CN105929165A - Magnetic particle-based quantitative chemiluminescent assay kit for anti-SLA/LP antibody IgG, and preparation and detection methods thereof - Google Patents
Magnetic particle-based quantitative chemiluminescent assay kit for anti-SLA/LP antibody IgG, and preparation and detection methods thereof Download PDFInfo
- Publication number
- CN105929165A CN105929165A CN201610249001.9A CN201610249001A CN105929165A CN 105929165 A CN105929165 A CN 105929165A CN 201610249001 A CN201610249001 A CN 201610249001A CN 105929165 A CN105929165 A CN 105929165A
- Authority
- CN
- China
- Prior art keywords
- concentration
- sla
- reagent
- igg antibody
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Food Science & Technology (AREA)
- Communicable Diseases (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a magnetic particle-based quantitative chemiluminescent assay kit for an anti-SLA/LP antibody IgG. The kit comprises an anti-SLA/LP antibody IgG calibrator, an anti-SLA/LP antibody IgG quality control product, a Tris buffer containing biotin-labeled SLA/LP 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.Magnetic particle more particularly, to a kind of anti-SLA/LP IgG antibody
Learn luminous quantitative determination reagent kit and preparation and detection method.
Background technology
Autoimmune hepatitis (autoimmune hepatitis, AIH) be a class based on autoimmune response, with
There are interface characteristics hepatitis and portal area slurry thin on multiple autoantibody, hyperimmunoglobulinemia, circulation autoantibody and histology
Born of the same parents infiltrate the chronic liver inflammatory diseases being characterized.
AIH can be divided into I type (ANA, SMA), II type (anti-LKM-1 antibody, anti-LC-1 antibody) according to detection of plasma inspection
With III type (anti-SLA/LP antibody), anti-soluble liver antigen/liver pancreas antigen (SLA/LP) antibody is autoimmune hepatitis (AIH)
The most special diagnosis indicates.Although its positive rate only has 10%～30%, but its positive advance notice value is almost 100%, if
Occurring that corresponding clinical symptoms substantially can be diagnosed as AIH, therefore, anti-SLA/LP IgG antibody detects autoimmune hepatitis
Diagnosis have important references be worth.
The detection of the most anti-SLA/LP IgG antibody at present has film bar immunization and enzyme linked immunosorbent assay.The immunity of film bar
Method application is film bar developing technology, and its feature measures at same film bar for fixing several projects, general by manual or partly
Automatically film bar instrument carries out experimental implementation, qualitatively judges eventually through naked eyes, and this sensitivity is low, and the response time is long, inspection
Survey project can only combine in regular collocation, very flexible.The sensitivity of enzyme linked immunosorbent assay on the basis of film bar immunization
Promote, but still relatively low, and the range of linearity is narrow, poor repeatability, and the response time is the longest, still can not meet clinic very well
Application.
Magnetic microparticle chemiluminescence immune assay method, film bar immunization than before and enzyme linked immunosorbent assay, in detection spirit
Having had in sensitivity, detection range, detection time and automation mechanized operation and be greatly improved, and do not polluted, clinical practice is wide.At present,
Magnetic microparticle chemiluminescence analytic process is used to have not yet to see in the application of anti-SLA/LP IgG antibody immunoassay product.
Summary of the invention
It is an object of the present invention to provide the magnetic microparticle chemiluminescence quantitative determination examination of a kind of anti-SLA/LP IgG antibody
Agent box, chemiluminescence analytical technique is combined by the test kit that the present invention provides with magnetic particle isolation technics, uses biotin
Labelled antigen and antibody is distinguished, with the super paramagnetic microsphere being coated Streptavidin of diameter 1-3 μm with alkali phosphatase (ALP)
As separation agent.After ALP catalytic substrate luminescence, calculate testing concentration by apparatus measures luminous intensity.This detection side
Sensitivity and linear measurement range, on the basis of traditional film bar immunization and enzyme linked immunosorbent assay, is improved 3-5 number by method again
Magnitude, realize the detection by quantitative of real meaning, be swift in response, reliable results, and Full-automatic chemiluminescence immunity can be coordinated to divide
Analyzer realizes full-automatic use, clinical diagnosis is had to the important value that can not be substituted.
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-SLA/LP IgG antibody, described test kit includes:
1) anti-SLA/LP IgG antibody calibration object, containing anti-SLA/LP IgG antibody and the Tris buffer of bovine serum albumin,
Described anti-SLA/LP IgG antibody calibration object is the liquid calibration object of 6 levels, anti-in the liquid calibration object of described 6 levels
The concentration of SLA/LP IgG antibody is respectively 0,5,20,50,100,200RU/mL；
2) anti-SLA/LP IgG antibody quality-control product, containing anti-SLA/LP IgG antibody and the Tris buffer of bovine serum albumin,
Described anti-SLA/LP IgG antibody quality-control product comprises the liquid quality control product of 2 levels, anti-in the liquid quality control product of described 2 levels
The target value concentration range of SLA/LP IgG antibody is respectively (20 ± 4) RU/mL and (100 ± 20) RU/mL；
3) reagent 1, containing biotin labeled SLA/LP antigen and the Tris buffer of bovine serum albumin；
4) reagent 2, the sheep anti-human polyclonal antibody containing alkali phosphatase enzyme mark and the Tris buffering of bovine serum albumin
Liquid；
5) Magneto separate reagent, the magnetic particle containing marked by streptavidin and the Tris buffer of bovine serum albumin；
6) cleanout fluid；
The content of described seminal plasma fructose detection kit 1, reagent 2 and Magneto separate reagent is than for 1:3:1, and described content is than for body
Long-pending ratio.
Further, the material of described magnetic particle is Fe2O3；Described magnetic particle pan coating has carboxylic group, is coated in thing
Carboxyl group content is more than 20wt%, and the size of described magnetic particle is 1-3 μm.
A kind of method of the magnetic microparticle chemiluminescence quantitative determination reagent kit preparing described anti-SLA/LP IgG antibody, the party
Method comprises the steps:
(1) anti-SLA/LP IgG antibody calibration object is prepared:
A. anti-SLA/LP IgG antibody calibration object diluent is prepared:
Purified water, Tris, sodium chloride and Proclin300 being added in container, be stirred well to be completely dissolved, Tris is dense
Degree is 1wt%, and sodium chloride concentration is 1wt%, and Proclin300 concentration is 0.2v%；With the HCL of 4M, the pH value of solution is adjusted to
7.0-7.5；Being added by bovine serum albumin in container, be 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 the filter that aperture is 0.2 μm filter anti-SLA/LP resist
Body IgG calibration object diluent, 2-8 DEG C of preservation is stand-by；
B. anti-SLA/LP IgG antibody calibration object is prepared:
It is 0 by anti-SLA/LP IgG antibody calibration object diluted to each concentration point by anti-SLA/LP IgG antibody, 5,20,
50,100,200RU/mL；
(2) anti-SLA/LP IgG antibody quality-control product is prepared:
It is 20 by above-mentioned anti-SLA/LP IgG antibody calibration object diluted to each concentration point by anti-SLA/LP IgG antibody,
100RU/mL；
(3) reagent preparation 1:
A. No. 1 diluent of reagent preparation:
Purified water, Tris, sodium chloride and Proclin300 are added in container, is stirred well to be completely dissolved, Tris's
Concentration is 1wt%, sodium chloride concentration be the concentration of 0.5wt%, Proclin300 be 0.2v%；Bovine serum albumin is added and holds
In device, being stirred well to be completely dissolved, 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；Filtering to obtain No. 1 diluent of reagent with the filter that aperture is 0.2 μm, 2-8 DEG C of preservation is stand-by；
B. reagent preparation 1:
By SLA/LP antigen with purified water dissolve, under the conditions of 2-8 DEG C with concentration be 0.2M, pH be the carbonate buffer of 9.0
Liquid dialysis 2h, be then concentrated into the antigenic solution that concentration is 2-4mg/mL, with concentration be 0.2M, pH be 8.5-9 carbonate delay
Rush the biotin solution that liquid compound concentration is 0.5-1.0mg/ml；According to the ratio that SLA/LP antigen and biotin mass ratio are 10:1
Example adds biotin solution, mix homogeneously in SLA/LP antigenic solution, and room temperature stands 12-18h, and reaction generates SLA/LP and resists
Former-biotin conjugate；By the reactant liquor containing SLA/LP 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 changing liquid, thus removes unreacted biotin, obtains
Solution containing SLA/LP antigen-biotin conjugate；To connect containing SLA/LP antigen-biotin with No. 1 diluent of reagent
The solution of thing is diluted to 0.1-0.3 μ g/mL, prepares reagent 1；
The reagent 1 of this step preparation can reduce experimental cost, and can efficiently separate free biotin and junctional complex,
The junctional complex arrived is purer, decreases non-specific responding；
(4) reagent preparation 2:
A. No. 2 diluents of reagent preparation:
By purified water, 4-hydroxyethyl piperazine ethanesulfonic acid, sodium chloride, bovine serum albumin, ZnCl2Add with Proclin300
In container, being stirred well to be completely dissolved, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 0.6wt%, and sodium chloride concentration is
0.8wt%, 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, the pH value of solution is adjusted to 7.5-8.0；With the filter that aperture is 0.2 μm
Device filters to obtain No. 2 diluents of reagent, and 2-8 DEG C of preservation is stand-by；
B. reagent preparation 2:
1mg sheep anti-human polyclonal antibody is joined the 2-iminothiolane HCI solution that 2-4 μ L concentration is 10mg/mL
In, room temperature stands 20min, adds the glycine solution 10 μ L of 0.1moL/L, and room temperature stands 5min, removes with G-25 gel column
Salt, collects the sheep anti-human polyclonal antibody after activation, and 2-8 DEG C saves backup；The alkali phosphatase of 1.5mg is joined 10-20 μ
The concentration of L is that in 4-(N-maleimidomethyl) hexamethylene-1-carboxylic acid butanimide ester solution of 5mg/mL, room temperature is quiet
Putting 30min, with G-25 gel column desalination, collect the alkali phosphatase after activation, 2-8 DEG C saves backup；Goat-anti people by activation
Polyclonal antibody mixes with the alkali phosphatase of activation, stands 12-24h, use Supperdex200 gel-purified under the conditions of 2-8 DEG C
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；By goat-anti
People's polyclonal antibody-alkali phosphatase junctional complex concentrated solution to 0.02-0.1 μ g/mL, prepares examination by No. 2 diluted of reagent
Agent 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 bovine serum albumin, new-born calf serum and Proclin300 are added in container, fully
Stirring is to being completely dissolved, and the concentration of bovine serum albumin is 0.5wt%, and new-born calf serum concentration is that 5v%, Proclin300 are dense
Degree is 0.2v ‰；With the HCL of 4M, the pH value of solution is adjusted to 7.9-8.1；With the filter that aperture is 0.2 μm filter magnetic particle delay
Rushing liquid, 2-8 DEG C of preservation is stand-by；
B. preparation Magneto separate reagent:
Taking 100mg magnetic particle, use magnetic frame absorption, suck supernatant after static 2min, adding concentration in magnetic particle is
0.025mol/L, pH are 2-(N-morpholine) the ethanesulfonic acid buffer 10mL of 4.5-5, fully mix；Add 0.5-1mL newly to join
The concentration of system is 1-(3-the dimethylamino-propyl)-3-ethyl carbodiimide of 10mg/mL and N-hydroxy-succinamide is water-soluble
Liquid, room temperature mixing 30-60min obtains magnetic bead suspending system；Using 2-(N-morpholine) ethanesulfonic acid buffer compound concentration is 5mg/
The solution of streptavidin of mL, the strepto-of the 0.8-1.6mL being then directly added into described concentration in magnetic bead suspending system is affine
Element, suspendible 16-20h under the conditions of 4 DEG C；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, suck after static 2min
Clearly, 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-ethyl carbon
Diimine plays Stabilization；Add after Streptavidin suspendible under the conditions of 4 degree, it is possible to preferably retaining protein is active,
Reduce the room temperature change impact on coupling effect simultaneously, make coupling result between batch more stable；Add ethanolamine, second can be made
Amino in alcohol amine molecule is not associated with the avtive spot of albumen and reacts after activating with magnetic bead, play termination reaction and sealing process,
Lower background values can be produced；
(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, is stirred well to mix completely,
The concentration of Tween-20 be the concentration of 0.5wt%, Triton100 be 0.5wt%；With the HCL of 4M, the pH value of solution is adjusted to
7.5-8.0, filters to obtain cleanout fluid, 2-8 DEG C of preservation with the filter that aperture is 0.2 μm.
The detection method of the magnetic microparticle chemiluminescence quantitative determination reagent kit of anti-SLA/LP IgG antibody, the method include as
Lower step:
1) anti-SLA/LP IgG antibody calibration object is put into Full-automatic chemiluminescence immunoassay analysis meter test position, obtain by
The matched curve of Full-automatic chemiluminescence immunoassay analysis meter output；
2) anti-SLA/LP IgG antibody quality-control product is put into above-mentioned analyser test position, obtains by Full-automatic chemiluminescence
Test luminous value and the matched curve matching obtained by step 1 of the described quality-control product of immunity analysis instrument output obtain anti-SLA/
The concentration value of LP IgG antibody 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,
Obtain 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-SLA/LP IgG antibody calibration object or quality-control product or 1:20 dilution is added to detecting pipe
In；
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 incubation 5min, enter
Row Magneto separate, removes supernatant；
4) cleanout fluid of 300 μ L is added to step 3) detect in 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) detect in pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min, carry out magnetic and divide
From, remove supernatant；
7) cleanout fluid of 300 μ L is added to step 6) detect in 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) detect in pipe, mixing, detects luminous intensity；
Described step 1), step 2) and step 3) all include Full-automatic chemiluminescence immunoassay analysis meter fully-automated synthesis step
Suddenly.
Beneficial effects of the present invention is as follows:
The invention discloses a kind of new technique measuring anti-SLA/LP IgG antibody so that course of reaction more fast and reliable,
Improve sensitivity and linear measurement range, it is achieved the quantitative determination of real meaning, and automatic chemiluminescence immunoassay of can arranging in pairs or groups
Instrument realizes full automatic use, is greatly improved work efficiency；Calibration object in test kit, biotin labeling reagent, enzyme labelling examination
Agent, Magneto separate reagent and cleanout fluid etc. are all the optimization formulas under this reaction system, imitate phase and detection to the use of this test kit
Performance provides powerful guarantee.
Accompanying drawing explanation
Fig. 1 a is concentration value between zero-dose calibration object with adjacent calibration object and luminous value during the blank limit of embodiment 7 is evaluated
Result carries out the linear function that 2 regression fits draw；
Fig. 1 b is concentration value between zero-dose calibration object with adjacent calibration object and luminous value during the blank limit of comparative example 1 is evaluated
Result carries out the linear function that 2 regression fits draw；
Fig. 2 is that in embodiment 7 range of linearity evaluation, concentration of specimens meansigma methods and dilution ratio method of least square are carried out directly
Line fit 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
Bright.
Embodiment 1
The preparation of anti-SLA/LP IgG antibody calibration object:
A. anti-SLA/LP IgG antibody calibration object diluent is prepared:
The purified water of 800ml, Tris, 8.6g sodium chloride of 11.2g and 2ml Proclin300 are added in container, fully
Stirring is to being completely dissolved；With the HCL of 4M, the pH value of solution is adjusted to 7.0-7.5；40g bovine serum albumin is added in container,
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 again；By purified water, solution is settled to 1L,
Filtering to obtain anti-SLA/LP IgG antibody calibration object diluent with 0.2 μm filter, 2-8 DEG C of preservation is stand-by；
B. anti-SLA/LP IgG antibody calibration object is prepared:
It is 0 by anti-SLA/LP IgG antibody calibration object diluted to each concentration point by anti-SLA/LP IgG antibody, 5,20,
50,100,200RU/mL.
Embodiment 2
The preparation of anti-SLA/LP IgG antibody quality-control product:
It is 20 by above-mentioned anti-SLA/LP IgG antibody calibration object diluted to each concentration point by anti-SLA/LP IgG antibody,
100RU/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, fully stirs
Mix to being completely dissolved；5g bovine serum albumin is added in container, is stirred well to be completely dissolved；With the HCL of 4M by solution
PH value is adjusted to 7.0-7.5；By purified water, solution is settled to 1L, filters to obtain No. 1 diluent of reagent, 2-8 DEG C of guarantor with 0.2 μm filter
Deposit stand-by；
B. reagent preparation 1:
By SLA/LP antigen with purified water dissolve, under the conditions of 2-8 DEG C with concentration be 0.2M, pH be the carbonate buffer of 9.0
Liquid dialysis 2h, be then concentrated into the antigenic solution that concentration is 2-4mg/mL, with concentration be 0.2M, pH be 8.5-9 carbonate delay
Rush the biotin solution that liquid compound concentration is 0.5-1.0mg/ml；According to the ratio that SLA/LP antigen and biotin mass ratio are 10:1
Example adds biotin solution, mix homogeneously in SLA/LP antigenic solution, and room temperature stands 12-18h, and reaction generates SLA/LP and resists
Former-biotin conjugate；By the reactant liquor containing SLA/LP 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 changing liquid, thus removes unreacted biotin, obtains
Solution containing SLA/LP antigen-biotin conjugate；To connect containing SLA/LP antigen-biotin with No. 1 diluent of reagent
The solution of thing is diluted to 0.1-0.3 μ g/mL, prepares reagent 1；
The reagent 1 of this step preparation can reduce experimental cost, and can efficiently separate free biotin and junctional complex,
The junctional complex arrived is purer, decreases non-specific responding.
Embodiment 4
The preparation that reagent 2:
A. No. 2 diluents of reagent preparation:
By 800ml purified water, the 4-hydroxyethyl piperazine ethanesulfonic acid of 6.06g, 8.5g sodium chloride, 5g bovine serum albumin,
0.1gZnCl2, 0.2ml Proclin300 and 0.1g MgCl2Add in container, be stirred well to be completely dissolved；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, filters to obtain reagent 2 dilution with 0.2 μm filter
Liquid, 2-8 DEG C of preservation is stand-by；
B. reagent preparation 2:
1mg sheep anti-human polyclonal antibody is joined the 2-iminothiolane HCI solution that 2-4 μ L concentration is 10mg/mL
In, room temperature stands 20min, adds the glycine solution 10 μ L of 0.1moL/L, and room temperature stands 5min, removes with G-25 gel column
Salt, collects the sheep anti-human polyclonal antibody after activation, and 2-8 DEG C saves backup；The alkali phosphatase of 1.5mg is joined 10-20 μ
The concentration of L is that in 4-(N-maleimidomethyl) hexamethylene-1-carboxylic acid butanimide ester solution of 5mg/mL, room temperature is quiet
Putting 30min, with G-25 gel column desalination, collect the alkali phosphatase after activation, 2-8 DEG C saves backup；Goat-anti people by activation
Polyclonal antibody mixes with the alkali phosphatase of activation, stands 12-24h, use Supperdex200 gel-purified under the conditions of 2-8 DEG C
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；By goat-anti
People's polyclonal antibody-alkali phosphatase junctional complex concentrated solution to 0.02-0.1 μ g/mL, prepares examination by No. 2 diluted of reagent
Agent 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 be completely dissolved；Again
5g bovine serum albumin, 50mL new-born calf serum and 0.2mL Proclin300 are added in container, is stirred well to the most molten
Solve；With the HCL of 4M, the pH value of solution is adjusted to 7.9-8.1；By purified water, solution is settled to 1L, filters with 0.2 μm filter
Magnetic particle buffer, 2-8 DEG C of preservation is stand-by；
B. preparation Magneto separate reagent:
Taking 100mg magnetic particle, use magnetic frame absorption, suck supernatant after static 2min, adding concentration in magnetic particle is
0.025mol/L, pH are 2-(N-morpholine) the ethanesulfonic acid buffer 10mL of 4.5-5, fully mix；Add 0.5-1mL newly to join
The concentration of system is 1-(3-the dimethylamino-propyl)-3-ethyl carbodiimide of 10mg/mL and N-hydroxy-succinamide is water-soluble
Liquid, room temperature mixing 30-60min obtains magnetic bead suspending system；Using 2-(N-morpholine) ethanesulfonic acid buffer compound concentration is 5mg/
The solution of streptavidin of mL, the strepto-of the 0.8-1.6mL being then directly added into described concentration in magnetic bead suspending system is affine
Element, suspendible 16-20h under the conditions of 4 DEG C；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, suck after static 2min
Clearly, 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-ethyl carbon
Diimine plays Stabilization；Add after Streptavidin suspendible under the conditions of 4 degree, it is possible to preferably retaining protein is active,
Reduce the room temperature change impact on coupling effect simultaneously, make coupling result between batch more stable；Add ethanolamine, second can be made
Amino in alcohol amine molecule is not associated with the avtive spot of albumen and reacts after activating with magnetic bead, play termination reaction and sealing process,
Lower background values can be produced.
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 be completely dissolved；Again
5g Tween-20 and 5g Triton100 is added in container, is stirred well to mix completely；With the HCL of 4M by the pH of solution
Value is adjusted to 7.5-8.0 purified water and solution is settled to 1L, filters to obtain cleanout fluid, 2-8 DEG C of preservation with 0.2 μm filter.
Embodiment 7
The magnetic microparticle chemiluminescence quantitative determination reagent kit of anti-SLA/LP IgG antibody:
This test kit includes:
The anti-SLA/LP IgG antibody calibration object prepared according to embodiment 1 method, the calibration object consumption of each level is
0.5ml；
The anti-SLA/LP IgG antibody 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 antagonism SLA/LP IgG antibody using embodiment 7 carries out detection by quantitative:
This detection method includes:
1) specimen to be measured after 20 μ L anti-SLA/LP IgG antibody calibration object or quality-control product or 1:20 dilution is added to detecting pipe
In；
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 incubation 5min, enter
Row Magneto separate, removes supernatant；
4) cleanout fluid of 300 μ L is added to step 3) detect in 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) detect in pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min, carry out magnetic and divide
From, remove supernatant；
7) cleanout fluid of 300 μ L is added to step 6) detect in 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) detect in pipe, mixing, detects luminous intensity；
Described step 1), step 2) and step 3) all include Full-automatic chemiluminescence immunoassay analysis meter fully-automated synthesis step
Suddenly.
Comparative example 1
Reagent 1 in test kit unlike the test kit of embodiment 7 preparation and Magneto separate reagent, remaining becomes split-phase
With.
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, fully stirs
Mix to being completely dissolved；5g bovine serum albumin is added in container, is stirred well to be completely dissolved；With the HCL of 4M by solution
PH value is adjusted to 7.0-7.5；By purified water, solution is settled to 1L, filters to obtain No. 1 diluent of reagent, 2-8 DEG C of guarantor with 0.2 μm filter
Deposit 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 SLA/LP antigen
Adding biotin solution, mix homogeneously in SLA/LP antigenic solution with the ratio that biotin mass ratio is 10:1, room temperature stands
18h, reaction generates SLA/LP antigen-biotin conjugate；To pass through containing the reactant liquor of SLA/LP antigen-biotin conjugate
G-25 gel column separates, and removes unreacted biotin, obtains the solution containing SLA/LP antigen-biotin conjugate；
With No. 1 diluent of reagent, the solution containing SLA/LP antigen-biotin conjugate is diluted to 0.1-0.3 μ g/mL, prepares reagent
No. 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 be completely dissolved；Again
5g bovine serum albumin, 50mL new-born calf serum and 0.2mL Proclin300 are added in container, is stirred well to the most molten
Solve；With the HCL of 4M, the pH value of solution is adjusted to 7.9-8.1；By purified water, solution is settled to 1L, filters with 0.2 μm filter
Magnetic particle buffer, 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-morpholine) of 4.5-5
Ethanesulfonic acid buffer 10mL is resuspended；Add the EDC aqueous solution that concentration is 10mg/mL that 0.5-1mL newly prepares, room temperature suspendible 30-
60min；Make magnetic bead fully activate, Magneto separate, remove supernatant, with concentration be 0.025mol/L, pH be 4.5-5 2-(N-morpholine)
Ethanesulfonic acid buffer 10mL is resuspended；Add the Streptavidin of 4-8mg, room temperature suspendible 16-20h；Carry out Magneto separate again, go
Clearly, it is resuspended to 0.5mg/mL with the dilution of magnetic particle buffer, prepares 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
The anti-SLA/LP IgG antibody sample A that concentration is about 200RU/mL (allowing its concentration deviation is ± 20%) adds
In the sample B of serum or other corresponding substrate, the volume of added A is less than the 10% of cumulative volume (A+B), according to formula
(1) calculating response rate R, the response rate of this method is in the range of 85-115%, and 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 the RLU value of 20 measurement results
(relative light unit), calculates its meansigma methods (M) and standard deviation (SD), draws the RLU value corresponding to M+2SD, according to zero-dose school
Concentration-RLU value result between quasi-product with adjacent calibration object carries out 2 regression fits and draws linear function, by right for M+2SD institute
The RLU value answered is brought in above-mentioned equation, obtains the concentration value of correspondence, is blank limit.The blank limit of this method is not more than 1RU/
ML, data see table 2, and A, B point even some matched curve and fit equation are shown in Fig. 1 a, the sky that the test kit of comparative example 1 preparation records
White limit data are shown in Table 2-1, and A, B point even some matched curve and fit equation are shown in Fig. 1 b, from data, embodiment 7 and comparative example 1
Comparing, the background values obtained is lower, thus the blank limit obtained is lower, and the sensitivity representing 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 of concentration will be diluted to by a certain percentage close to the high level sample of the range of linearity upper limit (200RU/mL), its
The sample of middle low value concentration must be close to the lower limit of the range of linearity.Operate by test kit description, the sample to each concentration
All duplicate detection 2 times, calculates its meansigma methods, result meansigma methods and dilution ratio method of least square is carried out fitting a straight line, and
Calculating linearly dependent coefficient r, the measurement scope of this method is [2,200] RU/mL, and correlation coefficient r answers >=0.9900.Data see
Table 3, matched curve 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 (20 ± 4) RU/mL and the sample of (100 ± 20) RU/mL is each
10 times, calculate meansigma methods M and standard deviation SD of 10 measurement results, draw the coefficient of variation according to formula CV=SD/M × 100%
CV, this method coefficient of variation (CV) is not more than 8%, and data see table 4, the repeated number that the test kit of comparative example 1 preparation records
According to being 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
It is good,
CV=SD/M × 100%...................................... (2)
In formula: the CV-coefficient of variation；The standard deviation of SD-10 measurement result；The meansigma methods of M-10 measurement result.
Table 4 reproducibility
| Measure serum-concentration (RU/mL) | Measure number of times | CV (%) in analyzing |
| 20 | 10 | 5.15% |
| 100 | 10 | 3.65% |
Table 4-1 reproducibility
| Measure serum-concentration (IU/mL) | Measure number of times | CV (%) in analyzing |
| 100 | 10 | 7.16% |
| 400 | 10 | 7.35% |
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 in (20 ± 4) RU/mL and (100 ± 20)
Sample in the range of RU/mL, the every batch of replication 10 times, calculate meansigma methods (M) and standard deviation (SD), the root of 30 measurement results
Calculating the coefficient of variation (CV) according to formula (3), this method coefficient of variation (CV) is not more than 15%, and data see table 5, and comparative example 1 is made
The difference between batch data that standby test kit records are 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 SD-30 measurement result；The meansigma methods of M-30 measurement result.
Table 5 difference between batch evaluation
Table 5-1 difference between batch is evaluated
| Measure serum-concentration (IU/mL) | Measure number of times | CV (%) between analysis |
| 100 | 30 | 9.18% |
| 400 | 30 | 10.65% |
6. the Evaluation on specificity of embodiment 7
Taking 1 part of Anti-SLA/LP IgG content is the sample of 0, and adding anti-LKM-1 IgG antibody concentration is 200RU/mL, makes
With this test kit, this sample is detected, measures the Anti-SLA/LP IgG content in sample, the results are shown in Table 6, this method with
The anti-equal no cross reaction of LKM-1 IgG antibody, data see table 6, and evaluation result meets the requirements.
Table 6 specificity experiments
7. the relativity evaluation of embodiment 7
With test kit and anti-SLA/LP IgG antibody detection kit (enzyme linked immunosorbent assay) of commercialization of embodiment 7
240 parts of human serum samples are detected simultaneously.Its testing result sees accompanying drawing 3, with anti-SLA/LP IgG antibody detection kit
The result that (enzyme linked immunosorbent assay) measures is abscissa, and the result of mensuration in the process of the present invention is that vertical coordinate makees recurrence point
Analysis, dependent equation is: y=0.9952x+0.4455, and correlation coefficient is R2: 0.9886, show through statistical procedures result, we
Method is good with the test kit clinical sample measured value dependency of additive method.
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 test kit mark
The change of quasi-product luminous intensity, batch in and betweenrun precision, accuracy index all within normal range, test kit effect duration
Up to 12 months.
Obviously, the above embodiment of the present invention is only for clearly demonstrating example of the present invention, and is not right
The restriction of embodiments of the present invention, for those of ordinary skill in the field, on the basis of the above description can also
Enough make other changes in different forms, cannot all of embodiment be given exhaustive here, every belong to this
What bright technical scheme was extended out obviously changes or changes the row still in protection scope of the present invention.
Claims (5)
1. the magnetic microparticle chemiluminescence quantitative determination reagent kit of an anti-SLA/LP IgG antibody, it is characterised in that described test kit
Including:
1) anti-SLA/LP IgG antibody calibration object, containing anti-SLA/LP IgG antibody and the Tris buffer of bovine serum albumin, described
Anti-SLA/LP IgG antibody calibration object is the liquid calibration object of 6 levels, anti-SLA/LP in the liquid calibration object of described 6 levels
The concentration of IgG antibody is respectively 0,5,20,50,100,200RU/mL；
2) anti-SLA/LP IgG antibody quality-control product, containing anti-SLA/LP IgG antibody and the Tris buffer of bovine serum albumin, described
Anti-SLA/LP IgG antibody quality-control product comprises the liquid quality control product of 2 levels, anti-SLA/ in the liquid quality control product of described 2 levels
The target value concentration range of LP IgG antibody is respectively (20 ± 4) RU/mL and (100 ± 20) RU/mL；
3) reagent 1, containing biotin labeled SLA/LP antigen and the Tris buffer of bovine serum albumin；
4) reagent 2, the sheep anti-human polyclonal antibody containing alkali phosphatase enzyme mark and the Tris buffer of bovine serum albumin；
5) Magneto separate reagent, the magnetic particle containing marked by streptavidin and the Tris buffer of bovine serum albumin；
6) cleanout fluid；
The content of described seminal plasma fructose detection kit 1, reagent 2 and Magneto separate reagent is than for 1:3:1, and described content is than for 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%, and the size of described magnetic particle 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 method includes walking as follows
Rapid:
(1) anti-SLA/LP IgG antibody calibration object is prepared:
A. anti-SLA/LP IgG antibody calibration object diluent is prepared:
Purified water, Tris, sodium chloride and Proclin300 being added in container, be 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；Being added by bovine serum albumin in container, be stirred well to be completely dissolved, the concentration of bovine serum albumin is 4wt%；Again
With the HCL of 4M, the pH value of solution is adjusted to 7.0-7.5；The calibration of anti-SLA/LP IgG antibody is filtered to obtain with the filter that aperture is 0.2 μm
Product diluent, 2-8 DEG C of preservation is stand-by；
B. anti-SLA/LP IgG antibody calibration object is prepared:
It is 0 by anti-SLA/LP IgG antibody calibration object diluted to each concentration point by anti-SLA/LP IgG antibody, 5,20,50,
100,200RU/mL；
(2) anti-SLA/LP IgG antibody quality-control product is prepared:
It is 20 by above-mentioned anti-SLA/LP IgG antibody calibration object diluted to each concentration point by anti-SLA/LP IgG antibody,
100RU/mL；
(3) reagent preparation 1:
A. No. 1 diluent of reagent preparation:
Purified water, Tris, sodium chloride and Proclin300 are added in container, is stirred well to be completely dissolved, the concentration of Tris
For 1wt%, sodium chloride concentration be the concentration of 0.5wt%, Proclin300 be 0.2v%；Bovine serum albumin is added container
In, it being stirred well to be completely dissolved, 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；Filtering to obtain No. 1 diluent of reagent with the filter that aperture is 0.2 μm, 2-8 DEG C of preservation is stand-by；
B. reagent preparation 1:
By SLA/LP 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 saturating
Analysis 2h, be then concentrated into the antigenic solution that concentration is 2-4mg/mL, with concentration be 0.2M, pH be the carbonate buffer solution of 8.5-9
Compound concentration is the biotin solution of 0.5-1.0mg/ml；Exist according to the ratio that SLA/LP antigen and biotin mass ratio are 10:1
Adding biotin solution, mix homogeneously in SLA/LP antigenic solution, room temperature stands 12-18h, and reaction generates SLA/LP antigen-life
Thing element junctional complex；It is that 0.2MpH be by concentration by the reactant liquor containing SLA/LP antigen-biotin conjugate under the conditions of 2-8 DEG C
The carbonate buffer solution of 9.0 is dialysed 2 days, and period carries out 4 times changing liquid, thus removes unreacted biotin, obtains containing SLA/
The solution of LP antigen-biotin conjugate；With No. 1 diluent of reagent by the solution containing SLA/LP antigen-biotin conjugate
It is diluted to 0.1-0.3 μ g/mL, prepares reagent 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, ZnCl2Container is added with Proclin300
In, it being stirred well to be completely dissolved, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 0.6wt%, and sodium chloride concentration is 0.8wt%,
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, the pH value of solution is adjusted to 7.5-8.0；Filter with the filter that aperture is 0.2 μm
No. 2 diluents of reagent, 2-8 DEG C of preservation is stand-by；
B. reagent preparation 2:
1mg sheep anti-human polyclonal antibody is joined in the 2-iminothiolane HCI solution that 2-4 μ L concentration is 10mg/mL,
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, receives
Sheep anti-human polyclonal antibody after collection activation, 2-8 DEG C saves backup；The alkali phosphatase of 1.5mg is joined the dense of 10-20 μ L
In degree 4-(N-maleimidomethyl) hexamethylene-1-carboxylic acid butanimide ester solution for 5mg/mL, room temperature stands
30min, with G-25 gel column desalination, collects the alkali phosphatase after activation, and 2-8 DEG C saves backup；By many for the goat-anti people of activation
Clonal antibody mixes with the alkali phosphatase of activation, stands 12-24h, with Supperdex200 gel-purified post under the conditions of 2-8 DEG C
Purification conjugate, it is thus achieved that sheep anti-human polyclonal antibody-alkali phosphatase junctional complex concentrated solution, 2-8 DEG C saves backup；By goat-anti people
Polyclonal antibody-alkali phosphatase junctional complex concentrated solution to 0.02-0.1 μ g/mL, prepares reagent 2 by No. 2 diluted of reagent
Number；
(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%, chlorination
The concentration of sodium is 0.8wt%；Again bovine serum albumin, new-born calf serum and Proclin300 are added in container, be sufficiently stirred for
To being completely dissolved, the concentration of bovine serum albumin is 0.5wt%, and new-born calf serum concentration is that 5v%, Proclin300 concentration is
0.2v‰；With the HCL of 4M, the pH value of solution is adjusted to 7.9-8.1；Magnetic particle buffering is filtered to obtain with the filter that aperture is 0.2 μm
Liquid, 2-8 DEG C preserves stand-by magnetic；
B. preparation Magneto separate reagent:
Taking 100mg magnetic particle, use magnetic frame absorption, suck supernatant after static 2min, adding concentration in magnetic particle is
0.025mol/L, pH are 2-(N-morpholine) the ethanesulfonic acid buffer 10mL of 4.5-5, fully mix；Add 0.5-1mL newly to join
The concentration of system is 1-(3-the dimethylamino-propyl)-3-ethyl carbodiimide of 10mg/mL and N-hydroxy-succinamide is water-soluble
Liquid, room temperature mixing 30-60min obtains magnetic bead suspending system；Using 2-(N-morpholine) ethanesulfonic acid buffer compound concentration is 5mg/
The solution of streptavidin of mL, the strepto-of the 0.8-1.6mL being then directly added into described concentration in magnetic bead suspending system is affine
Element, suspendible 16-20h under the conditions of 4 DEG C；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, suck after static 2min
Clearly, in magnetic particle, add the dilution of appropriate magnetic particle buffer make final concentration of 0.5mg/mL, prepare 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%, chlorination
The concentration of sodium is 0.8wt%；Again Tween-20 and Triton100 is added in container, be stirred well to mix completely, Tween-
The concentration of 20 be the concentration of 0.5wt%, Triton100 be 0.5wt%；With the HCL of 4M, the pH value of solution is adjusted to 7.5-8.0,
Cleanout fluid, 2-8 DEG C of preservation is filtered to obtain with the filter that aperture is 0.2 μm.
4. the detection method of test kit as claimed in claim 1, it is characterised in that the method comprises the steps:
1) anti-SLA/LP IgG antibody calibration object is put into Full-automatic chemiluminescence immunoassay analysis meter test position, obtains by the most certainly
The matched curve of dynamic chemical illumination immunity analysis instrument output；
2) anti-SLA/LP IgG antibody quality-control product is put into above-mentioned analyser test position, obtains by Full-automatic chemiluminescence immunity
The test luminous value of the described quality-control product of analyser output and the matched curve matching obtained by step 1 are obtained anti-SLA/LP and resist
The concentration value of body 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, obtain
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 steps:
1) specimen to be measured after 20 μ L anti-SLA/LP IgG antibody calibration object or quality-control product or 1:20 dilution is added to detecting in 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 incubation 5min, carry out magnetic
Separate, remove supernatant；
4) cleanout fluid of 300 μ L is added to step 3) detect in 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) detect in pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min, carry out Magneto separate, go
Supernatant；
7) cleanout fluid of 300 μ L is added to step 6) detect in 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) detect in pipe, mixing, detects luminous intensity；
Described step 1), step 2) and step 3) all include the fully-automated synthesis step of Full-automatic chemiluminescence immunoassay analysis meter.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610249001.9A CN105929165A (en) | 2016-04-20 | 2016-04-20 | Magnetic particle-based quantitative chemiluminescent assay kit for anti-SLA/LP antibody IgG, and preparation and detection methods thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610249001.9A CN105929165A (en) | 2016-04-20 | 2016-04-20 | Magnetic particle-based quantitative chemiluminescent assay kit for anti-SLA/LP antibody IgG, and preparation and detection methods thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105929165A true CN105929165A (en) | 2016-09-07 |
Family
ID=56839344
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610249001.9A Pending CN105929165A (en) | 2016-04-20 | 2016-04-20 | Magnetic particle-based quantitative chemiluminescent assay kit for anti-SLA/LP antibody IgG, and preparation and detection methods thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105929165A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109357697A (en) * | 2018-09-29 | 2019-02-19 | 广州市康软信息科技有限公司 | Medical instrument calibration method, system and device based on quality evaluation target value calculation |
| CN109633169A (en) * | 2018-12-29 | 2019-04-16 | 郑州安图生物工程股份有限公司 | A kind of kit of quantitative and qualitative detection smooth muscle antibody |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6475804B1 (en) * | 1998-02-13 | 2002-11-05 | Ansgar W. Lohse | Composition and method for diagnosing auto-immune hepatitis |
| CN101750492A (en) * | 2008-12-04 | 2010-06-23 | 上海裕隆生物科技有限公司 | Self-immunity hepatitis detection protein chip and kit thereof |
| CN102707055A (en) * | 2012-06-11 | 2012-10-03 | 郑州安图绿科生物工程有限公司 | Kit for joint or single detection of autoimmune liver disease related antibody and detection method of kit |
| CN105259344A (en) * | 2015-11-16 | 2016-01-20 | 北京中航赛维生物科技有限公司 | Kit for quantitative detection of Scl-70 resisting antibody IgG through chemiluminiscence of magnetic particles and preparing method and detection method of kit |
| CN105300965A (en) * | 2015-11-16 | 2016-02-03 | 北京中航赛维生物科技有限公司 | Kit for quantitatively determining anti-nRNP/Sm antibody IgG by using magnetic particle chemiluminescence and detection method |
| CN105301235A (en) * | 2015-11-16 | 2016-02-03 | 北京中航赛维生物科技有限公司 | Kit used for quantitative determination anti-nucleosome antibody Ig G via magnetic micro particle chemiluminiscence, and preparation method and detection method thereof |
| CN105352946A (en) * | 2015-11-16 | 2016-02-24 | 北京中航赛维生物科技有限公司 | Kit for magnetic particle chemiluminescent quantitative detection of anti-SS-B antibody IgG and its preparation method and detection method |
| 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 |
| CN105467129A (en) * | 2015-11-16 | 2016-04-06 | 北京中航赛维生物科技有限公司 | Kit for quantitative detection of anti-M2-3E antibody IgG by using magnetic particle chemiluminescence, and preparation method and detection method thereof |
| CN105466913A (en) * | 2015-11-16 | 2016-04-06 | 北京中航赛维生物科技有限公司 | Kit for quantitative detection of anti-Jo-1 antibody IgG by using magnetic particle chemiluminescence, and preparation method and detection method thereof |
| CN105467130A (en) * | 2015-11-16 | 2016-04-06 | 北京中航赛维生物科技有限公司 | Kit for quantitative detection of anti-Sm antibody IgG by using magnetic particle chemiluminescence, and preparation method and detection method thereof |
-
2016
- 2016-04-20 CN CN201610249001.9A patent/CN105929165A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6475804B1 (en) * | 1998-02-13 | 2002-11-05 | Ansgar W. Lohse | Composition and method for diagnosing auto-immune hepatitis |
| CN101750492A (en) * | 2008-12-04 | 2010-06-23 | 上海裕隆生物科技有限公司 | Self-immunity hepatitis detection protein chip and kit thereof |
| CN102707055A (en) * | 2012-06-11 | 2012-10-03 | 郑州安图绿科生物工程有限公司 | Kit for joint or single detection of autoimmune liver disease related antibody and detection method of kit |
| CN105259344A (en) * | 2015-11-16 | 2016-01-20 | 北京中航赛维生物科技有限公司 | Kit for quantitative detection of Scl-70 resisting antibody IgG through chemiluminiscence of magnetic particles and preparing method and detection method of kit |
| CN105300965A (en) * | 2015-11-16 | 2016-02-03 | 北京中航赛维生物科技有限公司 | Kit for quantitatively determining anti-nRNP/Sm antibody IgG by using magnetic particle chemiluminescence and detection method |
| CN105301235A (en) * | 2015-11-16 | 2016-02-03 | 北京中航赛维生物科技有限公司 | Kit used for quantitative determination anti-nucleosome antibody Ig G via magnetic micro particle chemiluminiscence, and preparation method and detection method thereof |
| CN105352946A (en) * | 2015-11-16 | 2016-02-24 | 北京中航赛维生物科技有限公司 | Kit for magnetic particle chemiluminescent quantitative detection of anti-SS-B antibody IgG and its preparation method and detection method |
| 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 |
| CN105467129A (en) * | 2015-11-16 | 2016-04-06 | 北京中航赛维生物科技有限公司 | Kit for quantitative detection of anti-M2-3E antibody IgG by using magnetic particle chemiluminescence, and preparation method and detection method thereof |
| CN105466913A (en) * | 2015-11-16 | 2016-04-06 | 北京中航赛维生物科技有限公司 | Kit for quantitative detection of anti-Jo-1 antibody IgG by using magnetic particle chemiluminescence, and preparation method and detection method thereof |
| CN105467130A (en) * | 2015-11-16 | 2016-04-06 | 北京中航赛维生物科技有限公司 | Kit for quantitative detection of anti-Sm antibody IgG by using magnetic particle chemiluminescence, and preparation method and detection method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109357697A (en) * | 2018-09-29 | 2019-02-19 | 广州市康软信息科技有限公司 | Medical instrument calibration method, system and device based on quality evaluation target value calculation |
| CN109633169A (en) * | 2018-12-29 | 2019-04-16 | 郑州安图生物工程股份有限公司 | A kind of kit of quantitative and qualitative detection smooth muscle antibody |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105954266A (en) | Magnetic particle-based quantitative chemiluminescent assay kit for anti-ribosome P protein antibody IgG, and preparation and detection methods thereof | |
| CN105954267A (en) | Magnetic particle-based quantitative chemiluminescent assay kit for anti-histone antibody IgG, and preparation and detection methods thereof | |
| CN105929156A (en) | Magnetic particle-based quantitative chemiluminescent assay kit for anti-double-stranded DNA antibody IgG, and preparation and detection methods thereof | |
| CN105929166A (en) | Magnetic particle-based quantitative chemiluminescent assay kit for anti-LKM-1 antibody IgG, and preparation and detection methods thereof | |
| CN101201354B (en) | Thyroxine chemiluminescence immune analysis quantitative measuring reagent kit and method for preparing the same | |
| CN102648413B (en) | Multisite biosensor and associated method | |
| US8956823B2 (en) | Anti-antibody reagent | |
| CN103698535B (en) | Platelet-activating factor acetylhydro-lase immue quantitative detection reagent box and preparation, method of operating | |
| CN110862881A (en) | Special cleaning solution or diluent for full-automatic chemiluminescence determinator and preparation method thereof | |
| CN102866249B (en) | Tetramethylbenzidine (TMB) developing system | |
| CN105352946A (en) | Kit for magnetic particle chemiluminescent quantitative detection of anti-SS-B antibody IgG and its preparation method and detection method | |
| CN105466913A (en) | Kit for quantitative detection of anti-Jo-1 antibody IgG by using magnetic particle chemiluminescence, and preparation method and detection method thereof | |
| CN105203748A (en) | Full-range quantitative detection kit for C-reactive protein | |
| CN107044977A (en) | A kind of tyrosine phosphatase antibody chemical luminescence immunity detection reagent and preparation method thereof | |
| CN102841206B (en) | TnT (Troponin-T, TNT) measures kit | |
| CN108761088A (en) | Composition, kit and method and purposes for the detection of sugar chain abnormal Protein Separation | |
| CN105929172A (en) | Magnetic particle-based quantitative chemiluminescent assay kit for anti-PM-Scl antibody IgG, and preparation and detection methods thereof | |
| CN105277717A (en) | Magnetic particle separation chemiluminescence immunoassay method for thyroglobulin | |
| CN104237513A (en) | Thyroid peroxidase antibody magnetic-particle chemiluminescence immune quantitative testing kit | |
| CN106501535A (en) | A kind of compound releasing agent of ANS salt and the kit for detecting Blood cortisol | |
| CN105445456A (en) | Kit for quantitatively detecting anti-SS-A antibody IgG by utilizing magnetic particle chemiluminescence, preparation method and detection method thereof | |
| CN1963512A (en) | Chemiluminescence method for qualitative and quantitative detection of hepatitis B virus | |
| CN109001471A (en) | Free beta-human chorionic gonadotropin chemiluminescence detection kit and preparation method thereof and application method | |
| CN105181694A (en) | Carcino-embryonic antigen latex enhanced immunoturbidimetric kit and application thereof | |
| CN106324254A (en) | Anti-insulin antibody detection kit and detection method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160907 |