CN106591240A

CN106591240A - Hybridoma cell strain 4F4 and antibody thereof

Info

Publication number: CN106591240A
Application number: CN201710028876.0A
Authority: CN
Inventors: 马良; 张宇昊; 钟红
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2017-01-16
Filing date: 2017-01-16
Publication date: 2017-04-26
Anticipated expiration: 2037-01-16
Also published as: CN106591240B

Abstract

The invention relates to a hybridoma cell strain 4F4 and an antibody thereof. The hybridoma cell strain 4F4 is preserved in China Center for Type Culture Collection on Dec. 27, 2016 with the preservation number of CCTCC NO:C201702. The hybridoma cell strain 4F4 generates an anti-tenuazonic acid monoclonal antibody, specifically detects tenuazonic acid, and is of great significance to detect the tenuazonic acid.

Description

Hybridoma cell strain 4F4 and its antibody

Technical field

The invention belongs to biological technical field, and in particular to hybridoma cell strain 4F4, further relate to by the hybridoma cell strain The antibody that 4F4 is produced.

Background technology

Thin Alternaria alternata bacterium keto acid (Tenuazonic acid, TA) be that rod method is mould, rice blast is mould, Sorghum vulgare Pers. point is mould etc. One of toxic metabolic products produced under specified conditions, are after alternariol (Alternariol, AOH), alternariol monomethyl ether A kind of rod method syphilis element that (Alternariol monomethyl ether, AME) has found afterwards.TA has acute toxicity, Asia Acute toxicity, potential carcinogenecity, cytotoxicity, cause mammal dizziness, sialorrhea, vomiting heart beating occur then and overrun, eat Road and the intestines and stomach large-area hemorrhage and circulatory failure, death.As TA has various biological characteristic, and in being rod method syphilis element The maximum one kind of toxicity, studies have reported that TA can cooperate with enhancing other mycotoxins, with huge potential hazard, in recent years Increasingly cause each side's concern and study.

TA synthesizing artificial antigens and polyclonal antibody prepare existing part and report that such as Jiang Tao etc. utilizes carbodlimide method TA structures are modified, is coupled with bovine serum albumin and is prepared artificial antigen；Ma Liang etc. carries out derivative drawing using oximate method to TA Enter to be coupled arm, be coupled with bovine serum albumin and hemocyanin, conjugate immunity BALB/c mouse prepares polyclonal antibody；Yang Xingxing Deng being derived to TA using hydrazine hydrate and glyoxalic acid successively, synthesizing nitrogen-containing hetero conjugated double bond is coupled arm, enters with bovine serum albumin Row coupling obtains complete antigen, prepares the polyclonal antibody of specific recognition TA derivant by immune new zealand white rabbit, and Set up ELISA detection method.

Polyclonal antibody preparation time is short, preparation cost is low, but poorer than monoclonal antibody in terms of specificity, different batches There is difference and the immunoreation of different animals between.Monoclonal antibody specificity is high, once target cell strain is screened, it is theoretical On can just produce on all four antibody, and the consistent antibody of substantial amounts of performance can be obtained by continuous culture method, be produced The differences between batches of product are little.The current preparation with regard to TA monoclonal antibodies and products thereof exploitation is currently in blank stage, there is no and appoints What is reported.

The content of the invention

In view of this, an object of the present invention is to provide a kind of hybridoma cell strain 4F4；The second object of the present invention It is the preparation method that hybridoma cell strain 4F4 is provided；The third object of the present invention is to provide to be produced by hybridoma cell strain 4F4 Raw monoclonal antibody；The fourth object of the present invention is the preparation method for providing said monoclonal antibody；The purpose of the present invention Five be provide monoclonal antibody application.

To reach above-mentioned purpose, the present invention provides following technical scheme：

The biological deposits numbering of the 1st, hybridoma cell strain 4F4, the hybridoma cell strain 4F4 is CCTCC NO： C201702。

2nd, the preparation method of the hybridoma cell strain 4F4, TAO couplings hemocyanin immunogen is mixed with Freund adjuvant Immune mouse, then takes potency more than 1：The splenocyte of 10000 immune mouse is merged with myeloma cell SP2/0, is melted ELISA detections are carried out after cultivating 10 days after conjunction, Jing screening sub-clones obtain hybridoma.

Preferably, splenocyte described in fusion and myeloma cell SP2/0 quantity ratio are 1：20.

3rd, the monoclonal antibody produced by hybridoma cell strain 4F4, the biological deposits numbering of the hybridoma cell strain 4F4 For CCTCC NO：C201702.

Hybridoma cell strain 4F4 is prepared ascites, then carries out ProteinA by the 4th, preparation method of the monoclonal antibody Purification, obtains monoclonal antibody.

5th, application of the monoclonal antibody in the reagent for preparing detection TAO antigens.

Preferably, the monoclonal antibody is in the reagent for preparing the thin Alternaria alternata bacterium keto acid of elisa competition laws detection Using.

Preferably, the monoclonal antibody is in the reagent for preparing the thin Alternaria alternata bacterium keto acid of elisa indirect methods detection Using.

The beneficial effects of the present invention is：Hybridoma cell strain 4F4, the hybridoma cell strain are exempted from by TAO-KLH immunogens Then the splenocyte of immune mouse is merged by epidemic disease mice with myeloma cell SP2/0, with TAO-BSA as antigen or chain The thin Alternaria alternata bacterium keto acid of the mould generation of lattice spore does the monoclonal antibody that competition antigen selection detection hybridoma is produced, screening Obtain the hybridoma that antibody titer is high, specificity is good；The cell can secrete anti-TAO-KLH monoclonal antibodies, Neng Gouyong In detection TAO antigens, significant is detected to thin Alternaria alternata bacterium keto acid.

Description of the drawings

In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below and carries out Explanation：

Fig. 1 is purifying protein SDS-PAGE electrophoresis result figures.

Biomaterial preservation

In the present invention, hybridoma cell strain 4F4 send China typical culture collection center preservation, and deposit number is CCTCC NO:C201702, address are located at Wuhan, China Wuhan University, and preservation date is on December 27th, 2016, and Classification And Nomenclature is hybridoma Cell strain 4F4.

Specific embodiment

Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.

Reagent and consumptive material used in the present invention is as follows：Protein Marker (bronze object is biological), Balb/c mices (are purchased from Yangzhou University), Acr, Bis, Tris (are purchased from Sigma companies), and SDS (is purchased from Amresco companies), and TEMED (is purchased from BIO-RAD Company), 0.22 μm of sterile filters and bag filter (are purchased from Millipore companies), and DMEM in high glucose culture medium is (public purchased from Gibco Department), cell counting count board (is purchased from Improved Neubauer), and liquid paraffin (bronze object is biological), PEG (are purchased from sigama), and 96 is thin Born of the same parents' culture plate (is purchased from Costar), and other reagents are domestic pure analysis pure.

Embodiment 1, animal immune and exempt from rear mice serum bioactivity

5 6～8 week old female BAl BIcs/c mices are chosen, hapten TAO is coupled into hemocyanin (Keyhole Limpet Hemocyanin, KLH) TAO-KLH immunogens and Freund adjuvant mixed immunity, 100 μ g of subcutaneous injection, 2-3 week booster immunization Once (first time immune original mixes with Freund's complete adjuvant, thereafter plus exempt to mix with incomplete Freund's adjuvant).Wherein, half Antigen TAO draws after carrying out deriving to thin Alternaria alternata bacterium keto acid (Tenuaconic acid, TA) by half hydrochlorate of carboxymethyl azanol Enter linking arm and obtain.

Four exempt from after blood sampling detection, determine that antiserum detects former potency, inspection for TAO-BSA by indirect ELISA method Survey condition：

Envelope antigen：TAO-BSA detections are former, the thin Alternaria alternata bacterium keto acid (competition antigen) of the mould generation of rod method；Coating Concentration：5 μ g/mL, 100 μ l/ holes；Coating buffer：(PBS, pH are 7.4) for phosphate buffer；Two resist：Mountain sheep anti mouse-HRP, 1/ 5000, testing result is as shown in table 1.

Table 1, antiserum and TAO-BSA Elisa results

Initial dilution:1:500

As a result show, 5 mice ELISA potency are all higher than 1：10000, can continue to arrange Mus serum and the mould product of rod method The competitive ELISA detection of raw thin Alternaria alternata bacterium keto acid, as a result as shown in table 2.

Table 2, antiserum and the thin Alternaria alternata bacterium keto acid competition Elisa results of the mould generation of rod method

Note：The thin Alternaria alternata bacterium keto acid initial dilution of the mould generation of rod method：100ug is initial, 3 times of dilutions

As a result show, substantially, final choice 1# mice carries out cell fusion experiment to 1#, 2#, 5# mice competition performance.

Embodiment 2, cell fusion

1st, prepared by myeloma cell

Fusion the last week, use the culture medium amplification culture SP2/0 cells of DMEM containing 10%FBS.When fusion, cell is covered with About 6 bottles of T25 Tissue Culture Flasks, collected SP2/0 cells in 50mL centrifuge tubes on the fusion same day, and 1000rpm is centrifuged 5min.Abandon Supernatant, then adds 20mL DMEM basal mediums, dispels cell and then counts.

2nd, prepared by splenocyte

Four Post-immunisation serum ELISA potency are 1：More than 10000 mice, exempts from for 3 days before fusion eventually, and lumbar injection resists 100 μ g of original.Merge the same day with cervical dislocation euthanasia mice to be merged.Spleen is taken with 75% alcohol-pickled 5min. is aseptic, Spleen is put in the interior culture dish for there are 10mL DMEM bases to cultivate.Take screen cloth to be put in another plate, spleen is transferred to On screen cloth, spleen is ground with syringe heart.Add DMEM on screen cloth, rinse screen cloth, splenocyte is more collected flat In ware.Cell being moved in 10mL centrifuge tubes, splenocyte being washed twice with the DMEM without serum, 1000rpm centrifugation 5min are collected Splenocyte is counted.

3rd, cell fusion

Mixing myeloma cell and splenocyte, makes myeloma cell with splenocyte quantity ratio 1：20 are advisable.Cell is put To in 50mL centrifuge tubes, diluted with DMEM basal mediums, then 1000rpm centrifugations 5min, abandon supernatant, shake centrifuge tube makes carefully Born of the same parents are uniform.It is slowly added to 0.8mL 50%PEG, reacts 90 seconds, be subsequently adding 20-30mL DMEM culture medium and terminate PEG, melts The cell of conjunction is reacted 10 minutes in being put into 37 DEG C of water-baths, 1000rpm centrifugation 5min, is abandoned supernatant and is subsequently adding HAT DMEM cultures Base.The cell of fusion is taped against in 96 orifice plates, per 100 μ L of hole, Tissue Culture Plate CO is put into into then₂Cultivate in incubator.Melt Check within 4 days after conjunction, hybridoma cell clone rate there are a small amount of cell debriss more than 50%, and cell growth state is good, fusion ELISA selective mechanisms are proceeded by after 10 days.Testing conditions：Envelope antigen：TAO-BSA detections are former, the mould generation of rod method thin Alternaria alternata bacterium keto acid (competition antigen)；Coating concentration：5 μ g/mL, 100 μ l/ holes；Coating buffer：Phosphate buffer (PBS,pH 7.4)；Two resist：Mountain sheep anti mouse-HRP, 1/5000, as a result as shown in Table 3 and Table 4.

Cell conditioned medium and TAO-BSA Elisa results after table 3, fusion

1. number plate

2. number plate

3. number plate

4. number plate

5. number plate

6. number plate

The thin Alternaria alternata bacterium keto acid Elisa results of cell conditioned medium and the mould generation of rod method after table 4, fusion

Cell strain title	1A2	1A5	1B5	1C4	1C8	1D4	1E1	1E2	1E4
										OD values	3.102	2.0833	1.807	0.248	2.549	0.260	0.235	0.266	0.279
Cell strain title	1E6	1F10	1F11	1G10	1G12	1H9	1H12	2A4	2A4
										OD values	0.221	0.258	0.256	0.323	0.222	0.429	3.407	0.181	2.037
Cell strain title	2B5	2C8	2D4	2D11	2F2	2F11	2G11	2H11	3A4
										OD values	0.191	0.283	0.251	0.248	0.221	0.219	0.232	0.228	1.499
Cell strain title	3A12	3B6	3F3	3F11	3G8	3H1	3H5	4A4	4A5
										OD values	0.863	0.179	3.085	3.152	0.172	0.244	0.233	0.204	0.387
Cell strain title	4A6	4B9	4B10	4D4	4E4	4F8	5C10	5E3	5E7
										OD values	0.264	3.527	0.189	1.412	0.252	0.253	0.540	0.662	0.170
Cell strain title	5G10	5H2	6C4	6C9	6E3	6H6	Positive control	Competition hole	Negative control
										OD values	1.631	1.988	0.175	2.929	0.340	0.252	3.129	0.617	0.101

Embodiment 3, fusion screening and sub-clone

1st, fusion screening

In the previous day of detection, 5 μ g/mL antigens are coated with elisa plate with PBS, overnight, next day draws 100 μ of cell conditioned medium L/ holes carry out ELISA detections according to ELISA results, judge that (sample well OD values/negative hole OD value >=2.1 item are judged in positive hole Positive hole.The positive hole that inspection imposite is detected is chosen with single track pipettor, second confirmation detection is carried out, is further confirmed that the positive Hole, it is determined that after positive hole cell carry out sub-clone.

2nd, sub-clone

Cell in the positive hole of piping and druming, counts, and adds N/4mL DMEM culture medium, take 100 μ l cell suspension in centrifuge tube To in centrifuge tube, blow it is even after stay 1mL, add DMEM to 4mL, blow even, stay 100 μ l (about 2 drop) in ttom of pipe.Add in centrifuge tube DMEM to 5mL, drops to the first three rows of 96 orifice plates after mixing, stay 1.8～2ml or so per one dropper bottom of hole, add DMEM extremely 5mL, blow it is even after drop to tri- row of D, E, F of 96 orifice plates, drip per hole one, ttom of pipe stays 1.5～1.8m or so, adds DMEM to 2.8 ～3mL or so, blow it is even after drop to G, H row of 96 orifice plates, drip per hole one, examine under a microscope after 7-10 days, detection has gram The hole of grand growth, marks monoclonal hole, and the positive monoclonal cell of picking as far as possible carries out sub-clone again, detect to After 100% positive, choose monoclonal hole amplification culture singling.Choosing 10 plants of positive and competitive fusion cell lines carries out Ya Ke Grand, Jing after ELISA screenings, fixed 10 plants of results are as follows：

Envelope antigen：TAO-BSA detections are former, the thin Alternaria alternata bacterium keto acid (competition antigen) of the mould generation of rod method；Coating Concentration：5 μ g/mL, 100 μ L/ holes；Coating buffer：(PBS, pH are 7.4) for phosphate buffer；Two resist：Mountain sheep anti mouse-HRP, 1/ 5000, as a result as shown in table 5.

Cell conditioned medium Elisa results after table 5, singling

Cell strain title	Indirect ELISA-OD is worth	Competitive ELISA-OD is worth
			3B3	2.924	0.712
3B4	2.950	0.752
			3D12	2.950	1.079
4A1	2.899	0.895
			4B9	2.876	0.883
4D5	3.236	0.846
			4F4	3.345	0.231
4G1	3.412	1.383
			7F11	3.412	0.524
7G11	3.236	0.450

According to the above results, 4F4 cell strains are selected to prepare ascites, because the cell strain competition performance is preferably, growing way is most fast. Hybridoma cell strain 4F4 send China typical culture collection center preservation, and deposit number is CCTCC NO:C201702, address bit In Wuhan, China Wuhan University, preservation date is on December 27th, 2016, and Classification And Nomenclature is hybridoma cell strain 4F4.

Embodiment 4, ProteinA antibody purifications and identification

By TAO-KLH 4F4 ascites, ProteinA purification is carried out.Protein A sepharose medium is taken, chromatographic column is loaded, by abdomen Water presses 1 with PBS:After 1 mixing, slow loading, uses glycine elution buffer solution elution after antibodies, that is, obtains required purification Antibody, carries out 4 DEG C of dialysed overnights immediately in PBS, the next day carry out purity, concentration and bioactivity.

Antibody indirect, competitive ELISA bioactivity, concrete operations are as follows：

(1) coating plate is designed according to experiment needs, and mark on lath；

(2) it is coated with：TAO-BSA detection antigens are diluted by 5 μ g/mL with PBS coating buffers, in adding lath after mixing, often 100 μ L of hole, 4 DEG C of refrigerator overnights；

(3) envelope antigen：TAO-BSA detects antigen；

(4) it is coated with concentration：Dilute by 5 μ g/mL, 100 μ L/ holes；

(5) it is coated with buffer：(PBS, pH are 7.4) for phosphate buffer；

(6) close：After coating is good, coating buffer is discarded, board-washing 3 times adds 200 μ l confining liquids (PBST+1%BSA) per hole, 37 DEG C of calorstat 1h, take out ELISA Plate, discard interior liquid, board-washing 1 time；

(7) one anti-reflective should：

Indirect ELISA：Antibody purification is diluted by 1/500,2 times, 37 DEG C of calorstat 1h；

Competitive ELISA：The thin 0.1 μ g/mL of Alternaria alternata bacterium keto acid of the mould generation of rod method are initial, 2 times of dilutions, per 50 μ of hole L, synchronous to add antibody purification, antibody purification is by 1/1000 dilution, every 50 μ L of hole, 37 DEG C of calorstat 1h；

(8) two anti-reflective should

ELISA Plate is taken out, interior liquid, board-washing 3 times, in every hole, add 100 μ L to dilute ELIAS secondary antibody, enzyme mark two is discarded It is anti-：Mountain sheep anti mouse-HRP, 1/5000,37 DEG C of calorstat 1 hour.

(9) develop the color

ELISA Plate is taken out, interior liquid is discarded, board-washing 4 times is initially charged 100 μ L TMB nitrite ions per hole, according to the depth of color Decision developing time, general 37 DEG C, 15min.

(10) terminating reaction

100 μ L 1M HCL solution, terminating reaction are added to be engraved in 450nm readings in microplate reader, OD values are more than per hole The corresponding dilution factor in hole of 2.1 times of the negative control OD value of setting, it is determined as the potency of the sample.

As a result as shown in table 6 and table 7.

The indirect Elisa results of table 6,4F4 antibody purifications

Note：Initial dilution:1:500；Potency is sample OD/ blank OD>=2.1 highest dilution

Drawn by testing result, the antibody titer of two concentration of 4F4 is all in 512K or so.

Table 7,4F4 antibody purifications competition Elisa results

Antibody concentration testing result shows that purified antibodies concentration is 0.4mg/mL or 0.31mg/mL；Correspondence antibody volume For 4.0mL or 6.0mL, its purity is then detected, as a result as shown in Figure 1.As a result show, present invention antibody purity after purification Height, can be used to detect correspondence antigen.

Finally illustrate, preferred embodiment above is only unrestricted to illustrate technical scheme, although logical Cross above preferred embodiment to be described in detail the present invention, it is to be understood by those skilled in the art that can be Various changes are made to which in form and in details, without departing from claims of the present invention limited range.

Claims

1. hybridoma cell strain 4F4, it is characterised in that：The biological deposits numbering of the hybridoma cell strain 4F4 is CCTCC NO： C201702。

2. the preparation method of hybridoma cell strain 4F4 described in claim 1, it is characterised in that：Hapten TAO is coupled into blood indigo plant egg White immunogen and Freund adjuvant mixed immunity mice, then take potency more than 1：The splenocyte and bone marrow of 10000 immune mouse Oncocyte SP2/0 is merged, and carries out ELISA detections after fusion after cultivating 10 days, and Jing screening sub-clones obtain hybridoma； The hapten TAO introduces linking arm after carrying out deriving to thin Alternaria alternata bacterium keto acid by half hydrochlorate of carboxymethyl azanol and obtains.

3. preparation method according to claim 2, it is characterised in that：Splenocyte described in fusion and myeloma cell SP2/ 0 quantity ratio is 1：20.

4. the monoclonal antibody for being produced by hybridoma cell strain 4F4, it is characterised in that：The biology of the hybridoma cell strain 4F4 Deposit number is CCTCC NO：C201702.

5. the preparation method of monoclonal antibody described in claim 4, it is characterised in that：Hybridoma cell strain 4F4 is prepared into ascites, Then ProteinA purification is carried out, monoclonal antibody is obtained.

6. application of the monoclonal antibody described in claim 4 in the reagent for preparing the thin Alternaria alternata bacterium keto acid of detection.

7. application according to claim 6, it is characterised in that：The monoclonal antibody is preparing the detection of elisa competition laws Application in the reagent of thin Alternaria alternata bacterium keto acid.

8. application according to claim 6, it is characterised in that：The monoclonal antibody is preparing the detection of elisa indirect methods Application in the reagent of thin Alternaria alternata bacterium keto acid.