CN105929166A - Magnetic particle-based quantitative chemiluminescent assay kit for anti-LKM-1 antibody IgG, and preparation and detection methods thereof - Google Patents
Magnetic particle-based quantitative chemiluminescent assay kit for anti-LKM-1 antibody IgG, and preparation and detection methods thereof Download PDFInfo
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Abstract
The invention discloses a magnetic particle-based quantitative chemiluminescent assay kit for an anti-LKM-1 antibody IgG. The kit comprises an anti-LKM-1 antibody IgG calibrator, an anti-LKM-1 antibody IgG quality control product, a Tris buffer containing biotin-labeled LKM-1 antigen and bovine serum albumin, a Tris buffer containing an alkaline phosphatase-labeled goat anti-human polyclonal antibody and bovine serum albumin, a Tris buffer containing streptavidin-labeled magnetic particles and bovine serum albumin, and a rinsing solution. The detection method using the kit improves sensitivity and a linearity range by 3 to 5 orders of magnitudes on the basis of a traditional membrane strip immunization method and a traditional enzyme linked immunosorbent assay method, realizes real quantitative determination, is rapid in reaction and reliable in results, can be automatically used in cooperation with an automatic chemiluminescence immunity analyzer and is of irreplaceable important value to clinical diagnosis.
Description
Technical field
The present invention relates to technical field of biological.More particularly, to a kind of anti-LKM-1 IgG antibody
Magnetic microparticle chemiluminescence quantitative determination reagent kit and preparation and detection method.
Background technology
Autoimmune hepatitis (autoimmune hepatitis, AIH) is that a class is with autoimmune response as base
Plinth, interface characteristics to have on multiple autoantibody, hyperimmunoglobulinemia, circulation autoantibody and histology
The chronic liver inflammatory diseases that hepatitis and portal area plasmocyte infiltrating are characterized.This disease is more common in women, men and women
Ratio is about 1:4, and any age all can fall ill.The pathogenesis of autoimmune liver disease is not yet clear and definite, mesh
Before think that genetic predisposition is principal element.There is obvious family member and concentrate morbidity phenomenon in AIH, and sick
Poison infection, medicine and environment are then probably the precipitating factor on the basis of inheritance susceptible.Nearly all AIH is
There is hepatic fibrosis in various degree, several cases may occur in which liver cirrhosis.AIH is according to detection of plasma inspection
I type (ANA, SMA), II type (anti-LKM-1 antibody) and III type (anti-SLA/LP antibody) can be divided into.
Anti-LKM-1 antibody is the serologic marker of autoimmune hepatitis II type, the most anti-LKM-1
The detection of IgG antibody has film bar immunization and enzyme linked immunosorbent assay.The application of film bar immunization is that film bar shows
Color technology, its feature measures at same film bar for fixing several projects, general by manual or semi-automatic film
Bar instrument carries out experimental implementation, qualitatively judges eventually through naked eyes, and this sensitivity is low, the response time
Long, detection project can only combine in regular collocation, very flexible.The sensitivity of enzyme linked immunosorbent assay is at film bar
Promote on the basis of immunization, but still relatively low, and the range of linearity is narrow, poor repeatability, the response time
The longest, still can not meet the application of clinic very well.
Magnetic microparticle chemiluminescence immune assay method, film bar immunization than before and enzyme linked immunosorbent assay,
Have in detection sensitivity, detection range, detection time and automation mechanized operation and be greatly improved, and do not polluted,
Clinical practice is wide.At present, use magnetic microparticle chemiluminescence analytic process at anti-LKM-1 IgG antibody immunoassay
The application of product has not yet to see.
Summary of the invention
The magnetic microparticle chemiluminescence that it is an object of the present invention to provide a kind of anti-LKM-1 IgG antibody is quantitative
Measuring test kit, the test kit that the present invention provides is by chemiluminescence analytical technique and magnetic particle isolation technics phase
In conjunction with, use biotin and alkali phosphatase (ALP) labelled antigen and antibody respectively, with diameter 1-3 μm
The super paramagnetic microsphere being coated Streptavidin as separation agent.After ALP catalytic substrate luminescence, pass through
Apparatus measures luminous intensity calculates testing concentration.This detection method joins at traditional film bar immunization and enzyme
On the basis of immunoabsorption, sensitivity and linear measurement range is improved 3-5 the order of magnitude again, realizes real meaning
Detection by quantitative, be swift in response, reliable results, and Full-automatic chemiluminescence immunoassay analysis meter can be coordinated real
The most automatically use, clinical diagnosis is had to the important value that can not be substituted.
Further object is that preparation method and the detection method that a kind of mentioned reagent box is provided.
For reaching above-mentioned purpose, the present invention uses following technical proposals:
1) anti-LKM-1 IgG antibody calibration object, containing anti-LKM-1 IgG antibody and bovine serum albumin
Tris buffer, described anti-LKM-1 IgG antibody calibration object is the liquid calibration object of 6 levels, described 6
In the liquid calibration object of individual level, the concentration of anti-LKM-1 IgG antibody is respectively 0, and 5,20,50,100,
200RU/mL;
2) anti-LKM-1 IgG antibody quality-control product, containing anti-LKM-1 IgG antibody and bovine serum albumin
Tris buffer, described anti-LKM-1 IgG antibody quality-control product comprises the liquid quality control product of 2 levels, described
In the liquid quality control product of 2 levels, the target value concentration range of anti-LKM-1 IgG antibody is respectively (20 ± 4)
RU/mL and (100 ± 20) RU/mL;
3) reagent 1, containing biotin labeled LKM-1 antigen and the Tris buffer of bovine serum albumin;
4) reagent 2, sheep anti-human polyclonal antibody containing alkali phosphatase enzyme mark and bovine serum albumin
Tris buffer;
5) Magneto separate reagent, the magnetic particle containing marked by streptavidin and the Tris of bovine serum albumin
Buffer;
6) cleanout fluid;
The content of described seminal plasma fructose detection kit 1, reagent 2 and Magneto separate reagent than for 1:3:1, described contains
Amount ratio is volume ratio.
Further, the material of described magnetic particle is Fe2O3;Described magnetic particle pan coating has carboxylic group,
Being coated carboxyl group content in thing and be more than 20wt%, the size of described magnetic particle is 1-3 μm.
A kind of side of the magnetic microparticle chemiluminescence quantitative determination reagent kit preparing described anti-LKM-1 IgG antibody
Method, the method comprises the steps:
(1) anti-LKM-1 IgG antibody calibration object is prepared:
A. anti-LKM-1 IgG antibody calibration object diluent is prepared:
Purified water, Tris, sodium chloride and Proclin300 are added in container, are stirred well to be completely dissolved,
Tris concentration is 1wt%, sodium chloride concentration be 1wt%, Proclin300 concentration be 0.2v%;With the HCL of 4M
The pH value of solution is adjusted to 7.0-7.5;Bovine serum albumin is added in container, is stirred well to be completely dissolved,
The concentration of bovine serum albumin is 4wt%;With the HCL of 4M, the pH value of solution is adjusted to 7.0-7.5 again;With
Aperture is that the filter of 0.2 μm filters to obtain anti-LKM-1 IgG antibody calibration object diluent, and 2-8 DEG C of preservation is stand-by;
B. anti-LKM-1 IgG antibody calibration object is prepared:
By anti-LKM-1 IgG antibody by anti-LKM-1 IgG antibody calibration object diluted to each concentration point
It is 0,5,20,50,100,200RU/mL;
(2) anti-LKM-1 IgG antibody quality-control product is prepared:
By anti-for anti-double-strand LKM-1 IgG antibody by above-mentioned anti-LKM-1 IgG antibody calibration object diluted
It is 20,100RU/mL to each concentration point;
(3) reagent preparation 1:
A. No. 1 diluent of reagent preparation:
Purified water, Tris, sodium chloride and Proclin300 are added in container, are stirred well to be completely dissolved,
The concentration of Tris is 1wt%, sodium chloride concentration be the concentration of 0.5wt%, Proclin300 be 0.2v%;By cattle
Serum albumin adds in container, is stirred well to be completely dissolved, and the concentration of bovine serum albumin is 0.5wt%;
With the HCL of 4M, the pH value of solution is adjusted to 7.0-7.5;Reagent is filtered to obtain with the filter that aperture is 0.2 μm
No. 1 diluent, 2-8 DEG C of preservation is stand-by;
B. reagent preparation 1:
By LKM-1 antigen with purified water dissolve, under the conditions of 2-8 DEG C with concentration be 0.2M, pH be 9.0
Carbonate buffer solution dialysis 2h, be then concentrated into the antigenic solution that concentration is 2-4mg/mL, use concentration
It is the biotin solution of 0.5-1.0mg/ml for the carbonate buffer solution compound concentration that 0.2M, pH are 8.5-9;
It is that the ratio of 10:1 adds biology in LKM-1 antigenic solution according to LKM-1 antigen and biotin mass ratio
Cellulose solution, mix homogeneously, room temperature stands 12-18h, and reaction generates LKM-1 antigen-biotin conjugate;
It is 0.2M by concentration by the reactant liquor containing LKM-1 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 and changes liquid, thus removes unreacted
Biotin, obtain the solution containing LKM-1 antigen-biotin conjugate;Will with No. 1 diluent of reagent
Solution containing LKM-1 antigen-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 company
Connecing thing, the junctional complex obtained is purer, decreases nonspecific reaction;
(4) reagent preparation 2:
A. No. 2 diluents of reagent preparation:
By purified water, 4-hydroxyethyl piperazine ethanesulfonic acid, sodium chloride, bovine serum albumin, ZnCl2And Proclin300
Adding in container, be stirred well to be completely dissolved, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 0.6wt%, chlorine
Change na concn is 0.8wt%, and the concentration of bovine serum albumin is 0.5wt%, ZnCl2Concentration be 0.1wt ‰,
The concentration of Proclin300 is 0.2v ‰, MgCl2Concentration be 0.1 ‰;With the HCL of 4M by the pH of solution
Value is adjusted to 7.5-8.0;Filtering to obtain No. 2 diluents of reagent with the filter that aperture is 0.2 μm, 2-8 DEG C of preservation is stand-by;
B. reagent preparation 2:
1mg sheep anti-human polyclonal antibody is joined the 2-iminothiolane salt that 2-4 μ L concentration is 10mg/mL
In acid salt solution, room temperature stands 20min, adds the glycine solution 10 μ L of 0.1moL/L, and room temperature stands
5min, with G-25 gel column desalination, collects the sheep anti-human polyclonal antibody after activation, and 2-8 DEG C of preservation is standby
With;The alkali phosphatase of 1.5mg is joined 4-(the N-maleimide that concentration is 5mg/mL of 10-20 μ L
Aminomethyl) in hexamethylene-1-carboxylic acid butanimide ester solution, room temperature stands 30min, uses G-25 gel
Post desalination, collects the alkali phosphatase after activation, and 2-8 DEG C saves backup;Goat-anti people's Anti-TNF-α by activation
Body mixes with the alkali phosphatase of activation, stands 12-24h under the conditions of 2-8 DEG C, pure with Supperdex200 gel
Change column purification conjugate, it is thus achieved that sheep anti-human polyclonal antibody-alkali phosphatase junctional complex concentrated solution, 2-8 DEG C of guarantor
Deposit standby;By sheep anti-human polyclonal antibody-alkali phosphatase junctional complex concentrated solution No. 2 diluted of reagent
To 0.02-0.1 μ g/mL, prepare reagent 2;
(5) preparation Magneto separate reagent:
A. preparation magnetic particle buffer:
Purified water, Tris and sodium chloride being added in container, be stirred well to be completely dissolved, the concentration of Tris is
1wt%, the concentration of sodium chloride is 0.8wt%;Again by bovine serum albumin, new-born calf serum and Proclin300
Adding in container, be stirred well to be completely dissolved, the concentration of bovine serum albumin is 0.5wt%, newborn Sanguis Bovis seu Bubali
Clear concentration be 5v%, Proclin300 concentration be 0.2v ‰;With the HCL of 4M, the pH value of solution is adjusted to
7.9-8.1;Filtering to obtain magnetic particle buffer with the filter that aperture is 0.2 μm, 2-8 DEG C of preservation is stand-by;
B. preparation Magneto separate reagent:
Take 100mg magnetic particle, use magnetic frame absorption, suck supernatant after static 2min, add in magnetic particle
Enter concentration be 0.025mol/L, pH be 2-(N-morpholine) the ethanesulfonic acid buffer 10mL of 4.5-5, fully
Mixing;Add the concentration that 0.5-1mL newly prepares and be 1-(3-the dimethylamino-propyl)-3-second of 10mg/mL
Base carbodiimide and N-hydroxy-succinamide aqueous solution, room temperature mixing 30-60min obtains magnetic bead suspending system;
Using 2-(N-morpholine) ethanesulfonic acid buffer compound concentration is the solution of streptavidin of 5mg/mL, then
The Streptavidin of the 0.8-1.6mL being formulated, 4 DEG C of conditions it are directly added in described magnetic bead suspending system
Lower suspendible 16-20h;Re-use magnetic frame absorption, after static 2min, suck supernatant, in magnetic particle, add 10mL
Concentration be 1M, pH be the ethanolamine solutions of 8.5, room temperature reaction 1-2h, re-use magnetic frame absorption, quiet
Only suck supernatant after 2min, in magnetic particle, add the dilution of appropriate magnetic particle buffer make final concentration of
0.5mg/mL, prepares Magneto separate reagent;
In this step, adding N-hydroxy-succinamide can be to coupling agent 1-(3-dimethylamino-propyl)-3-second
Base carbodiimide plays Stabilization;Add after Streptavidin suspendible under the conditions of 4 degree, it is possible to more preferable
Retaining protein activity, reduces the room temperature change impact on coupling effect simultaneously, makes coupling result between batch
More stable;Add ethanolamine, uncombined albumen after the amino in ethanolamine molecules can be made to activate with magnetic bead
Avtive spot reacts, and plays termination reaction and sealing process, it is possible to produce lower background values;
(6) preparation cleanout fluid:
Purified water, Tris and sodium chloride being added in container, be stirred well to be completely dissolved, the concentration of Tris is
1wt%, the concentration of sodium chloride is 0.8wt%;Again Tween-20 and Triton100 is added in container, fully
Stirring to mixing completely, the concentration of Tween-20 be the concentration of 0.5wt%, Triton100 be 0.5wt%;With
The pH value of solution is adjusted to 7.5-8.0 by the HCL of 4M, filters to obtain cleanout fluid with the filter that aperture is 0.2 μm,
2-8 DEG C of preservation.The detection method of the magnetic microparticle chemiluminescence quantitative determination reagent kit of anti-LKM-1 IgG antibody,
The method comprises the steps:
1) anti-LKM-1 IgG antibody calibration object is put into Full-automatic chemiluminescence immunoassay analysis meter test position,
Obtain the matched curve exported by Full-automatic chemiluminescence immunoassay analysis meter;
2) anti-LKM-1 IgG antibody quality-control product is put into above-mentioned analyser test position, obtains by full-automation
The test luminous value learning the described quality-control product of luminescence immunoassay instrument output and the matching song obtained by step 1
Line matching obtains the concentration value of anti-LKM-1 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, obtains the concentration value of the sample to be tested exported by Full-automatic chemiluminescence immunoassay analysis meter.
Further, this detection method specifically includes:
1) specimen to be measured after 20 μ L anti-LKM-1 IgG antibody calibration object or quality-control product or 1:20 dilution is added to detection
Guan Zhong;
2) reagent 1 of 50 μ L is added to step 1) in described detection pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min;
3) the Magneto separate reagent of 50 μ L is added to step 2) in described detection pipe, after mixing, 37 ± 0.5 DEG C of incubations
5min, carries out Magneto separate, removes supernatant;
4) cleanout fluid of 300 μ L is added to step 3) detect in pipe, mixing, carry out Magneto separate, remove supernatant;
5) step 4 is repeated) twice;
6) 150 μ L reagent 2 is added to step 5) detect in pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min, carry out
Magneto separate, removes supernatant;
7) cleanout fluid of 300 μ L is added to step 6) detect in pipe, mixing, carry out Magneto separate, remove supernatant;
8) step 7 is repeated) twice;
9) chemical luminous substrate of 200 μ L is added to step 8) detect in pipe, mixing, detects luminous intensity;
Described step 1), step 2) and step 3) all include Full-automatic chemiluminescence immunoassay analysis meter complete from
Dynamic detecting step.
Beneficial effects of the present invention is as follows:
The invention discloses a kind of new technique measuring anti-LKM-1 IgG antibody so that course of reaction is quicker
Reliably, improve sensitivity and linear measurement range, it is achieved the quantitative determination of real meaning, and it is full-automatic to arrange in pairs or groups
Chemical illumination immunity analysis instrument realizes full automatic use, is greatly improved work efficiency;Calibration in test kit
Product, biotin labeling reagent, enzyme labelling reagent, Magneto separate reagent and cleanout fluid etc. are all under this reaction system
Optimization formula, to this test kit use imitate the phase and detection performance provide powerful guarantee.
Accompanying drawing explanation
Fig. 1 a be during the blank limit of embodiment 7 is evaluated concentration value between zero-dose calibration object with adjacent calibration object with
Luminous value result carries out the linear function that 2 regression fits draw;
Fig. 1 b be during the blank limit of comparative example 1 is evaluated concentration value between zero-dose calibration object with adjacent calibration object with
Luminous value result carries out the linear function that 2 regression fits draw;
Fig. 2 is concentration of specimens meansigma methods and dilution ratio method of least square in embodiment 7 range of linearity evaluation
Carry out fitting a straight line equation;
Fig. 3 is the embodiment 7 test kit clinical sample measured value dependency scatterplot with existing commercialization.
Detailed description of the invention
In order to be illustrated more clearly that the present invention, below in conjunction with preferred embodiments and drawings, the present invention is done further
Explanation.
Embodiment 1
The preparation of anti-LKM-1 IgG antibody calibration object:
A. anti-LKM-1 IgG antibody calibration object diluent is prepared:
The purified water of 800ml, Tris, 8.6g sodium chloride of 11.2g and 2ml Proclin300 are added container
In, it is stirred well to be completely dissolved;With the HCL of 4M, the pH value of solution is adjusted to 7.0-7.5;By 40g
Bovine serum albumin adds in container, is stirred well to be completely dissolved;Again with the HCL of 4M by the pH of solution
Value is adjusted to 7.0-7.5;By purified water, solution is settled to 1L, with 0.2 μm filter filter anti-LKM-1 resist
Body IgG calibration object diluent, 2-8 DEG C of preservation is stand-by;
B. anti-LKM-1 IgG antibody calibration object is prepared:
By anti-LKM-1 IgG antibody by anti-LKM-1 IgG antibody calibration object diluted to each concentration point
It is 0,5,20,50,100,200RU/mL.
Embodiment 2
The preparation of anti-LKM-1 IgG antibody quality-control product:
By anti-LKM-1 IgG antibody by above-mentioned anti-LKM-1 IgG antibody calibration object diluted to each dense
Degree point is 20,100RU/mL.
Embodiment 3
The preparation that reagent 1:
A. No. 1 diluent of reagent preparation:
800ml purified water, Tris, 5.8g sodium chloride of 12.1g and 2ml Proclin300 are added in container,
It is stirred well to be completely dissolved;5g bovine serum albumin is added in container, is stirred well to be completely dissolved;
With the HCL of 4M, the pH value of solution is adjusted to 7.0-7.5;By purified water, solution is settled to 1L, uses 0.2 μm
Filter filters to obtain No. 1 diluent of reagent, and 2-8 DEG C of preservation is stand-by;
B. reagent preparation 1:
By LKM-1 antigen with purified water dissolve, under the conditions of 2-8 DEG C with concentration be 0.2M, pH be 9.0
Carbonate buffer solution dialysis 2h, be then concentrated into the antigenic solution that concentration is 2-4mg/mL, use concentration
It is the biotin solution of 0.5-1.0mg/ml for the carbonate buffer solution compound concentration that 0.2M, pH are 8.5-9;
It is that the ratio of 10:1 adds biology in LKM-1 antigenic solution according to LKM-1 antigen and biotin mass ratio
Cellulose solution, mix homogeneously, room temperature stands 12-18h, and reaction generates LKM-1 antigen-biotin conjugate;
It is 0.2M by concentration by the reactant liquor containing LKM-1 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 and changes liquid, thus removes unreacted
Biotin, obtain the solution containing LKM-1 antigen-biotin conjugate;Will with No. 1 diluent of reagent
Solution containing LKM-1 antigen-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 company
Connecing thing, the junctional complex obtained is purer, decreases the non-specific of subsequent reactions.
Embodiment 4
The preparation that reagent 2:
A. No. 2 diluents of reagent preparation:
By pure to 800ml purified water, the 4-hydroxyethyl piperazine ethanesulfonic acid of 6.06g, 8.5g sodium chloride, 5g Sanguis Bovis seu Bubali
Albumen, 0.1gZnCl2, 0.2ml Proclin300 and 0.1g MgCl2Add in container, be stirred well to completely
Dissolve;With the HCL of 4M, the pH value of solution is adjusted to 7.5-8.0;By purified water, solution is settled to 1L,
Filtering to obtain No. 2 diluents of reagent with 0.2 μm filter, 2-8 DEG C of preservation is stand-by;
B. reagent preparation 2:
1mg sheep anti-human polyclonal antibody is joined the 2-iminothiolane salt that 2-4 μ L concentration is 10mg/mL
In acid salt solution, room temperature stands 20min, adds the glycine solution 10 μ L of 0.1moL/L, and room temperature stands
5min, with G-25 gel column desalination, collects the sheep anti-human polyclonal antibody after activation, and 2-8 DEG C of preservation is standby
With;The alkali phosphatase of 1.5mg is joined 4-(the N-maleimide that concentration is 5mg/mL of 10-20 μ L
Aminomethyl) in hexamethylene-1-carboxylic acid butanimide ester solution, room temperature stands 30min, uses G-25 gel
Post desalination, collects the alkali phosphatase after activation, and 2-8 DEG C saves backup;Goat-anti people's Anti-TNF-α by activation
Body mixes with the alkali phosphatase of activation, stands 12-24h under the conditions of 2-8 DEG C, pure with Supperdex200 gel
Change column purification conjugate, it is thus achieved that sheep anti-human polyclonal antibody-alkali phosphatase junctional complex concentrated solution, 2-8 DEG C of guarantor
Deposit standby;By sheep anti-human polyclonal antibody-alkali phosphatase junctional complex concentrated solution No. 2 diluted of reagent
To 0.02-0.1 μ g/mL, prepare reagent 2.
Embodiment 5
The preparation of Magneto separate reagent:
A. preparation magnetic particle buffer:
800mL purified water, 12.1g Tris and 8.5g sodium chloride are added in container, is stirred well to completely
Dissolve;Again 5g bovine serum albumin, 50mL new-born calf serum and 0.2mL Proclin300 are added container
In, it is stirred well to be completely dissolved;With the HCL of 4M, the pH value of solution is adjusted to 7.9-8.1;Use purification
Solution is settled to 1L by water, filters to obtain magnetic particle buffer with 0.2 μm filter, and 2-8 DEG C of preservation is stand-by;
B. preparation Magneto separate reagent:
Take 100mg magnetic particle, use magnetic frame absorption, suck supernatant after static 2min, add in magnetic particle
Enter concentration be 0.025mol/L, pH be 2-(N-morpholine) the ethanesulfonic acid buffer 10mL of 4.5-5, fully
Mixing;Add the concentration that 0.5-1mL newly prepares and be 1-(3-the dimethylamino-propyl)-3-second of 10mg/mL
Base carbodiimide and N-hydroxy-succinamide aqueous solution, room temperature mixing 30-60min obtains magnetic bead suspending system;
Using 2-(N-morpholine) ethanesulfonic acid buffer compound concentration is the solution of streptavidin of 5mg/mL, then
The Streptavidin of the 0.8-1.6mL being formulated it is directly added in magnetic bead suspending system, mixed under the conditions of 4 DEG C
Outstanding 16-20h;Re-use magnetic frame absorption, after static 2min, suck supernatant, in magnetic particle, add 10mL
Concentration be 1M, pH be the ethanolamine solutions of 8.5, room temperature reaction 1-2h, re-use magnetic frame absorption, quiet
Only suck supernatant after 2min, in magnetic particle, add the dilution of appropriate magnetic particle buffer make final concentration of
0.5mg/mL, prepares Magneto separate reagent;
In this step, adding N-hydroxy-succinamide can be to coupling agent 1-(3-dimethylamino-propyl)-3-second
Base carbodiimide plays Stabilization;Add after Streptavidin suspendible under the conditions of 4 degree, it is possible to more preferable
Retaining protein activity, reduces the room temperature change impact on coupling effect simultaneously, makes coupling result between batch
More stable;Add ethanolamine, uncombined albumen after the amino in ethanolamine molecules can be made to activate with magnetic bead
Avtive spot reacts, and plays termination reaction and sealing process, it is possible to produce lower background values.
Embodiment 6
The preparation of cleanout fluid:
800mL purified water, 12.1g Tris and 8.5g sodium chloride are added in container, is stirred well to completely
Dissolve;Again 5g Tween-20 and 5g Triton100 is added in container, be stirred well to mix completely;With
The pH value of solution is adjusted to 7.5-8.0 purified water and solution is settled to 1L by the HCL of 4M, with 0.2 μm filter
Device filters to obtain cleanout fluid, 2-8 DEG C of preservation.
Embodiment 7
The magnetic microparticle chemiluminescence quantitative determination reagent kit of anti-LKM-1 IgG antibody:
This test kit includes:
The anti-LKM-1 IgG antibody calibration object prepared according to embodiment 1 method, the calibration object of each level is used
Amount is 0.5mL;
The anti-LKM-1 IgG antibody quality-control product prepared according to embodiment 2 method, quality-control product consumption is each 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 LKM-1 IgG antibody using embodiment 7 carries out detection by quantitative
Detection method comprises the steps:
1) specimen to be measured after 20 μ L anti-LKM-1 IgG antibody calibration object or quality-control product or 1:20 dilution is added to detection
Guan Zhong;
2) reagent 1 of 50 μ L is added to step 1) in described detection pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min;
3) the Magneto separate reagent of 50 μ L is added to step 2) in described detection pipe, after mixing, 37 ± 0.5 DEG C of incubations
5min, carries out Magneto separate, removes supernatant;
4) cleanout fluid of 300 μ L is added to step 3) detect in pipe, mixing, carry out Magneto separate, remove supernatant;
5) step 4 is repeated) twice;
6) 150 μ L reagent 2 is added to step 5) detect in pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min, carry out
Magneto separate, removes supernatant;
7) cleanout fluid of 300 μ L is added to step 6) detect in pipe, mixing, carry out Magneto separate, remove supernatant;
8) step 7 is repeated) twice;
9) chemical luminous substrate of 200 μ L is added to step 8) detect in pipe, mixing, detects luminous intensity.
Comparative example 1
Reagent 1 in test kit unlike the test kit of embodiment 7 preparation and Magneto separate reagent, remaining
Composition is identical.
Preparing of reagent 1 is as follows:
A. No. 1 diluent of reagent preparation:
800ml purified water, Tris, 5.8g sodium chloride of 12.1g and 2ml Proclin300 are added in container,
It is stirred well to be completely dissolved;5g bovine serum albumin is added in container, is stirred well to be completely dissolved;
With the HCL of 4M, the pH value of solution is adjusted to 7.0-7.5;By purified water, solution is settled to 1L, uses 0.2 μm
Filter filters to obtain No. 1 diluent of reagent, and 2-8 DEG C of preservation is stand-by;
B. reagent preparation 1:
Using concentration 0.2M, pH is the biotin solution of the carbonate buffer solution preparation 0.5mg/ml of 9;According to
LKM-1 antigen and biotin mass ratio are that the ratio of 10:1 adds life in LKM-1 antigen solution
Thing cellulose solution, mix homogeneously, room temperature stands 18h, and reaction generates LKM-1 antigen-biotin conjugate;
To be separated by G-25 gel column containing the reactant liquor of LKM-1 antigen-biotin conjugate, remove
Unreacted biotin, obtains the solution containing LKM-1 antigen-biotin conjugate;Dilute with reagent 1
Release liquid and the solution containing LKM-1 antigen-biotin conjugate is diluted 0.1-0.3 μ g/mL, prepare reagent 1
Number;
Preparing of Magneto separate reagent is as follows:
A. preparation magnetic particle buffer:
800mL purified water, 12.1g Tris and 8.5g sodium chloride are added in container, is stirred well to completely
Dissolve;Again 5g bovine serum albumin, 50mL new-born calf serum and 0.2mL Proclin300 are added container
In, it is stirred well to be completely dissolved;With the HCL of 4M, the pH value of solution is adjusted to 7.9-8.1;Use purification
Solution is settled to 1L by water, filters to obtain magnetic particle buffer with 0.2 μm filter, and 2-8 DEG C of preservation is stand-by;
B. preparation Magneto separate reagent:
Taking 100mg magnetic particle, Magneto separate removes supernatant, with concentration be 0.025mol/L, pH be the 2-(N-of 4.5-5
Morpholine) ethanesulfonic acid buffer 10mL is resuspended;Add the EDC that concentration is 10mg/mL that 0.5-1mL newly prepares
Aqueous solution, room temperature suspendible 30-60min;Magnetic bead is made fully to activate, Magneto separate, removes supernatant, by concentration is
0.025mol/L, pH are that 4.5-5 2-(N-morpholine) ethanesulfonic acid buffer 10mL is resuspended;Add the chain of 4-8mg
Mould Avidin, room temperature suspendible 16-20h;Carry out Magneto separate again, remove supernatant, with magnetic particle buffer dilution weight
Hang 0.5mg/mL, prepare Magneto separate reagent.
Embodiment 9
The test kit of embodiment 7 and comparative example 1 is carried out performance evaluation:
1. the accuracy estimating of embodiment 7
Concentration is about the anti-LKM-1 IgG antibody sample of 200RU/mL (allowing its concentration deviation is ± 20%)
Product 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 is at 85-115% model
In enclosing, data see table 1, and evaluation result meets the requirements,
R: the response rate;
V: add the volume of standard solution;
V0: the volume of people source sample;
C: people source sample adds the detectable concentration after standard solution;
C0: the detectable concentration of people source sample;
Cs: the concentration of standard solution.
Table 1 accuracy estimating
2. the blank limit of embodiment 7 and comparative example 1 is evaluated
Detect as sample with zero-dose calibration object, replication 20 times, draw 20 measurement results
RLU value (relative light unit), calculate its meansigma methods (M) and standard deviation (SD), draw M+2SD
Corresponding RLU value, enters according to the concentration-RLU value result between zero-dose calibration object with adjacent calibration object
2 regression fits of row draw linear function, bring in above-mentioned equation by the RLU value corresponding to M+2SD,
Obtain the concentration value of correspondence, be blank limit.The blank limit of this method is not more than 1RU/mL, and data see table
2, A, B point even some matched curve and fit equation is shown in Fig. 1 a, the sky that the test kit of comparative example 1 preparation records
White limit data are shown in Table 2-1, and A, B point even some matched curve and fit equation are shown in Fig. 1 b, from data, real
Executing example 7 compared with comparative example 1, the background values obtained is lower, thus the blank limit obtained is lower, represents examination
The sensitivity of agent box is more preferable.
The blank limit of table 2 is evaluated
Table 2-1 blank limit is evaluated
3. the range of linearity evaluation of embodiment 7
At least 5 kinds will be diluted to by a certain percentage close to the high level sample of the range of linearity upper limit (200RU/mL)
Concentration, wherein the sample of low value concentration must be close to the lower limit of the range of linearity.Operate by test kit description,
Sample standard deviation duplicate detection 2 times to each concentration, calculates its meansigma methods, by result meansigma methods and dilution ratio
Carry out fitting a straight line with method of least square, and calculate linearly dependent coefficient r, the measurement scope of this method be [2,
200] RU/mL, correlation coefficient r answers >=0.9900.Data see table 3, matched curve and correlation coefficient and see figure
2, evaluation result meets the requirements.
Table 3 range of linearity evaluation
4. embodiment 7 and the reproducibility of comparative example 1
Test kit duplicate detection concentration in Example 7 is (20 ± 4) RU/mL and (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% draws coefficient of variation CV, and this method coefficient of variation (CV) is not more than 8%, and data are joined
Being shown in Table 4, the repeated data that the test kit of comparative example 1 preparation records are shown in Table 4-1, from data, implement
Example 7 is compared with comparative example 1, and the CV value obtained is lower, represents the repeatability of test kit more preferably,
CV=SD/M × 100%...................................... (2)
In formula: the CV coefficient of variation;The standard deviation of 10 measurement results of SD;10 measurement results of M
Meansigma methods.
Table 4 reproducibility
Measure serum-concentration (RU/mL) | Measure number of times | CV (%) in analyzing |
20 | 10 | 5.50% |
100 | 10 | 5.52% |
Table 4-1 reproducibility
Measure serum-concentration (RU/mL) | Measure number of times | CV (%) in analyzing |
20 | 10 | 7.31% |
100 | 10 | 7.05% |
5. the difference between batch evaluation of embodiment 7 and comparative example 1
The test kit of embodiment 7 is 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 30 measurement results
Meansigma methods (M) and standard deviation (SD), calculate the coefficient of variation (CV) according to formula (3), and this method becomes
Different coefficient (CV) is not more than 15%, and data see table 5, between what the test kit of comparative example 1 preparation recorded criticizes
Difference data is shown in Table 5-1, and from data, embodiment 7 is compared with comparative example 1, and the CV value obtained is lower,
The difference between batch representing test kit is more preferable,
CV=SD/M × 100%...................................... (3)
In formula: the CV coefficient of variation;The standard deviation of 30 measurement results of SD;30 measurement results of M
Meansigma methods.
Table 5 difference between batch evaluation
Measure serum-concentration (RU/mL) | Measure number of times | CV (%) between analysis |
20 | 30 | 7.00% |
100 | 30 | 5.69% |
Table 5-1 difference between batch is evaluated
Measure serum-concentration (IU/mL) | Measure number of times | CV (%) between analysis |
100 | 30 | 8.93% |
400 | 30 | 11.41% |
6. the Evaluation on specificity of embodiment 7
Taking 1 part of Anti-LKM-1IgG content is the sample of 0, adds Anti-SLA/LP IgG, makes sample
Middle cross reaction substrate concentration is 200RU/mL, uses this test kit to detect this sample, measures in sample
Anti-LKM-1IgG content.The results are shown in Table 6, this method and anti-SLA/LP antibody no cross reaction.
Data see table 6, and evaluation result meets the requirements.
Table 6 specificity experiments
Test cross reaction thing | Concentration | RLU | Measurement result (RU/mL) |
Anti-SLA/LP IgG | 200RU/mL | 3785 | < 1 |
7. the relativity evaluation of embodiment 7
With test kit and the anti-LKM-1 IgG antibody detection kit (enzyme linked immunological of commercialization of embodiment 7
Absorption method) 240 parts of human serum samples are detected simultaneously.Its testing result sees accompanying drawing 3, with anti-LKM-1
The result that IgG antibody detection kit (enzyme linked immunosorbent assay) measures is abscissa, in the process of the present invention
The result of mensuration be that vertical coordinate makees regression analysis, dependent equation is: y=0.9911x+0.6454, correlation coefficient
For R2: 0.9875.Showing through statistical procedures result, this method is with the test kit clinical sample of additive method
Measured value dependency is good.
8. the estimation of stability of embodiment 7
Test kit carries out 4 DEG C of 12 months and the experiments of 37 DEG C of accelerated stabilities of 7 days respectively, and result shows
The change of kit standard product luminous intensity, batch in and betweenrun precision, accuracy index all at normal model
Within enclosing, test kit effect duration was up to 12 months.
Obviously, the above embodiment of the present invention is only for clearly demonstrating example of the present invention, and is not
Restriction to embodiments of the present invention, for those of ordinary skill in the field, in described above
On the basis of can also make other changes in different forms, here cannot be to all of embodiment
Give exhaustive, every belong to obvious change that technical scheme extended out or variation is still located
Row in protection scope of the present invention.
Claims (5)
1. a magnetic microparticle chemiluminescence quantitative determination reagent kit for anti-LKM-1 IgG antibody, its feature exists
In, described test kit includes:
1) anti-LKM-1 IgG antibody calibration object, containing anti-LKM-1 IgG antibody and bovine serum albumin
Tris buffer, described anti-LKM-1 IgG antibody calibration object is the liquid calibration object of 6 levels, described 6
In the liquid calibration object of individual level, the concentration of anti-LKM-1 IgG antibody is respectively 0, and 5,20,50,100,
200RU/mL;
2) anti-LKM-1 IgG antibody quality-control product, containing anti-LKM-1 IgG antibody and bovine serum albumin
Tris buffer, described anti-LKM-1 IgG antibody quality-control product comprises the liquid quality control product of 2 levels, described
In the liquid quality control product of 2 levels, the target value concentration range of anti-LKM-1 IgG antibody is respectively (20 ± 4)
RU/mL and (100 ± 20) RU/mL;
3) reagent 1, containing biotin labeled LKM-1 antigen and the Tris buffer of bovine serum albumin;
4) reagent 2, sheep anti-human polyclonal antibody containing alkali phosphatase enzyme mark and bovine serum albumin
Tris buffer;
5) Magneto separate reagent, the magnetic particle containing marked by streptavidin and the Tris of bovine serum albumin
Buffer;
6) cleanout fluid;
The content of described seminal plasma fructose detection kit 1, reagent 2 and Magneto separate reagent than for 1:3:1, described contains
Amount ratio is volume ratio.
Test kit the most according to claim 1, it is characterised in that: the material of described magnetic particle is Fe2O3;
Described magnetic particle pan coating has carboxylic group, is coated carboxyl group content in thing and is more than 20wt%, described magnetic
The size of microgranule is 1-3 μm.
3. prepare the method for test kit as described in any one of claim 1-2 for one kind, it is characterised in that the party
Method comprises the steps:
(1) anti-LKM-1 IgG antibody calibration object is prepared:
A. anti-LKM-1 IgG antibody calibration object diluent is prepared:
Purified water, Tris, sodium chloride and Proclin300 are added in container, are stirred well to be completely dissolved,
Tris concentration is 1wt%, sodium chloride concentration be 1wt%, Proclin300 concentration be 0.2v%;With the HCL of 4M
The pH value of solution is adjusted to 7.0-7.5;Bovine serum albumin is added in container, is stirred well to be completely dissolved,
The concentration of bovine serum albumin is 4wt%;With the HCL of 4M, the pH value of solution is adjusted to 7.0-7.5 again;With
Aperture is that the filter of 0.2 μm filters to obtain anti-LKM-1 IgG antibody calibration object diluent, and 2-8 DEG C of preservation is stand-by;
B. anti-LKM-1 IgG antibody calibration object is prepared:
By anti-LKM-1 IgG antibody by anti-LKM-1 IgG antibody calibration object diluted to each concentration point
It is 0,5,20,50,100,200RU/mL;
(2) anti-LKM-1 IgG antibody quality-control product is prepared:
By anti-for anti-double-strand LKM-1 IgG antibody by above-mentioned anti-LKM-1 IgG antibody calibration object diluted
It is 20,100RU/mL to each concentration point;
(3) reagent preparation 1:
A. No. 1 diluent of reagent preparation:
Purified water, Tris, sodium chloride and Proclin300 are added in container, are stirred well to be completely dissolved,
The concentration of Tris is 1wt%, sodium chloride concentration be the concentration of 0.5wt%, Proclin300 be 0.2v%;By cattle
Serum albumin adds in container, is stirred well to be completely dissolved, and the concentration of bovine serum albumin is 0.5wt%;
With the HCL of 4M, the pH value of solution is adjusted to 7.0-7.5;Reagent is filtered to obtain with the filter that aperture is 0.2 μm
No. 1 diluent, 2-8 DEG C of preservation is stand-by;
B. reagent preparation 1:
By LKM-1 antigen with purified water dissolve, under the conditions of 2-8 DEG C with concentration be 0.2M, pH be 9.0
Carbonate buffer solution dialysis 2h, be then concentrated into the antigenic solution that concentration is 2-4mg/mL, use concentration
It is the biotin solution of 0.5-1.0mg/ml for the carbonate buffer solution compound concentration that 0.2M, pH are 8.5-9;
It is that the ratio of 10:1 adds biology in LKM-1 antigenic solution according to LKM-1 antigen and biotin mass ratio
Cellulose solution, mix homogeneously, room temperature stands 12-18h, and reaction generates LKM-1 antigen-biotin conjugate;
It is 0.2M pH by concentration by the reactant liquor containing LKM-1 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 the solution containing LKM-1 antigen-biotin conjugate;To contain with No. 1 diluent of reagent
The solution of LKM-1 antigen-biotin conjugate is diluted to 0.1-0.3 μ g/mL, prepares reagent 1;
(4) reagent preparation 2:
A. No. 2 diluents of reagent preparation:
By purified water, 4-hydroxyethyl piperazine ethanesulfonic acid, sodium chloride, bovine serum albumin, ZnCl2And Proclin300
Adding in container, be stirred well to be completely dissolved, the concentration of 4-hydroxyethyl piperazine ethanesulfonic acid is 0.6wt%, chlorine
Change na concn is 0.8wt%, and the concentration of bovine serum albumin is 0.5wt%, ZnCl2Concentration be 0.1wt ‰,
The concentration of Proclin300 is 0.2v ‰, MgCl2Concentration be 0.1 ‰;With the HCL of 4M by the pH of solution
Value is adjusted to 7.5-8.0;Filtering to obtain No. 2 diluents of reagent with the filter that aperture is 0.2 μm, 2-8 DEG C of preservation is stand-by;
B. reagent preparation 2:
1mg sheep anti-human polyclonal antibody is joined the 2-iminothiolane salt that 2-4 μ L concentration is 10mg/mL
In acid salt solution, room temperature stands 20min, adds the glycine solution 10 μ L of 0.1moL/L, and room temperature stands
5min, with G-25 gel column desalination, collects the sheep anti-human polyclonal antibody after activation, and 2-8 DEG C of preservation is standby
With;The alkali phosphatase of 1.5mg is joined 4-(the N-maleimide that concentration is 5mg/mL of 10-20 μ L
Aminomethyl) in hexamethylene-1-carboxylic acid butanimide ester solution, room temperature stands 30min, uses G-25 gel
Post desalination, collects the alkali phosphatase after activation, and 2-8 DEG C saves backup;Goat-anti people's Anti-TNF-α by activation
Body mixes with the alkali phosphatase of activation, stands 12-24h under the conditions of 2-8 DEG C, pure with Supperdex200 gel
Change column purification conjugate, it is thus achieved that sheep anti-human polyclonal antibody-alkali phosphatase junctional complex concentrated solution, 2-8 DEG C of guarantor
Deposit standby;By sheep anti-human polyclonal antibody-alkali phosphatase junctional complex concentrated solution No. 2 diluted of reagent
To 0.02-0.1 μ g/mL, prepare reagent 2;
(5) preparation Magneto separate reagent:
A. preparation magnetic particle buffer:
Purified water, Tris and sodium chloride being added in container, be stirred well to be completely dissolved, the concentration of Tris is
1wt%, the concentration of sodium chloride is 0.8wt%;Again by bovine serum albumin, new-born calf serum and Proclin300
Adding in container, be stirred well to be completely dissolved, the concentration of bovine serum albumin is 0.5wt%, newborn Sanguis Bovis seu Bubali
Clear concentration be 5v%, Proclin300 concentration be 0.2v ‰;With the HCL of 4M, the pH value of solution is adjusted to
7.9-8.1;Filtering to obtain magnetic particle buffer with the filter that aperture is 0.2 μm, 2-8 DEG C preserves stand-by magnetic;
B. preparation Magneto separate reagent:
Take 100mg magnetic particle, use magnetic frame absorption, suck supernatant after static 2min, add in magnetic particle
Enter concentration be 0.025mol/L, pH be 2-(N-morpholine) the ethanesulfonic acid buffer 10mL of 4.5-5, fully
Mixing;Add the concentration that 0.5-1mL newly prepares and be 1-(3-the dimethylamino-propyl)-3-second of 10mg/mL
Base carbodiimide and N-hydroxy-succinamide aqueous solution, room temperature mixing 30-60min obtains magnetic bead suspending system;
Using 2-(N-morpholine) ethanesulfonic acid buffer compound concentration is the solution of streptavidin of 5mg/mL, then
The Streptavidin of the 0.8-1.6mL being formulated, 4 DEG C of conditions it are directly added in described magnetic bead suspending system
Lower suspendible 16-20h;Re-use magnetic frame absorption, after static 2min, suck supernatant, in magnetic particle, add 10mL
Concentration be 1M, pH be the ethanolamine solutions of 8.5, room temperature reaction 1-2h, re-use magnetic frame absorption, quiet
Only suck supernatant after 2min, in magnetic particle, add the dilution of appropriate magnetic particle buffer make final concentration of
0.5mg/mL, prepares Magneto separate reagent;
(6) preparation cleanout fluid:
Purified water, Tris and sodium chloride being added in container, be stirred well to be completely dissolved, the concentration of Tris is
1wt%, the concentration of sodium chloride is 0.8wt%;Again Tween-20 and Triton100 is added in container, fully
Stirring to mixing completely, the concentration of Tween-20 be the concentration of 0.5wt%, Triton100 be 0.5wt%;With
The pH value of solution is adjusted to 7.5-8.0 by the HCL of 4M, filters to obtain cleanout fluid with the filter that aperture is 0.2 μm,
2-8 DEG C of preservation.
4. the detection method of test kit as claimed in claim 1, it is characterised in that the method include as
Lower step:
1) anti-LKM-1 IgG antibody calibration object is put into Full-automatic chemiluminescence immunoassay analysis meter test position,
Obtain the matched curve exported by Full-automatic chemiluminescence immunoassay analysis meter;
2) anti-LKM-1 IgG antibody quality-control product is put into above-mentioned analyser test position, obtains by full-automation
The test luminous value learning the described quality-control product of luminescence immunoassay instrument output and the matching song obtained by step 1
Line matching obtains the concentration value of anti-LKM-1 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, obtains the concentration value of the sample to be tested exported by Full-automatic chemiluminescence immunoassay analysis meter.
Detection method the most according to claim 4, it is characterised in that the method specifically includes following step
Rapid:
1) specimen to be measured after 20 μ L anti-LKM-1 IgG antibody calibration object or quality-control product or 1:20 dilution is added to detection
Guan Zhong;
2) reagent 1 of 50 μ L is added to step 1) in described detection pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min;
3) the Magneto separate reagent of 50 μ L is added to step 2) in described detection pipe, after mixing, 37 ± 0.5 DEG C of incubations
5min, carries out Magneto separate, removes supernatant;
4) cleanout fluid of 300 μ L is added to step 3) detect in pipe, mixing, carry out Magneto separate, remove supernatant;
5) step 4 is repeated) twice;
6) 150 μ L reagent 2 is added to step 5) detect in pipe, after mixing, 37 ± 0.5 DEG C of incubation 10min, carry out
Magneto separate, removes supernatant;
7) cleanout fluid of 300 μ L is added to step 6) detect in pipe, mixing, carry out Magneto separate, remove supernatant;
8) step 7 is repeated) twice;
9) chemical luminous substrate of 200 μ L is added to step 8) detect in pipe, mixing, detects luminous intensity;
Described step 1), step 2) and step 3) all include Full-automatic chemiluminescence immunoassay analysis meter complete from
Dynamic detecting step.
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