CN105929156A - Magnetic particle-based quantitative chemiluminescent assay kit for anti-double-stranded DNA antibody IgG, and preparation and detection methods thereof - Google Patents
Magnetic particle-based quantitative chemiluminescent assay kit for anti-double-stranded DNA antibody IgG, and preparation and detection methods thereof Download PDFInfo
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- CN105929156A CN105929156A CN201610249121.9A CN201610249121A CN105929156A CN 105929156 A CN105929156 A CN 105929156A CN 201610249121 A CN201610249121 A CN 201610249121A CN 105929156 A CN105929156 A CN 105929156A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/104—Lupus erythematosus [SLE]
Abstract
The invention discloses a magnetic particle-based quantitative chemiluminescent assay kit for an anti-double-stranded DNA antibody IgG. The kit comprises an anti-double-stranded DNA antibody IgG calibrator, an anti-double-stranded DNA antibody IgG quality control product, a Tris buffer containing biotin-labeled double-stranded DNA antigen and bovine serum albumin, a Tris buffer containing an alkaline phosphatase-labeled goat anti-human polyclonal antibody and bovine serum albumin, a Tris buffer containing streptavidin-labeled magnetic particles and bovine serum albumin, and a rinsing solution. The detection method using the kit improves sensitivity and a linearity range by 3 to 5 orders of magnitudes on the basis of a traditional membrane strip immunization method and a traditional enzyme linked immunosorbent assay method, realizes real quantitative determination, is rapid in reaction and reliable in results, can be automatically used in cooperation with an automatic chemiluminescence immunity analyzer and is of irreplaceable important value to clinical diagnosis.
Description
Technical field
The present invention relates to technical field of biological.More particularly, to a kind of anti-dsDNA antibody IgG
Magnetic microparticle chemiluminescence quantitative determination reagent kit and preparation and detection method.
Background technology
Systemic lupus erythematosus (sle) (SLE) is the most relatively conventional a kind of autoimmune disease, and patient dashes forward
Going out performance is to there is multiple autoantibody, most important of which is that anti-dsDNA antibody, and it can for one
The autoantibody being combined with n DNA, the most only can detect that this antibody in SLE patient.Anti-double-strand
The growth and decline of DNA antibody titre are relevant to the change of illness state of SLE patient.11 ARA marks of the diagnosis base of SLE
Accurate (ARA, American Rheumatism Association), also referred to as 1988 ACR standards (ACR, U.S.'s rheumatology
Meeting), this standard was in revision in 1997.If occurring 4 in 11 standards, the probability of diagnosis SLE reaches
Between 80% to 90%.
The detection of anti-dsDNA antibody IgG has film bar immunization and enzyme linked immunosorbent assay the most clinically.
The application of film bar immunization is film bar developing technology, and its feature measures at same film bar for fixing several projects,
Typically carry out experimental implementation by manual or semi-automatic film bar instrument, qualitatively judge eventually through naked eyes,
This sensitivity is low, and the response time is long, and detection project can only combine in regular collocation, very flexible.Enzyme joins
The sensitivity of immunoabsorption has promoted on the basis of film bar immunization, but still relatively low, and linear model
Enclosing narrow, poor repeatability, the response time is the longest, still can not meet the application of clinic very well.
Magnetic microparticle chemiluminescence immune assay method, film bar immunization than before and enzyme linked immunosorbent assay,
Have in detection sensitivity, detection range, detection time and automation mechanized operation and be greatly improved, and do not polluted,
Clinical practice is wide.At present, use magnetic microparticle chemiluminescence analytic process at anti-dsDNA antibody IgG immunoassay
The application of product has not yet to see.
Summary of the invention
The magnetic microparticle chemiluminescence that it is an object of the present invention to provide a kind of anti-dsDNA antibody IgG is fixed
Measuring and determine test kit, the test kit that the present invention provides is by chemiluminescence analytical technique and magnetic particle isolation technics phase
In conjunction with, use biotin and alkali phosphatase (ALP) labelled antigen and antibody respectively, with diameter 1-3 μm
The superparamagnetic particles being coated Streptavidin as separation agent.After ALP catalytic substrate luminescence, pass through instrument
Device is measured luminous intensity and is calculated testing concentration.This detection method joins at traditional film bar immunization and enzyme to be exempted from
On the basis of epidemic disease absorption method, sensitivity and linear measurement range is improved 3-5 the order of magnitude again, realizes real meaning
Detection by quantitative, is swift in response, reliable results, and Full-automatic chemiluminescence immunoassay analysis meter can be coordinated to realize
Full-automatic use, has the important value that can not be substituted for clinical diagnosis.
Further object is that preparation method and the detection method that a kind of mentioned reagent box is provided.
For reaching above-mentioned purpose, the present invention uses following technical proposals:
A kind of magnetic microparticle chemiluminescence quantitative determination reagent kit of anti-dsDNA antibody IgG, described test kit
Including: 1) anti-dsDNA antibody IgG calibration object, pure containing anti-dsDNA antibody IgG and Sanguis Bovis seu Bubali
The Tris buffer of albumen, described anti-dsDNA antibody IgG calibration object comprises the liquid calibration of 6 levels
Product, in the liquid calibration object of described 6 levels, the concentration of anti-dsDNA antibody IgG is respectively 0, and 10,
100,200,400,800IU/mL；
2) anti-dsDNA antibody IgG quality-control product, containing anti-dsDNA antibody IgG and bovine serum albumin
White Tris buffer, described anti-dsDNA antibody IgG quality-control product comprises the liquid quality control product of 2 levels,
In the liquid quality control product of described 2 levels, the target value concentration range of anti-dsDNA antibody IgG is respectively
(100 ± 20) IU/mL and (400 ± 80) IU/mL.
3) reagent 1, the Tris buffering containing biotin labeled double-stranded DNA antigen and bovine serum albumin
Liquid；
4) reagent 2, sheep anti-human polyclonal antibody containing alkali phosphatase enzyme mark and bovine serum albumin
Tris buffer；
5) Magneto separate reagent, magnetic particle and the Tris of bovine serum albumin containing marked by streptavidin delay
Rush liquid；
6) cleanout fluid；
The content of described seminal plasma fructose detection kit 1, reagent 2 and Magneto separate reagent than for 1:3:1, described contains
Amount ratio is volume ratio.
Further, the material of described magnetic particle is Fe2O3；Described magnetic particle pan coating has carboxylic group,
Being coated carboxyl group content in thing and be more than 20wt%, the size of described magnetic particle is 1-3 μm.
A kind of side of the magnetic microparticle chemiluminescence quantitative determination reagent kit preparing described anti-dsDNA antibody IgG
Method, the method comprises the steps:
(1) preparation anti-dsDNA antibody IgG calibration object:
A. preparation anti-dsDNA antibody IgG calibration object diluent:
Purified water, Tris, sodium chloride and Proclin300 are added in container, are stirred well to be completely dissolved,
Tris concentration is 1wt%, sodium chloride concentration be 1wt%, Proclin300 concentration be 0.2v%；With the HCL of 4M
The pH value of solution is adjusted to 7.0-7.5；Bovine serum albumin is added in container, is stirred well to be completely dissolved,
The concentration of bovine serum albumin is 4wt%；With the HCL of 4M, the pH value of solution is adjusted to 7.0-7.5 again；With
Aperture is that the filter of 0.2 μm filters to obtain anti-dsDNA antibody IgG calibration object diluent, and 2-8 DEG C of preservation is treated
With；
B. preparation anti-dsDNA antibody IgG calibration object:
By anti-dsDNA antibody IgG anti-dsDNA antibody IgG calibration object diluted to each dense
Degree point is 0,10,100,200,400,800IU/mL；
(2) preparation anti-dsDNA antibody IgG quality-control product:
By anti-dsDNA antibody IgG by above-mentioned anti-dsDNA antibody IgG calibration object diluted extremely
Each concentration point is 100,400IU/mL；
(3) reagent preparation 1:
A. No. 1 diluent of reagent preparation:
Purified water, Tris, sodium chloride and Proclin300 are added in container, are stirred well to be completely dissolved,
The concentration of Tris is 1wt%, sodium chloride concentration be the concentration of 0.5wt%, Proclin300 be 0.2v%；By cattle
Serum albumin adds in container, is stirred well to be completely dissolved, and the concentration of bovine serum albumin is 0.5wt%；
With the HCL of 4M, the pH value of solution is adjusted to 7.0-7.5；Reagent is filtered to obtain with the filter that aperture is 0.2 μm
No. 1 diluent, 2-8 DEG C of preservation is stand-by；
B. reagent preparation 1:
By double-stranded DNA antigen with purified water dissolve, under the conditions of 2-8 DEG C with concentration be 0.2M, pH be 9.0
Carbonate buffer solution dialysis 2h, be then concentrated into the antigenic solution that concentration is 2-4mg/mL, use concentration
It is the biotin solution of 0.5-1.0mg/ml for the carbonate buffer solution compound concentration that 0.2M, pH are 8.5-9；
It is that the ratio of 10:1 adds in double-stranded DNA antigen solution according to double-stranded DNA antigen and biotin mass ratio
Biotin solution, mix homogeneously, room temperature stands 12-18h, and reaction generates double-stranded DNA antigen-biotin even
Connect thing；By the reactant liquor containing double-stranded DNA antigen-biotin conjugate under the conditions of 2-8 DEG C by concentration it is
0.2M, pH be 9.0 carbonate buffer solution dialyse 2 days, period carries out 4 times and changes liquid, thus removes
Unreacted biotin, obtains the solution containing double-stranded DNA antigen-biotin conjugate；With reagent 1
Solution containing double-stranded DNA antigen-biotin conjugate is diluted to 0.1-0.3 μ g/mL by diluent, prepares examination
Agent 1；
The reagent 1 of this step preparation can reduce experimental cost, and can efficiently separate free biotin and company
Connecing thing, the junctional complex obtained is purer, decreases the non-specific of reaction；
(4) reagent preparation 2:
A. No. 2 diluents of reagent preparation:
By purified water, 4-hydroxyethyl piperazine ethanesulfonic acid, sodium chloride, bovine serum albumin, ZnCl2And Proclin300
Adding in container, be stirred well to be completely dissolved, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 0.6wt%, chlorine
Change na concn is 0.8wt%, and the concentration of bovine serum albumin is 0.5wt%, ZnCl2Concentration be 0.1wt ‰,
The concentration of Proclin300 is 0.2v ‰, MgCl2Concentration be 0.1 ‰；With the HCL of 4M by the pH of solution
Value is adjusted to 7.5-8.0；Filtering to obtain No. 2 diluents of reagent with the filter that aperture is 0.2 μm, 2-8 DEG C of preservation is stand-by；
B. reagent preparation 2:
1mg sheep anti-human polyclonal antibody is joined the 2-iminothiolane salt that 2-4 μ L concentration is 10mg/mL
In acid salt solution, room temperature stands 20min, adds the glycine solution 10 μ L of 0.1moL/L, and room temperature stands
5min, with G-25 gel column desalination, collects the sheep anti-human polyclonal antibody after activation, and 2-8 DEG C of preservation is standby
With；The alkali phosphatase of 1.5mg is joined 4-(the N-maleimide that concentration is 5mg/mL of 10-20 μ L
Aminomethyl) in hexamethylene-1-carboxylic acid butanimide ester solution, room temperature stands 30min, uses G-25 gel
Post desalination, collects the alkali phosphatase after activation, and 2-8 DEG C saves backup；Goat-anti people's Anti-TNF-α by activation
Body mixes with the alkali phosphatase of activation, stands 12-24h under the conditions of 2-8 DEG C, pure with Supperdex200 gel
Change column purification conjugate, it is thus achieved that sheep anti-human polyclonal antibody-alkali phosphatase junctional complex concentrated solution, 2-8 DEG C of guarantor
Deposit standby；By sheep anti-human polyclonal antibody-alkali phosphatase junctional complex concentrated solution No. 2 diluted of reagent
To 0.02-0.1 μ g/mL, prepare reagent 2；
(5) preparation Magneto separate reagent:
A. preparation magnetic particle buffer:
Purified water, Tris and sodium chloride being added in container, be stirred well to be completely dissolved, the concentration of Tris is
1wt%, the concentration of sodium chloride is 0.8wt%；Again by bovine serum albumin, new-born calf serum and Proclin300
Adding in container, be stirred well to be completely dissolved, the concentration of bovine serum albumin is 0.5wt%, newborn Sanguis Bovis seu Bubali
Clear concentration be 5v%, Proclin300 concentration be 0.2v ‰；With the HCL of 4M, the pH value of solution is adjusted to
7.9-8.1；Filtering to obtain magnetic particle buffer with the filter that aperture is 0.2 μm, 2-8 DEG C of preservation is stand-by；
B. preparation Magneto separate reagent:
Take 100mg magnetic particle, use magnetic frame absorption, suck supernatant after static 2min, add in magnetic particle
Enter concentration be 0.025mol/L, pH be 2-(N-morpholine) the ethanesulfonic acid buffer 10mL of 4.5-5, fully
Mixing；Add the concentration that 0.5-1mL newly prepares and be 1-(3-the dimethylamino-propyl)-3-second of 10mg/mL
Base carbodiimide and N-hydroxy-succinamide aqueous solution, room temperature mixing 30-60min obtains magnetic bead suspending system；
Using 2-(N-morpholine) ethanesulfonic acid buffer compound concentration is the solution of streptavidin of 5mg/mL, then
The Streptavidin of the 0.8-1.6mL of described concentration it is directly added in magnetic bead suspending system, mixed under the conditions of 4 DEG C
Outstanding 16-20h；Re-use magnetic frame absorption, after static 2min, suck supernatant, in magnetic particle, add 10mL
Concentration be 1M, pH be the ethanolamine solutions of 8.5, room temperature reaction 1-2h, re-use magnetic frame absorption, quiet
Only suck supernatant after 2min, in magnetic particle, add the dilution of appropriate magnetic particle buffer make final concentration of
0.5mg/mL, prepares Magneto separate reagent；
In this step, adding N-hydroxy-succinamide can be to coupling agent 1-(3-dimethylamino-propyl)-3-second
Base carbodiimide plays Stabilization；Add after Streptavidin suspendible under the conditions of 4 degree, it is possible to more preferable
Retaining protein activity, reduces the room temperature change impact on coupling effect simultaneously, makes coupling result between batch
More stable；Add ethanolamine, uncombined albumen after the amino in ethanolamine molecules can be made to activate with magnetic bead
Avtive spot reacts, and plays termination reaction and sealing process, it is possible to produce lower background values；
(6) preparation cleanout fluid:
Purified water, Tris and sodium chloride being added in container, be stirred well to be completely dissolved, the concentration of Tris is
1wt%, the concentration of sodium chloride is 0.8wt%；Again Tween-20 and Triton100 is added in container, fully
Stirring to mixing completely, the concentration of Tween-20 be the concentration of 0.5wt%, Triton100 be 0.5wt%；With
The pH value of solution is adjusted to 7.5-8.0 by the HCL of 4M, filters to obtain cleanout fluid, 2-8 DEG C of guarantor with 0.2 μm filter
Deposit.
The magnetic microparticle chemiluminescence quantitative determination reagent kit detection anti-double-chain DNA of anti-dsDNA antibody IgG
The detection method of IgG antibody, the method comprises the steps:
1) anti-dsDNA antibody IgG calibration object is put into Full-automatic chemiluminescence immunoassay analysis meter test position,
Obtain the matched curve exported by Full-automatic chemiluminescence immunoassay analysis meter；
2) anti-dsDNA antibody IgG quality-control product is put into above-mentioned analyser test position, obtains by automatically
The test luminous value of the described quality-control product of chemical illumination immunity analysis instrument output and the matching obtained by step 1
Curve matching obtains the concentration value of anti-dsDNA antibody IgG quality-control product；
3) sample to be tested is put into above-mentioned analyser test position, described analyser automatically presses 1:20 by sample
Dilution, obtains the concentration value of the sample to be tested exported by Full-automatic chemiluminescence immunoassay analysis meter.
Further, this detection method specifically includes following steps:
1) specimen to be measured after 20 μ L anti-dsDNA antibody IgG calibration objects or quality-control product or 1:20 dilution is added extremely
In detection pipe；
2) reagent 1 of 50 μ L is added to step 1) in described detection pipe, after mixing, 37 ± 0.5 DEG C of warm 10min；
3) the Magneto separate reagent of 50 μ L is added to step 2) in described detection pipe, after mixing, 37 ± 0.5 DEG C of incubations
5min, carries out Magneto separate, removes supernatant；
4) cleanout fluid of 300 μ L is added to step 3) in described detection pipe, mixing, carry out Magneto separate, remove supernatant；
5) step 4 is repeated) twice；
6) 150 μ L reagent 2 is added to step 5) in described detection pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min,
Carry out Magneto separate, remove supernatant；
7) cleanout fluid of 300 μ L is added to step 6) in described detection pipe, mixing, carry out Magneto separate, remove supernatant；
8) step 7 is repeated) twice；
9) chemical luminous substrate of 200 μ L is added to step 8) in described detection pipe, mixing, detects luminous intensity.
Described step 1), step 2) and step 3) all include Full-automatic chemiluminescence immunoassay analysis meter complete from
Dynamic detecting step.
Beneficial effects of the present invention is as follows:
The invention discloses a kind of new technique measuring anti-dsDNA antibody IgG so that course of reaction is more accelerated
Speed is reliable, improves sensitivity and linear measurement range, it is achieved the quantitative determination of real meaning, and can arrange in pairs or groups the most certainly
Dynamic chemical illumination immunity analysis instrument realizes full automatic use, is greatly improved work efficiency；School in test kit
Quasi-product, biotin labeling reagent, enzyme labelling reagent, Magneto separate reagent and cleanout fluid etc. are all this reaction systems
Under optimization formula, to this test kit use imitate the phase and detection performance provide powerful guarantee.
Accompanying drawing explanation
Fig. 1 a be during the blank limit of embodiment 7 is evaluated concentration value between zero-dose calibration object with adjacent calibration object with
Luminous value result carries out the linear function that 2 regression fits draw；
Fig. 1 b be during the blank limit of comparative example 1 is evaluated concentration value between zero-dose calibration object with adjacent calibration object with
Luminous value result carries out the linear function that 2 regression fits draw；
Fig. 2 is concentration of specimens meansigma methods and dilution ratio method of least square in embodiment 7 range of linearity evaluation
Carry out fitting a straight line equation；
Fig. 3 is the embodiment 7 test kit clinical sample measured value dependency scatterplot with existing commercialization.
Detailed description of the invention
In order to be illustrated more clearly that the present invention, below in conjunction with preferred embodiments and drawings, the present invention is done further
Explanation.
Embodiment 1
The preparation of anti-dsDNA antibody IgG calibration object:
A. preparation anti-dsDNA antibody IgG calibration object diluent:
The purified water of 800ml, Tris, 8.6g sodium chloride of 11.2g and 2ml Proclin300 are added container
In, it is stirred well to be completely dissolved；With the HCL of 4M, the pH value of solution is adjusted to 7.0-7.5；By 40g
Bovine serum albumin adds in container, is stirred well to be completely dissolved；Again with the HCL of 4M by the pH of solution
Value is adjusted to 7.0-7.5；By purified water, solution is settled to 1L, filters to obtain anti-double-chain DNA with 0.2 μm filter
IgG antibody calibration object diluent, 2-8 DEG C of preservation is stand-by；
B. preparation anti-dsDNA antibody IgG calibration object:
By anti-dsDNA antibody IgG anti-dsDNA antibody IgG calibration object diluted to each dense
Degree point is 0,10,100,200,400,800IU/mL.
Embodiment 2
The preparation of anti-dsDNA antibody IgG quality-control product:
By anti-dsDNA antibody IgG by above-mentioned anti-dsDNA antibody IgG calibration object diluted extremely
Each concentration point is 100,400IU/mL.
Embodiment 3
The preparation that reagent 1:
A. No. 1 diluent of reagent preparation:
800ml purified water, Tris, 5.8g sodium chloride of 12.1g and 2ml Proclin300 are added in container,
It is stirred well to be completely dissolved；5g bovine serum albumin is added in container, is stirred well to be completely dissolved；
With the HCL of 4M, the pH value of solution is adjusted to 7.0-7.5；By purified water, solution is settled to 1L, uses 0.2 μm
Filter filters to obtain No. 1 diluent of reagent, and 2-8 DEG C of preservation is stand-by；
B. reagent preparation 1:
By double-stranded DNA antigen with purified water dissolve, under the conditions of 2-8 DEG C with concentration be 0.2M, pH be 9.0
Carbonate buffer solution dialysis 2h, be then concentrated into the antigenic solution that concentration is 2-4mg/mL, use concentration
It is the biotin solution of 0.5-1.0mg/ml for the carbonate buffer solution compound concentration that 0.2M, pH are 8.5-9；
It is that the ratio of 10:1 adds in double-stranded DNA antigen solution according to double-stranded DNA antigen and biotin mass ratio
Biotin solution, mix homogeneously, room temperature stands 12-18h, and reaction generates double-stranded DNA antigen-biotin even
Connect thing；By the reactant liquor containing double-stranded DNA antigen-biotin conjugate under the conditions of 2-8 DEG C by concentration it is
0.2M, pH be 9.0 carbonate buffer solution dialyse 2 days, period carries out 4 times and changes liquid, thus removes
Unreacted biotin, obtains the solution containing double-stranded DNA antigen-biotin conjugate；With reagent 1
Solution containing double-stranded DNA antigen-biotin conjugate is diluted to 0.1-0.3 μ g/mL by diluent, prepares examination
Agent 1；
The reagent 1 of this step preparation can reduce experimental cost compared with former method, and can efficiently separate trip
From biotin and junctional complex, the junctional complex obtained is purer, decreases the non-specific of subsequent reactions.
Embodiment 4
The preparation that reagent 2:
A. No. 2 diluents of reagent preparation:
By pure to 800ml purified water, the 4-hydroxyethyl piperazine ethanesulfonic acid of 6.06g, 8.5g sodium chloride, 5g Sanguis Bovis seu Bubali
Albumen, 0.1gZnCl2, 0.2ml Proclin300 and 0.1g MgCl2Add in container, be stirred well to completely
Dissolve；With the HCL of 4M, the pH value of solution is adjusted to 7.5-8.0；By purified water, solution is settled to 1L,
Filtering to obtain No. 2 diluents of reagent with 0.2 μm filter, 2-8 DEG C of preservation is stand-by；
B. reagent preparation 2:
1mg sheep anti-human polyclonal antibody is joined the 2-iminothiolane salt that 2-4 μ L concentration is 10mg/mL
In acid salt solution, room temperature stands 20min, adds the glycine solution 10 μ L of 0.1moL/L, and room temperature stands
5min, with G-25 gel column desalination, collects the sheep anti-human polyclonal antibody after activation, and 2-8 DEG C of preservation is standby
With；The alkali phosphatase of 1.5mg is joined 4-(the N-maleimide that concentration is 5mg/mL of 10-20 μ L
Aminomethyl) in hexamethylene-1-carboxylic acid butanimide ester solution, room temperature stands 30min, uses G-25 gel
Post desalination, collects the alkali phosphatase after activation, and 2-8 DEG C saves backup；Goat-anti people's Anti-TNF-α by activation
Body mixes with the alkali phosphatase of activation, stands 12-24h under the conditions of 2-8 DEG C, pure with Supperdex200 gel
Change column purification conjugate, it is thus achieved that sheep anti-human polyclonal antibody-alkali phosphatase junctional complex concentrated solution, 2-8 DEG C of guarantor
Deposit standby；By sheep anti-human polyclonal antibody-alkali phosphatase junctional complex concentrated solution No. 2 diluted of reagent
To 0.02-0.1 μ g/mL, prepare reagent 2.
Embodiment 5
The preparation of Magneto separate reagent:
A. preparation magnetic particle buffer:
800mL purified water, 12.1g Tris and 8.5g sodium chloride are added in container, is stirred well to completely
Dissolve；Again 5g bovine serum albumin, 50mL new-born calf serum and 0.2mL Proclin300 are added container
In, it is stirred well to be completely dissolved；With the HCL of 4M, the pH value of solution is adjusted to 7.9-8.1；Use purification
Solution is settled to 1L by water, filters to obtain magnetic particle buffer with 0.2 μm filter, and 2-8 DEG C of preservation is stand-by；
B. preparation Magneto separate reagent:
Take 100mg magnetic particle, use magnetic frame absorption, suck supernatant after static 2min, add in magnetic particle
Enter concentration be 0.025mol/L, pH be 2-(N-morpholine) the ethanesulfonic acid buffer 10mL of 4.5-5, fully
Mixing；Add the concentration that 0.5-1mL newly prepares and be 1-(3-the dimethylamino-propyl)-3-second of 10mg/mL
Base carbodiimide and N-hydroxy-succinamide aqueous solution, room temperature mixing 30-60min obtains magnetic bead suspending system；
Making magnetic particle fully activate, using 2-(N-morpholine) ethanesulfonic acid buffer compound concentration is the chain of 5mg/mL
Mould Avidin solution, is then directly added into the strepto-parent of the 0.8-1.6mL of described concentration in magnetic bead suspending system
And element, suspendible 16-20h under the conditions of 4 DEG C；Re-use magnetic frame absorption, after static 2min, suck supernatant, to
Magnetic particle adds 10mL concentration be 1M, pH be the ethanolamine solutions of 8.5, room temperature reaction 1-2h, then make
Adsorb with magnetic frame, after static 2min, suck supernatant, in magnetic particle, add the dilution of appropriate magnetic particle buffer
Make final concentration of 0.5mg/mL, prepare Magneto separate reagent；
In this step, adding N-hydroxy-succinamide can be to coupling agent 1-(3-dimethylamino-propyl)-3-second
Base carbodiimide plays Stabilization；Add after Streptavidin suspendible under the conditions of 4 degree, it is possible to more preferable
Retaining protein activity, reduces the room temperature change impact on coupling effect simultaneously, makes coupling result between batch
More stable；Add ethanolamine, uncombined albumen after the amino in ethanolamine molecules can be made to activate with magnetic bead
Avtive spot reacts, and plays termination reaction and sealing process, it is possible to produce lower background values.
Embodiment 6
The preparation of cleanout fluid:
800mL purified water, 12.1g Tris and 8.5g sodium chloride are added in container, is stirred well to completely
Dissolve；Again 5g Tween-20 and 5g Triton100 is added in container, be stirred well to mix completely；With
The pH value of solution is adjusted to 7.5-8.0 purified water and solution is settled to 1L by the HCL of 4M, with 0.2 μm filter
Device filters to obtain cleanout fluid, 2-8 DEG C of preservation.
Embodiment 7
The magnetic microparticle chemiluminescence quantitative determination reagent kit of anti-dsDNA antibody IgG:
This test kit includes:
The anti-dsDNA antibody IgG calibration object prepared according to embodiment 1 method, the calibration object of each level
Consumption is 0.5ml；
The anti-dsDNA antibody IgG quality-control product prepared according to embodiment 2 method, quality-control product consumption is 1mL；
The reagent 1 prepared according to embodiment 3 method, the consumption that reagent 1 is 5ml；
The reagent 2 prepared according to embodiment 4 method, the consumption that reagent 2 is 15ml；
The Magneto separate reagent prepared according to embodiment 5 method, the consumption of Magneto separate reagent is 5ml；
The cleanout fluid prepared according to embodiment 6 method, cleanout fluid consumption is 1L.
Embodiment 8
The test kit using embodiment 7 carries out detection by quantitative to anti-dsDNA antibody IgG
Detection method comprises the steps:
1) specimen to be measured after 20 μ L anti-dsDNA antibody IgG calibration objects or quality-control product or 1:20 dilution is added extremely
In detection pipe；
2) reagent 1 of 50 μ L is added to step 1) in described detection pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min；
3) the Magneto separate reagent of 50 μ L is added to step 2) in described detection pipe, after mixing, 37 ± 0.5 DEG C of incubations
5min, carries out Magneto separate, removes supernatant；
4) cleanout fluid of 300 μ L is added to step 3) in described detection pipe, mixing, carry out Magneto separate, remove supernatant；
5) step 4 is repeated) twice；
6) 150 μ L reagent 2 is added to step 5) in described detection pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min,
Carry out Magneto separate, remove supernatant；
7) cleanout fluid of 300 μ L is added to step 6) in described detection pipe, mixing, carry out Magneto separate, remove supernatant；
8) step 7 is repeated) twice；
9) chemical luminous substrate of 200 μ L is added to step 8) in described detection pipe, mixing, detects luminous intensity.
Comparative example 1
Reagent 1 in test kit unlike the test kit of embodiment 7 preparation and Magneto separate examination
Agent, remaining composition is identical.
Preparing of reagent 1 is as follows:
A. No. 1 diluent of reagent preparation:
800ml purified water, Tris, 5.8g sodium chloride of 12.1g and 2ml Proclin300 are added in container,
It is stirred well to be completely dissolved；5g bovine serum albumin is added in container, is stirred well to be completely dissolved；
With the HCL of 4M, the pH value of solution is adjusted to 7.0-7.5；By purified water, solution is settled to 1L, uses 0.2 μm
Filter filters to obtain No. 1 diluent of reagent, and 2-8 DEG C of preservation is stand-by；
B. reagent preparation 1:
Using concentration 0.2M, pH is the biotin solution of the carbonate buffer solution preparation 0.5mg/ml of 9；According to double-strand
It is molten that DNA antigen and the ratio that biotin mass ratio is 10:1 add biotin in double-stranded DNA antigen solution
Liquid, mix homogeneously, room temperature stands 18h, and reaction generates double-stranded DNA antigen-biotin conjugate；To contain
The reactant liquor of double-stranded DNA antigen-biotin conjugate is separated by G-25 gel column, removes unreacted
Biotin, obtains the solution containing double-stranded DNA antigen-biotin conjugate；To contain with No. 1 diluent of reagent
The solution having double-stranded DNA antigen-biotin conjugate is diluted to 0.1-0.3 μ g/mL, prepares reagent 1；
Preparing of Magneto separate reagent is as follows:
A. preparation magnetic particle buffer:
800mL purified water, 12.1g Tris and 8.5g sodium chloride are added in container, is stirred well to completely
Dissolve；Again 5g bovine serum albumin, 50mL new-born calf serum and 0.2mL Proclin300 are added container
In, it is stirred well to be completely dissolved；With the HCL of 4M, the pH value of solution is adjusted to 7.9-8.1；Use purification
Solution is settled to 1L by water, filters to obtain magnetic particle buffer with 0.2 μm filter, and 2-8 DEG C of preservation is stand-by；
B. preparation Magneto separate reagent:
Taking 100mg magnetic particle, Magneto separate removes supernatant, with concentration be 0.025mol/L, pH be the 2-(N-of 4.5-5
Morpholine) ethanesulfonic acid buffer 10mL is resuspended；Add the EDC that concentration is 10mg/mL that 0.5-1mL newly prepares
Aqueous solution, room temperature suspendible 30-60min；Magnetic bead is made fully to activate, Magneto separate, removes supernatant, by concentration is
0.025mol/L, pH are that 4.5-5 2-(N-morpholine) ethanesulfonic acid buffer 10mL is resuspended；Add the chain of 4-8mg
Mould Avidin, room temperature suspendible 16-20h；Carry out Magneto separate again, remove supernatant, with magnetic particle buffer dilution weight
Hang 0.5mg/mL, prepare Magneto separate reagent.
Embodiment 9
The test kit of embodiment 7 and comparative example 1 is carried out performance evaluation:
1. the accuracy estimating of embodiment 7
Concentration is about the anti-dsDNA antibody IgG of 200RU/mL (allowing its concentration deviation is ± 20%)
Sample A joins in the sample B of serum or other corresponding substrate, and the volume of added A is less than cumulative volume
(A+B) 10%, calculates response rate R according to formula (1), and the response rate of this method requires at 85-115%
In the range of, data see table 1, and evaluation result meets the requirements.
R: the response rate；
V: add the volume of standard solution；
V0: the volume of people source sample；
C: people source sample adds the detectable concentration after standard solution；
C0: the detectable concentration of people source sample；
Cs: the concentration of standard solution.
Table 1 accuracy estimating
2. the blank limit of embodiment 7 and comparative example 1 is evaluated
Detect as sample with zero-dose calibration object, replication 20 times, draw 20 measurement results
RLU value (relative light unit), calculate its meansigma methods (M) and standard deviation (SD), draw M+2SD
Corresponding RLU value, enters according to the concentration-RLU value result between zero-dose calibration object with adjacent calibration object
2 regression fits of row draw linear function, bring in above-mentioned equation by the RLU value corresponding to M+2SD,
Obtaining the concentration value of correspondence, be blank limit, the blank limit of this method requires to be not more than 1RU/mL, and data are joined
Being shown in Table 2, A, B point even some matched curve and fit equation are shown in that Fig. 1 a, the test kit of comparative example 1 preparation record
Blank limit data be shown in Table 2-1, A, B point even puts matched curve and fit equation is shown in Fig. 1 b, from data,
Embodiment 7 is compared with comparative example 1, and the background values obtained is lower, thus the blank limit obtained is lower, represents
The sensitivity of test kit is more preferable.
The blank limit of table 2 is evaluated
Table 2-1 blank limit is evaluated
3. the range of linearity evaluation of embodiment 7
At least 5 kinds will be diluted to by a certain percentage close to the high level sample of the range of linearity upper limit (800IU/mL)
Concentration, wherein the sample of low value concentration must be close to the lower limit of the range of linearity.Operate by test kit description,
Sample standard deviation duplicate detection 2 times to each concentration, calculates its meansigma methods, by result meansigma methods and dilution ratio
Carry out fitting a straight line with method of least square, and calculate linearly dependent coefficient r, the measurement scope of this method be [2,
800] IU/mL, it is desirable in this range, correlation coefficient r answers >=0.9900.Data see table 3, and matching is bent
Line and correlation coefficient are shown in that Fig. 2, evaluation result meet the requirements.
Table 3 range of linearity evaluation
4. embodiment 7 and the reproducibility of comparative example 1
Test kit duplicate detection concentration in Example 7 is (100 ± 20) IU/mL and (400 ± 80) IU/mL
Each 10 times of sample, calculate meansigma methods M and standard deviation SD of 10 measurement results, according to formula
CV=SD/M × 100% draws coefficient of variation CV, and this method coefficient of variation (CV) requires to be not more than 8%, number
According to seeing table 4, the repeated data that the test kit of comparative example 1 preparation records are shown in Table 4-1, from data,
Embodiment 7 is compared with comparative example 1, and the CV value obtained is lower, represents the repeatability of test kit more preferably,
CV=SD/M × 100%...................................... (2)
In formula: the CV coefficient of variation；The standard deviation of 10 measurement results of SD；10 measurement results of M
Meansigma methods.
Table 4 reproducibility
| Measure serum-concentration (IU/mL) | Measure number of times | CV (%) in analyzing |
| 100 | 10 | 5.37% |
| 400 | 10 | 5.00% |
Table 4-1 reproducibility
| Measure serum-concentration (IU/mL) | Measure number of times | CV (%) in analyzing |
| 100 | 10 | 7.25% |
| 400 | 10 | 7.04% |
5. the difference between batch evaluation of embodiment 7 and comparative example 1
The test kit of embodiment 7 takes three batches, and every batch of test kit all measures concentration at (100 ± 20) IU/mL
Sample in the range of (400 ± 80) IU/mL, the every batch of replication 10 times, calculate 30 measurement results
Meansigma methods (M) and standard deviation (SD), according to formula (3) calculate the coefficient of variation (CV), this method
The coefficient of variation (CV) requires to be not more than 15%, and data see table 5, and the test kit of comparative example 1 preparation records
Difference between batch data be shown in Table 5-1, from data, embodiment 7 compared with comparative example 1, the CV value obtained
Lower, the difference between batch representing test kit is more preferable,
CV=SD/M × 100%...................................... (3)
In formula: the CV coefficient of variation；The standard deviation of 30 measurement results of SD；30 measurement results of M
Meansigma methods.
Table 5 difference between batch evaluation
| Measure serum-concentration (IU/mL) | Measure number of times | CV (%) between analysis |
| 100 | 30 | 6.08% |
| 400 | 30 | 7.72% |
Table 5-1 difference between batch is evaluated
| Measure serum-concentration (IU/mL) | Measure number of times | CV (%) between analysis |
| 100 | 30 | 8.82% |
| 400 | 30 | 10.21% |
6. the Evaluation on specificity of embodiment 7
Taking 1 part of Anti-double-stranded DNA IgG content is the sample of 0, adds human serum albumin, makes in sample
Human serum albumin's concentration is 5000ng/mL, uses this test kit to detect this sample, measures in sample
Anti-double-stranded DNA IgG content.The results are shown in Table 6, this method and human serum albumin's no cross reaction.
Data see table 6, and evaluation result meets the requirements.
Table 6 specificity experiments
7. the relativity evaluation of embodiment 7
By the anti-dsDNA antibody IgG detection kit of the test kit of embodiment 7 and commercialization, (enzyme connection is exempted from
Epidemic disease absorption method) 240 parts of human serum samples are detected simultaneously.Its testing result sees accompanying drawing 3, with anti-double
The result that chain DNA IgG antibody detection kit (enzyme linked immunosorbent assay) measures is abscissa, with this
The result of the mensuration of bright method is that vertical coordinate makees regression analysis, and dependent equation is: y=0.9023x+7.3892, phase
Pass coefficient is R2: 0.9582.Showing through statistical procedures result, this method is faced with the test kit of additive method
Bed sample measured value dependency is good.
8. the estimation of stability of embodiment 7
Test kit carries out 4 DEG C of 12 months and the experiments of 37 DEG C of accelerated stabilities of 7 days respectively, and result shows
The change of kit standard product luminous intensity, batch in and betweenrun precision, accuracy index all at normal model
Within enclosing, test kit effect duration was up to 12 months.
Obviously, the above embodiment of the present invention is only for clearly demonstrating example of the present invention, and also
Non-is the restriction to embodiments of the present invention, for those of ordinary skill in the field, above-mentioned
Can also make other changes in different forms on the basis of explanation, here cannot be to all of enforcement
Mode gives exhaustive, every belongs to the obvious change or variation that technical scheme extended out
Row still in protection scope of the present invention.
Claims (5)
1. a magnetic microparticle chemiluminescence quantitative determination reagent kit of anti-dsDNA antibody IgG, its feature
Being, described test kit includes:
1) anti-dsDNA antibody IgG calibration object, containing anti-dsDNA antibody IgG and bovine serum albumin
White Tris buffer, described anti-dsDNA antibody IgG calibration object comprises the liquid calibration object of 6 levels,
In the liquid calibration object of described 6 levels, the concentration of anti-dsDNA antibody IgG is respectively 0, and 10,100,
200,400,800IU/mL；
2) anti-dsDNA antibody IgG quality-control product, containing anti-dsDNA antibody IgG and bovine serum albumin
White Tris buffer, described anti-dsDNA antibody IgG quality-control product comprises the liquid quality control product of 2 levels,
In the liquid quality control product of described 2 levels, the target value concentration range of anti-dsDNA antibody IgG is respectively
(100 ± 20) IU/mL and (400 ± 80) IU/mL；
3) reagent 1, the Tris buffering containing biotin labeled double-stranded DNA antigen and bovine serum albumin
Liquid；
4) reagent 2, sheep anti-human polyclonal antibody containing alkali phosphatase enzyme mark and bovine serum albumin
Tris buffer；
5) Magneto separate reagent, magnetic particle and the Tris of bovine serum albumin containing marked by streptavidin delay
Rush liquid；
6) cleanout fluid；
The content of described seminal plasma fructose detection kit 1, reagent 2 and Magneto separate reagent than for 1:3:1, described contains
Amount ratio is volume ratio.
Test kit the most according to claim 1, it is characterised in that: the material of described magnetic particle is Fe2O3；
Described magnetic particle pan coating has carboxylic group, is coated carboxyl group content in thing and is more than 20wt%, described magnetic
The size of microgranule is 1-3 μm.
3. prepare the method for test kit as described in any one of claim 1-2 for one kind, it is characterised in that the party
Method comprises the steps:
(1) preparation anti-dsDNA antibody IgG calibration object:
A. preparation anti-dsDNA antibody IgG calibration object diluent:
Purified water, Tris, sodium chloride and Proclin300 are added in container, are stirred well to be completely dissolved,
Tris concentration is 1wt%, sodium chloride concentration be 1wt%, Proclin300 concentration be 0.2v%；With the HCL of 4M
The pH value of solution is adjusted to 7.0-7.5；Bovine serum albumin is added in container, is stirred well to be completely dissolved,
The concentration of bovine serum albumin is 4wt%；With the HCL of 4M, the pH value of solution is adjusted to 7.0-7.5 again；With
Aperture is that the filter of 0.2 μm filters to obtain anti-dsDNA antibody IgG calibration object diluent, and 2-8 DEG C of preservation is treated
With；
B. preparation anti-dsDNA antibody IgG calibration object:
By anti-dsDNA antibody IgG by anti-dsDNA antibody IgG calibration object diluted to respectively
Concentration point is 0,10,100,200,400,800IU/mL；
(2) preparation anti-dsDNA antibody IgG quality-control product:
By anti-dsDNA antibody IgG by above-mentioned anti-dsDNA antibody IgG calibration object diluted extremely
Each concentration point is 100,400IU/mL；
(3) reagent preparation 1:
A. No. 1 diluent of reagent preparation:
Purified water, Tris, sodium chloride and Proclin300 are added in container, are stirred well to be completely dissolved,
The concentration of Tris is 1wt%, sodium chloride concentration be the concentration of 0.5wt%, Proclin300 be 0.2v%；By cattle
Serum albumin adds in container, is stirred well to be completely dissolved, and the concentration of bovine serum albumin is 0.5wt%；
With the HCL of 4M, the pH value of solution is adjusted to 7.0-7.5；Reagent is filtered to obtain with the filter that aperture is 0.2 μm
No. 1 diluent, 2-8 DEG C of preservation is stand-by；
B. reagent preparation 1:
By double-stranded DNA antigen with purified water dissolve, under the conditions of 2-8 DEG C with concentration be 0.2M, pH be 9.0
Carbonate buffer solution dialysis 2h, be then concentrated into the antigenic solution that concentration is 2-4mg/mL, use concentration
It is the biotin solution of 0.5-1.0mg/ml for the carbonate buffer solution compound concentration that 0.2M, pH are 8.5-9；
It is that the ratio of 10:1 adds in double-stranded DNA antigen solution according to double-stranded DNA antigen and biotin mass ratio
Biotin solution, mix homogeneously, room temperature stands 12-18h, and reaction generates double-stranded DNA antigen-biotin even
Connect thing；By the reactant liquor containing double-stranded DNA antigen-biotin conjugate under the conditions of 2-8 DEG C by concentration it is
0.2M pH be 9.0 carbonate buffer solution dialyse 2 days, period carries out 4 times and changes liquid, thus removes
Unreacted biotin, obtains the solution containing double-stranded DNA antigen-biotin conjugate；With reagent 1
Solution containing double-stranded DNA antigen-biotin conjugate is diluted to 0.1-0.3 μ g/mL by diluent, prepares examination
Agent 1；
(4) reagent preparation 2:
A. No. 2 diluents of reagent preparation:
By purified water, 4-hydroxyethyl piperazine ethanesulfonic acid, sodium chloride, bovine serum albumin, ZnCl2And Proclin300
Adding in container, be stirred well to be completely dissolved, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 0.6wt%, chlorine
Change na concn is 0.8wt%, and the concentration of bovine serum albumin is 0.5wt%, ZnCl2Concentration be 0.1wt ‰,
The concentration of Proclin300 is 0.2v ‰, MgCl2Concentration be 0.1 ‰；With the HCL of 4M by the pH of solution
Value is adjusted to 7.5-8.0；Filtering to obtain No. 2 diluents of reagent with the filter that aperture is 0.2 μm, 2-8 DEG C of preservation is stand-by；
B. reagent preparation 2:
1mg sheep anti-human polyclonal antibody is joined the 2-iminothiolane salt that 2-4 μ L concentration is 10mg/mL
In acid salt solution, room temperature stands 20min, adds the glycine solution 10 μ L of 0.1moL/L, and room temperature stands
5min, with G-25 gel column desalination, collects the sheep anti-human polyclonal antibody after activation, and 2-8 DEG C saves backup；
The alkali phosphatase of 1.5mg is joined 4-(the N-dimaleoyl imino that concentration is 5mg/mL of 10-20 μ L
Methyl) in hexamethylene-1-carboxylic acid butanimide ester solution, room temperature stands 30min, with G-25 gel column desalination,
Collecting the alkali phosphatase after activation, 2-8 DEG C saves backup；By sheep anti-human polyclonal antibody and the activation of activation
Alkali phosphatase mixing, under the conditions of 2-8 DEG C stand 12-24h, with Supperdex200 gel-purified column purification
Conjugate, it is thus achieved that sheep anti-human polyclonal antibody-alkali phosphatase junctional complex concentrated solution, 2-8 DEG C saves backup；Will
Sheep anti-human polyclonal antibody-alkali phosphatase No. 2 diluted of junctional complex concentrated solution reagent arrive
0.02-0.1 μ g/mL, prepares reagent 2；
(5) preparation Magneto separate reagent:
A. preparation magnetic particle buffer:
Purified water, Tris and sodium chloride being added in container, be stirred well to be completely dissolved, the concentration of Tris is
1wt%, the concentration of sodium chloride is 0.8wt%；Again by bovine serum albumin, new-born calf serum and Proclin300
Adding in container, be stirred well to be completely dissolved, the concentration of bovine serum albumin is 0.5wt%, newborn Sanguis Bovis seu Bubali
Clear concentration be 5v%, Proclin300 concentration be 0.2v ‰；With the HCL of 4M, the pH value of solution is adjusted to
7.9-8.1；Filtering to obtain magnetic particle buffer with the filter that aperture is 0.2 μm, 2-8 DEG C of preservation is stand-by；
B. preparation Magneto separate reagent:
Take 100mg magnetic particle, use magnetic frame absorption, suck supernatant after static 2min, add in magnetic particle
Enter concentration be 0.025mol/L, pH be 2-(N-morpholine) the ethanesulfonic acid buffer 10mL of 4.5-5, fully
Mixing；Add the concentration that 0.5-1mL newly prepares and be 1-(3-the dimethylamino-propyl)-3-second of 10mg/mL
Base carbodiimide and N-hydroxy-succinamide aqueous solution, room temperature mixing 30-60min obtains magnetic bead suspending system；
Using 2-(N-morpholine) ethanesulfonic acid buffer compound concentration is the solution of streptavidin of 5mg/mL, then
The Streptavidin of the 0.8-1.6mL of described concentration it is directly added in magnetic bead suspending system, mixed under the conditions of 4 DEG C
Outstanding 16-20h；Re-use magnetic frame absorption, after static 2min, suck supernatant, in magnetic particle, add 10mL
Concentration be 1M, pH be the ethanolamine solutions of 8.5, room temperature reaction 1-2h, re-use magnetic frame absorption, quiet
Only suck supernatant after 2min, in magnetic particle, add the dilution of appropriate magnetic particle buffer make final concentration of
0.5mg/mL, prepares Magneto separate reagent；
(6) preparation cleanout fluid:
Purified water, Tris and sodium chloride being added in container, be stirred well to be completely dissolved, the concentration of Tris is
1wt%, the concentration of sodium chloride is 0.8wt%；Again Tween-20 and Triton100 is added in container, fully
Stirring to mixing completely, the concentration of Tween-20 be the concentration of 0.5wt%, Triton100 be 0.5wt%；With
The pH value of solution is adjusted to 7.5-8.0 by the HCL of 4M, filters to obtain cleanout fluid with the filter that aperture is 0.2 μm,
2-8 DEG C of preservation.
4. use the detection of test kit as claimed in claim 1 detection anti-dsDNA antibody IgG content
Method, it is characterised in that the method comprises the steps:
1) anti-dsDNA antibody IgG calibration object is put into Full-automatic chemiluminescence immunoassay analysis meter test position,
Obtain the matched curve exported by Full-automatic chemiluminescence immunoassay analysis meter；
2) anti-dsDNA antibody IgG quality-control product is put into above-mentioned analyser test position, obtains by automatically
The test luminous value of the described quality-control product of chemical illumination immunity analysis instrument output and the matching obtained by step 1
Curve matching obtains the concentration value of anti-dsDNA antibody IgG quality-control product；
3) sample to be tested is put into above-mentioned analyser test position, described analyser automatically presses 1:20 by sample
Dilution, obtains the concentration value of the sample to be tested exported by Full-automatic chemiluminescence immunoassay analysis meter.
Detection method the most according to claim 4, it is characterised in that the method specifically includes following step
Rapid:
1) specimen to be measured after 20 μ L anti-dsDNA antibody IgG calibration objects or quality-control product or 1:20 dilution is added extremely
In detection pipe；
2) reagent 1 of 50 μ L is added to step 1) in described detection pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min；
3) the Magneto separate reagent of 50 μ L is added to step 2) in described detection pipe, after mixing, 37 ± 0.5 DEG C of incubations
5min, carries out Magneto separate, removes supernatant；
4) cleanout fluid of 300 μ L is added to step 3) in described detection pipe, mixing, carry out Magneto separate, remove supernatant；
5) step 4 is repeated) twice；
6) 150 μ L reagent 2 is added to step 5) in described detection pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min,
Carry out Magneto separate, remove supernatant；
7) cleanout fluid of 300 μ L is added to step 6) in described detection pipe, mixing, carry out Magneto separate, remove supernatant；
8) step 7 is repeated) twice；
9) chemical luminous substrate of 200 μ L is added to step 8) in described detection pipe, mixing, detects luminous intensity；
Described step 1), step 2) and step 3) all include Full-automatic chemiluminescence immunoassay analysis meter complete from
Dynamic detecting step.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106844760A (en) * | 2017-02-20 | 2017-06-13 | 安图实验仪器(郑州)有限公司 | The full-automatic particulate special aided analysis method of chemiluminescent analyzer |
| 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 |
| CN108333346A (en) * | 2017-01-19 | 2018-07-27 | 深圳市新产业生物医学工程股份有限公司 | DsDNA immune magnetic microspheres system, preparation method and application and preservation liquid |
| 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 |
| CN110426520A (en) * | 2019-06-18 | 2019-11-08 | 上海彧成生物科技有限公司 | A kind of magnetic particle storing liquid and its preparation |
| 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 |
| CN111812326A (en) * | 2020-07-23 | 2020-10-23 | 四川携光生物技术有限公司 | Kit for quantitatively detecting content of PD-L1 and PD-1 conjugate |
| CN113671195A (en) * | 2021-09-13 | 2021-11-19 | 迈克生物股份有限公司 | Anti-double-stranded DNA antibody detection kit and application thereof |
| WO2023155876A1 (en) * | 2022-02-21 | 2023-08-24 | 国家粮食和物资储备局科学研究院 | Mycotoxin magnetic chemiluminescence immunoassay kit based on bifunctional fusion protein, and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012021149A1 (en) * | 2010-08-12 | 2012-02-16 | Winters-Hilt Stephen N | Methods and systems for nanopore biosensing |
| CN103911345A (en) * | 2014-04-17 | 2014-07-09 | 山东师范大学 | Immunomagnetic microsphere used for capturing circulating tumor cells in peripheral blood |
| CN105445456A (en) * | 2015-11-16 | 2016-03-30 | 北京中航赛维生物科技有限公司 | Kit for quantitatively detecting anti-SS-A antibody IgG by utilizing magnetic particle chemiluminescence, preparation method and detection method thereof |
-
2016
- 2016-04-20 CN CN201610249121.9A patent/CN105929156A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012021149A1 (en) * | 2010-08-12 | 2012-02-16 | Winters-Hilt Stephen N | Methods and systems for nanopore biosensing |
| CN103911345A (en) * | 2014-04-17 | 2014-07-09 | 山东师范大学 | Immunomagnetic microsphere used for capturing circulating tumor cells in peripheral blood |
| CN105445456A (en) * | 2015-11-16 | 2016-03-30 | 北京中航赛维生物科技有限公司 | Kit for quantitatively detecting anti-SS-A antibody IgG by utilizing magnetic particle chemiluminescence, preparation method and detection method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 曾常茜 等: "化学发光免疫测定抗dsDNA抗体的实验条件分析和初步应用", 《中国免疫学杂志》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108333346A (en) * | 2017-01-19 | 2018-07-27 | 深圳市新产业生物医学工程股份有限公司 | DsDNA immune magnetic microspheres system, preparation method and application and preservation liquid |
| CN108333346B (en) * | 2017-01-19 | 2021-07-06 | 深圳市新产业生物医学工程股份有限公司 | dsDNA immunomagnetic microsphere system, preparation method and application thereof, and preservation solution |
| CN106844760A (en) * | 2017-02-20 | 2017-06-13 | 安图实验仪器(郑州)有限公司 | The full-automatic particulate special aided analysis method of chemiluminescent analyzer |
| 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 |
| CN110426520A (en) * | 2019-06-18 | 2019-11-08 | 上海彧成生物科技有限公司 | A kind of magnetic particle storing liquid and its preparation |
| 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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| CN113671195A (en) * | 2021-09-13 | 2021-11-19 | 迈克生物股份有限公司 | Anti-double-stranded DNA antibody detection kit and application thereof |
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Application publication date: 20160907 |