CN105784689A - Magnetic particle chemiluminescence quantitative determination reagent kit of anti-centromere antibody IgG and preparation and detection method - Google Patents
Magnetic particle chemiluminescence quantitative determination reagent kit of anti-centromere antibody IgG and preparation and detection method Download PDFInfo
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Abstract
The invention discloses a magnetic particle chemiluminescence quantitative determination reagent kit of anti-centromere antibody IgG. The reagent kit comprises an anti-centromere antibody IgG calibration material, an anti-centromere antibody IgG quality control product, a Tris buffering solution containing biotin-labeled centromere antigens and bovine serum albumin, a Tris buffering solution containing alkaline phosphatase-labeled goat-anti-human polyclonal antibodies and bovine serum albumin, a Tris buffering solution containing streptavidin-labeled magnetic particles and bovine serum albumin, and a cleaning solution. According to a detection method of the reagent kit, on the basis of a traditional membrane strip immunization method and enzyme linked immunosorbent assay, sensitivity and the linear range are improved by 3-5 magnitude orders, quantitative determination in real sense is achieved, responding is fast, the result is reliable, and the magnetic particle chemiluminescence quantitative determination reagent kit can be full automatically used in cooperation with a full-automatic chemiluminescence immunity analyzer and has irreplaceable important value on clinical diagnosis.
Description
Technical field
The present invention relates to technical field of biological, the magnetic microparticle chemiluminescence particularly relating to a kind of anti-centromere antibody IgG is fixed
Measure and determine test kit and preparation and detection method.
Background technology
Anti-centromere antibody (anticentromere antibody, ACA) refers to that a class identification is present in the albumen in centromere region
The antibody of matter antigen.It is currently known three kinds of dominant autoantigen that CENP-B, CENP-A and CENP-C are ACA.ACA
It is the biomarker of some autoimmune diseasees diagnosis, especially for local scleroderma (limited cutaneous
Systemic sclerosis, lcssc) diagnosis significant.The systemic sclerosis diagnosis that ACA is well recognized as at present is with pre-
The important biomolecule label of rear judgement.
The detection of anti-centromere antibody IgG has film bar immunization and enzyme linked immunosorbent assay the most clinically.Film bar immunization is applied
Be film bar developing technology, its feature measures at same film bar for fixing several projects, general by by hand or semi-automatic film bar
Instrument carries out experimental implementation, qualitatively judges eventually through naked eyes, and this sensitivity is low, and the response time is long, and detection project is only
Can regular collocation combine, very flexible.The sensitivity of enzyme linked immunosorbent assay has promoted on the basis of film bar immunization, but still
The most relatively low, and the range of linearity is narrow, poor repeatability, and the response time is long, still can not meet the application of clinic very well.
At present, magnetic microparticle chemiluminescence analytic process is used to have not yet to see in the application of anti-centromere antibody IgG immunoassay product.
Summary of the invention
It is an object of the present invention to provide the magnetic microparticle chemiluminescence quantitative determination reagent kit of a kind of anti-centromere antibody IgG,
Chemiluminescence analytical technique is combined by the test kit that the present invention provides with magnetic particle isolation technics, uses biotin and alkaline phosphatase
Enzyme (ALP) labelled antigen and antibody respectively, try using the superparamagnetic particles being coated Streptavidin of diameter 1-3 μm as separation
Agent.After ALP catalytic substrate luminescence, calculate testing concentration by apparatus measures luminous intensity.This detection method is traditional
On the basis of film bar immunization and enzyme linked immunosorbent assay, sensitivity and linear measurement range is improved again 3-5 the order of magnitude, it is achieved true
The detection by quantitative of positive meaning, is swift in response, reliable results, and it is full-automatic that Full-automatic chemiluminescence immunoassay analysis meter can be coordinated to realize
Use, clinical diagnosis is had to the important value that can not be substituted.
Further object is that the preparation method that a kind of mentioned reagent box is provided and the detection side using mentioned reagent box
Method.
For reaching above-mentioned purpose, the present invention uses following technical proposals:
A kind of magnetic microparticle chemiluminescence quantitative determination reagent kit of anti-centromere antibody IgG, described test kit includes:
(1) anti-centromere antibody IgG calibration object, the Tris buffer containing anti-centromere antibody IgG and bovine serum albumin,
Described anti-centromere antibody IgG calibration object comprises the liquid calibration object of 6 levels, anti-in the liquid calibration object of described 6 levels
The concentration of centromere IgG antibody is respectively 0,5,20,50,100,200RU/mL；
(2) anti-centromere antibody IgG quality-control product, the Tris buffer containing anti-centromere antibody IgG and bovine serum albumin,
Described anti-centromere antibody IgG 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 centromere IgG antibody is respectively (20 ± 4) RU/mL and (100 ± 20) RU/mL；
(3) reagent 1, the Tris buffer containing biotin labeled Kinetochore antigen and 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.
Further, the material of described magnetic particle is Fe2O3；Described magnetic particle pan coating has carboxylic group, is coated carboxylic in thing
Base group content is more than 20wt%, and the size of described magnetic particle is 1-3 μm.
A kind of method of magnetic microparticle chemiluminescence quantitative determination reagent kit preparing described anti-centromere antibody IgG, the method includes
Following steps:
(1) preparation anti-centromere antibody IgG calibration object:
Step 1) preparation anti-centromere antibody IgG calibration object diluent:
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%；Use 4M again
HCL the pH value of solution is adjusted to 7.0-7.5；Filter with the filter that aperture is 0.2 μm, obtain anti-centromere antibody IgG calibration
Product diluent, 2-8 DEG C of preservation is stand-by；
Step 2) preparation anti-centromere antibody IgG calibration object:
It is 0 by anti-centromere antibody IgG anti-centromere antibody IgG calibration object diluted to each concentration point, 5,20,50,
100,200RU/mL；
(2) preparation anti-centromere antibody IgG quality-control product:
It is 20 by above-mentioned anti-centromere antibody IgG calibration object diluted to each concentration point by anti-centromere antibody IgG,
100RU/mL；
(3) reagent preparation 1:
Step 1) No. 1 diluent of reagent preparation:
Purified water, Tris, sodium chloride and Proclin300 being added in container, be 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%；Bovine serum albumin is added in container, fill
Dividing stirring to being 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 with the filter that aperture is 0.2 μm, obtain No. 1 diluent of reagent, 2-8 DEG C of preservation is stand-by；
Step 2) reagent preparation 1:
By Kinetochore antigen with purified water dissolve, under the conditions of 2-8 DEG C with concentration be 0.2M, pH be 9.0 carbonate delay
Rush liquid dialysis 2h, be then concentrated into the antigenic solution that concentration is 2-4mg/mL, with concentration be 0.2M, pH be the carbon of 8.5-9
Phthalate buffer compound concentration is the biotin solution of 0.5-1.0mg/ml；It is 10:1 according to Kinetochore antigen and biotin mass ratio
Ratio in Kinetochore antigen solution, add biotin solution, mix homogeneously, room temperature stands 12-18h, and reaction generates centromere
Antigen-biotin conjugate；By the reactant liquor containing Kinetochore 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 the solution containing Kinetochore antigen-biotin conjugate；With No. 1 diluent of reagent will containing Kinetochore antigen-
The solution of biotin conjugate 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 Kinetochore antigen-biology
Element junctional complex, the Kinetochore antigen-biotin conjugate obtained is purer, decreases follow-up nonspecific reaction；
(4) reagent preparation 2:
Step 1) 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%, cattle
Sero-abluminous concentration is 0.5wt%, ZnCl2Concentration be 0.1wt ‰, the concentration of Proclin300 is 0.2v ‰, MgCl2
Concentration 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,
Obtaining No. 2 diluents of reagent, 2-8 DEG C of preservation is stand-by；
Step 2) 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,
Collecting the sheep anti-human polyclonal antibody after activation, 2-8 DEG C saves backup；The alkali phosphatase of 1.5mg is joined 10-20 μ L's
Concentration is in 4-(N-maleimidomethyl) hexamethylene-1-carboxylic acid butanimide ester solution of 5mg/mL, and 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, obtains reagent 2 by No. 2 diluted of reagent
Number；
(5) preparation Magneto separate reagent:
Step 1) 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 stirred well to
Being completely dissolved, the concentration of bovine serum albumin is 0.5wt%, new-born calf serum 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；Filter with the filter that aperture is 0.2 μm, obtain magnetic particle buffer,
2-8 DEG C of preservation is stand-by；
Step 2) 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
The concentration of new preparation 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, obtain magnetic bead suspending system；Using 2-(N-morpholine) ethanesulfonic acid buffer compound concentration is 5mg/mL
Solution of streptavidin, in magnetic bead suspending system, be then directly added into the Streptavidin of 4-8mg, suspendible under the conditions of 4 DEG C
16-20h；Re-using magnetic frame absorption, suck supernatant after static 2min, adding 10mL concentration in magnetic particle is 1M, pH
It is the ethanolamine solutions of 8.5, room temperature reaction 1-2h, re-use magnetic frame absorption, suck supernatant after static 2min, to magnetic particle
The dilution of middle addition appropriate magnetic particle buffer makes final concentration of 0.5mg/mL, prepares Magneto separate reagent；
In this step, adding N-hydroxy-succinamide can play coupling agent 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide
Stabilization；Add after Streptavidin suspendible under the conditions of 4 degree, can preferably retaining protein active, reduce room temperature simultaneously
The change impact on coupling effect, makes coupling result between batch more stable；Add ethanolamine, can make in ethanolamine molecules
Amino is not associated with the avtive spot of albumen and reacts after activating with magnetic bead, play termination reaction and sealing process, can produce lower basis
Floors.
(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-20
The concentration that concentration is 0.5wt%, Triton100 be 0.5wt%；With the HCL of 4M, the pH value of solution is adjusted to 7.5-8.0,
Filter with 0.2 μm filter, obtain cleanout fluid, 2-8 DEG C of preservation.
The detection side of the magnetic microparticle chemiluminescence quantitative determination reagent kit detection anti-centromere antibody IgG of anti-centromere antibody IgG
Method, the method comprises the steps:
(1) anti-centromere antibody IgG 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-centromere antibody IgG 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 anticentromere 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.
Further, the method specifically includes following steps:
(1) specimen to be measured after 20 μ L anti-centromere antibody IgG calibration objects or quality-control product or 1:20 dilution is added to detecting in pipe；
(2) reagent 1 of 50 μ L is added in step (1) described detection pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min；
(3) the Magneto separate reagent of 50 μ L is added in step (2) described detection pipe, after mixing, 37 ± 0.5 DEG C of incubation 5min,
Carry out Magneto separate, remove supernatant；
(4) cleanout fluid of 300 μ L is added in step (3) described detection pipe, mixing, carry out Magneto separate, remove supernatant；
(5) step (4) twice is repeated；
(6) 150 μ L reagent 2 is added in step (5) described detection pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min, enter
Row Magneto separate, removes supernatant；
(7) cleanout fluid of 300 μ L is added in step (6) described detection pipe, mixing, carry out Magneto separate, remove supernatant；
(8) step (7) twice is repeated；
(9) add in chemical luminous substrate extremely step (8) described detection pipe of 200 μ L, mixing, detects luminous intensity；
Described step (1), step (2) and step (3) all include the full-automatic inspection of Full-automatic chemiluminescence immunoassay analysis meter
Survey step.
Beneficial effects of the present invention is as follows:
The invention discloses a kind of new technique measuring anti-centromere antibody IgG 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 Full-automatic chemiluminescence immunoassay analysis meter realization of arranging in pairs or groups is complete
Use automatically, improve work efficiency；Calibration object in test kit, biotin labeling reagent, enzyme labelling reagent, Magneto separate examination
Agent and cleanout fluid etc. are all the more excellent formula under this reaction system, imitate the phase to the use of this test kit and detection performance provides effectively
Ensure.
Accompanying drawing explanation
Fig. 1 be during described in embodiment 7, test kit blank limit is evaluated concentration value between zero-dose calibration object with adjacent calibration object with send out
Light value result carries out the linear function that 2 regression fits draw；
Fig. 2 be during the test kit blank limit of comparative example 1 preparation 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. 3 is that in range of linearity evaluation, concentration of specimens meansigma methods and dilution ratio method of least square carry out fitting a straight line equation；
Fig. 4 is this method test kit clinical sample measured value dependency scatterplot with additive method.
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 described further.
Embodiment 1
The preparation of anti-centromere antibody IgG calibration object:
Step 1) preparation anti-centromere antibody IgG calibration object diluent:
The purified water of 800ml, Tris, 8.6g sodium chloride of 11.2g and 2ml Proclin300 are added in container, is sufficiently stirred for
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, filters to obtain anti-centromere antibody IgG calibration object diluent with 0.2 μm filter, and 2-8 DEG C of preservation is stand-by；
Step 2) preparation anti-centromere antibody IgG calibration object:
It is 0 by anti-centromere antibody IgG anti-centromere antibody IgG calibration object diluted to each concentration point, 5,20,50,
100,200RU/mL.
Embodiment 2
The preparation of anti-centromere antibody IgG quality-control product:
It is 20 by above-mentioned anti-centromere antibody IgG calibration object diluted to each concentration point by anti-centromere antibody IgG,
100RU/mL。
Embodiment 3
The preparation that reagent 1:
Step 1) No. 1 diluent of reagent preparation:
800ml purified water, Tris, 5.8g sodium chloride of 12.1g and 2ml Proclin300 are added in container, is stirred well to
It is 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 with 0.2 μm filter, 2-8 DEG C
Preserve stand-by；
Step 2) reagent preparation 1:
By Kinetochore antigen with purified water dissolve, under the conditions of 2-8 DEG C with concentration be 0.2M, pH be 9.0 carbonate delay
Rush liquid dialysis 2h, be then concentrated into the antigenic solution that concentration is 2-4mg/mL, with concentration be 0.2M, pH be the carbon of 8.5-9
Phthalate buffer compound concentration is the biotin solution of 0.5-1.0mg/ml；It is 10:1 according to Kinetochore antigen and biotin mass ratio
Ratio in Kinetochore antigen solution, add biotin solution, mix homogeneously, room temperature stands 12-18h, and reaction generates centromere
Antigen-biotin conjugate；By the reactant liquor containing Kinetochore 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 the solution containing Kinetochore antigen-biotin conjugate；With No. 1 diluent of reagent will containing Kinetochore antigen-
The solution of biotin conjugate is diluted to 0.1-0.3 μ g/mL, prepares reagent 1；
Embodiment 4
The preparation that reagent 2:
Step 1) 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 by solution
PH value is adjusted to 7.5-8.0；By purified water, solution is settled to 1L, filters to obtain No. 2 diluents of reagent with 0.2 μm filter, 2-8 DEG C
Preserve stand-by；
Step 2) reagent preparation 2:
1mg sheep anti-human polyclonal antibody is joined in the 2-imines Tetramethylene sulfide solution that 2-4 μ L concentration is 10mg/mL, room
Gentle and quiet putting 20min, add the glycine solution 10 μ L of 0.1moL/L, room temperature stands 5min, with G-25 gel column desalination,
Collecting the sheep anti-human polyclonal antibody after activation, 2-8 DEG C saves backup；The alkali phosphatase of 1.5mg is joined 10-20 μ L's
Concentration is in 4-(N-maleimidomethyl) hexamethylene-1-carboxylic acid butanimide ester solution of 5mg/mL, and 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.
Embodiment 5
The preparation of Magneto separate reagent:
Step 1) 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 by 5g
Bovine serum albumin, 50mL new-born calf serum and 0.2mL Proclin300 add in container, are stirred well to be completely dissolved；With
The pH value of solution is adjusted to 7.9-8.1 by the HCL of 4M；By purified water, solution is settled to 1L, filters to obtain magnetic with 0.2 μm filter
Particle buffer liquid, 2-8 DEG C of preservation is stand-by；
Step 2) 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
The concentration of new preparation 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, obtain magnetic bead suspending system；Make magnetic particle fully activate, use 2-(N-morpholine) ethyl sulfonic acid to delay
Rush the solution of streptavidin that liquid compound concentration is 5mg/mL, in magnetic bead suspending system, be then directly added into the strepto-of 4-8mg
Avidin, suspendible 16-20h under the conditions of 4 DEG C；Re-use magnetic frame absorption, suck supernatant after static 2min, add in magnetic particle
Enter 10mL concentration be 1M, pH be the ethanolamine solutions of 8.5, room temperature reaction 1-2h, re-use magnetic frame absorption, static 2min
After suck supernatant, add in magnetic particle appropriate magnetic particle buffer dilution make final concentration of 0.5mg/mL, prepare Magneto separate reagent；
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 by 5g
Tween-20 and 5g Triton100 adds in container, is stirred well to mix completely；With the HCL of 4M by the pH value of solution
It is adjusted to 7.5-8.0 purified water and solution is settled to 1L, filter 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-centromere antibody IgG
This test kit includes:
The anti-centromere antibody IgG calibration object prepared according to embodiment 1 method, the calibration object consumption of each level is 0.5ml；
The anti-centromere 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 antagonism centromere IgG antibody using embodiment 7 carries out detection by quantitative:
(1) specimen to be measured after 20 μ L anti-centromere antibody IgG calibration objects or quality-control product or 1:20 dilution is added to detecting in pipe；
(2) reagent 1 of 50 μ L is added in step (1) described detection pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min；
(3) the Magneto separate reagent of 50 μ L is added in step (2) described detection pipe, after mixing, 37 ± 0.5 DEG C of incubation 5min,
Carry out Magneto separate, remove supernatant；
(4) cleanout fluid of 300 μ L is added in step (3) described detection pipe, mixing, carry out Magneto separate, remove supernatant；
(5) step (4) twice is repeated；
(6) 150 μ L reagent 2 is added in step (5) described detection pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min, enter
Row Magneto separate, removes supernatant；
(7) cleanout fluid of 300 μ L is added in step (6) described detection pipe, mixing, carry out Magneto separate, remove supernatant；
(8) step (7) twice is repeated；
(9) add in chemical luminous substrate extremely step (8) described detection pipe of 200 μ L, mixing, detects luminous intensity；
Comparative example 1
Identical with the test kit of embodiment 7 preparation, the except for the difference that reagent in test kit 1 and Magneto separate reagent.
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, is stirred well to
It is 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 with 0.2 μm filter, 2-8 DEG C
Preserve 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 Kinetochore antigen with
Biotin mass ratio is that the ratio of 10:1 adds biotin solution, mix homogeneously in Kinetochore antigen solution, and room temperature stands 18h,
Reaction generates Kinetochore antigen-biotin conjugate；To be coagulated by G-25 containing the reactant liquor of Kinetochore antigen-biotin conjugate
Glue post separates, and removes unreacted biotin, obtains the solution containing Kinetochore antigen-biotin conjugate；With reagent 1
Solution containing Kinetochore antigen-biotin conjugate is diluted to 0.1-0.3 μ g/mL by number diluent, 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 be completely dissolved；Again by 5g
Bovine serum albumin, 50mL new-born calf serum and 0.2mL Proclin300 add in container, are stirred well to be completely dissolved；
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；Adding the EDC aqueous solution that concentration is 10mg/mL that 0.5-1mL newly prepares, room temperature is mixed
Outstanding 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 magnetic again
Separate, remove supernatant, be resuspended to 0.5mg/mL with the dilution of magnetic particle buffer, prepare Magneto separate reagent.
Embodiment 9
The test kit of embodiment 7 and comparative example 1 is carried out performance evaluation:
1) accuracy estimating
With the test kit in embodiment 7, concentration is about the anticentromere of 200RU/mL (allowing its concentration deviation is ± 20%)
IgG antibody 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%, according to formula (1) calculate response rate R, the response rate of this method is in the range of 85-115%, and data see table 1,
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) blank limit is evaluated
With the test kit in embodiment 7 and comparative example 1, detect as sample with zero-dose calibration object, replication 20 times,
Draw the RLU value (relative light unit) of 20 measurement results, calculate its meansigma methods (M) and standard deviation (SD), draw
RLU value corresponding to M+2SD, carries out 2 points according to the concentration-RLU value result between zero-dose calibration object with adjacent calibration object
Regression fit draws linear function, the RLU value corresponding to M+2SD is brought in above-mentioned equation, obtains the concentration value of correspondence,
It is blank limit.The blank limit of this method is not more than 1RU/mL.Data see table 2, A, B point even some matched curve and matching
Equation is shown in that the blank limit data that Fig. 1, the test kit of comparative example 1 preparation record are shown in Table 2-1, A, B point even some matched curve and plan
Closing equation and see Fig. 2, from data, embodiment 7 is compared with comparative example 1, and the background values obtained is lower, thus the sky obtained
White limit 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) range of linearity evaluation
With the test kit in embodiment 7, the high level sample close to the range of linearity upper limit (200RU/mL) is diluted in proportion to
Few 5 kinds of 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, to often
The sample standard deviation duplicate detection of one concentration 2 times, calculates its meansigma methods, result meansigma methods and dilution ratio method of least square is carried out
Fitting a straight line, and calculate 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. 3, evaluation result meet the requirements.
Table 3 range of linearity evaluation
4) reproducibility
Test kit duplicate detection concentration in Example 7 and comparative example 1 is (20 ± 4) RU/mL and (100 ± 20) RU/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%
Going out coefficient of variation CV, this method coefficient of variation (CV) is not more than 8%.Data see table 4, the test kit of comparative example 1 preparation
The repeated data recorded are shown in Table 4-1, and from data, embodiment 7 is compared with comparative example 1, and the CV value obtained is lower, generation
The repeatability of table test kit is more preferably.
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 (%) between analysis |
| 20 | 10 | 5.73% |
| 100 | 10 | 5.54% |
Table 4-1 reproducibility
| Measure serum-concentration (RU/mL) | Measure number of times | CV (%) between analysis |
| 20 | 10 | 7.11% |
| 100 | 10 | 7.64% |
5) difference between batch evaluation
The test kit of embodiment 7 and comparative example 1 is respectively taken three batches, the every batch of test kit all measure concentration at (20 ± 4) RU/mL and
Sample in the range of (100 ± 20) RU/mL, the every batch of replication 10 times, calculate the meansigma methods (M) of 30 measurement results
With standard deviation (SD), calculating the coefficient of variation (CV) according to formula (3), this method coefficient of variation (CV) is not more than 15%.
Data see table 5, and the difference between batch data that the test kit of comparative example 1 preparation records are shown in Table 5-1, from data, embodiment 7 with
Comparative example 1 is compared, and the CV value obtained is lower, and 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
| Measure serum-concentration (RU/mL) | Measure number of times | CV (%) between analysis |
| 20 | 30 | 7.07% |
| 100 | 30 | 7.23% |
Table 5-1 difference between batch is evaluated
| Measure serum-concentration (RU/mL) | Measure number of times | CV (%) between analysis |
| 20 | 30 | 9.33% |
| 100 | 30 | 10.54% |
6) Evaluation on specificity
With the test kit in embodiment 7, taking 6 parts of Anti-Centromeres IgG content is the sample of 0, is separately added into anti-ribose
Body P protein antibodies, anti-nRNP/Sm antibody, anti-Sm antibody, Anti SS-B antibody, Anti-Scl-70, anti-Jo-1 are anti-
Body, making cross reaction substrate concentration in sample is 200RU/mL, uses this test kit to detect this sample, measures in sample
Anti-Centromeres IgG content.The results are shown in Table 6, this method resists with anti-ribosomal P protein antibody, anti-nRNP/Sm
Body, anti-Sm antibody, Anti SS-B antibody, Anti-Scl-70, anti-Jo-1 antibody no cross reaction.Data see table 6,
Evaluation result meets the requirements.
Table 6 specificity experiments
7) relativity evaluation
By the test kit of embodiment 7 and the anti-centromere antibody IgG detection kit (enzyme linked immunosorbent assay) of commercialization to 240
Part human serum sample detect simultaneously.Its testing result sees Fig. 4, and with anti-centromere antibody IgG detection kit, (enzyme joins
Immunoabsorption) result that measures is abscissa, the result of mensuration in the process of the present invention is that vertical coordinate makees regression analysis, relevant
Equation is: y=0.9831x+0.4727, and correlation coefficient is R2: 0.9896.Showing through statistical procedures result, this method is with other
The test kit clinical sample measured value dependency of method is good.
8) estimation of stability
The test kit of embodiment 7 carries out 4 DEG C of 12 months and the 37 DEG C of accelerated stabilities of 7 days experiments respectively, and result shows examination
The change of agent box standard substance luminous intensity, batch in and betweenrun precision, accuracy index all within normal range, test kit
Effect duration can reach 12 months.
Obviously, the above embodiment of the present invention is only for clearly demonstrating example of the present invention, and is not to the present invention
The restriction of embodiment, for those of ordinary skill in the field, can also be made other on the basis of the above description
The change of multi-form or variation, cannot give exhaustive to all of embodiment here, every belongs to technical scheme
That is 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-centromere antibody IgG, it is characterised in that described test kit
Including:
(1) anti-centromere antibody IgG calibration object, the Tris buffer containing anti-centromere antibody IgG and bovine serum albumin,
Described anti-centromere antibody IgG calibration object comprises the liquid calibration object of 6 levels, anti-in the liquid calibration object of described 6 levels
The concentration of centromere IgG antibody is respectively 0,5,20,50,100,200RU/mL；
(2) anti-centromere antibody IgG quality-control product, the Tris buffer containing anti-centromere antibody IgG and bovine serum albumin,
Described anti-centromere antibody IgG 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 centromere IgG antibody is respectively (20 ± 4) RU/mL and (100 ± 20) RU/mL；
(3) reagent 1, the Tris buffer containing biotin labeled Kinetochore antigen and 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 is micro-
Grain 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. preparing a method for test kit as described in any one of claim 1-2, it comprises the steps:
(1) preparation anti-centromere antibody IgG calibration object:
Step 1) preparation anti-centromere antibody IgG calibration object diluent:
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%；Use 4M again
HCL the pH value of solution is adjusted to 7.0-7.5；Filter with the filter that aperture is 0.2 μm, obtain anti-centromere antibody IgG calibration
Product diluent, 2-8 DEG C of preservation is stand-by；
Step 2) preparation anti-centromere antibody IgG calibration object:
It is 0 by anti-centromere antibody IgG anti-centromere antibody IgG calibration object diluted to each concentration point, 5,20,50,
100,200RU/mL；
(2) preparation anti-centromere antibody IgG quality-control product:
It is 20 by above-mentioned anti-centromere antibody IgG calibration object diluted to each concentration point by anti-centromere antibody IgG,
100RU/mL；
(3) reagent preparation 1:
Step 1) No. 1 diluent of reagent preparation:
Purified water, Tris, sodium chloride and Proclin300 being added in container, be 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%；Bovine serum albumin is added in container, fill
Dividing stirring to being 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 with the filter that aperture is 0.2 μm, obtain No. 1 diluent of reagent, 2-8 DEG C of preservation is stand-by；
Step 2) reagent preparation 1:
By Kinetochore 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 the carbonate of 8.5-9
Buffer concentration is the biotin solution of 0.5-1.0mg/ml；According to the ratio that Kinetochore antigen and biotin mass ratio are 10:1
Example adds biotin solution in Kinetochore antigen solution, mix homogeneously, and room temperature stands 12-18h, reaction generate Kinetochore antigen-
Biotin conjugate；It is 0.2M by concentration by the reactant liquor containing Kinetochore antigen-biotin conjugate under the conditions of 2-8 DEG C,
PH be 9.0 carbonate buffer solution dialyse 2 days, period carries out 4 times changing liquid, thus removes unreacted biotin,
Obtain the solution containing Kinetochore antigen-biotin conjugate；Will containing Kinetochore antigen-biotin even with No. 1 diluent of reagent
The solution connecing thing is diluted to 0.1-0.3 μ g/mL, prepares reagent 1；
(4) reagent preparation 2:
Step 1) 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%, cattle
Sero-abluminous concentration is 0.5wt%, ZnCl2Concentration be 0.1wt ‰, the concentration of Proclin300 is 0.2v ‰, MgCl2
Concentration 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,
Obtaining No. 2 diluents of reagent, 2-8 DEG C of preservation is stand-by；
Step 2) 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, 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
In the 4-that concentration is 5mg/mL (N-maleimidomethyl) hexamethylene-1-carboxylic acid butanimide ester solution of 10-20 μ L, room
Gentle and quiet putting 30min, with G-25 gel column desalination, collect the alkali phosphatase after activation, 2-8 DEG C saves backup；By activation
Sheep anti-human polyclonal antibody mixes with the alkali phosphatase of activation, stands 12-24h, coagulate with Supperdex200 under the conditions of 2-8 DEG C
Glue purification 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 sheep anti-human polyclonal antibody-alkali phosphatase junctional complex concentrated solution No. 2 diluted of reagent to 0.02-0.1 μ g/mL, system
Obtain reagent 2；
(5) preparation Magneto separate reagent:
Step 1) 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 stirred well to
Being completely dissolved, the concentration of bovine serum albumin is 0.5wt%, new-born calf serum 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；Filter with the filter that aperture is 0.2 μm, obtain magnetic particle buffer,
2-8 DEG C of preservation is stand-by；
Step 2) 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
The concentration of new preparation 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, obtain magnetic bead suspending system；Using 2-(N-morpholine) ethanesulfonic acid buffer compound concentration is 5mg/mL
Solution of streptavidin, in magnetic bead suspending system, be then directly added into the Streptavidin of the 0.8-1.6mL of above-mentioned concentration, 4 DEG C
Under the conditions of suspendible 16-20h；Re-use magnetic frame absorption, after static 2min, suck supernatant, in magnetic particle, add 10mL concentration
It is the ethanolamine solutions of 8.5 for 1M, pH, room temperature reaction 1-2h, re-use magnetic frame absorption, 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；
(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-20
The concentration that concentration is 0.5wt%, Triton100 be 0.5wt%；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, obtain cleanout fluid, 2-8 DEG C of preservation.
4. using the detection method of test kit as claimed in claim 1 detection anti-centromere antibody IgG content, its feature exists
In, the method comprises the steps:
(1) anti-centromere antibody IgG 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-centromere antibody IgG 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 anticentromere 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-centromere antibody IgG calibration objects or quality-control product or 1:20 dilution is added to detecting in pipe；
(2) reagent 1 of 50 μ L is added in step (1) described detection pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min；
(3) the Magneto separate reagent of 50 μ L is added in step (2) 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 in step (3) described detection pipe, mixing, carry out Magneto separate, remove supernatant；
(5) step (4) twice is repeated；
(6) 150 μ L reagent 2 is added in step (5) described detection pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min, carry out magnetic
Separate, remove supernatant；
(7) cleanout fluid of 300 μ L is added in step (6) described detection pipe, mixing, carry out Magneto separate, remove supernatant；
(8) step (7) twice is repeated；
(9) add in chemical luminous substrate extremely step (8) described detection pipe of 200 μ L, mixing, detects luminous intensity；
Described step (1), step (2) and step (3) all include the full-automatic inspection of Full-automatic chemiluminescence immunoassay analysis meter
Survey step.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108872210A (en) * | 2018-09-18 | 2018-11-23 | 贵州大学 | A kind of electrochemical luminescence reagent of macrocyclic compound preparation and its 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 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030044799A1 (en) * | 2001-08-31 | 2003-03-06 | Matson Robert S. | Analysis using a distributed sample |
| CN101377514A (en) * | 2008-03-25 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Insulin autoantibody chemiluminescence immune analysis determination reagent kit and preparing method thereof |
| WO2013140349A1 (en) * | 2012-03-20 | 2013-09-26 | Università Degli Studi Di Milano - Bicocca | A method and a kit for the detection of anti-beta amyloid antibodies |
| CN103901203A (en) * | 2014-04-11 | 2014-07-02 | 苏州浩欧博生物医药有限公司 | Chemiluminescence quantitative detection kit for procalcitonin, and preparation method and detection method thereof |
| CN105277690A (en) * | 2015-11-17 | 2016-01-27 | 苏州浩欧博生物医药有限公司 | Reagent kit and method for full-automatically measuring antiprotease 3 antibody IgG |
| 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 |
-
2016
- 2016-04-20 CN CN201610248741.0A patent/CN105784689A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030044799A1 (en) * | 2001-08-31 | 2003-03-06 | Matson Robert S. | Analysis using a distributed sample |
| CN101377514A (en) * | 2008-03-25 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Insulin autoantibody chemiluminescence immune analysis determination reagent kit and preparing method thereof |
| WO2013140349A1 (en) * | 2012-03-20 | 2013-09-26 | Università Degli Studi Di Milano - Bicocca | A method and a kit for the detection of anti-beta amyloid antibodies |
| CN103901203A (en) * | 2014-04-11 | 2014-07-02 | 苏州浩欧博生物医药有限公司 | Chemiluminescence quantitative detection kit for procalcitonin, 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 |
| CN105277690A (en) * | 2015-11-17 | 2016-01-27 | 苏州浩欧博生物医药有限公司 | Reagent kit and method for full-automatically measuring antiprotease 3 antibody IgG |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108872210A (en) * | 2018-09-18 | 2018-11-23 | 贵州大学 | A kind of electrochemical luminescence reagent of macrocyclic compound preparation and its application |
| CN108872210B (en) * | 2018-09-18 | 2020-08-07 | 贵州大学 | Electrochemical luminescence reagent prepared from macrocyclic compound and application thereof |
| 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 |
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