CN105954267A - Magnetic particle-based quantitative chemiluminescent assay kit for anti-histone antibody IgG, and preparation and detection methods thereof - Google Patents
Magnetic particle-based quantitative chemiluminescent assay kit for anti-histone antibody IgG, and preparation and detection methods thereof Download PDFInfo
- Publication number
- CN105954267A CN105954267A CN201610249004.2A CN201610249004A CN105954267A CN 105954267 A CN105954267 A CN 105954267A CN 201610249004 A CN201610249004 A CN 201610249004A CN 105954267 A CN105954267 A CN 105954267A
- Authority
- CN
- China
- Prior art keywords
- concentration
- reagent
- histone
- solution
- antibody igg
- 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
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
Abstract
The invention discloses a magnetic particle-based quantitative chemiluminescent assay kit for an anti-histone antibody IgG. The kit comprises an anti-histone antibody IgG calibrator, an anti-histone antibody IgG quality control product, a Tris buffer containing biotin-labeled histone 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, the magnetic particle chemistry particularly relating to a kind of anti-histone antibody IgG is sent out
Light quantitative determination reagent kit and preparation and detection method.
Background technology
Histone is a kind of basonuclin, is made up of 5 subunits (H1, H2A, H2B, H3, H4).Anti-histone antibody
Can occur in multiple connective tissue disease that not there is specificity, but systemic lupus erythematosus (sle) and Drug erythema wolf
The positives rate of skin ulcer patient is higher.
Anti-histone antibody is relevant to various autoimmune diseasees, at systemic lupus erythematosus (SLE) patient, anti-group of egg
White Antibody positive rate is 30%-70%, at uncomplicated patient with rheumatoid arthritis positive rate 15%-50%, and young type
Patient with rheumatoid arthritis positive rate is 60%, the recall rate the highest (> 95% of drug induced lupus patient's anti-histone).These
On SLE patients clinical common with nephritis person.
Systemic lupus erythematosus (sle) (SLE) is a kind of systematic autoimmune disease.Its pathogeny is relevant with autoimmune,
Serological feature is to have multiple autoantibody.Primary disease can occur at any age, the most common for the women of child-bearing age, clinical manifestation
Big frequently-occurring disease is anxious slow to differ, can be heating, erythra, bald head, pleuritis, pericarditis, nephritis, hemolytic anemia, leukopenia,
Thrombocytopenia and central nervous system (CNS) get involved, the most often by mistaken diagnosis or fail to pinpoint a disease in diagnosis.The most immunologic develop rapidly also
It is greatly promoted the development of clinical medical development especially rheumatology.Along with immunologic development, the inspection of autoantibody
Survey the important means having become diagnosis SLE.
The detection of anti-histone antibody IgG has film bar immunization and enzyme linked immunosorbent assay the most clinically.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 long, still can not meet answering of clinic very well
With.
At present, use magnetic microparticle chemiluminescence analytic process anti-histone antibody IgG immunoassay product application not yet
See.
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-histone antibody IgG
Agent box, the present invention provide test kit chemiluminescence analytical technique is combined with magnetic particle isolation technics, use biotin with
Alkali phosphatase (ALP) respectively labelled antigen and antibody, 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 exists
On the basis of traditional film bar immunization and enzyme linked immunosorbent assay, sensitivity and linear measurement range is improved again 3-5 the order of magnitude,
Realize the detection by quantitative of real meaning, be swift in response, reliable results, and Full-automatic chemiluminescence immunoassay analysis meter can be coordinated to realize
Full-automatic use, has the important value that can not be substituted for clinical diagnosis.
Further object is that and the preparation method of a kind of mentioned reagent box is provided and uses mentioned reagent box
Detection 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-histone antibody IgG, described test kit includes:
(1) anti-histone antibody IgG calibration object, the Tris buffer containing anti-histone antibody IgG and bovine serum albumin,
Described anti-histone 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 HAB IgG is respectively 0,5,20,50,100,200RU/mL；
(2) anti-histone antibody IgG quality-control product, the Tris buffer containing anti-histone antibody IgG and bovine serum albumin,
Described anti-histone 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 HAB IgG is respectively (20 ± 4) RU/mL and (100 ± 20) RU/mL；
(3) reagent 1, containing biotin labeled histone 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-histone antibody IgG, the party
Method comprises the steps:
(1) preparation anti-histone antibody IgG calibration object:
Step 1) preparation anti-histone antibody IgG calibration object diluent:
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；Filter with the filter that aperture is 0.2 μm, obtain anti-histone and resist
Body IgG calibration object diluent, 2-8 DEG C of preservation is stand-by；
Step 2) preparation anti-histone antibody IgG calibration object:
It is 0 by anti-histone antibody IgG anti-histone antibody IgG calibration object diluted to each concentration point, 5,20,
50,100,200RU/mL；
(2) preparation anti-histone antibody IgG quality-control product:
It is 20 by above-mentioned anti-histone antibody IgG calibration object diluted to each concentration point by anti-histone antibody IgG,
100RU/mL；
(3) reagent preparation 1:
Step 1) 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 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 histone 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 histone antigen and biotin mass ratio are 10:1
Example adds biotin solution, mix homogeneously in histone antigenic solution, and room temperature stands 12-18h, and reaction generates histone and resists
Former-biotin conjugate；By the reactant liquor containing histone 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 histone antigen-biotin conjugate；To connect containing histone 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 histone resists
Former-biotin conjugate, the histone obtained antigen-biotin conjugate 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, 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, and obtains No. 2 diluents of reagent, and 2-8 DEG C of preservation is stand-by；
Step 2) 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, obtains examination by No. 2 diluted of reagent
Agent 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%,
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；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 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, obtain magnetic bead suspending system；Using 2-(N-morpholine) ethanesulfonic acid buffer compound concentration is 5mg/
The solution of streptavidin of mL, is then directly added into the Streptavidin of 4-8mg in magnetic bead suspending system, mixed under the conditions of 4 DEG C
Outstanding 16-20h；Re-using magnetic frame absorption, suck supernatant after static 2min, adding 10mL concentration in magnetic particle is that 1M, pH are
The ethanolamine solutions of 8.5, room temperature reaction 1-2h, re-use magnetic frame absorption, suck supernatant after static 2min, add in magnetic particle
Enter the dilution of appropriate magnetic particle buffer and 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 two
Imines plays Stabilization；Add after Streptavidin suspendible under the conditions of 4 degree, can preferably retaining protein active, simultaneously
Reduce the room temperature change impact on coupling effect, make coupling result between batch more stable；Add ethanolamine, ethanolamine can be made
Amino in molecule is not associated with the avtive spot of albumen and reacts after activating with magnetic bead, play termination reaction and sealing process, can produce
Raw lower background values.
(6) preparation cleanout fluid:
Purified water, Tris and sodium chloride being added in container, be stirred well to be completely dissolved, the concentration of Tris is 1wt%,
The concentration of sodium chloride is 0.8wt%；Again Tween-20 and Triton100 is added in container, 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 with 0.2 μm filter, obtains cleanout fluid, 2-8 DEG C of preservation.
The inspection of the magnetic microparticle chemiluminescence quantitative determination reagent kit detection anti-histone antibody IgG of anti-histone antibody IgG
Survey method, the method comprises the steps:
(1) anti-histone antibody IgG 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-histone antibody IgG 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-group
The concentration value of protein antibodies 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-histone antibody IgG calibration objects or quality-control product or 1:20 dilution is added to detecting pipe
In；
(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, carry out
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-histone 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 automatic chemiluminescence immunoassay of arranging in pairs or groups
Instrument realizes full automatic use, improves work efficiency；Calibration object in test kit, biotin labeling reagent, enzyme labelling reagent, magnetic
Separation 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 carries
Supply powerful guarantee.
Accompanying drawing explanation
Fig. 1 is concentration value between zero-dose calibration object with adjacent calibration object during the test kit blank limit of embodiment 7 is evaluated
The linear function that 2 regression fits draw is carried out with luminous value result；
Fig. 2 is dense between zero-dose calibration object with adjacent calibration object during the test kit blank limit of comparative example 1 preparation is evaluated
Angle value carries out, with luminous value result, 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 side
Journey；
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 done further
Bright.
Embodiment 1
The preparation of anti-histone antibody IgG calibration object:
Step 1) preparation anti-histone 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, 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-histone antibody IgG calibration object diluent with 0.2 μm filter, 2-8 DEG C of preservation is stand-by；
Step 2) preparation anti-histone antibody IgG calibration object:
It is 0 by anti-histone antibody IgG anti-histone antibody IgG calibration object diluted to each concentration point, 5,20,
50,100,200RU/mL.
Embodiment 2
The preparation of anti-histone antibody IgG quality-control product:
It is 20 by above-mentioned anti-histone antibody IgG calibration object diluted to each concentration point by anti-histone 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, 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；
Step 2) reagent preparation 1:
By histone 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 histone antigen and biotin mass ratio are 10:1
Example adds biotin solution, mix homogeneously in histone antigenic solution, and room temperature stands 12-18h, and reaction generates histone and resists
Former-biotin conjugate；By the reactant liquor containing histone 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 histone antigen-biotin conjugate；To connect containing histone 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；
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
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；
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 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.
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
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；
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 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, 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, after static 2min, suck supernatant, add in magnetic particle
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
Supernatant, adds the dilution of appropriate magnetic particle buffer in magnetic particle and makes 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
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-histone antibody IgG
This test kit includes:
The anti-histone antibody IgG calibration object prepared according to embodiment 1 method, the calibration object consumption of each level is
0.5ml；
The anti-histone antibody IgG quality-control product prepared according to embodiment 2 method, quality-control product consumption is 1mL；
The reagent 1 prepared according to embodiment 3 method, the consumption that reagent 1 is 5ml；
The reagent 2 prepared according to embodiment 4 method, the consumption that reagent 2 is 15ml；
The Magneto separate reagent prepared according to embodiment 5 method, the consumption of Magneto separate reagent is 5ml；
The cleanout fluid prepared according to embodiment 6 method, cleanout fluid consumption is 1L.
Embodiment 8
The test kit using embodiment 7 carries out detection by quantitative to anti-histone antibody IgG:
(1) specimen to be measured after 20 μ L anti-histone antibody IgG calibration objects or quality-control product or 1:20 dilution is added to detecting pipe
In；
(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, carry out
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, 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 histone antigen
Adding biotin solution, mix homogeneously in histone antigenic solution with the ratio that biotin mass ratio is 10:1, room temperature stands
18h, reaction generates histone antigen-biotin conjugate；To pass through containing the reactant liquor of histone antigen-biotin conjugate
G-25 gel column separates, and removes unreacted biotin, obtains the solution containing histone antigen-biotin conjugate；
With No. 1 diluent of reagent, the solution containing histone 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. accuracy estimating
With the test kit of embodiment 7, concentration is about anti-group of egg of 200RU/mL (allowing its concentration deviation is ± 20%)
White 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%, calculates response rate R according to formula (1), and the response rate of this method requires 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
Detect as sample with the zero-dose calibration object of embodiment 7 and the test kit of comparative example 1, replication 20
Secondary, draw the RLU value (relative light unit) of 20 measurement results, calculate its meansigma methods (M) and standard deviation (SD), draw M+2SD
Corresponding RLU value, carries out at 2 according to the concentration-RLU value result between zero-dose calibration object with adjacent calibration object and returns plan
Conjunction draws linear function, the RLU value corresponding to M+2SD is brought in above-mentioned equation, obtains the concentration value of correspondence, is blank
Limit.The blank limit of this method requires to be not more than 1RU/mL, and data see table 2, and A, B point even some matched curve and fit equation are shown in figure
1, the blank limit data that the test kit of comparative example 1 preparation records are shown in Table 2-1, and A, B point even some matched curve and fit equation are shown in figure
2, from data, embodiment 7 is compared with comparative example 1, and the background values obtained is lower, thus the blank limit obtained is lower, represents
The sensitivity of test kit is more preferable.
The blank limit of table 2 is evaluated
Table 2-1 blank limit is evaluated
3. range of linearity evaluation
With the test kit of embodiment 7, will be the dilutest close to the high level sample of the range of linearity upper limit (200RU/mL)
Being interpreted as at least 5 kinds of concentration, wherein the sample of low value concentration must be close to the lower limit of the range of linearity.Grasp by test kit description
Make, the sample standard deviation duplicate detection 2 times to each concentration, calculate its meansigma methods, by result meansigma methods and dilution ratio with a young waiter in a wineshop or an inn
Multiplication carries out fitting a straight line, and calculates linearly dependent coefficient r, and the measurement scope of this method is [2,200] RU/mL, it is desirable at this
In the range of, correlation coefficient r answers >=0.9900.Data see table 3, matched curve and correlation coefficient and see that Fig. 3, evaluation result conform to
Ask.
Table 3 range of linearity evaluation
4. reproducibility
It is (20 ± 4) RU/mL and (100 ± 20) RU/ by the test kit duplicate detection concentration in embodiment 7 and comparative example 1
Each 10 times of the sample of mL, calculates meansigma methods M and standard deviation SD of 10 measurement results, obtains according to formula CV=SD/M × 100%
Going out coefficient of variation CV, this method coefficient of variation (CV) requires to be not more than 8%, and 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, represent examination
The repeatability of agent box is more preferable,
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 | 4.97% |
| 100 | 10 | 3.11% |
Table 4-1 reproducibility
| Measure serum-concentration (RU/mL) | Measure number of times | CV (%) in analyzing |
| 20 | 10 | 7.24% |
| 100 | 10 | 7.06% |
5. difference between batch evaluation
The test kit of embodiment 7 and comparative example 1 is taken three batches respectively, and every batch of test kit all measures concentration at (20 ± 4) RU/
Sample in the range of mL and (100 ± 20) RU/mL, the every batch of replication 10 times, calculate 30 measurement results meansigma methods (M) and
Standard deviation (SD), calculates the coefficient of variation (CV) according to formula (3), and this method coefficient of variation (CV) requires to be not more than 15%, data
Seeing table 5, 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 and comparative example
1 compares, 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 | 4.86% |
| 100 | 30 | 3.25% |
Table 5-1 difference between batch is evaluated
| Measure serum-concentration (RU/mL) | Measure number of times | CV (%) between analysis |
| 20 | 30 | 8.49% |
| 100 | 30 | 9.55% |
6. Evaluation on specificity
With the test kit of embodiment 7, taking 1 part of anti-histone antibody IgG content is the sample of 0, adds human serum albumin,
Making human serum albumin's concentration in sample is 5000ng/mL, uses this test kit to detect this sample, measures in sample
Anti-histone antibody IgG content.The results are shown in Table 6, this method and human serum albumin's no cross reaction.Data see table 6, evaluate
Result meets the requirements.
Table 6 specificity experiments
7. relativity evaluation
By test kit and the anti-histone antibody IgG 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 4, with anti-histone antibody IgG 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=1.0187x+0.0565, and correlation coefficient is R2: 0.9846.Show through statistical procedures result, we
Method is good with the test kit clinical sample measured value dependency of additive method.
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
The change of kit standard product luminous intensity, batch in and betweenrun precision, accuracy index all within normal range, reagent
Box effect duration was up to 12 months.
Obviously, the above embodiment of the present invention is only for clearly demonstrating example of the present invention, and is not right
The restriction of embodiments of the present invention, for those of ordinary skill in the field, the most also may be used
To 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-histone antibody IgG, it is characterised in that described test kit
Including:
(1) anti-histone antibody IgG calibration object, the Tris buffer containing anti-histone antibody IgG and bovine serum albumin, described
Anti-histone antibody IgG calibration object comprises the liquid calibration object of 6 levels, anti-group of egg in the liquid calibration object of described 6 levels
The concentration of white IgG antibody is respectively 0,5,20,50,100,200RU/mL；
(2) anti-histone antibody IgG quality-control product, the Tris buffer containing anti-histone antibody IgG and bovine serum albumin, described
Anti-histone antibody IgG quality-control product comprises the liquid quality control product of 2 levels, anti-group of egg in the liquid quality control product of described 2 levels
The target value concentration range of white IgG antibody is respectively (20 ± 4) RU/mL and (100 ± 20) RU/mL；
(3) reagent 1, containing biotin labeled histone 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. preparing a method for test kit as described in any one of claim 1-2, it comprises the steps:
(1) preparation anti-histone antibody IgG calibration object:
Step 1) preparation anti-histone 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%；Again
With the HCL of 4M, the pH value of solution is adjusted to 7.0-7.5；Filter with the filter that aperture is 0.2 μm, obtain anti-histone antibody IgG school
Quasi-product diluent, 2-8 DEG C of preservation is stand-by；
Step 2) preparation anti-histone antibody IgG calibration object:
It is 0 by anti-histone antibody IgG anti-histone antibody IgG calibration object diluted to each concentration point, 5,20,50,
100,200RU/mL；
(2) preparation anti-histone antibody IgG quality-control product:
It is 20 by above-mentioned anti-histone antibody IgG calibration object diluted to each concentration point by anti-histone antibody IgG,
100RU/mL；
(3) reagent preparation 1:
Step 1) 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 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 histone 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 histone antigen and biotin mass ratio are 10:1
Adding biotin solution, mix homogeneously in histone antigenic solution, room temperature stands 12-18h, and reaction generates histone antigen-life
Thing element junctional complex；It is 0.2M, pH by concentration by the reactant liquor containing histone antigen-biotin conjugate under the conditions of 2-8 DEG C
Be 9.0 carbonate buffer solution dialyse 2 days, period carries out 4 times changing liquid, thus removes unreacted biotin, obtains containing group
The solution of proteantigen-biotin conjugate；With molten by containing histone antigen-biotin conjugate of No. 1 diluent of reagent
Liquid 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%,
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,
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, 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:
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 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；Filter with the filter that aperture is 0.2 μm, obtain magnetic particle buffering
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 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, obtain 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 above-mentioned 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,
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-histone antibody IgG content, its feature exists
In, the method comprises the steps:
(1) anti-histone 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-histone antibody IgG quality-control product is put into above-mentioned analyser test position, obtains by Full-automatic chemiluminescence immunity
Test luminous value and the matched curve matching obtained by step (1) of the described quality-control product of analyser output obtain anti-histone
The concentration value of 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.
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-histone 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, 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 and divide
From, 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 fully-automated synthesis step of Full-automatic chemiluminescence immunoassay analysis meter
Suddenly.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610249004.2A CN105954267A (en) | 2016-04-20 | 2016-04-20 | Magnetic particle-based quantitative chemiluminescent assay kit for anti-histone antibody IgG, and preparation and detection methods thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610249004.2A CN105954267A (en) | 2016-04-20 | 2016-04-20 | Magnetic particle-based quantitative chemiluminescent assay kit for anti-histone antibody IgG, and preparation and detection methods thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105954267A true CN105954267A (en) | 2016-09-21 |
Family
ID=56917704
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610249004.2A Pending CN105954267A (en) | 2016-04-20 | 2016-04-20 | Magnetic particle-based quantitative chemiluminescent assay kit for anti-histone antibody IgG, and preparation and detection methods thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105954267A (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107402306A (en) * | 2017-09-15 | 2017-11-28 | 江苏浩欧博生物医药股份有限公司 | A kind of chemical luminescent analysis reagent kid of phosphatide IgM antibody and preparation method thereof and detection method |
| CN107402309A (en) * | 2017-09-15 | 2017-11-28 | 江苏浩欧博生物医药股份有限公司 | A kind of chemical luminescent analysis reagent kid of phosphatide IgG antibody and preparation method thereof and detection method |
| CN108037285A (en) * | 2017-11-29 | 2018-05-15 | 北京美联泰科生物技术有限公司 | A kind of magnetic microparticle chemiluminescence quantitatively detects kit of UCHL-1 and preparation method thereof |
| CN108051586A (en) * | 2017-11-29 | 2018-05-18 | 北京美联泰科生物技术有限公司 | A kind of magnetic microparticle chemiluminescence quantitatively detects kit of GFAP and preparation method thereof |
| CN109142750A (en) * | 2018-08-14 | 2019-01-04 | 江苏浩欧博生物医药股份有限公司 | A kind of kit and detection method measuring histonic antibody IgG |
| 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 |
| CN112255416A (en) * | 2020-09-29 | 2021-01-22 | 北京利德曼生化股份有限公司 | Kit for quantitatively detecting HBP (hepatitis B protein) by using magnetic particle chemiluminescence as well as preparation and detection methods thereof |
| CN113373156A (en) * | 2021-06-30 | 2021-09-10 | 四川携光生物技术有限公司 | NMDAR recombinant protein related to autoimmune encephalitis, and coding sequence, preparation method and application thereof |
| CN113533734A (en) * | 2021-07-22 | 2021-10-22 | 泰州泽成生物技术有限公司 | Kit for detecting Golgi protein 73 by magnetic particle chemiluminescence method and preparation method thereof |
| CN113721009A (en) * | 2021-03-05 | 2021-11-30 | 郑州如飞生物技术有限公司 | Calibration method for optical system of immunoassay analyzer |
| CN113804896A (en) * | 2021-09-14 | 2021-12-17 | 北京利德曼生化股份有限公司 | Magnetic particle chemiluminescence kit for measuring interleukin-8 content in human serum |
| CN115327137A (en) * | 2022-10-11 | 2022-11-11 | 苏州惠中生物科技有限公司 | Human immunoglobulin G4 subtype chemiluminescence immunoassay kit |
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 CN201610249004.2A patent/CN105954267A/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 (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107402306A (en) * | 2017-09-15 | 2017-11-28 | 江苏浩欧博生物医药股份有限公司 | A kind of chemical luminescent analysis reagent kid of phosphatide IgM antibody and preparation method thereof and detection method |
| CN107402309A (en) * | 2017-09-15 | 2017-11-28 | 江苏浩欧博生物医药股份有限公司 | A kind of chemical luminescent analysis reagent kid of phosphatide IgG antibody and preparation method thereof and detection method |
| CN108037285A (en) * | 2017-11-29 | 2018-05-15 | 北京美联泰科生物技术有限公司 | A kind of magnetic microparticle chemiluminescence quantitatively detects kit of UCHL-1 and preparation method thereof |
| CN108051586A (en) * | 2017-11-29 | 2018-05-18 | 北京美联泰科生物技术有限公司 | A kind of magnetic microparticle chemiluminescence quantitatively detects kit of GFAP and preparation method thereof |
| CN109142750B (en) * | 2018-08-14 | 2021-10-22 | 江苏浩欧博生物医药股份有限公司 | Kit for determining anti-histone antibody IgG and detection method |
| CN109142750A (en) * | 2018-08-14 | 2019-01-04 | 江苏浩欧博生物医药股份有限公司 | A kind of kit and detection method measuring histonic antibody IgG |
| 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 |
| CN112255416A (en) * | 2020-09-29 | 2021-01-22 | 北京利德曼生化股份有限公司 | Kit for quantitatively detecting HBP (hepatitis B protein) by using magnetic particle chemiluminescence as well as preparation and detection methods thereof |
| CN113721009A (en) * | 2021-03-05 | 2021-11-30 | 郑州如飞生物技术有限公司 | Calibration method for optical system of immunoassay analyzer |
| CN113721009B (en) * | 2021-03-05 | 2024-01-30 | 郑州如飞生物技术有限公司 | Optical system calibration method for immunity analyzer |
| CN113373156A (en) * | 2021-06-30 | 2021-09-10 | 四川携光生物技术有限公司 | NMDAR recombinant protein related to autoimmune encephalitis, and coding sequence, preparation method and application thereof |
| CN113533734A (en) * | 2021-07-22 | 2021-10-22 | 泰州泽成生物技术有限公司 | Kit for detecting Golgi protein 73 by magnetic particle chemiluminescence method and preparation method thereof |
| CN113804896A (en) * | 2021-09-14 | 2021-12-17 | 北京利德曼生化股份有限公司 | Magnetic particle chemiluminescence kit for measuring interleukin-8 content in human serum |
| CN115327137A (en) * | 2022-10-11 | 2022-11-11 | 苏州惠中生物科技有限公司 | Human immunoglobulin G4 subtype chemiluminescence immunoassay kit |
| CN115327137B (en) * | 2022-10-11 | 2023-01-17 | 苏州惠中生物科技有限公司 | Human immunoglobulin G4 subtype chemiluminescence immunoassay kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105954267A (en) | Magnetic particle-based quantitative chemiluminescent assay kit for anti-histone antibody IgG, and preparation and detection methods thereof | |
| CN105954266A (en) | Magnetic particle-based quantitative chemiluminescent assay kit for anti-ribosome P protein 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 | |
| US8956823B2 (en) | Anti-antibody reagent | |
| CN101201354A (en) | Thyroxine chemiluminescence immune analysis quantitative measuring reagent kit and method for preparing the same | |
| CN105352946B (en) | A kind of kit quantitatively detecting Anti SS-B antibody IgG using magnetic microparticle chemiluminescence and preparation method thereof and detection method | |
| CN102841206B (en) | TnT (Troponin-T, TNT) measures kit | |
| CN105203748A (en) | Quantitative detection kit for full-measuring-range C reaction protein | |
| CN105466913A (en) | Kit for quantitative detection of anti-Jo-1 antibody IgG by using magnetic particle chemiluminescence, and preparation method and detection method thereof | |
| 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 and chemiluminescence immunoassay method of thyroglobulin | |
| CN105353139A (en) | Parathyroid hormone quantitative detection kit | |
| CN104237513A (en) | Thyroid peroxidase antibody magnetic-particle chemiluminescence immune quantitative testing kit | |
| CN107044977A (en) | A kind of tyrosine phosphatase antibody chemical luminescence immunity detection reagent and preparation method thereof | |
| CN106501535A (en) | A kind of compound releasing agent of ANS salt and the kit for detecting Blood cortisol | |
| CN111175494A (en) | Thyroglobulin antibody detection kit and use method thereof | |
| CN105445456A (en) | Kit for quantitatively detecting anti-SS-A antibody IgG by utilizing magnetic particle chemiluminescence, preparation method and detection method thereof | |
| CN109001471A (en) | Free beta-human chorionic gonadotropin chemiluminescence detection kit and preparation method thereof and application method | |
| CN103901188B (en) | A kind of chemical luminescent analysis reagent kid sucking anaphylactogen and preparation method thereof and detection method | |
| CN105259344A (en) | Kit for quantitative detection of Scl-70 resisting antibody IgG through chemiluminiscence of magnetic particles and preparing method and detection method of kit | |
| CN102890083B (en) | Chemical luminous substrate solution and containing its test kit and the detection method applying it | |
| CN105467130A (en) | Kit for quantitative detection of anti-Sm antibody IgG by using magnetic particle chemiluminescence, and preparation method and detection method thereof | |
| CN109212193A (en) | Kit for clinical detection abnormal prothrombin | |
| CN109239359A (en) | β 2-MG chemical luminescence immune assay determination reagent kit and preparation 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: 20160921 |