CN101906159B - Hybridoma cell line D6D - Google Patents

Abstract

The invention relates to the development of a hybridoma cell line D6D, and belongs to the field of biotechnology. OTA-BSA obtained by coupling BSA and OTA is used to immunize a BALB/c mice; splenic cells of the mice are fused with Sp2/0 myeloma cells; and the fused cells are selectively cultured by an HAT culture medium. A complete antigen OTA-KLH obtained by coupling KLH and OTA is taken as a coating antigen of ELISA; antibody content in hybridoma cell culture supernatant is detected by an indirect ELISA method; the hybridoma cell line D6D stably secreting anti-OTA monoclonal antibodies is screened; the detected D6D ascite ELISA titer is 1:3,200; identification shows that the monoclonal antibody can be specifically combined with the OTA, and an SDS-PAG electrophoretogram of the purified monoclonal antibody has two protein bands which are respectively about 50 KD and 25 KD and coincide with the molecular weight magnitudes of a light chain and a heavy chain denatured from an antibody protein, so that the fact that an anti-OTA specific antibody is obtained is proved again, the subtype of the monoclonal antibody is IgG2b, and the light chain is a kappa chain. The specific monoclonal antibody can be used for researching the OTA and establishing an OTA immunological detection method.

Description

Hybridoma cell line D 6 D

Technical field

(Ochratoxin A, OTA) hybridoma cell line D 6 D of monoclonal antibody belongs to biological technical field, relates to the antibody engineering technology to the present invention relates to secrete ochratoxin A.

Background technology

Ochratoxin (Ochratoxins) be one type mainly by aspergillus fungi (for example, Aspergillus ochraceus Aspergillus Ochraceus, sulfuraspergillus Aspergillus Fresenii etc.) and the mycetogenetic mycotoxins of penicillium.OTA is the main ingredient of ochracin, also is the major cause that causes ochracin to be poisoned, and it urgees carcinous and carinogenicity is confirmed in rat.Epidemiology survey finds that OTA possibly have certain dependency with area, human Balkan ephrosis to the pollution of grain products.IARC (IARC) is divided into 2B class carcinogens to OTA.

The structure of OTA is the phenylformic acid Isocoumarin >97.It is a kind of stable colourless crystallization compound, is slightly acidic, is dissolved in polar solvent and sodium hydrogen carbonate solution, is slightly soluble in water, and its methanol solution is preserved in refrigerator and can not be decomposed in 1 year.OTA molecular formula C ₂₀H ₁₈ClNO ₆, molecular weight is 404, is a kind of haptin material.

The chemical structural formula of OTA

OTA extensively is present in agricultural-food such as cereal such as wheat, barley, oat, corn and coffee berry, grape and the goods, and human health and animal husbandry development are constituted potential threat.(The Codex Committee on Food Additives and Contaminants, CCFAC) etc. international organization thinks that OTA is a kind of mycotoxins of the public health security that counts for much for the foodstuff additive and the pollutent code council.The limit standard of OTA is formulated in the 56th the FAO/WHO foodstuff additive joint specialist council (JECFA) meeting, thinks in cereal such as wheat, barley and rye and the goods thereof that it is 5 μ g kg that OTA limits the quantity of ^-1

Harm in view of OTA; Through preparing its monoclonal antibody; The foundation that can be research of ochratoxin antigenicity and OTA immunological detection method provides necessary component and technique means, to the biological function of exploring each structural region of OTA with set up OTA special, the sensitive detection method is significant.

Summary of the invention

Technical problem

The hybridoma cell strain that the purpose of this invention is to provide secretion OTA monoclonal antibody.Monoclonal antibody of the present invention is that bovine serum albumin (BSA) is prepared complete antigen as carrier proteins and OTA coupling, and immune BALB/c mouse utilizes hybridoma technology to obtain the monoclonal antibody D6D of ability specific recognition OTA.

Technical scheme

A kind of OTA monoclonal antibody; It is characterized in that: this monoclonal antibody is that hybridoma cell line D 6 D produces; Hybridoma cell line D 6 D is preserved in Chinese typical culture collection center on May 27th, 2010; Address: Wuhan Wuhan University postcode: 430072, deposit number is CCTCC NO:C201052.The preparation method comprises:

1) preparation of immunizing antigen OTA-BSA

DMSO 99.8MIN. (DMSO) solution of preparation OTA adds N-hydroxyl succinic diamide (NHS) and 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) and carries out the activation of OTA.Above-mentioned solution slowly dripped in bovine serum albumin (BSA) solution carry out the coupling of OTA and BSA, reaction product obtains the conjugate of OTA and carrier proteins with phosphate buffered saline buffer (PBS) dialysis.

2) mouse immune

Low dose of macrocyclic immunization protocol is adopted in this experiment.Use coupling protein OTA-BSA as immunizing antigen, the female BALB/c mouse (available from Yangzhou University comparative medicine center) in 7 ages in week is carried out immunity.By 200 μ g/ immunizing dose only BALB/c mouse is carried out 8 immunity, last immunity was won mouse spleen after three days, did cell and merged.

3) cytogamy

Get the BALB/c mouse splenocyte uniform mixing in proportion of Sp2/0 myeloma cell's (be so kind as to give the academy of agricultural sciences in Jiangsu Province) and immunity; Under 50% Macrogol 4000 (PEG 4000) effect, carry out cytogamy, use 1640 substratum (available from Gibico company) that contain 20%FCS (purchasing the biological scientific and technological Graduate School of Engineering of intelligent people) and HAT (purchasing company) at 5%CO in Invitrogen in Shanghai ₂Cultivate in the incubator; After cultivating 14d; Progressively use 1640 substratum of 20%FCS and HT (purchasing company) and 1640 culture medium culturing of 20%FCS in Invitrogen, when the cell that merges grow to 96 orifice bore floorages 1/5 the time, get supernatant and carry out anti-OTA monoclonal antibody and detect.

4) hybridoma screening

Carbonate buffer solution with OTA-hemocyanin (KLH) is done coating antigen, does confining liquid with 1% gelatin, and 1: 10000 horseradish peroxidase target sheep anti-mouse igg is as two anti-(available from doctor's moral biotech companies), TMB-H ₂O ₂Be substrate, 2mol L ^-1Sulphuric acid soln is a stop buffer; PBST does washing lotion; The negative reference serum of the positive reference serum of dilution in 1: 800 and dilution in 1: 800 is done the indirect enzyme-linked immunosorbent assay (ELISA) of contrast; Anti-OTA antibody-secreting situation in the hybridoma supernatant detects, the positive hybridoma cell of the anti-OTA antibody of screening secretion.Measure OD through ELIASA _450nmValue: with the blank zeroing, as the OD of positive reference serum _450nmThe OD of value and negative reference serum _450nmRatio>=2.1 of value are detected the hole and are judged to the positive, as the OD of positive reference serum _450nmThe OD of value and negative reference serum _450nmThe detection hole of the ratio of value＜1.5 is judged to feminine gender, and ratio is judged to suspicious between the centre.

5) limiting dilution assay is to the hybridoma subclone

At first positive hole viable cell is expected that with platform orchid dyes and count, be diluted to 100 cells/15mL substratum, the cell suspension of dilution is added 96 porocyte culture plates, reach 1 cell in every hole, at 37 ℃, 5%CO with 1640 perfect mediums ₂Cultivate in the incubator.Get cell conditioned medium when cultivating 8～9d, in time carry out ELISA and detect.Select the male monoclonal cell to carry out same subclone again more than 3 times, detect all positive and to detect OD value more approaching in each hole, with the positive cell enlarged culturing after the subclone cultivation repeatedly and frozen until this cell institute foraminous supernatant.

6) anti-OTA MONOCLONAL ANTIBODIES SPECIFIC FOR

With 5 * 10 of PBS dilution ⁶～1 * 10 ⁷Individual positive hybridoma cell injects the BALB/c mouse abdominal cavity of growing up, and gathers belly behind 5～10d and obviously heaves the ascites of mouse, and is centrifugal, collects supernatant.

7) evaluation-denaturing polyacrylamide gel electrophoresis (SDS-PAGE) of anti-OTA monoclonal antibody purity

The monoclonal antibody ascites solution of anti-OTA mixes with 5 * sample-loading buffer, and sex change is boiled in water-bath, and through denaturing polyacrylamide gel electrophoresis (SDS-PAGE), coomassie brilliant blue R250 dyeing is according to electrophoretic antibody protein band quality size, the existence of checking antibody.

(6) evaluation of antibody subtype

(Rache Iso strip, cat.NO.14932027) antagonism OTA monoclonal antibody is carried out the hypotype test with Mouse Monoclonal Antibody Isotyping Kit.

Beneficial effect characteristics of the present invention and advantage are following:

1, antigen prepd method provided by the invention is simple and feasible.

2, OTA monoclonal antibody high specificity provided by the invention, the preparation method is simple.

3, monoclonal antibody specific provided by the invention can be used for the research of OTA and the foundation of OTA immunological detection method.

4, obtain the hybridoma cell line D 6 D of stably excreting OTA monoclonal antibody, recording that D6D ascites ELISA tires is 1: 3200.Through identifying that this monoclonal antibody is the IgG2b antibody-like, light chain of antibody is the κ hypotype.

Description of drawings

The continuous wave spectrum scanning of Fig. 1 coupled antigen ultraviolet detects

The electrophorogram of monoclonal antibody behind Fig. 2 purifying

Biological preservation

Hybridoma cell line D 6 D is preserved in Chinese typical culture collection center, address: Wuhan Wuhan University postcode on May 27th, 2010: 430072, and deposit number is CCTCC C201052.

Embodiment

(1) preparation of immunizing antigen OTA-BSA and detection

The activation of OTA: get 1mg OTA and be dissolved in the 0.25mL DMSO solution, measure NHS and EDC respectively and add (OTA: NSH: the EDC mol ratio is 1: 2: 4) in the above-mentioned solution, lucifuge is acutely shaken 2h under the room temperature, and 4 ℃ of reactions are afterwards spent the night.The coupling of OTA: above-mentioned solution dripped slowly (2mg albumen is dissolved in 1mL 0.1mol L in BSA solution ^-1In the sodium hydrogen carbonate solution), add the magnetic force rotor, place on the magnetic force vibrator lucifuge vibration 2h under the room temperature; Reaction product is in 0.05mol L ^-14 ℃ of following lucifuge dialysis 72h in the PBS damping fluid, every 5h changes dialyzate once, obtains the conjugate of OTA and carrier proteins.

The complete antigen that coupling is good is verified through the ultraviolet continuous sweep method.Configuration OTA standardized solution, BSA standardized solution and coupling complete antigen diluent.The BSA standardized solution maximum absorption band occurs at 280nm, and the OTA standardized solution maximum absorption band occurs at 379nm.In 200～400nm wavelength, 5nm carries out continuous wavelength scanning at interval.Result such as Fig. 1.

(2) foundation of hybridoma cell strain

1) mouse immune

Low dose of macrocyclic immunization protocol is adopted in this experiment.Use coupling protein OTA-BSA as immunizing antigen, the female BALB/c mouse in 7 ages in week is carried out immunity.By 200 μ g/ immunizing dose only, 1: 1 (V/V) mixes with Freund's complete adjuvant, and emulsification 30min reaches the water-in-oil shape, in water, does not dissolve or 4 ℃ of not stratified effective emulsifications that are regarded as of spending the night.With the emulsive complete antigen, nape portion multiple spot subcutaneous injection immunity BALB/c mouse (200 μ g/ are only); After 2 weeks, with same antigen and isopyknic Freund's incomplete adjuvant emulsification, 200 μ g/ only carry out booster immunization; After 7 immunity, do not add adjuvant and only carry out the abdominal injection booster immunization with 200 μ g/.From beginning for the third time, each one week of immunity back tail vein or eyeball blood sampling, 37 ℃ leave standstill 1h, 4 ℃ are spent the night and separate out serum, 3000r min ^-1Centrifugal 10min, separation of serum test sera titre.Last immunity was won mouse spleen after three days, did cell and merged.

2) cytogamy

The BALB/c mouse splenocyte of getting Sp2/0 myeloma cell and immunity by 1: 5～1: 10 mixed in the 50mL centrifuge tube, abundant mixing, the centrifugal 10min of 1000rpm; Abandon supernatant, touch the pipe end, make cell loose evenly with palm; Put 40 ℃ of water-bath preheatings, in 45s, add the 1mL 50%PEG 4000 that is preheated to 40 ℃ with the 1mL suction pipe, the limit edged vibrates gently; In 90s, add 30mL then and be preheated to 37 ℃ 1640 incomplete substratum, room temperature leaves standstill 10min, the centrifugal 10min of 1000rpm; Abandon supernatant, adding 20mL, to contain 1640 substratum of 20%FCS and HAT resuspended.Divide on 96 orifice plates that install to existing feeder cell, in 5%CO ₂Incubator is cultivated, and behind the 7d, the upgrowth situation of observation of cell is with 1640 substratum that contain 20%FCS and HT, 1/2 substratum that swaps out; Behind the 14d, use 20%FCS and HT 1640 culture medium culturing instead, when the cell that merges grow to 96 orifice bore floorages 1/5 the time, get supernatant and carry out antibody test.

3) anti-OTA hybridoma screening

Use coating buffer to be 0.05mol L ^-1PH 9.6 carbonate buffer solutions, the envelope antigen that coupling is good (OTA-KLH) is done doubling dilution and is encapsulated elisa plate, and 100 μ L/ holes are put 4 ℃ and are encapsulated and spend the night; PBST washing 3 times, each 5min, the last bat done; Seal every hole with 1% gelatin, 2h is placed for 37 ℃ in 300 μ L/ holes; PBST washing 3 times, each 5min, the last bat done; With the positive reference serum of cell conditioned medium, dilution in 1: 800 and the negative reference serum of dilution in 1: 800, add in the respective aperture 100 μ L/ holes, 37 ℃ of effect 60min; PBST washing 3 times, each 5min, the last bat done; Add the enzyme mark sheep anti-mouse igg of dilution in 1: 10000,60min is placed for 37 ℃ in 100 μ L/ holes; PBST washing 5 times, each 5min, the last bat done; Add substrate TMB-H ₂O ₂, 100 μ L/ holes, room temperature lucifuge colour developing 15min; Every hole adds 100 μ L 2mol L ^-1The sulphuric acid soln termination reaction.Measure OD through ELIASA _450nmValue: with the blank zeroing, as the OD of positive reference serum _450nmThe OD of value and negative reference serum _450nmRatio>=2.1 of value are detected the hole and are judged to the positive, as the OD of positive reference serum _450nmThe OD of value and negative reference serum _450nmThe detection hole of the ratio of value＜1.5 is judged to feminine gender, and ratio is judged to suspicious between the centre.

4) limiting dilution assay carries out subclone to positive hybridoma cell

Positive hybridoma cell for the secretion monoclonal antibody specific that screens gained in time adopts limiting dilution assay to carry out subclone.At first positive hole viable cell is expected that with platform orchid dyes and count, be diluted to 100 cells/15mL substratum with 1640 perfect mediums, the cell suspension of dilution is added 96 porocyte culture plates, the 0.15mL/ hole is at 37 ℃, 5%CO ₂Cultivate in the incubator.Behind 4～5d, microscopically can be observed the formation of clone cell, and record has only single clonal growth hole, gets cell conditioned medium during 8～9d, in time carries out ELISA and detects.Select the male monoclonal cell to carry out same subclone again more than 3 times, detect all positive and to detect OD value more approaching in each hole until all cells hole supernatant; With the positive cell enlarged culturing after repeatedly subclone is cultivated and frozen, obtain the hybridoma cell strain called after D6D of stably excreting OTA monoclonal antibody.

(3) ascites of the anti-OTA monoclonal antibody of production

With the adult BALB/c mouse of Yellow Protopet 2A injection, 0.5mL/, sensitization was used 0.01mol L with positive hybridoma cell after 7 days ^-1Mouse peritoneal, every injected in mice 5 * 10 are injected in PBS dilution back ⁶～1 * 10 ⁷, 0.2mL takes belly obviously to heave the ascites of mouse behind 5～10d, 10000rpm, and centrifugal 5min collects supernatant, packing in a small amount ,-70 ℃ of preservations.

(4) the ascites titration of anti-OTA monoclonal antibody

With PBS damping fluid dilution in 1: 100 monoclonal antibody ascites, carry out doubling dilution then, add the enzyme plate that KLH-OTA encapsulates, 60min, is hatched in 100 μ L/ holes by 37 ℃; PBST washing 3 times, each 5min, the last bat done; Add the HRP mark sheep anti-mouse igg of dilution in 1: 10000,60min, is hatched in 100 μ L/ holes by 37 ℃; PBST washing 5 times, each 5min, the last bat done; Add substrate TMB-H ₂O ₂, 100 μ L/ holes, room temperature lucifuge colour developing 15min; Every hole adds 100 μ L2mol L ^-1The sulfuric acid termination reaction; Measure OD with ELIASA _450nmValue.With negative OD _450nmValue detects hole and negative OD less than 0.2 _450nmThe ratio of value is judged to the positive greater than 2.1, tires as the ascites of monoclonal antibody with the greatest dilution in positive hole.The result shows that D6D cell strain ascites indirect ELISA titer is 1: 3200.

(5) evaluation-denaturing polyacrylamide gel electrophoresis (SDS-PAGE) of anti-OTA monoclonal antibody purity

Preparation 10mL 12% separation gel solution.Between two sheet glass, pour into separation gel rapidly, use water-lute above.Behind about 30min, treat the complete polymerization of separation gel, pour out the water on rubber cover upper strata, blot with filter paper.Preparation 5% concentrates glue, is poured on the separation gel, inserts comb afterwards immediately, careful bubble.After waiting to concentrate gelling admittedly, carefully extract comb, with electrophoresis buffer solution for cleaning hole.Sample and 5 * sample-loading buffer proportional mixing, heated and boiled 10min is centrifugal in the water, the about 20 μ L of appearance on every hole.Fill it up with electrophoretic buffer, the 80V electrophoresis, when sample was pressed into straight line in concentrated glue after, regulating voltage arrived separation gel bottom, powered-down to 120V up to tetrabromophenol sulfonphthalein.Unload lower glass plate, knock sheet glass open, Yi Bian after marking, scrape gel with spatula.Coomassie brilliant blue R250 dye liquor dyeing 2h.Decolouring is spent the night high-visible to band, takes pictures, like Fig. 2.

(6) evaluation of antibody subtype

Test with Mouse Monoclonal Antibody Isotyping Kit.At first ascites is diluted with PBS, to AC be 0.1～1 μ g mL ^-1, get 150 μ L and add testing tube.15～25 ℃, 30s shakes the vortex pipe then, makes that blue colloid fully suspends in the pipe, and blackhead inserts test strip down, behind 5～10min, with reference to positive control, blue band place occurs and is this antibody subtype classification.The hypotype of this monoclonal antibody is IgG2b, and light chain is the κ chain.

Claims (1)

1. OTA monoclonal antibody is characterized in that: this monoclonal antibody is that hybridoma cell line D 6 D produces, and hybridoma cell line D 6 D is preserved in Chinese typical culture collection center on May 27th, 2010, and deposit number is CCTCC NO:C201052.

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