CN101509031A - Method for preparing short and naked dinoflagellate toxin monoclone antibody - Google Patents

Abstract

A method for preparing a short-Gymnodinium toxin monoclonal antibody relates to the preparation of a monoclonal antibody in the immune safety detection technology, which is finished by the following steps: (1) the preparation of a complete antigen, comprising the preparation of an immune antigen (BTX-IgG) and a detection antigen (BTX-BSA); (2) animal immunization; (3) and the preparation of the monoclonal antibody by blending an immune mouse spleen cell with the serum titer greater than 10<4> and an SP2/0 myeloma cell according to a conventional method. The ascites titer of the monoclonal antibody which is prepared by the immune method can be up to 2.4*10<5>, the affinity constant thereof is up to 0.6*10<9>M<-1>, and the ascites titer and the affinity constant thereof are not lower than the ascites titer and the affinity constant of the antibody which is prepared by the traditional immune method; the immune speed is fast, thus the cost for preparing the monoclonal antibody is effectively reduced.

Description

A kind of method for preparing short and naked dinoflagellate toxin monoclone antibody

[technical field]

The present invention relates to MONOCLONAL ANTIBODIES SPECIFIC FOR in the immune safety detection technology, further relate to the preparation that can be applicable to short and naked dinoflagellate toxin monoclone antibody in water surrounding and the fishery products.

[background technology]

Body eutrophication causes the algae abnormality proliferation, and discharges biotoxin---algae toxin (AlgaeToxin), these secondary metabolite serious harms human water safety and other hydrobiological safety.(Brevetoxins is that toxicity is stronger BTX), endangers very big a kind of algae toxin wherein to cause the short and naked dinoflagellate toxin of neural system paralysis.Toxin poisons hydrocoles such as fish, shellfish by food chain, the mankind poison because of these animals that ingest.

BTX is the ladder type polyether compound with unique pharmacological function that is produced by the dino flagellate (dinoflagellate) that Gymnodinium breve belongs to.BTX belongs to the neurotoxic shellfish poison, and acceptor target site VI combines with the sodium channel.BTX is the heat-resisting solid of low melting point, and the ester dissolubility can be dissolved in organic solvent and NaOH solution such as methyl alcohol, ethanol, acetone, chloroform, ether, benzene, is not soluble in neutrality and acidic aqueous solution.Usually the mass mortality of the shoal of fish can be caused, poisoning can be caused behind this algae of other animal ingestion or the contaminated fish.Understand producing putting upside down of gastrointestinal upset and neurological symptom such as nausea,vomiting,diarrhea, nerve numb and cold and hot consciousness etc. after the poisoning, understand irregular pulse when serious, suffocate.This toxin also can cause the respiratory tract acute poisoning by the aerosol in the ocean except causing the poisoning by food chain, and toxicity symptom is dizzy, platycoria, asthma, dry cough, have a running nose and eye keratitis etc.Kind of analogue was in series by ether ring surplus BTX had 10.I type toxin is the main toxin that Gymnodinium breve produces.Molecular formula is C ₅₀H ₇₀O ₁₄, molecular weight is about 894.The America and Europe waits some developed countries, and the highest to allow to limit the quantity of be 0.8 μ g/g to this toxin in the refrigeration scallop meat.At present, the national standard that China does not also have BTX to detect, this not only has influence on human consumer's health and lives safety, also can have influence on the outlet of fishery products.

About the detection method of BTX mainly contains (1) red, orange, green, blue, yellow (ROGBY): comprise tlc (TLC) and high performance liquid chromatography (HPLC) method.The TLC method is mainly used in separation and the purifying of BTX.The HPLC method adopts ultraviolet detection relatively poor aspect sensitivity and selectivity, and adopts mass spectrum (MS) to detect owing to be total to the effluent background interference, can improve selectivity and sensitivity, detects and is limited to 0.9ng; Adopt high performance liquid chromatography/quadrupole flight time mass spectrum (HPLC/Q TOFMS) coupling technique detectability can reach 0.5ng.Shortcoming is a sample pretreatment requirement height; Lack standard substance, equipment is huge, expensive; Need the professional.(2) tissue culture method: its ultimate principle is to utilize mouse neuroma clone easily by toxin blocking-up Na ⁺This Characteristics Detection toxin of passage.After adding channel activator, Na ⁺The excessively interior stream of ion causes cellular swelling, even dead.But added the antagonist toxin, can make cell survival, thereby can determine the existence of toxin and then the amount of definite toxin.The characteristics of this method are sensitivity, cheapness, do not need a large amount of animals.Shortcoming is to lack specificity; Processing requirements height to sample; Has tissue culture technique and equipment.

Immunological detection is able to widespread use with advantages such as stability, high sensitivity and the speed of its detection are fast in some algae toxins detect.

MONOCLONAL ANTIBODIES SPECIFIC FOR is one of committed step of setting up immunological detection method.But, do not see the report of BTX Monoclonal Antibody so far both at home and abroad as yet because the BTX standard substance cost an arm and a leg, are difficult to obtain.

At present, MONOCLONAL ANTIBODIES SPECIFIC FOR adopts abdominal cavity and subcutaneous injection immunization protocol more.Treat to be tired and reach 4 * 10 by immune serum ⁴After above, get mouse boosting cell and sp2/0 cytogamy, the limiting dilution assay clone, the strain of ELISA screening positive hybridoma cell, after the positive cell enlarged culturing, mouse ascites prepares monoclonal antibody.This method has been a very proven technique, is widely used in MONOCLONAL ANTIBODIES SPECIFIC FOR in food and the environment immunity safety detection technology.But traditional method prepares monoclonal antibody and needs at least just can carry out cytogamy after the immunity more than 4 times, needs the time more than 3.5 months just can produce monoclonal antibody at least; Traditional method is big to antigenic demand simultaneously, generally needs 200-475 μ g/ mouse.This has increased the cost of Monoclonal Antibody widely, particularly to expensive marine biotoxinses such as BTX, prepares tens thousand of yuans of strain monoclonal antibody needs.

[summary of the invention]

The technical problem to be solved in the present invention provides a kind of method for preparing short and naked dinoflagellate toxin monoclone antibody, and it is fast to have speed, the few characteristics of required antigen amount, thus effectively reduce the cost of Monoclonal Antibody.

The scheme that technical solution problem of the present invention is adopted adopts following steps:

1. the preparation of complete antigen

Concrete preparation method is as follows for immunizing antigen (BTX-IgG) and detection antigen (BTX-BSA):

(1) preparation of mixed solution: get 0.5mg BTX, 0.08mg N-maloyl imines, 0.15mgN, N dicyclo ethane carbodiimide is mixed in 0.5mL DMF, hatches 2h and make mixed solution under 10-30 ℃ of condition, and mixed solution is divided into two equal portions; (2) preparation of carrier proteins reaction solution: get 0.4mg IgG and be dissolved in 0.5mL 0.1mol/L NaHCO ₃In the damping fluid, behind the mixing as the carrier proteins reaction solution; (3) the carrier proteins reaction solution with step (2) preparation adds in the mixed solution of step (1) preparation, hatches 2h under 10-30 ℃ of condition, and products therefrom is immunizing antigen (BTX-IgG), the centrifugal removal unreacted matters of ultrafiltration, and-20 ℃ of preservations are standby after the packing; (4) get 0.4mg BSA, be dissolved in 0.5mL 0.1mol/L NaHCO ₃In the damping fluid, behind the mixing as the carrier proteins reaction solution; (5)

The carrier proteins reaction solution of step (4) preparation is added in the mixed solution of step (1) preparation, hatch 2h under 10-30 ℃ of condition, products therefrom is detection antigen (BTX-BSA), the centrifugal removal unreacted matters of ultrafiltration, and-20 ℃ of preservations are standby after the packing.

2. animal immune

(1) immune animal is male BALB/C mice in 8 ages in week, and the docking blood sampling is as negative serum before the immunity;

(2) preparation immunizing antigen reference liquid 2 μ g/ μ L.First immunisation is used Freund's complete adjuvant emulsification immunizing antigen, and antigen adjuvant is than 1:1, abdominal injection 100 μ L/ mouse.Adopt the injection of intrasplenic injection method after (3) 18 days, concrete grammar is as follows: the mouse fasting was prohibited water 24 hours, use every 100 μ L of 2% vetanarcol with mouse anesthesia, after treating that eye blink response disappears, be fixed on the ease of Use platform mouse is prostrate, to the sterilization of back fur,, cut off the long otch of 1cm downwards along the body axis at the last rib 0.1cm of back center line left side 0.5cm place, with have gentle hands light press both sides stomach wall, make spleen expose the abdominal cavity, use the 1mL syringe, inject 10 μ L holoantigen reference liquids, marginal not is penetrated the limit withdraw of the needle makes it evenly inject in the spleen as far as possible, carefully spleen is put back to the abdominal cavity, layer-by-layer suture abdominal cavity and skin, head are exempted from after 21 days docking and are got blood examination and survey and tire.

3, get serum titer greater than 10 ⁴Merged according to a conventional method by immune mouse spleen cell and SP2/0 myeloma cell, the preparation monoclonal antibody.

Adopt the prepared monoclonal antibody ascites of this immunization method to tire and to reach 2.4 * 10 ⁵, affinity costant reaches 0.6 * 10 ⁹M ^-1, ascites antibody is tired and affinity costant is not less than traditional immunization method gained antibody.This programme is compared with traditional method, and it is fast to have immune speed, saves 30 days time at least; Required antigen amount few (only needing 120 μ g/ mouse).Thereby reduce the cost of Monoclonal Antibody effectively.

[embodiment]

The concrete preparation of [embodiment 1] immunizing antigen (BTX-IgG) and detection antigen (BTX-BSA)

(1) preparation of mixed solution: get 0.5mg BTX, 0.08mg N-maloyl imines, 0.15mgN, N dicyclo ethane carbodiimide is mixed in 0.5mL DMF, hatches 2h and make mixed solution under 10-30 ℃ of condition, and mixed solution is divided into two equal portions; (2) preparation of carrier proteins reaction solution: get 0.4mg IgG and be dissolved in 0.5mL 0.1mol/L NaHCO ₃In the damping fluid, behind the mixing as the carrier proteins reaction solution; (3) the carrier proteins reaction solution with step (2) preparation adds in the mixed solution of step (1) preparation, hatches 2h under 10-30 ℃ of condition, and products therefrom is immunizing antigen (BTX-IgG), the centrifugal removal unreacted matters of ultrafiltration, and-20 ℃ of preservations are standby after the packing; (4) get 0.4mg BSA, be dissolved in 0.5mL 0.1mol/L NaHCO ₃In the damping fluid, behind the mixing as the carrier proteins reaction solution; (5) the carrier proteins reaction solution with step (4) preparation adds in the mixed solution of step (1) preparation, hatches 2h under 10-30 ℃ of condition, and products therefrom is detection antigen (BTX-BSA), the centrifugal removal unreacted matters of ultrafiltration, and-20 ℃ of preservations are standby after the packing.

[embodiment 2]

2. animal immune

(1) immune animal is male BALB/C mice in 8 ages in week, and the docking blood sampling is as negative serum before the immunity;

(2) preparation immunizing antigen reference liquid 2 μ g/ μ L.First immunisation is used Freund's complete adjuvant emulsification immunizing antigen, and antigen adjuvant is than 1:1, abdominal injection 100 μ L/ mouse.Adopt the injection of intrasplenic injection method after (3) 18 days, concrete grammar is as follows: the mouse fasting was prohibited water 24 hours, use every 100 μ L of 2% vetanarcol with mouse anesthesia, after treating that eye blink response disappears, be fixed on the ease of Use platform mouse is prostrate, to the sterilization of back fur,, cut off the long otch of 1cm downwards along the body axis at the last rib 0.1cm of back center line left side 0.5cm place, with have gentle hands light press both sides stomach wall, make spleen expose the abdominal cavity, use the 1mL syringe, inject 10 μ L holoantigen reference liquids, marginal not is penetrated the limit withdraw of the needle makes it evenly inject in the spleen as far as possible, carefully spleen is put back to the abdominal cavity, layer-by-layer suture abdominal cavity and skin, head are exempted from after 21 days docking and are got blood examination and survey and tire.

The preparation of [embodiment 3] reagent source and SP2/0 myeloma cell's suspension

BTX is Express Technology Co., the Ltd. product; Human IgG is the Thermo product; Polyoxyethylene glycol (polythylene glycol-4000, PEG), RPMI1640 substratum, HAT selective medium, bovine serum albumin (BSA), glutaraldehyde, Mercamine Cysteamine, Fu Shi Freund's complete adjuvant (FCA), freund 's incomplete adjuvant (FICA), O-Phenylene Diamine (OPD), N-maloyl imines (N-hydroxysuccinimide), N, N dicyclo ethane carbodiimide (N, N-dimethyformamide DMF) waits available from Sigma company; The centrifugal ultrafiltration pipe is the MILLPORE product; The horseradish peroxidase-labeled sheep anti-mouse igg is purchased in ancient cooking vessel state Bioisystech Co., Ltd (Beijing), and methyl alcohol, acetate, yellow soda ash, sodium bicarbonate, the used chemical reagent of ELISA etc. are homemade analytical pure.

In preceding 15 days recovery SP2/0 myeloma cells of fusion and enlarged culturing.Take out SP2/0 cell cryopreservation pipe, put into the insulated tank that 40 ℃ of water are housed immediately, rock gently, dissolve fully, in 1-2min, finish until moving liquid storage.To move liquid storage and move on to the 15mL centrifuge tube, add the 5mL nutrient solution, blow gently evenly, 1000rpm, 5min abandons supernatant.Add 1 pipe nutrient solution with pasteur pipet, piping and druming evenly moves in the 50mL culturing bottle, adds 2 suction pipe nutrient solutions again, and piping and druming is evenly assigned in 2 new 50mL culturing bottles, so just a frozen tube cell is assigned in 3 50mL culturing bottles.Being put in 37 ℃ contains in 5% the CO2gas incubator and cultivates.Change liquid behind the 24h, every bottle 4 pasteur pipe.Just can be used for behind the 48h merging, change liquid once, make cell when merging, be in the logarithm division stage at preceding 24 hours needs of fusion.

The preparation of [embodiment 4] feeder cell

Get more than 12 ages in week, with myelomatosis homology mouse 1-2.Cut out an eyeball bloodletting, treat that drop of blood does not go out till.Draw neck to put to death, mouse is immersed in 75% alcohol 5min sterilization.Aseptic condition is down cut off mouse web portion outer skin (be sure not peritonaeum broken and make intraperitoneal fluid loss) with sterilize tweezers and scissors.Two side directions draw back mouse skin up and down then, expose peritonaeum.Extract 3mL serum-free HT1640 nutrient solution with sterile syringe, peritonaeum is mentioned, solution is injected in the mouse peritoneal with tweezers.Hand is syringe fixedly, and syringe needle rests on intraperitoneal, massages stomach wall 1-2min (scavenger cell is swum out of) gently, draws back intraperitoneal liquid with syringe then, and carries out 3 flushings.Collect washing fluid, the centrifugal 5min of 1000rpm abandons supernatant.With the cell of HAT nutrient solution dilution centrifugation, and adjust cell concn to 1 * 10 ⁵Individual/mL.Cell suspension is pressed every hole 100 μ l (about two) bed board.Being put in 37 ℃ contains in 5% the CO2gas incubator and cultivates.This cell is the 24h preparation before fusion.

The preparation of [embodiment 5] immune lymphoglandula B cell suspension

The BALB/C mice of immunity is plucked eye get blood and cause death, collect blood in the EP pipe, place centrifugation serum behind the 2h for 37 ℃ ,-20 ℃ of storages are standby.The back mouse that will die suddenly is immersed the clear middle 3min of 75% wine.Aseptic Qu lymphonodi poplitei is put into the aseptic plate that is loaded with 200 order copper mesh.Add the 2mL basic medium lymphoglandula is soaked into, lymphoglandula is ground to form pulpous state with the glue rod.Pasteur pipet piping and druming cell dispersion is transferred in the 50mL glass centrifuge tube, and the centrifugal 5min of 1000g/min repeats to wash 2 times, and counting cells.Use an amount of RPMI-1640 suspension cell at last, make into 5 * 10 ⁷/ mL, volume should be more than the 2mL.

[embodiment 6] cytogamy and the cultivation of merging the back cell

Embodiment 5 De popliteal lymphocyte and embodiment 3 gained SP2/0 cells (ratio of cell count is 10:1) are moved on in the round bottom glass centrifuge tube, and it is even to be mixed, the centrifugal 10min of 1000rpm.After discarding supernatant, exert oneself slightly to make precipitation be the oar pasty state in attack test tube bottom with finger.Splash into 50% PEG (about 0.7mL) rapidly, with suction pipe pressure-vaccum 4-5 time gently, behind the 30s, the centrifugal 3min of 700rpm; Slowly add 20mL RPMI-1640 substratum (action is soft as far as possible), the test tube bottom is done draw circular motion; The centrifugal 5min of 1000g/min abandons supernatant behind the 2min, washes once with method again; Slowly add 50mL and contain the HT substratum gently behind the mixing, join on 96 orifice plates that are ready to nurse cell, every hole adds 100 μ L fused cell suspensions, places 37 ℃ 5% CO ₂Cultivate in the incubator.Merge after 24 hours, every hole is added 50 μ L and is contained the HAT substratum.And respectively at 4 days, 7 days and 10 days, change or add the HAT substratum according to the cell growing state, changed with 20% calf serum HT substratum in the 14th, 18,22,25 day.In culturing process, adopt half amount to change the liquid method, promptly sucking-off 0.1mL nutrient solution from each aperture at every turn adds the 0.1mL fresh medium again.

[embodiment 7] hybridoma screening

Observe the growing state of fused cell, the cell of visible cloning growth after general 3 days treats that the fused cell colony grows to 1/2 visual field (* 100) when above, just can detect the antibody in the supernatant liquor, filters out the hybridoma of secretory antibody, and clones.Detect for the first time antibody and changing liquid two days later for the second time, carried out in the 10th day after promptly merging.Be decided to be the positive with what the OD value of measured hole was higher than oncocyte supernatant liquor contrast OD value more than 2 times.

The clone again and the enlarged culturing of [embodiment 8] positive hybridoma cell

Treat positive porocyte when covering with, cell blown afloat gently that be drawn onto in the sterile vials, cell counting is diluted among per 100 μ l and contains 0.5-1 cell density with the RPMI1640 substratum that contains 20% foetal calf serum.Cell suspension with dilution splashes in the feeder cell culture plate of embodiment 4 methods preparation every hole 100 μ l then.Plate is inserted 37 ℃ contain 5% CO ₂Incubator is cultivated, and observe once every day, changes liquid once about 3 days, observe every hole several clone strains are arranged, to there being two or do not have the hole of cell to make mark respectively, when treating that clone cell grows to 8-10 days, detect antibody, mark positive hole, liquid is changed in all clone cell holes, change liquid after 2 days, detect again, detecting the high hole of OD value with twice and contrast, all is male hole time cloning again to twice detection.All clone cell holes are all positive up to being cloned into, and select the hybridoma that the OD value is high, cell viability is good to carry out enlarged culturing at last.

The preparation of [embodiment 9] ascites antibody

Get 8 age in week BALB/C mice, with high pressure (113 ℃, 15min) Mie Jun paraffin oil is expelled to mouse peritoneal, 0.5mL/ only, the 8d pneumoretroperitoneum is injected embodiment 8 gained hybridomas 2 * 10 ⁶/ only, observe the mouse ascites production every day behind the inoculation 7d, obviously heaves as the abdominal cavity, and mouse moving difficulty and skin have nervous sense, promptly gather ascites with No. 16 syringe needles, with the centrifugal 10min of ascites 3000rpm, get supernatant, discard fat, clarification ascites in the middle of collecting.Adopt sad-saturated ammonium sulphate method preliminary purification ,-20 ℃ of packing are frozen standby.

The mensuration of [embodiment 10] ascites antibody avidity

Ascites is diluted to the preceding equal volume of purifying behind the purifying, does contrast with the equal extension rate of the ascites of SP2/0 cell inoculation simultaneously.Adopt the noncompetitive enzyme immunoassay to measure affinity of antibody.Detect antigen (BSA-BTX) by 1,0.5,0.25,0.125 μ g/ml coated elisa plate, 100 μ l/ holes, 37 ℃, 2h; After the sealing of 1% gelatin, with monoclonal antibody since 2000 times of doubling dilutions.Logarithmic value with antibody concentration (mol/L) is an abscissa, is ordinate with its corresponding OD value, can make 4 sigmoid curves in a set of coordinates.Find out the top of S curve, be set at ODmax.In curve, find out 4 curves antibody concentration of 50% ODmax correspondence separately respectively.With one group in twos of 4 concentration, calculate the affinity costant of monoclonal antibody according to formula.

Ka＝(n-1)/2(n[Ab`]t-[Ab]t)

Annotate: two bags were by the multiple of concentration during n was every group, and [Ab`] t and [Ab] t are respectively the antibody concentration (mol/L) of two 50% ODmax correspondences in every group, and Ka is an affinity costant.

Claims (9)

1, a kind of method for preparing short and naked dinoflagellate toxin monoclone antibody is characterized in that: adopt following steps to finish:

1) preparation of complete antigen

Concrete preparation method is as follows for immunizing antigen (BTX-IgG) and detection antigen (BTX-BSA):

(1) preparation of mixed solution: get 0.5mg BTX, 0.08mg N-maloyl imines, 0.15mg N, N dicyclo ethane carbodiimide is mixed in 0.5mL DMF, hatches 2h and make mixed solution under 10-30 ℃ of condition, and mixed solution is divided into two equal portions; (2) preparation of carrier proteins reaction solution: get 0.4mg IgG and be dissolved in 0.5mL0.1mol/L NaHCO ₃In the damping fluid, behind the mixing as the carrier proteins reaction solution; (3) the carrier proteins reaction solution with step (2) preparation adds in the mixed solution of step (1) preparation, hatches 2h under 10-30 ℃ of condition, and products therefrom is immunizing antigen (BTX-IgG), the centrifugal removal unreacted matters of ultrafiltration, and-20 ℃ of preservations are standby after the packing; (4) get 0.4mg BSA, be dissolved in 0.5mL0.1mol/L NaHCO ₃In the damping fluid, behind the mixing as the carrier proteins reaction solution; (5) the carrier proteins reaction solution with step (4) preparation adds in the mixed solution of step (1) preparation, hatches 2h under 10-30 ℃ of condition, and products therefrom is detection antigen (BTX-BSA), the centrifugal removal unreacted matters of ultrafiltration, and-20 ℃ of preservations are standby after the packing;

2) animal immune

(1) immune animal is male BALB/C mice in 8 ages in week, and the docking blood sampling is as negative serum before the immunity;

(2) preparation immunizing antigen reference liquid 2 μ g/ μ L, first immunisation is used Freund's complete adjuvant emulsification immunizing antigen, and antigen adjuvant is than 1:1, and abdominal injection 100 μ L/ mouse are adopted the injection of intrasplenic injection method after (3) 18 days, and head exempts from after 21 days docking and gets blood examination and survey and tire;

3) get serum titer greater than 10 ⁴Merged according to a conventional method by immune mouse spleen cell and SP2/0 myeloma cell, the preparation monoclonal antibody.

2, method according to claim 1, it is characterized in that: the intrasplenic injection method is as follows, the mouse fasting was prohibited water 24 hours, use every 100 μ L of 2% vetanarcol with mouse anesthesia, after treating that eye blink response disappears, be fixed on the ease of Use platform mouse is prostrate, to the sterilization of back fur, at the last rib 0.1cm of back center line left side 0.5cm place, cut off the long otch of 1cm downwards along the body axis, with have gentle hands light press both sides stomach wall, make spleen expose the abdominal cavity, use the 1mL syringe, inject 10 μ L holoantigen reference liquids, marginal not is penetrated the limit withdraw of the needle makes it evenly inject in the spleen as far as possible, spleen is put back to the abdominal cavity, layer-by-layer suture abdominal cavity and skin.

3, method according to claim 1, it is characterized in that: the preparation of feeder cell is to get more than 12 ages in week, with myelomatosis homology mouse 1-2, cut out an eyeball bloodletting, till treating that drop of blood does not go out, draw neck to put to death, mouse is immersed in 75% alcohol 5min sterilization, aseptic condition is cut off the mouse web portion outer skin with sterilization tweezers and scissors down, two side directions draw back mouse skin up and down then, expose peritonaeum, extract 3mL serum-free HT1640 nutrient solution, peritonaeum is mentioned with tweezers with sterile syringe, solution is injected in the mouse peritoneal, hand is syringe fixedly, and syringe needle rests on intraperitoneal, massages stomach wall 1-2min gently, scavenger cell is swum out of, draw back intraperitoneal liquid with syringe then, and carry out 3 flushings, collect washing fluid, the centrifugal 5min of 1000rpm, abandon supernatant,, and adjust cell concn to 1 * 10 with the cell of HAT nutrient solution dilution centrifugation ⁵Individual/mL, cell suspension by every hole 100 μ l bed boards, is put in 37 ℃ and contains in 5% the CO2gas incubator and cultivate.This cell is the 24h preparation before fusion.

4, method according to claim 1, it is characterized in that: the preparation of immune lymphoglandula B cell suspension is the BALB/C mice of immunity to be plucked eye get blood and cause death, collect blood in the EP pipe, centrifugation serum behind 37 ℃ of placement 2h,-20 ℃ of storages are standby, the back mouse that will die suddenly is immersed the clear middle 3min of 75% wine, aseptic Qu lymphonodi poplitei, put into the aseptic plate that is loaded with 200 order copper mesh, add the 2mL basic medium lymphoglandula is soaked into, lymphoglandula is ground to form pulpous state, pasteur pipet piping and druming cell dispersion with the glue rod, be transferred in the 50mL glass centrifuge tube, the centrifugal 5min of 1000g/min repeats to wash 2 times, and counting cells, use an amount of RPMI-1640 suspension cell at last, make into 5 * 10 ⁷/ mL, volume are more than the 2mL.

5, according to claim 1 and 4 described methods, it is characterized in that: the cultivation Shi of cytogamy and fusion back cell is Jiang popliteal lymphocyte and SP2/0 cell, and the ratio of cell count is 10:1, move on in the round bottom glass centrifuge tube, it is even to be mixed, the centrifugal 10min of 1000rpm, discard supernatant after, with pointing slightly firmly attack test tube bottom, make precipitation be the oar pasty state, splash into 50% PEG 0.7mL, use suction pipe pressure-vaccum 4-5 time, behind the 30s, the centrifugal 3min of 700rpm; Slowly add 20mL RPMI-1640 substratum, the test tube bottom is done draw circular motion; The centrifugal 5min of 1000g/min abandons supernatant behind the 2min, washes once with method again; Slowly add 50mL and contain the HT substratum gently behind the mixing, join on 96 orifice plates that are ready to nurse cell, every hole adds 100 μ L fused cell suspensions, places 37 ℃ 5% CO ₂Cultivate in the incubator, merge after 24 hours, every hole is added 50 μ L and is contained the HAT substratum, and respectively at 4 days, 7 days and 10 days, change or add the HAT substratum according to the cell growing state, changed with 20% calf serum HT substratum in the 14th, 18,22,25 day, in culturing process, adopt half amount to change the liquid method, promptly sucking-off 0.1mL nutrient solution from each aperture at every turn adds the 0.1mL fresh medium again.

6, method according to claim 1, it is characterized in that: hybridoma screening is to observe the growing state of fused cell, the cell of visible cloning growth after 3 days, treat that the fused cell colony grows to 1/2 visual field when above, detect the antibody in the supernatant liquor, filter out the hybridoma of secretory antibody, and clone, detect for the first time antibody and changing liquid two days later for the second time, carried out in the 10th day after promptly merging, and be decided to be the positive more than 2 times with what the OD value of measured hole was higher than that the oncocyte supernatant liquor contrasts the OD value.

7, method according to claim 1, it is characterized in that: the clone again of positive hybridoma cell and enlarged culturing are to treat that positive porocyte is when covering with, with the RPMI1640 substratum that contains 20% foetal calf serum cell is blown afloat gently, be drawn onto in the sterile vials, cell counting, be diluted among per 100 μ l and contain 0.5-1 cell density, the cell suspension with dilution splashes in the feeder cell culture plate every hole 100 μ l then.Plate is inserted 37 ℃ contain 5%CO ₂Incubator is cultivated, observe once every day, changed liquid once in 3 days, observe every hole several clone strains are arranged, to there being two or do not have the hole of cell to make mark respectively, when treating that clone cell grows to 8-10 days, detect antibody, mark positive hole, liquid is changed in all clone cell holes, change liquid after 2 days, detecting, detect the high hole of OD value with twice and contrast, all is male hole time cloning again to twice detection, all clone cell holes are all positive up to being cloned into, and select the hybridoma that the OD value is high, cell viability is good to carry out enlarged culturing at last.

8, method according to claim 1 is characterized in that: the preparation of ascites antibody be get 8 age in week BALB/C mice, 113 ℃, the autoclaved paraffin oil of 15min condition are expelled to mouse peritoneal, 0.5mL/ only, 8 days pneumoretroperitoneum injection hybridomas 2 * 10 ⁶/ only, inoculate after 7 days and to observe the mouse ascites production every day, obviously heave as the abdominal cavity, mouse moving difficulty and skin have nervous sense, promptly gather ascites with No. 16 syringe needles, with the centrifugal 10min of ascites 3000rpm, get supernatant, discard fat, clarification ascites in the middle of collecting, adopt sad-saturated ammonium sulphate method preliminary purification ,-20 ℃ of packing are frozen standby.

9, method according to claim 1, it is characterized in that: the mensuration of ascites antibody avidity is that ascites is diluted to the preceding equal volume of purifying behind the purifying, simultaneously do contrast with the equal extension rate of the ascites of SP2/0 cell inoculation, adopt the noncompetitive enzyme immunoassay to measure affinity of antibody, detect antigen BSA-BTX by 1,0.5,0.25,0.125 μ g/ml coated elisa plate, 100 μ l/ holes, 37 ℃, 2h; After 1% the gelatin sealing, with monoclonal antibody since 2000 times of doubling dilutions, logarithmic value with antibody concentration mol/L is an abscissa, with its corresponding OD value is ordinate, makes 4 sigmoid curves in a set of coordinates, finds out the top of S curve, be set at ODmax, in curve, find out 4 curves antibody concentration of 50%ODmax correspondence separately respectively,, calculate the affinity costant of monoclonal antibody according to formula one group in twos of 4 concentration.