CN107402306A

CN107402306A - A kind of chemical luminescent analysis reagent kid of phosphatide IgM antibody and preparation method thereof and detection method

Info

Publication number: CN107402306A
Application number: CN201710829906.8A
Authority: CN
Inventors: 李庆春; 徐顺澍; 李永红; 吴荣桂; 周斌; 万文琴
Original assignee: Jiangsu Hao Bo Biomedical Ltd By Share Ltd
Current assignee: Jiangsu Hao Bo Biomedical Ltd By Share Ltd
Priority date: 2017-09-15
Filing date: 2017-09-15
Publication date: 2017-11-28

Abstract

The present invention relates to a kind of chemical luminescent analysis reagent kid of phosphatide IgM 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 phosphatidylinositols antigen of biotin labeling for 5 ~ 100ng/mL, 5 ~ 100ng/mL biotin labeling phosphatidylserine antigen mixed solution；Concentration is the anti-human IgM monoclonal antibody solution of mouse 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

A kind of chemical luminescent analysis reagent kid of phosphatide IgM antibody and preparation method thereof And detection method

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 IgM 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 IgM 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 IgM 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 mixed solution；

Concentration is the anti-human IgM monoclonal antibody solution of mouse 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 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；Described alkaline phosphatase and the mass ratio that feeds intake of the described anti-human IgM monoclonal antibody of mouse are 1 ~10：1；The carboxyl-content of described nanometer magnetic particle 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；Described alkaline phosphatase and the mass ratio that feeds intake of the described anti-human IgM monoclonal antibody of mouse are 1 ~2：1.

Preferably, it is molten also to include the horizontal anti-phosphatide IgM antibody of two or more various concentrations for described kit Calibration object, standard curve, the sample of the horizontal anti-phosphatide IgM 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 IgM 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 IgM 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% preservative.

It is further preferable that described quality-control product includes anti-phosphatide IgM 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 IgM 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 IgM antibody in quality-control product described in one is 4~21RU/mL, separately The concentration of anti-phosphatide IgM 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 IgM antibody is 10~40RU/mL, and the concentration of the anti-phosphatide IgM antibody in the calibration object described in another is 100~300RU/mL.It is further preferable that the concentration of the anti-phosphatide IgM antibody in quality-control product described in one is 9~11RU/mL, The concentration of anti-phosphatide IgM antibody in quality-control product described in another is 72~88RU/mL；In calibration object described in one The concentration of anti-phosphatide IgM antibody is 18~22RU/mL, and the concentration of the anti-phosphatide IgM 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 IgM 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 The preparation method of the mixed solution of the phosphatidylserine antigen of element mark comprises the following steps：

Step (1), described cuorin antigen, described phosphatidyl-ethanolamine antigen, described phosphatidylinositols resisted Former, described phosphatidylserine antigen respectively with 0.01~0.03mol/L, pH be 7~7.5 phosphate buffer, 2 Dialysed overnight at~8 DEG C, cardiolipin antigenic solution, phosphatidyl-ethanolamine antigenic solution, phosphatidylinositols antigen are prepared respectively Solution, phosphatidylserine 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 are well mixed with described biotin solution respectively, 15 Placed 20~40 minutes at~40 DEG C, be then respectively adding 0.05~1.1M TRIS buffer, terminated anti- Should, place 8~12 minutes at 15~40 DEG C, then be 0.1~5% containing mass ratio bovine serum albumin(BSA), pH be 7~8, Substance withdrawl syndrome is that 0.005~0. 1mol/L phosphate buffer is diluted the biotin labeling described in respectively obtaining Cuorin antigenic solution, phosphatidyl-ethanolamine antigenic solution, the phosphorus of described biotin labeling of described biotin labeling The phosphatidylserine antigenic solution of acyl inositol 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 be mixed to get the cuorin antigen of the biotin labeling containing 5~100ng/mL, 5~ The phosphatidyl-ethanolamine antigen of 100ng/mL biotin labeling, the phosphatidylinositols of 5~100ng/mL biotin labeling resist The mixed solution of the phosphatidylserine antigen of former, 5~100ng/mL biotin labeling；

The preparation method of the anti-human IgM monoclonal antibody solution of mouse of described alkali phosphatase enzyme mark comprises the following steps：

Step (1), the anti-human IgM monoclonal antibody of mouse 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 anti-human IgM monoclonal antibody of mouse 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 anti-human IgM monoclonal antibody of mouse after described activation mixed with alkaline phosphatase after described activation 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 anti-human IgM monoclonal antibody solution of mouse 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 preparation method of the mixed solution of the phosphatidylserine antigen of mL biotin labeling comprises the following steps：

Step (3), by described cuorin antigenic solution, described phosphatidyl-ethanolamine antigenic solution, described phosphatide Acyl inositol antigenic solution, described phosphatidylserine antigenic solution are well mixed with described biotin solution respectively, 15 Placed 20~40 minutes at~40 DEG C, be then respectively adding 0.9~1.1M TRIS buffer, terminated anti- Should, place 8~12 minutes at 15~40 DEG C, then be 0.9~1.1% containing mass ratio bovine serum albumin(BSA), pH be 7~ 7.5th, the phosphate buffer that substance withdrawl syndrome is 0.009~0.011mol/L, which is diluted, respectively obtains described biology Cuorin antigenic solution, phosphatidyl-ethanolamine antigenic solution, the described biotin mark of described biotin labeling of element mark The phosphatidylinositols antigenic solution of note, the phosphatidylserine antigenic solution of 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 be mixed to get the cuorin antigen of the biotin labeling containing 5~100ng/mL, 5~ The phosphatidyl-ethanolamine antigen of 100ng/mL biotin labeling, the phosphatidylinositols of 5~100ng/mL biotin labeling resist The mixed solution of the phosphatidylserine antigen of former, 5~100ng/mL biotin labeling.

Preferably, the preparation method of the anti-human IgM monoclonal antibody solution of the mouse of described alkali phosphatase enzyme mark is included such as Lower step：

Step (1), the anti-human IgM monoclonal antibody of mouse 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 anti-human IgM monoclonal antibody of mouse 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 anti-human IgM monoclonal antibody of mouse after described activation mixed with alkaline phosphatase after described activation 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 anti-human IgM monoclonal antibody solution of mouse 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 IgM 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 life containing 5~100ng/mL The cuorin antigen of thing element mark, the 5~100ng/mL phosphatidyl-ethanolamine antigen of biotin labeling, 5~100ng/mL The phosphatidylinositols antigen of biotin labeling, 5~100ng/mL biotin labeling phosphatidylserine antigen mixing it is molten The nano magnetic microparticle suspending liquid of liquid and described marked by streptavidin, 10~30min of incubation obtains first at 25~40 DEG C Solution；Wherein, the addition of sample to be tested is 10~60 μ L after described dilution, and sample to be tested after described dilution is described The phosphatidyl-ethanolamine of the cuorin antigen of biotin labeling containing 5~100ng/mL, 5~100ng/mL biotin labeling Antigen, the 5~100ng/mL phosphatidylinositols antigen of biotin labeling, 5~100ng/mL biotin labeling phosphatidyl The volume ratio that feeds intake of the nano magnetic microparticle suspending liquid of the mixed solution of serine antigen and described marked by streptavidin 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 anti-human IgM monoclonals of mouse for adding into the second described solution described alkali phosphatase enzyme mark Antibody-solutions, 10~30min of incubation obtains the 3rd solution at 25~40 DEG C；Wherein, sample to be tested and institute after described dilution The volume ratio that feeds intake of the anti-human IgM monoclonal antibody solution of mouse for the alkali phosphatase enzyme mark stated is 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 cuorin containing the biotin labeling Antigenic solution, the phosphatidyl-ethanolamine antigen of biotin labeling, the phosphatidylinositols antigen of biotin labeling, biotin labeling The nano magnetic microparticle suspending liquid of the mixed solution of phosphatidylserine antigen and described marked by streptavidin, at 36~38 DEG C 10~20min of lower incubation obtains the first solution；Wherein, the addition of described sample to be tested dilution is 18~22 μ L, described Dilution after sample to be tested, the cuorin antigenic solution of described biotin labeling, the phosphatidyl second of described biotin labeling Hydramine antigenic solution, the phosphatidylinositols antigenic solution of described biotin labeling, the phosphatidyl silk of described biotin labeling The volume ratio that feeds intake of the nano magnetic microparticle suspending liquid of propylhomoserin antigenic solution and described marked by streptavidin is 1：2.4~2.6： 2.4~2.6；

Step (4), the anti-human IgM monoclonals of mouse 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 anti-human IgM monoclonal antibody solution of mouse 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), the APL IgM in sample and coating Streptavidin magnetic particle By immune response, immune complex is formed, by magnetic separation system, after the uncombined antigen of washing removal, antibody and impurity, The anti-human IgM monoclonal antibody of mouse for adding alkali phosphatase enzyme mark is combined with above-mentioned immune complex.By magnetic separation system, Washing adds luminous substrate after removing uncombined antibody, and the complex catalysts luminous substrate sends photon, luminous intensity and APL IgM content is directly proportional.

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 and cardiolipin antibody are selected in kind phospholipid antibody It is used together, 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 sour antigen；

(2), concentration is the anti-human IgM monoclonal antibody solution of mouse 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 IgM that anti-phosphatide IgM antibody solution that concentration is 10RU/mL, concentration are 80RU/mL Antibody-solutions；

(2), calibration object：The anti-phosphatide that anti-phosphatide IgM antibody solution that concentration is 20RU/mL, concentration are 200RU/mL IgM 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 preparation method of the phosphatidylserine antigenic solution of biotin labeling are identical, under The preparation method of the cuorin antigenic solution of biotin labeling is described in detail in face, and the preparation method of other solution repeats no more.

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 propylhomoserin antigen is：Will the biotin labeling as made from the method for above-mentioned steps (one) cuorin Antigenic solution, the phosphatidyl-ethanolamine antigenic solution of biotin labeling, phosphatidylinositols antigenic solution, the biology of biotin labeling The phosphatidylserine antigenic solution of element mark is mixed to prepare.

(3), the preparation of the anti-human IgM monoclonal antibody solution of the mouse of alkali phosphatase enzyme mark comprises the following steps：

1. taking the anti-human IgM monoclonal antibody of 1mg mouse, 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, 5min is stored at room temperature, with G-25 gels 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 and lives Antibody after change, 4 DEG C save backup；

3. the anti-human IgM monoclonal antibody of the mouse 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 anti-human IgM monoclonal antibody solutions of mouse 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-IgM antibody, and concentration of the anti-APL-IgM 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-IgM antibody, and concentration of the anti-APL-IgM 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-IgM antibody, and concentration of the anti-APL-IgM 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-IgM antibody, and concentration of the anti-APL-IgM 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：(sample stoste is 1 with Sample dilution volume ratio to sample to be tested after 20ul dilutions are added in detection pipe: 20, mix), the cuorin antigen that 50ul is mixed with biotin labeling is then added, the phosphatidyl-ethanolamine of biotin labeling resists Original, the phosphatidylinositols antigenic solution of biotin labeling, the solution and 50ul chains of the phosphatidylserine antigen of biotin labeling The nano magnetic microparticle suspending liquid of mould Avidin mark, mixes, is 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 anti-human IgM Dan Ke of mouse of 100ul alkali phosphatase enzyme marks are 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=32589 of A points

SD=1850

X+2SD=34439

(2) B points luminous value, as a result referring to table 2.

Table 2

APL gG-STD-B(RLU)
	113362
107396
	113260
118489
	108697

The luminous average X=112241 of B points

(3) sensitivity=0.232RU/mL.

(2) precision is evaluated

(1) precision in analysis

Kit 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 parallel Measure, as a result referring to table 3, it is 3.11%~5.69% to draw variation within batch coefficient.

Table 3

Determine serum-concentration (RU/mL)	Determine number	CV (%) in analysis
			10	10	3.11
20	10	4.32
			80	10	5.69

(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 5.36%~6.78%, it the results are shown in Table 4.

Table 4

Determine serum-concentration (RU/mL)	Determine number	CV (%) in analysis
			10	80	5.36
20	80	5.81
			80	80	6.78

(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

With the kit of embodiment 1, the cuorin detection kit of certain common commercial companies market, biotin is only added The kit of the cuorin antigen of mark and the phosphatidyl-ethanolamine antigen of biotin labeling (other reagents with embodiment 1 Box is identical), only add biotin labeling cuorin antigen, the phosphatidyl-ethanolamine antigen and biotin labeling of biotin labeling Phosphatidylinositols antigen kit (other are identical with the kit of embodiment 1) and meanwhile detection 254 made a definite diagnosis APS The patients serum of positive or negative.Its testing result is shown in Table 6 respectively, it is seen that sensitivity 94.73% of the present invention, specificity 98.74%, total coincidence rate 97.24%；And the cuorin detection kit sensitivity 42.11% that certain companies market is common, specifically Property 98.74%, total coincidence rate 77.56%.Compared with traditional antiphospholipid antibody syndrome detection project cardiolipin antibody, this hair The bright other three kinds of phospholipid antibodies (phosphatidyl-ethanolamine antibody, phosphatidylinositols antibody, phosphatidylserine antibody) of adding Detection 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

A kind of 1. chemical luminescent analysis reagent kid of phosphatide IgM 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 The mixed solution of serine antigen；

Concentration is the anti-human IgM monoclonal antibody solution of mouse 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 IgM 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 IgM 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 IgM 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 Alkaline phosphatase and the mass ratio that feeds intake of the anti-human IgM monoclonal antibody of described mouse are 1 ~ 10：1；Described nanometer magnetic particle Carboxyl-content is not less than 0.4mmol/g.
5. the chemical luminescent analysis reagent kid of phosphatide IgM 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 IgM 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 IgM antibody solution of individual above various concentrations Thing.
6. the chemical luminescent analysis reagent kid of phosphatide IgM antibody according to claim 5, it is characterised in that：It is described Quality-control product include anti-phosphatide IgM 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 IgM 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 IgM antibody according to claim 5 or 6, it is characterised in that：One The concentration of anti-phosphatide IgM 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 IgM antibody is 50 ~ 100RU/mL；The concentration of anti-phosphatide IgM antibody in calibration object described in one is 10 ~ 40RU/ ML, the concentration of the anti-phosphatide IgM 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 IgM 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 The preparation method of the mixed solution of phosphatidylserine antigen comprises the following steps：

Step（1）, by described cuorin antigen, described phosphatidyl-ethanolamine antigen, described phosphatidylinositols antigen, institute The phosphatidylserine antigen stated is saturating at 2 ~ 8 DEG C respectively with the phosphate buffer that 0.01 ~ 0.03mol/L, pH are 7 ~ 7.5 Analysis overnight, prepares cardiolipin antigenic solution, phosphatidyl-ethanolamine antigenic solution, phosphatidylinositols antigenic solution, phosphatide respectively Acyl serine 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 are well mixed with described biotin solution respectively, at 15 ~ 40 DEG C It is lower to place 20 ~ 40 minutes, it is then respectively adding 0.05 ~ 1.1M TRIS buffer, terminating reaction, 15 ~ Placed at 40 DEG C 8 ~ 12 minutes, be then 7 ~ 8, substance withdrawl syndrome with bovine serum albumin(BSA), pH that mass ratio is 0.1 ~ 5% is contained For 0.005 ~ 0. 1mol/L phosphate buffer be diluted respectively obtain described biotin labeling cuorin antigen it is molten Liquid, the phosphatidyl-ethanolamine antigenic solution of described biotin labeling, the phosphatidylinositols antigen of described biotin labeling are molten The phosphatidylserine antigenic solution of liquid, described biotin labeling；

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 be mixed to get the cuorin antigen of the described biotin labeling containing 5 ~ 100ng/mL, 5 ~ 100ng/mL Phosphatidylinositols antigen, the 5 ~ 100ng/ of the phosphatidyl-ethanolamine antigen of biotin labeling, 5 ~ 100ng/mL biotin labeling The mixed solution of the phosphatidylserine antigen of mL biotin labeling；

The preparation method of the anti-human IgM monoclonal antibody solution of mouse of described alkali phosphatase enzyme mark comprises the following steps：

Step（1）, that the anti-human IgM monoclonal antibody of mouse 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 anti-human IgM monoclonal antibody of mouse 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 anti-human IgM monoclonal antibody of mouse 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 anti-human IgM monoclonal antibody solution of mouse 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 nanometer of phosphate buffer that 7 ~ 8, substance withdrawl syndrome is 0.005 ~ 0. 1mol/L to marked by streptavidin Magnetic particle be resuspended and is made.
A kind of 9. inspection of the chemical luminescent analysis reagent kid of phosphatide IgM 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 described biotin mark containing 5 ~ 100ng/mL The cuorin antigen of note, the phosphatidyl-ethanolamine antigen of 5 ~ 100ng/mL biotin labeling, 5 ~ 100ng/mL biotin mark The phosphatidylinositols antigen of note, the mixed solution of phosphatidylserine antigen of 5 ~ 100ng/mL biotin labeling and described The nano magnetic microparticle suspending liquid of marked by streptavidin, 10 ~ 30min of incubation obtains the first solution at 25 ~ 40 DEG C；Wherein, institute The addition of sample to be tested is 10 ~ 60 μ L after the dilution stated；Sample to be tested after described dilution, 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 mixing it is molten The volume ratio that feeds intake of the nano magnetic microparticle suspending liquid of liquid and described marked by streptavidin 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 the anti-human IgM monoclonal antibody of mouse 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 anti-human IgM monoclonal antibody solution of mouse 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.