 1mmol/L EDTA
(2) experimental implementation step
1. get 2g thalline 10ml lysate, concuss.
2. add 20ml lysate II and put upside down fast 5 times, the mixed content thing is positioned over centrifuge tube on ice.
3. add the 15ml lysate III of precooling, put upside down repeatedly for several times, lysate III is uniformly dispersed in sticky bacterial lysate, afterwards pipe is placed 5min on ice.
4.15000g, 4 ℃, centrifugal 5min.Supernatant is transferred in another centrifuge tube.
5. get the phenol that supernatant adds equivalent volumes: chloroform, concussion mixes organic phase and water, and 4 ℃, 15000g, centrifugal 15min transfers to supernatant in another centrifuge tube.
With 2 times of volume of ethanol in precipitation at room temperature nucleic acid, concussion mixes, and places 2min in room temperature.
7.4 ℃, 15000g, centrifugal 5min, the nucleic acid of collecting precipitation.
8. the ethanol suspension with 1ml 70% precipitates 15000g, centrifugal 2min.Supernatant discarded in the air dries up precipitation.
9. with the TE that contains RNase A (pH8.0) dissolution precipitation of 200ul, gentleness is shaken several seconds.Be stored in-20 ℃.
The electrophoretogram of the plasmid DNA that obtains of traditional method for extracting is presented among Fig. 2 according to this.Arrow among the figure shows that RNA does not eliminate.
Embodiment 2: the biotin labeling of double-stranded DNA
Get the double-stranded DNA antigen that embodiment 1 makes and directly mix, carry out the illumination mark, with preparation biotinylation plasmid dsDNA with the signal lamp with long arm photobiotin (Jia Kang source, Beijing development in science and technology company limited).Concrete preparation method is as follows: add DNA 5 μ g/10 μ l to be marked in a sterilization EP pipe, add 5 μ g/ μ l photobiotins in the darkroom, fully mixing is put in the ice bath.
Embodiment anti-and ELIAS secondary antibody preparation in 3: two
(1) two anti-preparation
With the purifying human IgG is immunogene, and immunized mice or sheep prepare anti-human IgG polyclonal antibody or monoclonal antibody; Buy commercialization SPA or Protein G (PROSPEC company), preferred commercialization SPA.
(2) ELIAS secondary antibody mark
Adopt horseradish peroxidase, by the glutaraldehyde method or the crosslinked SPA of mistake salt compounded of iodine acid system of routine.
Embodiment 4: the enzyme linked immunological kit that detects double-stranded-DNA antibody
(1) enzyme linked immunological kit that detects double-stranded-DNA antibody is formed
Kit is by the vacuum-packed 96 hole ELISA Plate of box body, aluminium film, 3 bottles of accurate product solution of double-stranded-DNA antibody, 1 bottle of enzyme labeling thing working fluid, 1 bottle of sample diluting liquid, 1 bottle of substrate colour developing liquid A liquid, 1 bottle of substrate develop the color liquid B liquid, 1 bottle of stop buffer, 1 bottle of concentrated cleaning solution, foam bracket, be shaped on shrinkage pool on the foam bracket, the mentioned reagent bottle is placed in the shrinkage pool of foam bracket, and foam bracket and ELISA Plate are placed in the box body.Wherein ELISA Plate is made up of plastic stent and detachable plastic strip.
(2) required reagent
1) double-stranded-DNA antibody calibration object solution: the calibration substance with anti-dsDNA antibody international standard substance WO/80 has traceability comprises three kinds of different concentration.
2) bag is cushioned liquid: pH9.6, the phosphate buffer of 0.05mol/L.
3) confining liquid: the phosphate buffer of 3-10% calf serum.
4) concentrated cleaning solution: the phosphate buffer (0.01M pH7.4) that contains the .8%-1.2% tween.
5) enzyme labeling thing working fluid: anti-human IgG of enzyme labeling or SPA or Protein G 10-15ml/ bottle.
6) colour developing liquid A liquid: principal ingredient is hydrogen peroxide or urea peroxide.
7) substrate colour developing liquid B liquid: principal ingredient is o-phenylenediamine (OPD) or tetramethyl benzidine (TMB)
8) stop buffer: 1-2mol/L sulfuric acid or hydrochloric acid.
9) sample diluting liquid: the phosphate buffer that contains 0.8-1.2% tween and 0.1～0.3%BSA.
(3) preparation of ELISA Plate
With the sterilization deionized water SA is diluted to 10-40 μ g/ml earlier, every hole adds 100 μ l, 37 ℃ of incubation 1h, the liquid that inclines is crossed once with cleansing solution, pats dry, be cushioned biotin-double-stranded DNA antigen that liquid makes embodiment 2 with bag then and be diluted to 5-7 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h, and 4 ℃ are spent the night, coating buffer inclines, with cleansing solution washing 3 times, each 1min pats dry, in every hole, add 150-200 μ l confining liquid then, 37 ℃ of incubation 1-2h, liquid in the hole of inclining, preserve with the vacuum seal of aluminium film dry back.
Embodiment 5: kit sensitivity, specificity, accuracy, precision and stability test
(1) kit sensitivity, specificity, accuracy
From 100 parts of the Longhua Hospital affiliated Shanghai University Of Chinese Traditional Medicine collection system lupus erythematosus patients serums of cooperation unit, as the sensitivity determination sample.Totally 50 parts of other autoimmune diseases (comprising Sjogren syndrome, rheumatoid arthritis, vasculitis, systemic sclerosis etc.), 50 parts of normal human serums are as the specific assay sample.Gained the results are shown in Table 1.
Table 1 diagnostic test is estimated the data compilation table
| Kit is tested oneself | The SLE patients serum | Non-SLE patient and normal human serum | Add up to |
| Positive | True positives (68) | False positive (3) | ??71 |
| Negative | False negative (32) | True negative (97) | ??129 |
| Add up to | ??100 | ?100 | ??200 |
Sensitivity (True Positive Rate)=68/100 * 100%=68%
Specificity (true negative rate)=97/100 * 100%=97%
False positive rate=3/100 * 100%=3%
False negative rate=32/100 * 100%=32%
Accuracy=(A+D)/(A+B+C+D) * 100%=(68+97)/200=165/200=82.5%
200 parts in clinical after measured fresh sample, comprise that SLE patient and control group (non-SLE patient and normal human serum) result shows that this kit is 68% to the diagnostic sensitivity of SLE, specificity is 97%, accuracy is 82.5%, shows that the clinical analysis performance of this kit is higher.
(2) kit precision test
Detect 3 parts of variable concentrations positive serums, each measures sample with a collection of kit replication 10 times, measure finish after, calculate the measurement result average respectively
Standard deviation (SD) is passed through average at last
/ standard deviation (SD) is calculated its variation within batch coefficient (CV%), weighs batch interior repeatability of this kit according to this.All CV values are better than the CV of similar kit 10% all less than 5%, the results are shown in Table 3.
Table 3 withinrun precision testing result
(3) kit stability test
1) 37 ℃ of accelerated stabilities
Place 37 ℃ to carry out accelerated tests each component of kit, every day, taking-up was tested with indoor quality-control product, and the yin and yang attribute coincidence rate by the yin and yang attribute reference material and the withinrun precision of precision reference material are judged the stability of kit.The result shows after 4 days, and the yin and yang attribute coincidence rate of each yin and yang attribute reference material is that the withinrun precision of 100%, 3 part of precision reference material is all less than 5%.The yin and yang attribute reference material is 10 parts of double-stranded-DNA antibody positive serums and 10 parts of double-stranded-DNA antibody negative serums.
2) 4 ℃ of stability experiments
Place 4 ℃ to carry out conventional stability experiment each component of kit, took out indoor quality-control product test in every month, same yin and yang attribute coincidence rate and the withinrun precision of the precision reference material stability of judging kit by the yin and yang attribute reference material with above-mentioned accelerated tests.The result shows after six months, and the yin and yang attribute coincidence rate of each yin and yang attribute reference material is that the withinrun precision of 100%, 3 part of precision reference material is all less than 5%.The result shows after 7 months, and the yin and yang attribute coincidence rate of each yin and yang attribute reference material is still 100%.The result shows after 8 months, an official holiday feminine gender occurs.Kit being described based on the above results 2～8 ℃ of storages, is stable at least in half a year.
Above with the formal description of preferred implementation the present invention.It should be understood that scope of the present invention is not limited to above-mentioned embodiment.Those skilled in the art can make suitable modification and change to technical scheme of the present invention under the situation that does not depart from spirit and scope of the invention.And each the concrete technical characterictic in each preferred implementation described in the invention, numerical range etc. also can make up mutually, and formed technical scheme is also within protection scope of the present invention.Scope of the present invention wants appended claim to limit.
List of references
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2.Feltkamp，T.E.W.et?al.The?first?international?standard?for?antibodies?todouble?stranded?DNA.Annals?of?the?Rheumatic?Diseases.1988，47：740-746。
3.Egner?W.The?use?of?laboratory?tests?in?the?diagnosis?of?SLE.J.Clin.Pathol.2000，53：424-432。
4.Harley，John?B.Gaither，Kimberley?K.et?al.Autoantibodies.RheumaticDisease?Clincs?of?North?America.1988，14：43-56。
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7.Winkler?T.H.et?al.IgG?human?monoclonal?anti-DNA?autoantibodies?frompatients?with?systemic?lupus?erythematosus.Clinical?and?ExperimentalImmunology?1991，85：379-385。
8.Pisetsky?D?S，Reich?C?F.The?influence?of?DNA?size?on?the?binding?ofanti-DNA?antibodies?in?the?solid?and?liquid?phase[J].Clinical?Immunol?andImmunopathology?1994，72：350-356。
9. Xu Lu booth, Yu Naichang, Wang Hong, etc. use the dna sequence dna [J] that immuno-PCR method research anti-dsDNA antibody is discerned. Chinese Journal of Immunology .1998,14:207-208.
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Claims (10)
1. a systemic loupus erythematosus detects the preparation method who uses double-stranded DNA antigen, it is characterized in that, said method comprising the steps of:
(1) use the damping fluid P1 contain Tris, edta salt and RNase A to handle plasmid DNA, centrifugal removing adds this damping fluid P1 again behind the supernatant and suspends;
(2) use alkaline buffer P2 treatment step (1) the gained suspension that contains SDS and alkali metal ion; With
(3) use contains the product of the damping fluid P3 treatment step (2) of potassium acetate, centrifugal acquisition supernatant, and supernatant filters the back and handles with isopropyl alcohol, centrifugal acquisition precipitation.
2. the method for claim 1 is characterized in that, described step also comprises, in step (3) afterwards, uses the precipitation with alcohol mycoprotein respectively, uses PEG8000 deposit D NA, carries out phenol-chloroform extracting DNA then.
3. adopt the systemic loupus erythematosus detection double-stranded DNA antigen that each described method makes among the claim 1-2.
4. a kit that detects anti-double-stranded-DNA antibody of human serum is characterized in that, this kit comprises the described systemic loupus erythematosus detection of claim 3 double-stranded DNA antigen.
5. kit as claimed in claim 4 is characterized in that described double-stranded DNA is through biotin labeling.
6. as claim 4 or 5 described kits, it is characterized in that described kit comprises that also two of horseradish peroxidase or alkali phosphatase enzyme mark resists, and ELISA Plate;
Wherein, described ELISA Plate contains through Streptavidin SA and biotin system and has adsorbed the solid phase carrier of described double-stranded DNA antigen absorption.
7. kit as claimed in claim 6 is characterized in that, described kit also comprises calibration object, sample diluting liquid, concentrated cleaning solution, colour developing liquid A, shows liquid B and stop buffer.
8. kit as claimed in claim 7 is characterized in that, described colour developing liquid A is hydrogen peroxide or urea peroxide, and described colour developing liquid B is o-phenylenediamine or tetramethyl benzidine; Described concentrated cleaning solution is the phosphate buffer that contains 0.8～1.2% tween; Described sample diluting liquid is the phosphate buffer of 0.8～1.2% tween and 0.1～0.3%BSA; Described stop buffer is 1M～3M sulfuric acid or hydrochloric acid solution.
9. method for preparing the kit that detects anti-double-stranded-DNA antibody of human serum, the described systemic loupus erythematosus of claim 5 that comprises described kit detects two anti-and ELISA Plate with double-stranded DNA antigen, horseradish peroxidase or alkali phosphatase enzyme mark, it is characterized in that described method comprises:
(1) prepares described systemic loupus erythematosus detection by each described method among the claim 1-2 and use double-stranded DNA antigen, so that described double-stranded DNA antigen to be provided;
(2) provide two of horseradish peroxidase or alkali phosphatase enzyme mark to resist; With
(3) through Streptavidin SA and biotin system with described double-stranded DNA antigen bag by on the solid phase carrier of ELISA Plate;
(4) use above-mentioned (1)～(a 3) described composition to dispose described kit.
10. adopt the kit of the detection anti-double-stranded-DNA antibody of human serum that the described method of claim 9 makes.
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| CN200910051586A CN101776682A (en) | 2009-05-20 | 2009-05-20 | Preparation method for double-stranded DNA antigen and kit for testing anti-double-stranded-DNA antibody of human serum |
| PCT/CN2010/072113 WO2010133120A1 (en) | 2009-05-20 | 2010-04-23 | Methods for preparing double-stranded dna antigens and kits for detecting anti-double-stranded dna antibodies in human serum |
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| CN200910051586A CN101776682A (en) | 2009-05-20 | 2009-05-20 | Preparation method for double-stranded DNA antigen and kit for testing anti-double-stranded-DNA antibody of human serum |
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| CN200910051586A Pending CN101776682A (en) | 2009-05-20 | 2009-05-20 | Preparation method for double-stranded DNA antigen and kit for testing anti-double-stranded-DNA antibody of human serum |
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| WO (1) | WO2010133120A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108254571A (en) * | 2018-01-02 | 2018-07-06 | 江苏浩欧博生物医药股份有限公司 | A kind of detection kit and its detection method of anti-dsDNA antibody IgG |
| CN108469526A (en) * | 2018-02-08 | 2018-08-31 | 深圳市新产业生物医学工程股份有限公司 | Double-stranded DNA antigen, preparation method, the reagent comprising it, kit and application |
| CN110632290A (en) * | 2019-09-27 | 2019-12-31 | 昆山迪安医学检验实验室有限公司 | Anti-double-chain DNA antibody detection reagent |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111621497A (en) * | 2020-05-29 | 2020-09-04 | 广西大学 | Rapid extraction method and application of chicken blood DNA |
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| US5183735A (en) * | 1989-02-27 | 1993-02-02 | Reaads Medical Products, Inc. | Method and diagnostic test kit for detection of anti-dsDNA antibodies |
| US20010034435A1 (en) * | 1996-07-19 | 2001-10-25 | Valentis, Inc. | Process and equipment for plasmid purification |
| CN101165491A (en) * | 2006-10-19 | 2008-04-23 | 陕西西大北美基因股份有限公司 | Method for detecting double-chain DNA resistant antibody using gold magnetic particle as carrier |
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2009
- 2009-05-20 CN CN200910051586A patent/CN101776682A/en active Pending
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2010
- 2010-04-23 WO PCT/CN2010/072113 patent/WO2010133120A1/en active Application Filing
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| US5183735A (en) * | 1989-02-27 | 1993-02-02 | Reaads Medical Products, Inc. | Method and diagnostic test kit for detection of anti-dsDNA antibodies |
| US20010034435A1 (en) * | 1996-07-19 | 2001-10-25 | Valentis, Inc. | Process and equipment for plasmid purification |
| CN101165491A (en) * | 2006-10-19 | 2008-04-23 | 陕西西大北美基因股份有限公司 | Method for detecting double-chain DNA resistant antibody using gold magnetic particle as carrier |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108254571A (en) * | 2018-01-02 | 2018-07-06 | 江苏浩欧博生物医药股份有限公司 | A kind of detection kit and its detection method of anti-dsDNA antibody IgG |
| CN108469526A (en) * | 2018-02-08 | 2018-08-31 | 深圳市新产业生物医学工程股份有限公司 | Double-stranded DNA antigen, preparation method, the reagent comprising it, kit and application |
| CN110632290A (en) * | 2019-09-27 | 2019-12-31 | 昆山迪安医学检验实验室有限公司 | Anti-double-chain DNA antibody detection reagent |
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| WO2010133120A1 (en) | 2010-11-25 |
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