CN104745538A

CN104745538A - Anti-tilmicosin specific monoclonal antibody hybrid tumor cell strain and application thereof

Info

Publication number: CN104745538A
Application number: CN201510166612.2A
Authority: CN
Inventors: 宋月鸿; 胥传来
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2015-04-10
Filing date: 2015-04-10
Publication date: 2015-07-01

Abstract

The invention discloses an anti-tilmicosin specific monoclonal antibody hybrid tumor cell strain and an application thereof, and belongs to the field of food safety immunological detection. The anti-tilmicosin specific monoclonal antibody hybrid tumor cell strain N is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No.9803. An anti-tilmicosin specific monoclonal antibody is secreted by the hybrid tumor cell strain N with the preservation number of CGMCC No.9803. The invention further discloses the application of the anti-tilmicosin specific monoclonal antibody hybrid tumor cell strain to tilmicosin analysis and detection. The monoclonal antibody secreted by the cell strain has relatively good specificity (IC50 value being 2.7ng/mL) on tilmicosin, wherein the tylosin IC50 value is measured to be 42.1ng/mL, and the cross-reaction rate is 6.41%. The anti-tilmicosin specific monoclonal antibody hybrid tumor cell strain can be used for specificity detection of tilmicosin in food safety.

Description

One strain anti-tilmicosin monoclonal antibody specific hybridoma cell strain and application thereof

Technical field

One strain anti-tilmicosin monoclonal antibody specific hybridoma cell strain and application thereof, relates to the anti-tilmicosin monoclonal antibody specific of hybridoma cell strain N and generation thereof, belongs to food safety field of immunodetection.

Background technology

Tilmicosin (TIM), English name is Tilmicosin, and molecular formula is C ₄₆h ₈₀n ₂o ₁₃molecular weight is 869.15, chemical name is 4A-O-de-(2,6-dideoxy-3-C-methyl-L-ribose-pyrans hexyl)-20-deoxidation-20-(3,5-dimethyl-piperidino)-[20 (cis: trans)] tylosin, have another name called 20-deoxidation-20-(3,5-dimethyl-piperidino) desmycosin.CAS 108050-54-0.Tylosin also known as safe agriculture, tylosin (TYL), a kind of macrolide antibiotics that to be Hamill etc. obtain the nutrient solution of nineteen fifty-nine from streptomyces fradiae (Streptomycesfradiae).

Tilmicosin is the semi-synthetic macrolide antibiotics derived by tylosin.There is the anti-microbial activity similar to Macrocyclolactone lactone kind medicine, to gram-positive microorganism and some Gram-negative bacterias and mycoplasma effective.Especially stronger to the specific activity tylosin of actinobacillus pleuropneumoniae, pasteurella, streptococcus aureus, streptococcus pyogenes, streptococcus pneumoniae, corynebacterium pyogenes and livestock and poultry mycoplasma.Be mainly used in the animals such as treatment ox, goat, sheep, milk cow, pig, chicken clinically by the microbial infectious diseases of sensitivity, particularly livestock and poultry respiratory tract infection and responsive microbial mammitis of cow.

Tilmicosin is to the toxic action of animal mainly cardiovascular systems, and it can cause tachycardia and convergent force to weaken.Its residual meeting in animal product causes sensitization, toxic reaction to the mankind, ediblely also can make human intestine's flora imbalance for a long time, produces bacterial drug resistance.No. 235 bulletin that the Ministry of Agriculture of China issues, is specified animal food herbal medicine maximum residue limit(MRL).Wherein, the maximum residue limit(MRL) of regulation tilmicosin in sheep milk must not more than 50 μ g/kg.So be necessary the method for quick setting up tilmicosin.

Detection method mainly instrument detection method and the immunoassay detection method of current tilmicosin.Instrument detection method instrument cost is high, complicated operation, needs full-time staff to operate, and immune analysis method have low cost, high-throughput, highly sensitive, to features such as technician's relative requirement are low, be therefore applicable to the rapid screening of a large amount of sample.The invention reside in and a kind of monoclonal antibody hybridoma cell strain tilmicosin to higher affinity and detection sensitivity is provided, for the research and development popularization of indirect competitive ELISA test kit and colloidal gold strip lays the foundation.

Summary of the invention

Object of the present invention provides a kind of anti-tilmicosin monoclonal antibody specific hybridoma cell strain, the antibody prepared by this cell strain has good avidity and detection sensitivity to tilmicosin, can be used for setting up tilmicosin enzyme-linked immune detection method, or set up colloidal gold immunochromatographimethod technology method for quick.

Technical scheme of the present invention a: strain anti-tilmicosin monoclonal antibody specific hybridoma cell strain N, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.9803.

Anti-tilmicosin monoclonal antibody specific, it is produced secreted by the hybridoma cell strain N of CGMCC No.9803 by described deposit number.

The application of described anti-tilmicosin monoclonal antibody specific, its application in tilmicosin analyzing and testing.

The preparation basic step of hybridoma cell strain N provided by the invention is: (1) immunogenic preparation and qualification: tylosin tartrate and carboxymethyl azanol oximation reaction are prepared containing carboxyl haptens, be connected with the amino of protein carrier by active ester method, after reaction terminates, be separated the small haptens of complete antigen and non-coupling by dialysis, complete antigen is identified by uv-absorbing scan method; (2) immunity of mouse: after antigen and freund adjuvant emulsification completely, by subcutaneous multi-point injection immunity BALB/c mouse.First immunisation adopts Freund's complete adjuvant, and booster immunization uses freund 's incomplete adjuvant, and during spurt immunity, immunizing dose is the half of a front immunizing dose, mixes directly to carry out abdominal injection afterwards with physiological saline; Each time immunization interval is three weeks.After third time immunity, interval blood sampling in a week detects serum titer and suppression; (3) cytogamy and cell strain are set up: by polyoxyethylene glycol (PEG4000) method, mouse boosting cell and murine myeloma cell are merged, by HAT culture medium culturing, indirect ELISA is utilized to detect positive cell hole, and utilize indirect competitive ELISA method to measure the inhibition in positive cell hole further, carry out three subclones by limiting dilution assay to there being the positive cell hole preferably suppressed, final screening obtains hybridoma cell strain N; (4) qualification of hybridoma cell strain character: adopt mouse monoclonal Ig class/subgroup identification ELIAS secondary antibody suit to measure; IC ₅₀the mensuration of value, cross reacting rate and avidity passes through ELISA method.

Beneficial effect of the present invention is: the anti-tilmicosin cell strain of monoclonal antibody that (1) the present invention obtains, and not only has good detection sensitivity and avidity, and low to the cross reacting rate of tylosin, can reach the object of specific detection tilmicosin.(2) the new immunogenic method of synthesis tilmicosin, haptenic synthesis step simplifies more, effectively.Research for people from now on provides the thoughts and methods of synthetic immunogen.

Biological material specimens preservation a: strain anti-tilmicosin monoclonal antibody specific hybridoma cell strain N, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.9803, preservation date on October 15th, 2014.

Accompanying drawing explanation

Fig. 1. immunogenic ultra-violet absorption spectrum characterizes.

Fig. 2. SDS-polyacrylamide gel electrophoresis characterizes: 1, BSA, and 2, TYL-CMO ︰ BSA=120,3, TYL-CMO ︰ BSA=100,4, TYL-CMO ︰ BSA=80,5, TYL-CMO ︰ BSA=60.

Fig. 3. hybridoma cell strain N monoclonal antibody is to the typical curve of TIM.

Embodiment

The following examples of the present invention further illustrating only as content of the present invention, perhaps scope in not as limiting to the invention.Below by embodiment, the invention will be further described.

The present invention passes through TYL complete antigen immune mouse, pass through cytogamy, HAT selective medium is cultivated, and by indirect ELISA and indirect competitive ELISA screening cell conditioned medium, finally obtains monoclonal antibody hybridoma cell strain N TIM being had to better avidity and sensitivity.

Embodiment 1: the preparation of hybridoma cell strain N

1, the synthesis of complete antigen

Get 100mg TYL(tylosin), be dissolved in the methyl alcohol-0.02mol/L NaHCO of 5 mL ₃in (1:1, V/V) mixed solution, add 15mg carboxymethyl hydroxylamine hydrochloride, 60 DEG C of backflow 12h.Rotary evaporation, obtains solid mixture.Add 3mL methyl alcohol, make it dissolve, cross and filter insoluble particles, get supernatant liquor, use nitrogen to dry up.Final reaction gained solid is derivative rear required haptens.Get 4.5mg haptens, then add 2.0mg EDC and 1.0mg NHS, use DMF to dissolve, stirring at room temperature, activates 4 h; Separately get 5 mg BSA and be dissolved in 2mL, in the CB solution of 0.05 M, pH9.6, dropwise added at a slow speed in BSA solution by the tylosin solution after above-mentioned activation, after stirring at room temperature reaction is spent the night, dialyse three days for 4 DEG C ,-20 DEG C of packing are preserved.

2, animal immune

The Balb/C mouse in 6 ~ 8 week healthy age is selected to carry out immunity.After getting tylosin complete antigen (1 mg/mL) and equivalent freund adjuvant emulsification evenly, by subcutaneous multi-point injection immunity BALB/c mouse, every only 100 μ L.First immunisation adopts Freund's complete adjuvant, and booster immunization uses freund 's incomplete adjuvant, and during spurt immunity, immunizing dose is the half of a front immunizing dose, mixes directly to carry out abdominal injection afterwards with physiological saline; Each time immunization interval is three weeks.After third time immunity, interval blood sampling in a week detects serum titer and suppression; Select to suppress best mouse, after exempting from five, spurt immunity in 21 days, prepares to merge.

3, cytogamy

In spurt immunity after three days, conveniently PEG(polyoxyethylene glycol, molecular weight is 4000) method carries out cytogamy, and concrete steps are as follows: (1) is aseptic gets mouse spleen, and grinding also obtains splenocyte suspension by 200 order cell screen clothes, and carries out cell counting; (2) collect SP2/0 cell, be suspended in RPMI-1640 basic culture solution, carry out cell counting; (3) splenocyte and SP2/0 cell are mixed according to the ratio of 5 ~ 10:1, centrifugal rear PEG merges, time 1 min, afterwards according to from slowly to soon, add RPMI-1640 basic culture solution, be suspended in after centrifugal containing 20% foetal calf serum, 2% 50 × HAT RPMI-1640 screening and culturing liquid in, be added to 96 porocyte culture plates, be placed in 37 DEG C, 5% CO ₂incubator in cultivate.

4, cell screening and cell strain are set up

RPMI-1640 screening and culturing liquid was carried out to fused cell in the 3rd day and partly changes liquid in cytogamy, within the 5th day, carry out with containing 20% foetal calf serum, 1% the RPMI-1640 transition nutrient solution of 100 × HT entirely change liquid, got cell conditioned medium at the 7th day and screen.Screen in two steps: the first step first filters out positive cell hole with indirect ELISA, and second step selects TIM, and TYL is standard substance, carries out inhibition mensuration with indirect competitive ELISA to positive cell.Select to have TIM better suppress and suppress bad cell hole to TYL, adopt limiting dilution assay to carry out subclone, use the same method and detect.In triplicate, cell strain N is obtained.

5, the preparation of monoclonal antibody and qualification

Get 8-10 BALB/c mouse in age in week, every mouse peritoneal injection paraffin oil 1 mL; Every mouse peritoneal injection 1 × 10 afterwards in 7 days ⁶hybridoma, collected ascites from the 7th day, and by ascites by sad-saturated ammonium sulphate method purifying, the monoclonal antibody of acquisition is placed in-20 DEG C of preservations.

Use mouse monoclonal hypotype identification kit to carry out the qualification of immunoglobulin (Ig) hypotype to the monoclonal antibody that ascites purifying obtains, its hypotype is IgG2b type.

Use indirect competitive ELISA and indirect ELISA, measure monoclonal antibody to the IC of TIM, TYL ₅₀be respectively 2.7ng/mL, 42.1ng/mL, cross reacting rate is 6.41%.Can be used for the specificity rapid detection of tilmicosin.

The anti-tilmicosin monoclonal antibody specific application of embodiment 2

Hybridoma cell strain N is applied to tilmicosin ELISA by monoclonal antibody prepared by ascites in body and adds recovery test, concrete steps are as follows:

(1) the 0.1 μ g/mL using carbonate buffer solution (CBS) to dilute is as coating antigen bag by 96 hole enzyme plates, and every hole 100 μ L, 37 DEG C of bags, by after 2 h, wash plate three times by PBST washing lotion, and each every hole 250 μ L, each 3 min, pat dry.

(2) close with the CBS containing 0.2% gelatin, every hole 200 μ L, close 2 h, wash plate three times by PBST washing lotion for 37 DEG C, each every hole 250 μ L, each 3 min, pat dry.

(3) the tilmicosin standardized solution of 0,0.2,0.5,1,2,5,10,20ng/mL is configured respectively with phosphate buffered saline buffer (PBS).By standardized solution and detected sample extracting solution, join in the enzyme plate closed respectively, every hole 50 μ L, each sample repeats 3 holes, every hole adds the anti-tilmicosin monoclonal antibody that 50 μ L 1 ︰ 16000 dilute again, and 37 DEG C of reactions, after 0.5 hour, are washed plate and patted dry.

(4) to add the sheep anti-mouse igg two of the HRP mark that PBS 1 ︰ 3000 of 100 μ L containing 0.1% gelatin dilute anti-in every hole, and 37 DEG C are reacted after 0.5 hour, wash plate and pat dry.

(5) every hole adds 100 μ L TMB nitrite ions, and after 37 DEG C of colour developing 15 min, every hole adds 50 μ L 2M H ₂sO ₄stop buffer, 450 nm survey light absorption value.

(6) recovery and sample pre-treatments is added: take 1g honey sample and insert in 50mL centrifuge tube, add 2ng, 4 ng and 10ng TIM respectively.After adding the concussion evenly of 1mL CBS damping fluid to sample, then add the extraction of 2mL acetonitrile, fully shake 5min, the centrifugal 10min of 3000rpm, get upper organic phase, nitrogen dries up.Then redissolve to 2mL with PBS, as ELISA sample extracting solution, diluted sample multiple is 2 times again.Adopt indirect competitive ELISA to carry out interpolation recovery test, its rate of recovery is respectively 112.71%, and 97.74%, 95.40%.

The configuration of solution:

Carbonate buffer solution (CBS): take Na ₂cO ₃1.59g, NaHCO ₃2.93g, mixes after being dissolved in a small amount of distilled water respectively, and add distilled water and mix to about 800mL, adjust pH to 9.6, add distilled water and be settled to 1000mL, 4 DEG C of storages are for subsequent use.

Phosphate buffered saline buffer (PBS): 8.00 g NaCl, 0.2 g KCl, 0.24 g KH ₂pO ₄, 3.62 g Na ₂hPO ₄12 H ₂o, is dissolved in 800 mL pure water, adjusts pH to 7.2 ~ 7.4, be settled to 1000 mL with NaOH or HCl;

PBST: containing the PBS of 0.05% Tween 20;

TMB nitrite ion: A liquid: Na ₂hPO ₄12H ₂o 18.43g, citric acid 9.33g, pure water is settled to 1000 mL; B liquid: 60 mg TMB are dissolved in 100 mL ethylene glycol.A, B liquid is TMB nitrite ion by 1 ︰ 5 mixing, existing with existing mixed.

The hypotype qualification of embodiment 3 hybridoma cell strain N monoclonal antibody.

Mouse monoclonal Ig class/subgroup identification ELIAS secondary antibody suit is used to measure the hypotype of monoclonal antibody.

The hypotype qualification of table 1 hybridoma cell strain N monoclonal antibody

Learnt by kit measurement result, antibody subtype is IgG2b.

Embodiment 4

Hybridoma cell strain N monoclonal antibody to TIM, TYL, the IC of acetylisovaleryl tylosin (ATYL) ₅₀and cross reacting rate.As shown in table 2.

Table 2 hybridoma cell strain N monoclonal antibody is to the IC of TIM, TYL, ATYL ₅₀and cross reacting rate

	IC ₅₀	Cross reacting rate
			TIM	2.7	100.00%
TYL	42.1	6.41%
			ATYL	28.2	9.57%

Be only preferred embodiment of the present invention in sum, be not used for limiting practical range of the present invention.Namely all equivalences done according to the content of the present patent application scope change and modify, and all should be technology category of the present invention.

Claims

1. a strain anti-tilmicosin monoclonal antibody specific hybridoma cell strain N, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.9803.

2. anti-tilmicosin monoclonal antibody specific, is characterized in that: it is produced secreted by the hybridoma cell strain N of CGMCC No.9803 by described deposit number.

3. the application of anti-tilmicosin monoclonal antibody specific according to claim 2, is characterized in that: its application in tilmicosin analyzing and testing.