CN107402309A - A kind of chemical luminescent analysis reagent kid of phosphatide IgG antibody and preparation method thereof and detection method - Google Patents
A kind of chemical luminescent analysis reagent kid of phosphatide IgG antibody and preparation method thereof and detection method Download PDFInfo
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- CN107402309A CN107402309A CN201710829885.XA CN201710829885A CN107402309A CN 107402309 A CN107402309 A CN 107402309A CN 201710829885 A CN201710829885 A CN 201710829885A CN 107402309 A CN107402309 A CN 107402309A
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- antigen
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- biotin labeling
- phosphatide
- biotin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The present invention relates to a kind of chemical luminescent analysis reagent kid of phosphatide IgG antibody, including following reagent：The cuorin antigen of biotin labeling containing 5 ~ 100ng/mL, the phosphatidyl-ethanolamine antigen of 5 ~ 100ng/mL biotin labeling, the 5 ~ 100ng/mL phosphatidylinositols antigen of biotin labeling, the 5 ~ 100ng/mL phosphatidylserine antigen of biotin labeling, 1 ~ 10 μ g/mL biotin labeling beta 2 glycoprotein I antigen mixed solution；Concentration is the mouse anti-human igg monoclonal antibody solution of 10 ~ 1000ng/mL alkali phosphatase enzyme mark；Concentration is the nano magnetic microparticle suspending liquid of 0.1 ~ 1 mg/mL marked by streptavidin.The high sensitivity of this kit, precision is high, the range of linearity is wide, stability is good, term of validity length, and environmental-protecting performance greatly improves.
Description
Technical field
The invention belongs to medicine equipment external diagnosis reagent chemiluminescence immune assay field, more particularly to a kind of phosphatide
Chemical luminescent analysis reagent kid of IgG antibody and preparation method thereof and detection method.
Background technology
Phosphatide in human body mainly includes negatively charged cuorin (Cardiolipin, CL), phosphatidylserine
(phosphatidylserine, PI), phosphatidylinositols (phosphatidylinositol, PS), the phosphatide with neutral charge
Acyl monoethanolamine (phosphatidylethanolamine, PE) etc..Anti-phospholipid antibody (Anti-phospholipids, APL) is
Family includes phosphatide or the pathologic antibody of negatively charged phosphatide and carrier protein complex for autoantigen.Anti-phospholipid antibody
Syndrome (antiphospholipidsyndrome, APS), it is one group of autoimmune disease relevant with anti-phospholipid antibody.
Typical clinical manifestation has arterial-venous thrombus, decrease of platelet and habitual abortion etc., is more common in young man, 60%-
80% is female patient, and women the median age is 30 years old.APS can be divided into primary APS and Secondary cases APS, and Secondary cases APS is common
In the autoimmunity disease such as systemic loupus erythematosus (SLE) or rheumatoid arthritis (RA).It is in addition, also a kind of rare pernicious
APS (catastrophic APS), shows as the extensive thrombosis of progressive in a short time, causes MOF even dead
Die.It is the necessary condition for establishing APS diagnosis that APL (IgG types and/or IgM types) is detected in APS patient's blood.
High radioactivity, the term of validity of radiommunoassay (RIA) are short, are brought to operator healthy hidden danger and to environment
Pollution, common microwell plate ELISA (EIA) precision is poor, poor sensitivity, can not realize quantitative detection, is also not easy reality
Existing full-automation.Traditional ELISA sensitivity is relatively low.
Traditional antiphospholipid antibody syndrome detection project is cardiolipin antibody, and it has the problem of sensitivity is low.
The content of the invention
The chemiluminescence that the technical problems to be solved by the invention are to provide a kind of phosphatide IgG antibody of high sensitivity quantifies
Detection kit.
Another technical problem to be solved by this invention is to provide the preparation method of mentioned reagent box.
Another technical problem to be solved by this invention is to provide the detection method detected using mentioned reagent box.
To solve above technical problem, the present invention adopts the following technical scheme that：
It is an object of the invention to a kind of chemical luminescent analysis reagent kid of phosphatide IgG antibody, including following examination
Agent：
The phosphatide of the cuorin antigen of biotin labeling containing 5~100ng/mL, 5~100ng/mL biotin labeling
Acyl monoethanolamine antigen, 5~100ng/mL biotin labeling phosphatidylinositols antigen, 5~100ng/mL biotin labeling
Phosphatidylserine antigen, 1~10 μ g/mL biotin labeling beta 2 glycoprotein I antigenic solution mixed solution；
Concentration is the mouse anti-human igg monoclonal antibody solution of 10~1000ng/mL alkali phosphatase enzyme mark；
Concentration is the nano magnetic microparticle suspending liquid of 0.1~1mg/mL marked by streptavidin.
Preferably, the concentration of the cuorin antigen of the biotin labeling described in described mixed solution is 10~20ng/
mL。
Preferably, the concentration of the phosphatidyl-ethanolamine antigen of the biotin labeling described in described mixed solution be 10~
20ng/mL。
Preferably, the concentration of the phosphatidylinositols antigen of the biotin labeling described in described mixed solution be 10~
20ng/mL。
Preferably, the concentration of the phosphatidylserine antigen of the biotin labeling described in described mixed solution be 10~
20ng/mL。
Preferably, the concentration of the beta 2 glycoprotein I antigen of the biotin labeling described in described mixed solution be 2.5~
3.5μg/mL。
Preferably, the concentration of the anti-human IgM monoclonal antibody solution of the mouse of described alkali phosphatase enzyme mark be 90~
110ng/mL.Preferably, described cuorin antigen, described phosphatidyl-ethanolamine antigen, described phosphatidylinositols antigen,
Described phosphatidylserine antigen is respectively by amido modified.
Preferably, described biotin is n-hydroxysuccinimide.
Preferably, the molar ratio of described cuorin antigen and described biotin is 1：10~100；Described phosphorus
The molar ratio of acyl monoethanolamine antigen and described biotin is 1：10~100；Described phosphatidylinositols antigen and institute
The molar ratio for the biotin stated is 1：10~100；Described phosphatidylserine antigen feeds intake with described biotin
Mol ratio is 1：10~100；The molar ratio of described beta 2 glycoprotein I antigen and described biotin is 1：10~100；Institute
The alkaline phosphatase stated and the mass ratio that feeds intake of described mouse anti-human igg monoclonal antibody are 1~10：1；Described nano magnetic is micro-
The carboxyl-content of grain is not less than 0.4mmol/g.
It is further preferred that the molar ratio of described cuorin antigen and described biotin is 1：18~22；Institute
The molar ratio of the phosphatidyl-ethanolamine antigen stated and described biotin is 1：18~22；Described phosphatidylinositols antigen
Molar ratio with described biotin is 1：18~22；The throwing of described phosphatidylserine antigen and described biotin
It is 1 to expect mol ratio：18~22；The molar ratio of described beta 2 glycoprotein I antigen and described biotin is 1：18~22；Institute
The alkaline phosphatase stated and the mass ratio that feeds intake of described mouse anti-human igg monoclonal antibody are 1~2：1.
Preferably, it is molten also to include the horizontal anti-phosphatide IgG antibody of two or more various concentrations for described kit
Calibration object, standard curve, the sample of the horizontal anti-phosphatide IgG antibody solution of the quality-control product of liquid, two or more various concentrations
This dilution and chemical luminous substrate.
It is further preferred that described quality-control product includes anti-phosphatide IgG antibody, 0.005~0.1mol/L, pH are 7~8
Phosphate buffer, the preservative that the bovine serum albumin and mass concentration that mass concentration is 0.1~5% are 0.01~1%；
Described calibration object includes anti-phosphatide IgG antibody, and 0.005~0.1mol/L, pH are 7~8 phosphate buffer,
The preservative that the bovine serum albumin and mass concentration that mass concentration is 0.1~5% are 0.01~1%；
Described Sample dilution includes the phosphate buffer that 0.005~0.1mol/L, pH are 7~8, and mass concentration is
0.1~5% bovine serum albumin and mass concentration is 0.01~1% 0.01~1% preservative.
It is further preferable that described quality-control product includes anti-phosphatide IgG antibody, 0.009~0.011mol/L, pH be 7~
7.5 phosphate buffer, the anti-corrosion that the bovine serum albumin and mass concentration that mass concentration is 0.9~1.1% are 0.01~1%
Agent；
Described calibration object includes anti-phosphatide IgG antibody, and the phosphate that 0.009~0.011mol/L, pH are 7~7.5 delays
Fliud flushing, the preservative that the bovine serum albumin and mass concentration that mass concentration is 0.9~1.1% are 0.01~1%；
Described Sample dilution includes the phosphate buffer that 0.009~0.011mol/L, pH are 7~7.5, and quality is dense
Spend the preservative for being 0.01~1% for 0.9~1.1% bovine serum albumin and mass concentration.
It is further preferred that the concentration of the anti-phosphatide IgG antibody in quality-control product described in one is 4~21RU/mL, separately
The concentration of anti-phosphatide IgG antibody in quality-control product described in one is 50~100RU/mL；It is anti-in calibration object described in one
The concentration of phosphatide IgG antibody is 10~40RU/mL, and the concentration of the anti-phosphatide IgG antibody in the calibration object described in another is
100~300RU/mL.
It is further preferable that the concentration of the anti-phosphatide IgG antibody in quality-control product described in one is 9~11RU/mL, it is another
The concentration of anti-phosphatide IgG antibody in individual described quality-control product is 72~88RU/mL；Anti- phosphorus in calibration object described in one
The concentration of fat IgG antibody is 18~22RU/mL, and the concentration of the anti-phosphatide IgG antibody in the calibration object described in another is 180
~220RU/mL.
It is a further object to provide a kind of chemical luminescent analysis reagent kid of described phosphatide IgG antibody
Preparation method,
Cuorin antigen, the 5~100ng/mL biotin labeling of the described biotin labeling containing 5~100ng/mL
Phosphatidyl-ethanolamine antigen, 5~100ng/mL biotin labeling phosphatidylinositols antigen, 5~100ng/mL biology
Element mark phosphatidylserine antigen, 1~10 μ g/mL biotin labeling beta 2 glycoprotein I antigen mixed solution system
Preparation Method comprises the following steps：
Step (1), described cuorin antigen, described phosphatidyl-ethanolamine antigen, described phosphatidylinositols resisted
Former, described phosphatidylserine antigen, described beta 2 glycoprotein I antigen respectively with 0.01~0.03mol/L, pH be 7~
7.5 phosphate buffer, the dialysed overnight at 2~8 DEG C, prepare cardiolipin antigenic solution respectively, phosphatidyl-ethanolamine resists
Original solution, phosphatidylinositols antigenic solution, phosphatidylserine antigenic solution, beta 2 glycoprotein I antigenic solution,
Step (2), described biotin is dissolved in substance withdrawl syndrome is configured in dimethyl sulfoxide (DMSO) is 8~12mM's
Biotin solution,
Step (3), by described cuorin antigenic solution, described phosphatidyl-ethanolamine antigenic solution, described phosphatide
Acyl inositol antigenic solution, described phosphatidylserine antigenic solution, described beta 2 glycoprotein I antigenic solution respectively with it is described
Biotin solution is well mixed, and is placed 20~40 minutes at 15~40 DEG C, is then respectively adding 0.05~1.1M three hydroxyl first
Base aminomethane buffer solution, terminating reaction, placed at 15~40 DEG C 8~12 minutes, be then 0.1~5% with containing mass ratio
Bovine serum albumin(BSA), the phosphate buffer that pH is 7~8, substance withdrawl syndrome is 0.005~0.1mol/L is diluted
Cuorin antigenic solution, the phosphatidyl-ethanolamine antigen of described biotin labeling for respectively obtaining described biotin labeling are molten
Liquid, the phosphatidylinositols antigenic solution of described biotin labeling, the phosphatidylserine antigen of described biotin labeling are molten
The beta 2 glycoprotein I antigenic solution of liquid, described biotin labeling；
Step (4), the phosphatidyl second by the cuorin antigenic solution of described biotin labeling, described biotin labeling
Hydramine antigenic solution, the phosphatidylinositols antigenic solution of described biotin labeling, the phosphatidyl silk of described biotin labeling
Propylhomoserin antigenic solution, the beta 2 glycoprotein I antigenic solution of described biotin labeling be mixed to get it is described containing 5~
The cuorin antigen of 100ng/mL biotin labeling, the phosphatidyl-ethanolamine antigen of 5~100ng/mL biotin labeling, 5
The phosphatidylserine of the phosphatidylinositols antigen of~100ng/mL biotin labeling, 5~100ng/mL biotin labeling
Antigen, 1~10 μ g/mL biotin labeling beta 2 glycoprotein I antigen mixed solution；
The preparation method of the mouse anti-human igg monoclonal antibody solution of described alkali phosphatase enzyme mark comprises the following steps：
Step (1), mouse anti-human igg monoclonal antibody is added to the 2- imido grpup sulfane hydrochloric acid that concentration is 8~12mg/ml
In salt coupling agent, 18~25 minutes are stood at 15~40 DEG C, adds 0.01~0.11mol/L glycine solution, 15~
4~6 minutes are stood at 40 DEG C, with G-25 gel column desalinations, collects the mouse anti-human igg monoclonal antibody after activation, 2~8 DEG C of guarantors
Deposit it is standby,
Step (2), 4- (N- maleimidomethyls) hexamethylene that alkaline phosphatase enzyme solutions are added to 1~10mg/ml
In alkane -1- carboxylic acid succinimide ester solutions, 10~100 minutes are stood at 15~40 DEG C, with G-25 gel column desalinations, is collected
Alkaline phosphatase after activation, 2~8 DEG C save backup,
Step (3), the mouse anti-human igg monoclonal antibody after described activation and alkaline phosphatase after described activation mixed
Close, 12~24h is stood at 2~8 DEG C, is purified with Supperdex200 gel-purified posts, attachment concentrated solution is obtained, 2~8
DEG C save backup,
Step (4), by described attachment concentrated solution be 0.1~5% containing mass ratio bovine serum albumin(BSA), pH be
5.8~8.2, substance withdrawl syndrome is that 0.01~0.1mol/L MES buffer solution is diluted the alkali for being made described
The mouse anti-human igg monoclonal antibody solution of acid phosphatase mark；
The nano magnetic microparticle suspending liquid of described marked by streptavidin is by using the ox blood that mass ratio is 0.1~5%
The phosphate buffer that pure albumen, pH are 7~8, substance withdrawl syndrome is 0.005~0.1mol/L is to marked by streptavidin
Nanometer magnetic particle carry out be resuspended be made.
Preferably, the life of the cuorin antigen, 5~100ng/mL of the biotin labeling containing 5~100ng/mL
Phosphatidylinositols antigen, the 5~100ng/ of the phosphatidyl-ethanolamine antigen of thing element mark, 5~100ng/mL biotin labeling
The phosphatidylserine antigen of mL biotin labeling, 1~10 μ g/mL biotin labeling beta 2 glycoprotein I antigen mixing
The preparation method of solution comprises the following steps：
Step (1), described cuorin antigen, described phosphatidyl-ethanolamine antigen, described phosphatidylinositols resisted
Former, described phosphatidylserine antigen, described beta 2 glycoprotein I antigen respectively with 0.01~0.03mol/L, pH be 7~
7.5 phosphate buffer, the dialysed overnight at 2~8 DEG C, prepare cardiolipin antigenic solution respectively, phosphatidyl-ethanolamine resists
Original solution, phosphatidylinositols antigenic solution, phosphatidylserine antigenic solution, beta 2 glycoprotein I antigenic solution,
Step (2), described biotin is dissolved in substance withdrawl syndrome is configured in dimethyl sulfoxide (DMSO) is 8~12mM's
Biotin solution,
Step (3), by described cuorin antigenic solution, described phosphatidyl-ethanolamine antigenic solution, described phosphatide
Acyl inositol antigenic solution, described phosphatidylserine antigenic solution, described beta 2 glycoprotein I antigenic solution respectively with it is described
Biotin solution is well mixed, and is placed 20~40 minutes at 15~40 DEG C, is then respectively adding 0.9~1.1M three hydroxyl first
Base aminomethane buffer solution, terminating reaction place 8~12 minutes at 15~40 DEG C, then be 0.9 containing mass ratio~
1.1% bovine serum albumin(BSA), the phosphate buffer that pH is 7~7.5, substance withdrawl syndrome is 0.009~0.011mol/L
It is diluted cuorin antigenic solution, the phosphatidyl ethanol of described biotin labeling for respectively obtaining described biotin labeling
Amine antigenic solution, the phosphatidylinositols antigenic solution of described biotin labeling, the phosphatidyl silk ammonia of described biotin labeling
The beta 2 glycoprotein I antigenic solution of sour antigenic solution, described biotin labeling；
Step (4), the phosphatidyl second by the cuorin antigenic solution of described biotin labeling, described biotin labeling
Hydramine antigenic solution, the phosphatidylinositols antigenic solution of described biotin labeling, the phosphatidyl silk of described biotin labeling
Propylhomoserin antigenic solution, the beta 2 glycoprotein I antigenic solution of described biotin labeling be mixed to get it is described containing 5~
The cuorin antigen of 100ng/mL biotin labeling, the phosphatidyl-ethanolamine antigen of 5~100ng/mL biotin labeling, 5
The phosphatidylserine of the phosphatidylinositols antigen of~100ng/mL biotin labeling, 5~100ng/mL biotin labeling
Antigen, 1~10 μ g/mL biotin labeling beta 2 glycoprotein I antigen mixed solution.
Preferably, the preparation method of the mouse anti-human igg monoclonal antibody solution of described alkali phosphatase enzyme mark is included such as
Lower step：
Step (1), mouse anti-human igg monoclonal antibody is added to the 2- imido grpup sulfane hydrochloric acid that concentration is 8~12mg/ml
In salt coupling agent, 18~25 minutes are stood at 15~40 DEG C, adds 0.09~0.11mol/L glycine solution, 15~
4~6 minutes are stood at 40 DEG C, with G-25 gel column desalinations, collects the mouse anti-human igg monoclonal antibody after activation, 2~8 DEG C of guarantors
Deposit it is standby,
Step (2), 4- (N- maleimidomethyls) hexamethylene that alkaline phosphatase enzyme solutions are added to 4~6mg/ml
In alkane -1- carboxylic acid succinimide ester solutions, 25~35 minutes are stood at 15~40 DEG C, with G-25 gel column desalinations, is collected
Alkaline phosphatase after activation, 2~8 DEG C save backup,
Step (3), the mouse anti-human igg monoclonal antibody after described activation and alkaline phosphatase after described activation mixed
Close, 12~24h is stood at 2~8 DEG C, is purified with Supperdex200 gel-purified posts, attachment concentrated solution is obtained, 2~8
DEG C save backup,
Step (4), by described attachment concentrated solution be 0.9~1.1% containing mass ratio bovine serum albumin(BSA), pH be
5.8~6.2, substance withdrawl syndrome is diluted obtained institute for 0.045~0.0.055mol/L MES buffer solution
The mouse anti-human igg monoclonal antibody solution for the alkali phosphatase enzyme mark stated.
Preferably, the nano magnetic microparticle suspending liquid of described marked by streptavidin by using mass ratio be 0.9~
1.1% bovine serum albumin(BSA), the phosphate-buffered that pH is 7~7.5, substance withdrawl syndrome is 0.009~0.0.011mol/L
Liquid to the nanometer magnetic particle of marked by streptavidin be resuspended and is made.
Third object of the present invention is to provide a kind of chemical luminescent analysis reagent kid of described phosphatide IgG antibody
Detection method, comprise the following steps：
Step (1), by sample stoste and Sample dilution it is 1 by volume：After 10~100 ratio is mixed to get dilution
Sample to be tested；
Step (2), add in detection pipe sample to be tested after described dilution, the heart phosphorus containing biotin labeling
Fat antigen, the phosphatidyl-ethanolamine antigen of biotin labeling, the phosphatidylinositols antigen of biotin labeling, the phosphorus of biotin labeling
Acyl serine antigen, the mixed solution of beta 2 glycoprotein I antigen of biotin labeling and receiving for described marked by streptavidin
Rice magnetic particle suspension, 10~30min of incubation obtains the first solution at 25~40 DEG C；Wherein, test sample is treated after described dilution
This addition is 10~60 μ L；Sample to be tested after described dilution, the cuorin antigen containing biotin labeling
Molten, biotin labeling phosphatidyl-ethanolamine antigen, the phosphatidylinositols antigen of biotin labeling, the phosphatidyl of biotin labeling
The nano magnetic of serine antigen, the beta 2 glycoprotein I antigen mixed solution of biotin labeling and described marked by streptavidin is micro-
The volume ratio that feeds intake of grain suspension is 1：0.5~4:0.5~4；
Step (3), addition magnetic field, make the first described solution be settled in magnetic field, remove supernatant, then cleaned liquid
After being cleaned multiple times, magnetic field is removed, obtains the second solution；
Step (4), the mouse anti-human igg monoclonal for adding into the second described solution described alkali phosphatase enzyme mark
Antibody-solutions, 10~30min is incubated at 25~40 DEG C and obtains the 3rd solution；Wherein, after described dilution sample to be tested with it is described
Alkali phosphatase enzyme mark mouse anti-human igg monoclonal antibody solution the volume ratio that feeds intake for 1：1.5~10.5；
Step (5), addition magnetic field, make the 3rd described solution be settled in magnetic field, remove supernatant, then cleaned liquid
After being cleaned multiple times, supernatant is removed, adds described chemical luminous substrate, magnetic field is removed, after being fully suspended, at 25~40 DEG C
1~10min is incubated, detects the relative luminous intensity value in 3 seconds；Wherein, sample to be tested and described chemistry after described dilution
The volume ratio that feeds intake of luminous substrate is 1：1~15.
Preferably, described detection method, comprises the following steps：
Step (1), by sample stoste and Sample dilution it is 1 by volume：After 18~22 ratio is mixed to get dilution
Sample to be tested；
Step (2), add in detection pipe sample to be tested after described dilution, the heart phosphorus containing biotin labeling
Fat antigen, the phosphatidyl-ethanolamine antigen of biotin labeling, the phosphatidylinositols antigen of biotin labeling, the phosphorus of biotin labeling
Acyl serine antigen, the mixed solution of beta 2 glycoprotein I antigen of biotin labeling and receiving for described marked by streptavidin
Rice magnetic particle suspension, 10~20min of incubation obtains the first solution at 36~38 DEG C；Wherein, test sample is treated after described dilution
This addition is 18~22 μ L, sample to be tested after described dilution, the cuorin antigen containing biotin labeling,
The phosphatidyl-ethanolamine antigen of biotin labeling, the phosphatidylinositols antigen of biotin labeling, the phosphatidyl silk of biotin labeling
Propylhomoserin antigen, biotin labeling beta 2 glycoprotein I antigen mixed solution and described marked by streptavidin nano magnetic it is micro-
The volume ratio that feeds intake of grain suspension is 1：2.4~2.6：2.4~2.6；
Step (3), addition magnetic field, make the first described solution be settled in magnetic field, remove supernatant, then cleaned liquid
After being cleaned multiple times, magnetic field is removed, obtains the second solution；
Step (4), the mouse anti-human igg monoclonal for adding into the second described solution described alkali phosphatase enzyme mark
Antibody-solutions, 10~20min of incubation obtains the 3rd solution at 36~38 DEG C；Wherein, sample to be tested and institute after described dilution
The volume ratio that feeds intake of the mouse anti-human igg monoclonal antibody solution for the alkali phosphatase enzyme mark stated is 1：4.5~5.5；
Step (5), addition magnetic field, make the 3rd described solution be settled in magnetic field, remove supernatant, then cleaned liquid
After being cleaned multiple times, supernatant is removed, adds described chemical luminous substrate, magnetic field is removed, after being fully suspended, at 36~38 DEG C
4~6min is incubated, detects the relative luminous intensity value in 3 seconds；Wherein, sample to be tested and described chemistry hair after described dilution
The volume ratio that feeds intake of light substrate is 1：9~11.
Preferably, described detection method automatic detection or detection manually on Full-automatic chemiluminescence apparatus.
The present invention Cleaning Principle be：Using indirect method principle：Phospholipid antigen (including cuorin, the phosphorus of biotin labeling
Acyl monoethanolamine, phosphatidylinositols, phosphatidylserine) and the glycoprotein I antigens of β 2 of biotin labeling, sample in APL
Magnetic particles of the IgG with being coated with Streptavidin forms immune complex, by magnetic separation system, washing is gone by immune response
After uncombined antigen, antibody and impurity, the mouse anti-human igg monoclonal antibody for adding alkali phosphatase enzyme mark is immunized with above-mentioned
Compound combines.By magnetic separation system, washing adds luminous substrate after removing uncombined antibody, and the complex catalysts light
Substrate sends photon, and luminous intensity is directly proportional to APL IgG content.
Due to the implementation of above technical scheme, the present invention has the following advantages that compared with prior art：
Instant invention overcomes the high pollution of radiommunoassay, the short and common microwell plate ELISA of the term of validity are accurate
The shortcomings that degree is poor, poor sensitivity, it is of the invention from more compared with traditional antiphospholipid antibody syndrome detection project cardiolipin antibody
Phosphatidyl-ethanolamine antibody, phosphatidylinositols antibody, phosphatidylserine antibody are selected in kind phospholipid antibody, and selects 2 sugared eggs of β
White I antibody is used together with cardiolipin antibody, so as to substantially increase the sensitivity of clinical detection.
The present invention is further by using alkaline phosphatase enzyme-catalyzed chemical luminescence system, magnetic particle piece-rate system, unique
Antigen-antibody coupling technology, and the composition to kit and the optimization of the various key technologies of each preparation of reagents technique etc. so that
The high sensitivity of this kit, precision is high, the range of linearity is wide, stability is good, term of validity length, and environmental-protecting performance greatly improves.
Brief description of the drawings
Accompanying drawing 1 is built-in canonical plotting of the invention.
Embodiment
With reference to specific embodiment, the present invention will be further described in detail, but the present invention is not limited to following implementation
Example.The implementation condition used in embodiment can do further adjustment, unreceipted implementation according to specifically used different requirements
Condition is the normal condition in the industry.What those of ordinary skill in the art were obtained under the premise of creative work is not made
All other embodiment, belongs to the scope of protection of the invention.
The commercially available acquisition of agents useful for same of the present invention.
Embodiment 1：The present invention utilizes the carrier and isolation technics of magnetic particle, using kit A with kit B is supporting makes
With, naturally it is also possible to all reagents are put in a kit and sold jointly.
Kit A includes following reagent：
(1), the cuorin antigen of the biotin labeling containing 15ng/mL, 15ng/mL biotin labeling phosphatidyl second
Hydramine antigen, the 15ng/mL phosphatidylinositols antigen of biotin labeling, 15ng/mL biotin labeling phosphatidyl silk ammonia
The mixed solution of the beta 2 glycoprotein I antigen of sour antigenic solution and 3 μ g/mL biotin labeling；
(2), concentration is the mouse anti-human igg monoclonal antibody solution of 100ng/mL alkali phosphatase enzyme mark；
(3), concentration is the nano magnetic microparticle suspending liquid of 0.4mg/mL marked by streptavidin.
Kit B includes following reagent：
(1), quality-control product：The anti-phosphatide IgG that anti-phosphatide IgG antibody solution that concentration is 10RU/mL, concentration are 80RU/mL
Antibody-solutions；
(2), calibration object：The anti-phosphatide that anti-phosphatide IgG antibody solution that concentration is 20RU/mL, concentration are 200RU/mL
IgG antibody solution；
(3), standard curve, referring to accompanying drawing 1；
(4), Sample dilution；
(5), chemical luminous substrate.
Embodiment 2：The preparation of kit shown in embodiment 1
(1), phosphatidyl-ethanolamine antigenic solution, the biology of the cuorin antigenic solution of biotin labeling, biotin labeling
The phosphatidylinositols antigenic solution of element mark, the phosphatidylserine antigenic solution of biotin labeling, the sugar of β 2 of biotin labeling
The preparation method of protein I antigenic solution is essentially identical.
The preparation method of the cuorin antigenic solution of biotin labeling is explained in detail below, the preparation method of other solution is no longer superfluous
State.
The preparation method of the cuorin antigenic solution of biotin labeling comprises the following steps：
1. by amido modified cuorin antigen with 0.02mol/L, pH7.4 PBS (phosphate buffer) 4
Liquid is changed once in dialysed overnight at DEG C, centre.
2. by BNHS (n-hydroxysuccinimide) balances to room temperature (20 DEG C), with a ten thousandth assay balance, (maximum claims
Amount 200g) weigh 1.5mg BNHS and be dissolved in 326 μ L DMSO (dimethyl sulfoxide (DMSO)), the BNHS solution for being configured to 10mM is (current
Now match somebody with somebody)；
3. it is 1 according to amido modified cuorin antigen and BNHS mol ratios:20 ratio is in the solution obtained by step 1
The middle solution added obtained by step 2, is well mixed, room temperature placing response 30 minutes；
4. adding 1M TRIS (trishydroxymethylaminomethane) buffer solutions into step 3 to final concentration of 10mM, terminate anti-
Should, room temperature placing response 10min, that is, obtain biotin labeling cuorin antigen concentrated solution, with containing 1% bovine serum albumin(BSA),
The cuorin antigen concentrate of biotin labeling is diluted to 15ng/ml by PH 7.4,0.01mol/L phosphate buffer, complete
Into the preparation of the cuorin antigenic solution of biotin labeling.
(2), the cuorin antigen of the biotin labeling containing 15ng/mL, 15ng/mL biotin labeling phosphatidyl
Monoethanolamine antigen, the 15ng/mL phosphatidylinositols antigen of biotin labeling, 15ng/mL biotin labeling phosphatidyl silk
The preparation method of the mixed solution of the beta 2 glycoprotein I antigen of propylhomoserin antigenic solution and 3 μ g/mL biotin labeling is：Will be by upper
State cuorin antigenic solution, the phosphatidyl-ethanolamine antigen of biotin labeling of biotin labeling made from the method for step (1)
Solution, the phosphatidylinositols antigenic solution of biotin labeling, the phosphatidylserine antigenic solution of biotin labeling, biotin mark
The beta 2 glycoprotein I antigenic solution of note is mixed to prepare.
(3), the preparation of the mouse anti-human igg monoclonal antibody solution of alkali phosphatase enzyme mark comprises the following steps：
1. taking 1mg mouse anti-human igg monoclonal antibodies, 10mg/ml coupling agent 2-IT (2- imido grpup sulfane hydrochloric acid is added
Salt) 3 μ l of solution, 20min is stored at room temperature, the 0.1mol/L μ l of glycine solution 10 is added, is stored at room temperature 5min, is coagulated with G-25
Glue post desalination, antibody after activation is collected, 2-8 DEG C saves backup；
2. taking 1.5mg ALP (alkaline phosphatase) solution, 5mg/ml SMCC (4- (N- dimaleoyl imino first is added
Base) hexamethylene -1- carboxylic acids succinimide ester) 15 μ l of solution, 30min is stored at room temperature, with G-25 gel column desalinations, collects activation
Antibody afterwards, 4 DEG C save backup；
3. the mouse anti-human igg monoclonal antibody of activation is mixed with the ALP activated, 18h is stood under the conditions of 4 DEG C, is used
Supperdex200 gel-purifieds post purifies conjugate, obtains attachment concentrated solution, 4 DEG C save backup.
4. attachment concentrated solution is used to MES (the 2- morpholine second sulphurs containing 1% bovine serum albumin(BSA), PH6.0,0.05mol/L
Acid) fliud flushing is diluted to 100ng/ml, complete the preparation of the mouse anti-human igg monoclonal antibody solution of alkali phosphatase enzyme mark.
(4), the nano magnetic microparticle suspending liquid of marked by streptavidin is prepared as：
With the bovine serum albumin(BSA) containing mass ratio 1%, PH7.4,0.01mol/L phosphate buffer by Streptavidin
Mark magnetic particle is resuspended to 0.4mg/ml, completes the preparation of the nano magnetic microparticle suspending liquid of marked by streptavidin.Strepto- is affine
Element mark magnetic particle is purchased in Life Technologies
(5), the preparation of quality-control product
Quality-control product 1：Business serum containing anti-APL-IgG antibody, and concentration of the anti-APL-IgG antibody in quality-control product is
10RU/mL, pH 7.4,0.01mol/L phosphate buffer, mass concentration are 1% bovine serum albumin(BSA), and mass concentration is
0.5% preservative.
Quality-control product 2：Business serum containing anti-APL-IgG antibody, and concentration of the anti-APL-IgG antibody in quality-control product is
80RU/mL, pH 7.4,0.01mol/L phosphate buffer, mass concentration are 1% bovine serum albumin(BSA), and mass concentration is
0.5% preservative.
(6), the preparation of calibration object
Calibration object 1：Business serum containing anti-APL-IgG antibody, and concentration of the anti-APL-IgG antibody in quality-control product is
00RU/mL, pH 7.4,0.01mol/L phosphate buffer, mass concentration are 1% bovine serum albumin(BSA), and mass concentration is
0.5% preservative.
Calibration object 2：Business serum containing anti-APL-IgG antibody, and concentration of the anti-APL-IgG antibody in quality-control product is
200RU/mL, pH 7.4,0.01mol/L phosphate buffer, mass concentration be 1% bovine serum albumin(BSA), mass concentration
For 0.5% preservative.
(7), the preparation of Sample dilution
PH 7.4,0.01mol/L phosphate buffer, mass concentration are 1% bovine serum albumin(BSA), and mass concentration is
0.5% preservative.
Embodiment 3：Detection method, it is automatically performed by Full-automatic chemiluminescence analyzer, also manually actuated completion.
Step 1：20ul diluted samples to be measured are added in detection pipe, and (sample stoste is 1 with Sample dilution volume ratio:
20, mix), then add 50ul contain the cuorin antigen of biotin labeling, biotin labeling phosphatidyl-ethanolamine antigen,
The phosphatidylinositols antigen of biotin labeling, the phosphatidylserine antigen of biotin labeling, the beta 2 glycoprotein I of biotin labeling
The mixed solution of antigen and the nano magnetic microparticle suspending liquid of 50ul marked by streptavidin, mix, incubated 15 minutes at 37 DEG C；
Step 2：Magnetic field is added, the system after step 1 incubation is settled in magnetic field, removes supernatant, cleaned liquid is more
After secondary cleaning, magnetic field is removed；
Step 3：Then the mouse anti-human igg Dan Ke of 100ul alkali phosphatase enzyme marks is added in the system after being washed to step 2
Grand antibody-solutions, mix, incubated 15 minutes at 37 DEG C；
Step 4：Magnetic field is added, the system after step 3 incubation is settled in magnetic field, removes supernatant, cleaned liquid is more
After secondary cleaning, supernatant is removed；
Step 5：200ul chemical luminous substrates are added, magnetic field is removed, after being fully suspended, is incubated 5 minutes at 37 DEG C, examined
Survey relative luminous intensity value (RLU) in 3 seconds.
Embodiment 4：
The performance detection of kit prepared by the present invention：
(1) sensitivity evaluation (minimum detection limit LOD)
" 0 " concentration samples are detected, repeat detection 20 times, calculate the average value (M) and standard deviation of relative luminous intensity (RLU)
(SD), and M+2SD values are calculated, 2 regression fits is carried out according to the concentration-RLU between zero-dose calibration object and adjacent calibration object
Linear function is drawn, M+2SD values are brought into above-mentioned equation, obtains corresponding concentration value, as minimum detection limit.This method
Sensitivity is not more than 1RU/mL.
(1) A points luminous value, as a result referring to table 1.
Table 1
The luminous average X=31958 of A points
SD=2347
X+2SD=36652
(2) B points luminous value, as a result referring to table 2.
Table 2
| APL gG-STD-B(RLU) |
| 120354 |
| 123083 |
| 117818 |
| 105439 |
| 106697 |
The luminous average X=114678 of B points
(3) sensitivity=0.284RU/mL
(2) precision is evaluated
(1) precision in analysis
The kit prepared in embodiment 1 is a collection of, the serum of basic, normal, high three kinds of various concentrations is determined respectively, and 10 holes are put down
Row measure, as a result referring to table 3, it is 4.55%~5.97% to draw variation within batch coefficient.
Table 3
| Determine serum-concentration (RU/mL) | Determine number | CV (%) in analysis |
| 10 | 10 | 4.55 |
| 20 | 10 | 5.12 |
| 80 | 10 | 5.97 |
(2) precision between analyzing
The serum of the basic, normal, high three kinds of various concentrations of kit measurement prepared in Example 1,4 hole parallel determinations, on
Afternoon at noon, respectively once, follow-on test 10 days, every part of serum obtained 80 concentration measured values for test.The coefficient of variation is between statistical analysis
5.15%~6.24%, it the results are shown in Table 4.
Table 4
(3) accuracy estimating
Not same amount people APL IgM standard items are added in 2 pooled serum samples, the serum for forming 3 concentration levels adds
Originally, additive volume is less than the 10% of cumulative volume to sample-adding.Concentration of specimens is detected, according to the following formula the rate of recovery.This method blood
The clear matrix rate of recovery is between 85-115%.Data are referring to table 5.
R：The rate of recovery；
V：Add the volume of standard liquid；
V0：The volume of people source sample；
C：The detectable concentration that people source sample is added after standard liquid；
C0：The detectable concentration of people source sample；
Cs：The concentration of standard liquid.
Table 5
(4) methodology compares
The kit being prepared into embodiment 1, the cuorin detection kit of certain common commercial companies market, is only added
The kit of the cuorin antigen of biotin labeling and the phosphatidyl-ethanolamine antigen of biotin labeling (other with embodiment 1
Kit it is identical), only add biotin labeling cuorin antigen, the phosphatidyl-ethanolamine antigen and biology of biotin labeling
The kit (other are identical with the kit of embodiment 1) of the phosphatidylinositols antigen of element mark, only adds biotin labeling
Cuorin antigen, the phosphatidyl-ethanolamine antigen of biotin labeling, the phosphatidylinositols antigen of biotin labeling and biotin
The kit (other are identical with the kit of embodiment 1) of the phosphatidylserine of mark while detection 226 has been made a definite diagnosis
The patients serum of APS positive or negatives.Its testing result is shown in Table 6, sensitivity 95.60% of the present invention, specificity 98.52%, always
Coincidence rate 97.34%；And the cuorin detection kit sensitivity 39.56% that certain companies market is common, specificity 98.52%,
Total coincidence rate 63.53%.Compared with traditional antiphospholipid antibody syndrome detection project cardiolipin antibody, present invention adds another
Outer three kinds of phospholipid antibodies (phosphatidyl-ethanolamine antibody, phosphatidylinositols antibody, phosphatidylserine antibody) and beta 2 glycoprotein I
The detection of antibody can greatly improve Clinical Sensitivity.
Table 6
(5) Evaluation of Thermal Stability
Carry out 4 DEG C of 12 months and 37 DEG C of stability experiments of 7 days respectively to the kit of embodiment 1, the results showed that reagent
The index such as precision, accuracy is within normal range (NR) between analysis in the change of box standard items luminous intensity, analysis, reagent
The box term of validity was up to 12 months.
The present invention is described in detail above, its object is to allow the personage for being familiar with this art to understand this
The content of invention is simultaneously carried out, and it is not intended to limit the scope of the present invention, all Spirit Essence institutes according to the present invention
The equivalent change or modification of work, it should all cover within the scope of the present invention.
Claims (10)
- A kind of 1. chemical luminescent analysis reagent kid of phosphatide IgG antibody, it is characterised in that：Including following reagent：The phosphatidyl ethanol of the cuorin antigen of biotin labeling containing 5 ~ 100ng/mL, 5 ~ 100ng/mL biotin labeling Amine antigen, the 5 ~ 100ng/mL phosphatidylinositols antigen of biotin labeling, 5 ~ 100ng/mL biotin labeling phosphatidyl Serine antigen, 1 ~ 10 μ g/mL biotin labeling beta 2 glycoprotein I antigen mixed solution；Concentration is the mouse anti-human igg monoclonal antibody solution of 10 ~ 1000ng/mL alkali phosphatase enzyme mark；Concentration is the nano magnetic microparticle suspending liquid of 0.1 ~ 1 mg/mL marked by streptavidin.
- 2. the chemical luminescent analysis reagent kid of phosphatide IgG antibody according to claim 1, it is characterised in that：It is described Cuorin antigen, described phosphatidyl-ethanolamine antigen, described phosphatidylinositols antigen, described phosphatidylserine resist Original is respectively by amido modified.
- 3. the chemical luminescent analysis reagent kid of phosphatide IgG antibody according to claim 1, it is characterised in that：It is described Biotin be n-hydroxysuccinimide.
- 4. the chemical luminescent analysis reagent kid of phosphatide IgG antibody according to claim 1, it is characterised in that：It is described Cuorin antigen and the molar ratio of described biotin be 1：10~100；Described phosphatidyl-ethanolamine antigen with it is described Biotin molar ratio be 1：10~100；The molar ratio of described phosphatidylinositols antigen and described biotin For 1：10~100；The molar ratio of described phosphatidylserine antigen and described biotin is 1：10~100；Described β The molar ratio of 2 glycoprotein I antigens and described biotin is 1：10~100；Described alkaline phosphatase resists with described mouse The mass ratio that feeds intake of human IgG monoclonal antibody is 1 ~ 10：1；The carboxyl-content of described nanometer magnetic particle is not less than 0.4mmol/g。
- 5. the chemical luminescent analysis reagent kid of phosphatide IgG antibody according to claim 1, it is characterised in that：It is described Kit also include the quality-control product of the horizontal anti-phosphatide IgG antibody solution of two or more various concentrations, two or two Calibration object, standard curve, Sample dilution and the chemiluminescence bottom of the horizontal anti-phosphatide IgG antibody solution of individual above various concentrations Thing.
- 6. the chemical luminescent analysis reagent kid of phosphatide IgG antibody according to claim 5, it is characterised in that：It is described Quality-control product include anti-phosphatide IgG antibody, 0.005 ~ 0.1 mol/L, pH is 7 ~ 8 phosphate buffer, mass concentration 0.1 ~ 5 % bovine serum albumin and mass concentration is 0.01 ~ 1% preservative；Described calibration object includes anti-phosphatide IgG antibody, and 0.005 ~ 0.1 mol/L, pH is 7 ~ 8 phosphate buffer, quality The bovine serum albumin that concentration is 0.1 ~ 5% and the preservative that mass concentration is 0.01 ~ 1%；Described Sample dilution include 0.005 ~ 0.1 mol/L, pH be 7 ~ 8 phosphate buffer, mass concentration be 0.1 ~ 5% bovine serum albumin and mass concentration is 0.01 ~ 1% preservative.
- 7. the chemical luminescent analysis reagent kid of the phosphatide IgG antibody according to claim 5 or 6, it is characterised in that：One The concentration of anti-phosphatide IgG antibody in individual described quality-control product is 4 ~ 21RU/mL, the anti-phosphatide in the quality-control product described in another The concentration of IgG antibody is 50 ~ 100RU/mL；The concentration of anti-phosphatide IgG antibody in calibration object described in one is 10 ~ 40RU/ ML, the concentration of the anti-phosphatide IgG antibody in the calibration object described in another is 100 ~ 300RU/mL.
- A kind of 8. system of the chemical luminescent analysis reagent kid of phosphatide IgG antibody as any one of claim 1 to 7 Preparation Method, it is characterised in that：The phosphatide of the cuorin antigen of the described biotin labeling containing 5 ~ 100ng/mL, 5 ~ 100ng/mL biotin labeling Acyl monoethanolamine antigen, the 5 ~ 100ng/mL phosphatidylinositols antigen of biotin labeling, 5 ~ 100ng/mL biotin labeling Phosphatidylserine antigen, 1 ~ 10 μ g/mL preparation method of mixed solution of beta 2 glycoprotein I antigen of biotin labeling include Following steps：Step（1）, by described cuorin antigen, described phosphatidyl-ethanolamine antigen, described phosphatidylinositols antigen, institute The phosphatidylserine antigen stated, described beta 2 glycoprotein I antigen are respectively with the phosphoric acid that 0.01 ~ 0.03mol/L, pH are 7 ~ 7.5 Salt buffer, the dialysed overnight at 2 ~ 8 DEG C, cardiolipin antigenic solution, phosphatidyl-ethanolamine antigenic solution, phosphatide is prepared respectively Acyl inositol antigenic solution, phosphatidylserine antigenic solution, beta 2 glycoprotein I antigenic solution,Step（2）, described biotin is dissolved in the biotin for being configured to that substance withdrawl syndrome is 8 ~ 12mM in dimethyl sulfoxide (DMSO) Solution,Step（3）, by described cuorin antigenic solution, described phosphatidyl-ethanolamine antigenic solution, described phosphatidyl-4 Alcohol antigenic solution, described phosphatidylserine antigenic solution, described beta 2 glycoprotein I antigenic solution respectively with described biology Plain solution is well mixed, and is placed 20 ~ 40 minutes at 15 ~ 40 DEG C, is then respectively adding 0.05 ~ 1.1M trihydroxy methyl amino first Alkane buffer solution, terminating reaction, placed at 15 ~ 40 DEG C 8 ~ 12 minutes, then with containing the bovine serum albumin that mass ratio is 0.1 ~ 5% In vain, pH be phosphate buffer that 7 ~ 8, substance withdrawl syndrome is 0.005 ~ 0.1mol/L be diluted respectively obtain it is described The cuorin antigenic solution of biotin labeling, the phosphatidyl-ethanolamine antigenic solution of described biotin labeling, described biology Phosphatidylinositols antigenic solution, phosphatidylserine antigenic solution, the described biology of described biotin labeling of element mark The beta 2 glycoprotein I antigenic solution of element mark；Step（4）, by the cuorin antigenic solution of described biotin labeling, the phosphatidyl-ethanolamine of described biotin labeling Antigenic solution, the phosphatidylinositols antigenic solution of described biotin labeling, the phosphatidylserine of described biotin labeling Antigenic solution, the beta 2 glycoprotein I antigenic solution of described biotin labeling, which be mixed to get, described contains 5 ~ 100ng/mL The cuorin antigen of biotin labeling, 5 ~ 100ng/mL biotin labeling phosphatidyl-ethanolamine antigen, 5 ~ 100ng/mL The phosphatidylinositols antigen of biotin labeling, 5 ~ 100ng/mL biotin labeling phosphatidylserine antigen, 1 ~ 10 μ g/ The mixed solution of the beta 2 glycoprotein I antigen of mL biotin labeling；The preparation method of the mouse anti-human igg monoclonal antibody solution of described alkali phosphatase enzyme mark comprises the following steps：Step（1）, that mouse anti-human igg monoclonal antibody is added to the 2- imido grpup sulfanes hydrochloride that concentration is 8 ~ 12mg/ml is even Join in agent, 18 ~ 25 minutes are stood at 15 ~ 40 DEG C, add 0.01 ~ 0.11mol/L glycine solution, it is quiet at 15 ~ 40 DEG C Put 4 ~ 6 minutes, with G-25 gel column desalinations, collect the mouse anti-human igg monoclonal antibody after activation, 2 ~ 8 DEG C save backup,Step（2）, alkaline phosphatase enzyme solutions are added to 1 ~ 10mg/ml 4- (N- maleimidomethyls) hexamethylene -1- In carboxylic acid succinimide ester solution, 10 ~ 100 minutes are stood at 15 ~ 40 DEG C, with G-25 gel column desalinations, is collected after activating Alkaline phosphatase, 2 ~ 8 DEG C save backup,Step（3）, the mouse anti-human igg monoclonal antibody after described activation mixed with alkaline phosphatase after described activation, 12 ~ 24h is stood at 2 ~ 8 DEG C, is purified with Supperdex200 gel-purified posts, obtains attachment concentrated solution, in 2 ~ 8 DEG C of preservations It is standby,Step（4）, by the described attachment concentrated solution bovine serum albumin(BSA) for being 0.1 ~ 5% containing mass ratio, pH be 5.8 ~ 8.2, Substance withdrawl syndrome is that 0.01 ~ 0.1mol/L MES buffer solution is diluted the alkaline phosphatase mark for being made described The mouse anti-human igg monoclonal antibody solution of note；The nano magnetic microparticle suspending liquid of described marked by streptavidin is by using the bovine serum albumin that mass ratio is 0.1 ~ 5% In vain, pH is the nano magnetic of phosphate buffer that 7 ~ 8, substance withdrawl syndrome is 0.005 ~ 0.1mol/L to marked by streptavidin Particulate be resuspended and is made.
- A kind of 9. inspection of the chemical luminescent analysis reagent kid of phosphatide IgG antibody as any one of claim 1 to 7 Survey method, it is characterised in that：Comprise the following steps：Step（1）, by sample stoste and Sample dilution be 1 by volume：10 ~ 100 ratio be mixed to get dilution after it is to be measured Sample；Step（2）, add in detection pipe sample to be tested after described dilution, the cuorin containing biotin labeling resists Original, the phosphatidyl-ethanolamine antigen of biotin labeling, the phosphatidylinositols antigen of biotin labeling, the phosphatidyl of biotin labeling The mixed solution of beta 2 glycoprotein I antigen and the nano magnetic of described marked by streptavidin of serine antigen, biotin labeling Microparticle suspending liquid, 10 ~ 30min of incubation obtains the first solution at 25 ~ 40 DEG C；Wherein, sample to be tested adds after described dilution Dosage is 10 ~ 60 μ L, sample to be tested after described dilution, the cuorin antigen containing biotin labeling, biotin mark The phosphatidyl-ethanolamine antigen of note, the phosphatidylinositols antigen of biotin labeling, biotin labeling phosphatidylserine antigen, The mixed solution of the beta 2 glycoprotein I antigen of biotin labeling and the nano magnetic microparticle suspending liquid of described marked by streptavidin The volume ratio that feeds intake is 1：0.5~4:0.5~4；Step（3）, addition magnetic field, the first described solution is settled in magnetic field, remove supernatant, then cleaned liquid is multiple After cleaning, magnetic field is removed, obtains the second solution；Step（4）, add into the second described solution described alkali phosphatase enzyme mark mouse anti-human igg monoclonal antibody it is molten Liquid, 10 ~ 30min of incubation obtains the 3rd solution at 25 ~ 40 DEG C；Wherein, sample to be tested and described alkalescence after described dilution The volume ratio that feeds intake of the mouse anti-human igg monoclonal antibody solution of phosphatase enzyme mark is 1：1.5~10.5；Step（5）, addition magnetic field, the 3rd described solution is settled in magnetic field, remove supernatant, then cleaned liquid is multiple After cleaning, supernatant is removed, adds described chemical luminous substrate, magnetic field is removed, after being fully suspended, 1 is incubated at 25 ~ 40 DEG C ~ 10min, detect the relative luminous intensity value in 3 seconds；Wherein, sample to be tested and described chemiluminescence bottom after described dilution The volume ratio that feeds intake of thing is 1：1~15.
- 10. detection method according to claim 9, it is characterised in that：Described detection method is in Full-automatic chemiluminescence Automatic detection or detection manually on instrument.
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