CN102816238A - IgG1 anti-human beta2-glycoprotein I monoclonal antibody, and preparation and application thereof - Google Patents

Abstract

The invention relates to an IgG1 anti-human beta2-glycoprotein I (beta2GPI) monoclonal antibody. The IgG1 anti-human beta2-glycoprotein I monoclonal antibody is secreted by the hybridoma cell line AB2-F6. The monoclonal antibody can react with beta2GPI prepared in the invention and is comparable with a standard anti-beta2GPI monoclonal antibody; the IgG1 anti-human beta2-glycoprotein I monoclonal antibody provided by the invention has strong specificity and is used for preparation of a double-antibody sandwich ELISA assay kit for human plasma beta2 glycoprotein I.

Description

The anti-people β of IgG1 type 2Glycoprotein I monoclonal antibody and preparation thereof and application

Technical field

The invention belongs to the antibody production techniques category, be used for the research field of relevant autoimmune disorder, be specifically related to anti-people β ₂Glycoprotein I (β ₂GPI) MONOCLONAL ANTIBODIES SPECIFIC FOR, evaluation and application.

Background note

Antiphospholipid syndrome (APS) is to be a kind of non-organ specificity autoimmune disorder of principal character to occur high titre anti-phospholipid antibody (APL) in the patient body, can fall ill separately, also can occur together with other autoimmune diseases, especially SLE.Its main clinical characters is repeatedly the artery and vein thrombosis, many organs ischemic and habitual abortion.APL is one group of cyclicity autoantibody that directly is directed against the heterogencity of negative charge phosphatide and albumen cofactor, mainly comprises lupus anticoagulant (LA), ACLA (aCL), anti-phosphatidylserine antibody and anti-phosphatidylethanolamine antibody.Discover that APL mainly discerns phospholipids incorporate albumen, rather than phosphatide itself, wherein, most important phospholipids incorporate albumen is β ₂Glycoprotein I (β ₂GPI), itself and corresponding antibodies (anti-β ₂GPI) form mixture, in the APS pathologic process, play a significant role.International thrombus hemostasis association (ISTH) meeting that hold in Sydney in 2005 is augmented anti-β 2GPI antibody positive first as one of APS laboratory diagnosis index.

Anti-β ₂GPI/ β ₂The GPI mixture can make its secretion and discharge cell adhesion molecule, inflammatory factor, TF etc., and can stimulate blood mononuclear cell to express TF through activating vascular endothelial cell.TF is the strand gp through cytolemma, is the acceptor of serum prothrombin conversion accelerator(SPCA)/VIIa, and TF/VIIa activates plasma thromboplastin component, X with the form of mixture, thereby causes blood coagulation.Therefore, anti-β ₂GPI/ β ₂It is the thrombotic important mechanism of APS that GPI mixture inducing cell is expressed TF, but concrete mechanism of action is also demanded urgently inquiring into.β mainly is engaged in this laboratory ₂GPI and antibody thereof stimulate the research of monocytic intracellular signal transmission mechanism, and then illustrate anti-β ₂GPI/ β ₂The keying action of GPI in APS thrombosis mechanism.Need use a large amount of β under study for action ₂GPI and anti-β ₂GPI, but also not anti-β on the present home market ₂GPI sells, and abroad this antibody selling price is too high, and the Buying Cycle is long, restricts carrying out smoothly of research work.Therefore, prepare anti-β ₂The GPI monoclonal antibody will be practiced thrift the time cost and the Financial cost of research greatly, establish good basis smoothly for the research work in this field, have the important use meaning.

β among the human normal plasma of domestic and international different bibliographical informations ₂The level of GPI has difference slightly: 200 ± 35m g/L, 150 ~ 300m g/L, 50 ~ 150mgL etc. are arranged; The bibliographical information result very big reason that differs is that used detection method is different; Have plenty of with ELISA and detect, also have with radioimmunology, immunoelectrophoresis etc.Expect that unified result so at first must unify detection method.The present domestic β that also do not detect ₂The test kit of GPI, so anti-β among the present invention ₂The GPI MONOCLONAL ANTIBODIES SPECIFIC FOR is β ₂Certain basis has been established in the preparation of GPI test kit, so that provide a unification to detect β for domestic ₂The method of GPI.Bibliographical information is arranged recently, total β in APS patient's body ₂The level of GPI is apparently higher than healthy and other autoimmune disorders collator, and compares β in APS patient's body with the normal healthy controls group ₂GPI content raises, and β is explained in the also corresponding increase of its thrombotic risk ₂The level of GPI and thrombotic risk have very big dependency.According to going back some bibliographical information, β ₂GPI and numerous disease exist certain relation, various disease β ₂GPI can show different the variation, like β in liver cirrhosis patient and disseminated intravascular coagulation patient body ₂GPI reduces, and has family to assemble the patients with cerebral apoplexy β of tendency ₂Increasing of GPI serum level, but concrete mechanism is not quite clear.To sum up, test kit according to the invention not only can be used for the prediction of APS patient's thrombosis risk but also can be used for Chinese population β ₂Relation between the generaI investigation of GPI level and convenient research itself and other disease.

This laboratory is purified into β from human plasma ₂GPI has also prepared anti-β ₂The GPI polyclonal antibody (yellow loud and clear, Zhou Hong, Yu Ying, etc. the anti-people β of rabbit ₂Gp I Polyclonal Antibody Preparation and evaluation [J]. Clinical Laboratory magazine .2009,27 (3): 192-194.), and carrying out the discussion work of APS mechanism of causing a disease in recent years always.Have high specificity in view of monoclonal antibody (MAbs), advantages such as highly sensitive and easy stdn, the present invention has prepared mouse-anti people β ₂The GPI monoclonal antibody is also analyzed its characteristic, for deep Mechanism Study is laid a good foundation.In view of the existing anti-β in this laboratory ₂The monoclonal antibody of GPI and how anti-the invention provides a kind of human plasma β ₂GPI double-antibody sandwich elisa detection method and test kit.

Summary of the invention

β ₂GPI is the gp of the about 45～50KD of molecular weight, and (I～V) form, liver are main synthesising parts to its structure by 5 CCP spline structure districts.Anti-β ₂GPI/ β ₂The effect of GPI mixture in the APS pathomechanism receives very big concern.Have high specificity in view of MAbs, advantages such as highly sensitive and easy stdn the object of the present invention is to provide anti-β ₂The GPI monoclonal antibody, and at preparation detection β ₂The double antibodies sandwich ELISA of GPI measures the application in the test kit.

The anti-people β of IgG1 type of the present invention ₂The GPI monoclonal antibody is that the preserving number of said hybridoma cell line AB2-F6 is CCTCCNO:C201132 by hybridoma cell line AB2-F6 secretion.

The anti-people β of above-mentioned IgG1 type ₂GPI MONOCLONAL ANTIBODIES SPECIFIC FOR method is: with the human plasma β of purifying ₂GPI immunity BALB/c mouse adopts the PEG integration technology, indirect elisa method and limiting dilution assay, preparation and screening hybridoma.Be injected into BALB/c mouse after the hybridoma continuation enlarged culturing and prepare ascites, through ammonium sulfate precipitation and Protein G purifying MAbs.NST-757 prevents method to identify the hybridoma cell strain chromosome number, and ELISA measures titre, and Ig subclass test kit is identified the Ig subclass, and Western blot identifies specificity.

The present invention is with the human plasma β of purifying ₂Mouse boosting cell after the GPI immunity and murine myeloma cell merge the hybridoma AB2-F6 that the back produces, and the excretory monoclonal antibody is the IgG1/Kappa type, its can with the β of this chamber preparation ₂GPI reaction, and with the anti-β of standard ₂The GPI monoclonal antibody has comparability, and the present invention has stronger specificity.

The invention also discloses the anti-people β of said IgG1 type ₂The monoclonal antibody of glycoprotein I is at preparation human plasma β ₂The double-antibody sandwich elisa of glycoprotein I is measured the application in the test kit.

A kind of human plasma β ₂The double-antibody sandwich elisa of glycoprotein I is measured test kit, by anti-β ₂The elisa plate that the GPI polyclonal antibody encapsulates, 10% foetal calf serum confining liquid, anti-β ₂GPI monoclonal antibody, HRP-sheep anti-mouse igg, colour developing liquid and stop buffer are formed.The liquid that wherein develops the color comprises A liquid: H ₂O ₂B liquid: TMB (TMB).

Above-mentioned detection β ₂The preparation method that the double antibodies sandwich ELISA of GPI measures test kit is: through to chessboard titration confirm β ₂The confirming of GPI list/polyclonal antibody and HRP-sheep anti-mouse igg best effort concentration, best confining liquid and off-period, optimum antibody incubation time and best β to be checked ₂Confirming of GPI concentration finally set up β ₂The detection method of GPI and test kit.

Detection β according to the invention ₂The double antibodies sandwich ELISA measuring method of GPI.Its step comprises:

(1) with coating buffer CBS with the anti-people β of the rabbit of this prepared in laboratory ₂The polyclonal antibody of GPI is diluted to working concentration (0.08 μ g/ μ l) and adds in the elisa plate hole, and every hole 100 μ l place 4 ℃ of refrigerator overnight;

(2) discard liquid in the hole, wash enzyme plate 5 times with washings PBST, each 5min claps dried with thieving paper;

(3) the foetal calf serum 250 μ l of adding confining liquid 10% in the hole in each elisa plate hole, 37 ℃ of effect 2h, the method for (2) washing set by step;

(4) standard substance after will diluting with the PBS diluent and plasma specimen to be measured add and are coated with in the elisa plate hole of polyclonal antibody every hole 50 μ l, 37 ℃ of effect 1h, the method for (2) washing set by step;

(5) will resist people β with the PBS diluent ₂The monoclonal antibody of GPI is diluted to working concentration (0.06 μ g/ hole), and every hole adds 50 μ l, 37 ℃ of effect 1h, the method washing of repeating step (2);

(6) with washings the HRP-sheep anti-mouse igg is pressed working concentration dilution (1:10000 by volume), every hole adds 50 μ l, 37 ℃ of effect 1h, the method washing of repeating step (2);

(7) in every hole in enzyme plate hole, add A, each 50 μ l of B colour developing liquid, 37 ℃ of lucifuge colour developing 15min;

(8) in every hole in enzyme plate hole, add 50 μ l stop buffer termination reactions;

(9) on ELIASA in predominant wavelength 450nm, reference wavelength 630nm place carries out the dual wavelength colorimetric, the absorbance in the determination step (8) (OD value);

(10) drawing standard curve calculates β in the sample to be checked ₂The content of GPI.Wherein:

The described elisa plate of step (1) is available from the removable enzyme plate of 96 hole single holes of Costar company;

The described coating buffer CBS of step (1) (0.05M) prepares as follows: yellow soda ash 1.59g, and sodium hydrogencarbonate 2.93g is settled to 1000ml with zero(ppm) water, transfers pH to 9.6 with HCL/NaOH;

The preparation of the said lavation buffer solution PBST of step (2) (0.15M) is following: sodium-chlor 8.0g, and Repone K 0.2g, Sodium phosphate, dibasic 0.2g, potassium primary phosphate 2.9g, polysorbas20 0.5ml is settled to 1000ml with zero(ppm) water, transfers pH to 7.4;

The said confining liquid of step (3) is prepared as follows: washings 100ml, and foetal calf serum 10ml, foetal calf serum is available from GIBCO company;

The preparation of step (4), (5) described antigen, antibody diluent PBS (0.01M) is following: SODIUM PHOSPHATE, MONOBASIC 0.39g, and Sodium phosphate, dibasic 1.27g, sodium-chlor 0.85g, zero(ppm) water is settled to 1000ml, transfers pH to 7.2;

HRP-sheep anti-mouse igg described in the step (6) is available from doctor's moral biotechnology ltd;

The said colour developing liquid of step (7) A, B, and stop buffer described in the step (8) is all from clinical laboratory of Affiliated Hospital of Jiangsu University.

Description of drawings

Fig. 1 is the anti-β of purifying ₂The 12%SDS-PAGE of GPI monoclonal antibody analyzes;

Fig. 2 is the hybridoma chromosome analysis;

Fig. 3 is the subgroup identification (test strip method) of MAbs; Wherein 1,3,5 is calibration tape, and 2,4,6 is index zone, detected result IgG1/Kappa.

Fig. 4 is anti-β ₂GPI monoclonal antibody specificity analyses (Western blot).

Fig. 5 is human plasma β ₂The double-antibody sandwich elisa of glycoprotein I is measured the typical curve of test kit.

Hybridoma cell line AB2-F6 of the present invention delivers Chinese typical culture collection center preservation on June 14th, 2011, and deposit number is CCTCC NO:C201132; Depositary institution address: Chinese Wuhan University.

Embodiment

Following employed β ₂GPI and anti-β ₂The GPI polyclonal antibody is prepared by the contriver, and the preparation method sees reference respectively, and (Huang is loud and clear, Zhou Hong for document; Wang Ting, etc. beta 2 glycoprotein I purifying and stability analysis thereof [J]. the journal .2008 of Jiangsu University, 18 (4): 344-347.) with (yellow loud and clear; Zhou Hong, Yu Ying, etc. the anti-people β of rabbit ₂Gp I Polyclonal Antibody Preparation and evaluation [J]. Clinical Laboratory magazine .2009,27 (3): 192-194.), the β of preparation ₂GPI has identified itself and standard β ₂The GPI characteristic is identical, the anti-β of preparation ₂Methods such as the immune double diffusion test of GPI polyclonal antibody (IDD), EL ISA and western-bloting identify to have specificity.

Embodiment 1, anti-β ₂The GPI MONOCLONAL ANTIBODIES SPECIFIC FOR

(1) mouse immune is with β ₂GPI processes immunogen with saline water dilution back and equivalent Fu Shi Freund's complete adjuvant (Sigma company) after fully emulsified, nape portion subcutaneous multi-point injection BALB/c female mices in 6 age in week (50 μ g/ only, available from Yangzhou medical science Correlation Centre).Whenever all at a distance from 2 later on freund 's incomplete adjuvant (Sigma company) Isodose β ₂Carry out booster immunization after the GPI emulsification 2～3 times (50 μ g/, abdominal injection).Behind the last booster immunization 7 days, blood was got in the mouse docking, surveys immunizing potency, selected the high mouse of immunizing potency in last booster immunization 20 days, with saline water dilution Isodose β ₂GPI carries out the tail vein and strengthens (12.5 μ g/ only), impacts in the immunity back 3～5 days the eyeball blood sampling and keeps sample and tire with ELISA method mensuration immune serum; Extracting spleen cell is used for merging.

(2) cytogamy and hybridoma cloning SP2/0 myeloma cell (available from Shanghai biocytology institute) are in merging a few days ago enlarged culturing, and the cell of selecting logarithmic phase is used for merging.Mouse is plucked eyeball and gets blood, stays serum to make positive control, and mouse is put to death the back extracting spleen cell; Splenocyte and SP2/0 cell are mixed in fusion pipe with 4:1, centrifugal, remove supernatant; Be deposited in and drip 1mL50%PEG1450 (Sigma company) in 37 ℃ of water-baths, evenly add in the 60s, in 90s, drip 30mL serum-free RPMI1640 substratum (Gibco company) termination reaction immediately; Leave standstill 10min in 37 ℃ of water-baths, the centrifugal supernatant that goes, fused cell go into to be added with 96 orifice plates (Greiner company) of feeder cell with shop, HAT substratum (Gibco company) dilution back; Place 37 ℃, 5%CO ₂Cultivate in the incubator.Begin to observe the clonal growth situation after 3 days, changed liquid with HAT substratum half amount on the 4th day, about 7 days with HT substratum (Gibco company) the HAT substratum in the hole that swaps out fully.Adopt indirect elisa method to screen, it is frozen that positive hole changes 24 well culture plate enlarged culturing over to, carries out subclone 3～4 times with limiting dilution assay simultaneously, reaches 100% enlarged culturing to positive rate, builds strain and preserve this hybridoma cell line called after AB2-F6.

(3) indirect elisa method detects anti-β ₂Definite β of antigen and two anti-concentration during the GPI monoclonal antibody ₂GPI is with the carbonate solution dilution back coated elisa plate of 0.05mol/L pH9.6, and confining liquid is for containing the 0.02mol/LpH7.4PBS of 10% calf serum (Gibco company).With normal mouse serum 1:1000 as negative control, with β ₂The polyvalent antibody 1:2000 of GPI immunized mice is as positive control, confirms envelope antigen and the enzymic-labelled antibody (best of breed of sheep anti-mouse igg-HRP) with the square formation volumetry.The result judges: sample absorbancy (A) value is judged to the positive greater than 2.1 times of negative control A value.Top condition is: 1 μ g/ml β ₂The GPI coated elisa plate, sheep anti-mouse igg-HRP working concentration 1: 10000.

(4) anti-β ₂GPI MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying are got 10～12 BALB/c mouse abdominal injection whiterusss in age in week (0.5mL/ only), 7～10 days pneumoretroperitoneum injection hybridoma AB2-F6 (0.5～1 * 10 ⁶/ only), extract ascites about 7～14 days, be stored in-70 ℃ of refrigerators after the packing.Adopt ammonium sulfate precipitation and Protein G purifying ascites, identify purity with SDS-PAGE.Press experimental formula: protein concentration (mgmL)=1.45 * A ₂₈₀-0.74 * A ₂₆₀Calculate the protein concentration and the yld of purifying front and back.SDS-PAGE operates by ordinary method, 12% separation gel, and 5% concentrates glue, and AC 1mgmL mixes with the reduced form sample loading buffer of equivalent, 100 ℃ of 5min, appearance on the 25 μ L/ holes, constant current 50mA electrophoresis, 0.25% coomassie brilliant blue staining carries out albumen scanning.Analytical results is seen Fig. 1, and swimming lane is an electrophoresis result under the reductive condition, is respectively heavy chain immunoglobulin (about 50KD) and light chain (about 25KD), and the purity of ascites monoclonal antibody is more than 90%.

Embodiment 2, anti-β ₂The evaluation of GPI monoclonal antibody

(1) evaluation of monoclonal antibody prevents method to obtain the hybridoma (AB2-F6) of more metaphase with NST-757, and again through hypotonic, after fixing the processing, with the dyeing of 10%Giemsa dye liquor, mirror is observed karyomit(e) down.Show that like Fig. 2 the hybridoma chromosome number is more than 51 of the SP2/0 cell, between the 99-108 bar.Tire with indirect elisa method mensuration cells and supernatant and ascites, the indirect elisa method result shows that the ascites monoclonal antibody behind the cleer and peaceful purifying is to β on the Hybridoma Cell Culture ₂The titre of GPI was respectively 1: 2 ⁹With 1: 2 ¹⁵Identify Ig subclass (by specification operation), showed cell IgG secretion 1/Kappa type antibody (Fig. 3) as a result with monoclonal antibody subgroup identification test kit.Identify anti-β with Western blot ₂The specificity of GPI monoclonal antibody: with sample loading buffer with β ₂GPI is mixed with 1mg/mL, and 12%SDS-PAGE electrophoretic separation albumen is transferred to pvdf membrane with the 350mA constant current again; Film places the TBS/T room temperature sealing 1h that contains 5% skimmed milk; With film respectively with the β of the present invention of 1:1000 dilution ₂GPI monoclonal antibody and standard β ₂GPI monoclonal antibody (MAB1066, U.S. Chemicon company) reaction, inferior daily TBS/T washing 3 times, 15min/ time; Adopt the sheep anti-mouse igg antibody (1:2000) of HRP mark, hatch 1h for 37 ℃, TBS/T washing 3 times; 15min/ time, the hybridization band is observed in ECL Color Appearance System photographic fixing colour developing.The result sees Fig. 4: figure A is anti-β of the present invention ₂GPI monoclonal antibody reactive result, result show anti-β of the present invention ₂The β of about 45KD on GPI monoclonal antibody and the pvdf membrane ₂GPI antigen reacts, and does not have significant reaction with the BSA at 67KD place, explains that monoclonal antibody only discerns β ₂GPI, specificity is good, the standard anti-β of figure B for buying ₂GPI monoclonal antibody reactive (positive control).

(2) frozen 6 months cell strain AB2-F6 in the evaluation recovery liquid nitrogen container of hybridoma cell strain passes through the abdominal injection BALB/c mouse with cell after enlarged culturing, collect ascites and survey it to β ₂The ELISA titre of GPI is observed hybridoma and is secreted anti-β ₂The stability of GPI monoclonal antibody; Chromosome examination is undertaken by ordinary method.The result shows the anti-β of AB2-F6 stably excreting ₂The GPI monoclonal antibody, and the hybridoma chromosome number is between the 99-108 bar.

Embodiment 3, human plasma β ₂The foundation of GPI double-antibody sandwich elisa test kit

(1) chooses suitable encapsulant

Experiment is divided into three groups, and wherein every group is divided into again and does not encapsulate a closed group and add closed group with encapsulating, and chooses three confining liquids commonly used and is mixed with 5% skim-milk with PBST respectively; 2% bovine serum albumin, 10% foetal calf serum (FBS) seal above three groups respectively, and the result shows; Preceding two confining liquid sealing effects are not good; Stronger non-specific colour developing is arranged, and the 10%FBS sealing effect is better, does not almost have non-specific colour developing.

(2) anti-people β ₂Confirming of GPI list/polyclonal antibody and HRP-sheep anti-mouse igg best effort concentration

Utilize the chessboard titration to confirm the best effort concentration of each component.Because present method belongs to indirect double antibodies sandwich method, need to confirm the best effort concentration of three components, so chessboard titration at twice:

1. for the first time titration, the fixedly concentration of coated antibody and ELIAS secondary antibody: with the conservative relatively anti-β that measures 0.05 μ g/ μ l ₂GPI polyclonal antibody 100 μ l encapsulate elisa plate, and 4 ℃ encapsulate and spend the night; Next day, with PBST washing 5 times, 1min/ time, thieving paper is clapped and is done; Every hole adds 250 μ l10%FBS, 37 ℃ of sealing 2h; After the same washing, elisa plate lateral variation antigen β ₂The concentration of GPI adds a blank again with 1 μ g/ μ l, 0.2 μ g/ μ l, 0.04 μ g/ μ l, 0.008 μ g/ μ l and l5 gradient of 0.0016 μ g/ μ respectively and promptly only adds PBS by in every hole 50 μ l adding micropore, 37 ℃ of incubation 1h; After the washing; Vertically change the monoclonal anti bulk concentration; Add one 0 concentration again with 0.04 μ g/ μ l, 0.02 μ g/ μ l, 0.01 μ g/ μ l, 0.005 μ g/ μ l, 0.0025 μ g/ μ l, 0.0012 μ g/ μ l, l7 gradient of 0.0006 μ g/ μ respectively and add micropore, 37 ℃ of incubation 1h by every hole 50 μ l; After the washing, every hole adds the HRP-sheep anti-mouse igg 50 μ l with 8000 times of PBST dilutions, 37 ℃ of incubation 0.5h; Behind the thorough washing, every hole adds A, each 50 μ l of B substrate solution, and behind 37 ℃ of reaction 15min, every hole adds 50 μ l stop buffer termination reactions, and it is 450nm that ELIASA reads predominant wavelength, and reference wavelength is the absorbance at 630nm place.With SNR (containing measured OD value of analyte and the ratio that does not contain the measured OD value of analyte) the pairing antigen-antibody concentration of the maximum is best effort concentration.The result sees table 1

Table 1 is the chessboard titration results for the first time

2. carry out titration for the second time according to antigen and an anti-best effort concentration that the first time, the chessboard titration drew: the polyclonal antibody concentration that lateral variation encapsulates; Encapsulate elisa plate with 0.1 μ g/ μ l, 0.08 μ g/ μ l, 0.06 μ g/ μ l, 0.04 μ g/ μ l, 0.02 μ g/ μ l, 0 μ g/ μ l respectively; 100 μ l/ holes, 4 ℃ are spent the night; Next day, after the same washing of PBST, every hole adds 250 μ l10%FBS, 37 ℃ of sealing 2h; After the washing, every hole adds the antigen β of 50 μ l0.2 μ g/ μ l ₂GPI, 37 ℃ of incubation 1h; After the washing, every hole adds the monoclonal antibody of 50 μ l0.0012 μ g/ μ l, 37 ℃ of incubation 1h; After the washing, vertically change the concentration of HRP-sheep anti-mouse igg, add the HRP-sheep anti-mouse igg of 50 μ l respectively, 37 ℃ of incubation 0.5h with 6000,8000,10000,12000 times of PBST dilutions; Behind the thorough washing, every hole adds A, each 50 μ l of B substrate solution, and behind 37 ℃ of reaction 15min, every hole adds 50 μ l stop buffer termination reactions, and it is 450nm that ELIASA reads predominant wavelength, and reference wavelength is the absorbance at 630nm place.Obtain much more best anti-and two anti-concentration according to above result treatment mode.The result sees the following form 2

Table 2 is the chessboard titration results for the second time

After twice chessboard titration, draw top condition and be: the anti-β of 0.08 μ g/ μ l ₂The GPI polyclonal antibody encapsulates elisa plate, and the monoclonal antibody working concentration is 0.0012 μ g/ μ l, and HRP-sheep anti-mouse igg working concentration is 1:10000.

Embodiment 4, human plasma β ₂The preparation of GPI double-antibody sandwich elisa test kit typical curve

With the anti-β of 50 μ l0.08 μ g/ μ l ₂The GPI polyclonal antibody encapsulates elisa plate, and 4 ℃ encapsulate and spend the night; Next day, with PBST washing 5 times, 1min/ time, every hole adds 250 μ l10%FBS, 37 ℃ of sealing 2h; After the same washing, add the standard substance β of different weaker concns ₂GPI is with standard substance β ₂It is 10ng/ μ l, 20ng/ μ l, 40ng/ μ l, 80ng/ μ l, 160ng/ μ l that GPI is diluted to concentration, establishes negative control simultaneously and (carries β ₂Cross post liquid during GPI) and blank (PBS), 37 ℃ of incubation 1h; After the washing, add the anti-β of 50 μ l0.0012 μ g/ μ l ₂The GPI monoclonal antibody, 37 ℃ of incubation 1h; After the washing, every hole adds the HRP-sheep anti-mouse igg of 50 μ l1:10000 dilution, 37 ℃ of incubation 0.5h; Behind the thorough washing, every hole adds A, each 50 μ l of B substrate solution, and behind 37 ℃ of reaction 15min, every hole adds 50 μ l stop buffer termination reactions, and it is 450nm that ELIASA reads predominant wavelength, and reference wavelength is the absorbance at 630nm place.Based on colorimetric drawing standard curve as a result, see Fig. 5.

Embodiment 5, human plasma β ₂The application of GPI double-antibody sandwich elisa test kit

With human plasma β of the present invention ₂The GPI double-antibody sandwich elisa is measured test kit and is detected clinical samples, is divided into 2 test set: normal healthy controls and APS patient's group.In advance with sample with 5 times of PBS dilutions, then with test kit detection according to the invention, concrete steps are with described in the embodiment 2, ELIASA colorimetric result such as table 3:

Table 3 human plasma β ₂The double-antibody sandwich elisa of glycoprotein I is measured the result of kit measurement sample

Claims (4)

1. anti-people β of IgG1 type ₂The monoclonal antibody of glycoprotein I is characterized in that said monoclonal antibody by hybridoma cell line AB2-F6 secretion, and the preserving number of said hybridoma cell line AB2-F6 is CCTCC NO:C201132.

2. one kind prepares the anti-people β of IgG1 type ₂The glycoprotein I monoclonal antibody method is characterized in that may further comprise the steps: prepare ascites with being injected into BALB/c mouse after the hybridoma AB2-F6 enlarged culturing, ascites obtains anti-β behind ammonium sulfate precipitation and Protein G purifying ₂The glycoprotein I monoclonal antibody.

3. the anti-people β of the said IgG1 type of claim 1 ₂The monoclonal antibody of glycoprotein I is at preparation human plasma β ₂The double-antibody sandwich elisa of glycoprotein I is measured the application in the test kit.

4. human plasma β ₂The double-antibody sandwich elisa of glycoprotein I is measured test kit, it is characterized in that by anti-β ₂The elisa plate that the GPI polyclonal antibody encapsulates, 10% foetal calf serum confining liquid, the described anti-β of claim 1 ₂GPI monoclonal antibody, HRP-sheep anti-mouse igg, colour developing liquid and stop buffer are formed.

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