CN109705220A - One plant of hybridoma cell strain for secreting anti-chlorine promazine monoclonal antibody and its application - Google Patents

Abstract

One plant of hybridoma cell strain for secreting anti-chlorine promazine monoclonal antibody and its application, belong to food safety field of immunodetection.Hybridoma cell strain D of secretion anti-chlorine promazine monoclonal antibody prepared by the present invention, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.17304.The hybridoma cell strain D, which can secrete generation, has preferable affinity and higher sensitivity to chlorpromazine, to 50% inhibition concentration IC of chlorpromazine₅₀For 17.2 μ g/L, it can be used for preparing the immunity detection reagent and colloidal gold strip of chlorpromazine, the detection for chlorpromazine in animal derived food provides strong detection method and means.

Description

One plant of hybridoma cell strain for secreting anti-chlorine promazine monoclonal antibody and its application

Technical field

The hybridoma cell strain for secreting anti-chlorine promazine monoclonal antibody the present invention relates to one plant and its application belong to food peace Panimmunity detection field.

Background technique

Chlorpromazine (chlorpromazine, CPZ), also known as wintermin, belong to phenothiazines, are central dopamine Receptor blocking pharmacon has the effects that calmness, antipsychotic, reduces body temperature and basic metabolism.This medicine is mainly used to town on animal doctor Quiet and antiemetic.Chlorpromazine is added in daily ration so that animal is played the role of fattening indirectly, some are illegal during animal transport Usually large dosage uses chlorpromazine to retailer, this is because chlorpromazine can reduce the maintenance needs of animal, reduces weightless and dead on the way Die rate.Large dosage uses before butchering, and can lead to a large amount of chlorpromazines and remains in animal product, the storage of chlorpromazine in animal body Product property residual can constitute potential threat to the safety of eater, can cause eater that oligoleukocythemia and agranulocytosis occurs Disease, and other adverse reactions.Therefore there are rule in European Union, Japan and China etc. to residual quantity of the chlorpromazine in animal food It is fixed.

At present detection chlorpromazine remain more conventional method have spectrophotometer method, fluorescent spectrometry, liquid chromatography, Liquid Chromatography-Mass Spectrometry and electrochemical process etc., these methods or expensive equipment or sample treatment are complicated, it is more difficult to answer extensively For in extensive high-throughput detection.And immunoassay method is with inexpensive, high-throughput, highly sensitive, opposite to technical staff It is required that the features such as low, therefore it is suitable for the rapid screening of a large amount of samples.

Summary of the invention

The hybridoma cell strain for secreting anti-chlorine promazine monoclonal antibody the purpose of the present invention is to provide one plant and its application, There is preferable affinity and sensitivity to chlorpromazine by monoclonal antibody prepared by the cell strain, can be used to establish chlorpromazine Enzyme-linked immune detection method, or establish colloidal gold immuno-chromatography test paper strip rapid detection method.

Technical solution of the present invention, has been preserved in by hybridoma cell strain D of one plant of secretion anti-chlorine promazine monoclonal antibody China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3 Institute of Microorganism, Academia Sinica, classification naming are monoclonal cell strain, preservation date on May 24th, 2018, and preservation is compiled Number CGMCC No.17304.

Anti-chlorine promazine monoclonal antibody is produced by No. D secretion of deposit number CGMCC No.17304 hybridoma cell strain It is raw.

The application of the anti-chlorine promazine monoclonal antibody, for the remaining detection of chlorpromazine in food.

Secrete hybridoma cell strain D preparation basic step of anti-chlorine promazine monoclonal antibody are as follows:

(1) preparation of haptens: by the reaction of half hydrochloride of acetylpromazine maleate and carboxylic methoxamine, synthesis one is had The chlorpromazine haptens of two carbochains.

(2) preparation and identification of immunogene: using chlorpromazine haptens as raw material, pass through the ammonia of active ester method and protein carrier Base phase connects, and after reaction, by small haptens dialysis separation comlete antigen and be not coupled, comlete antigen passes through ultraviolet Absorb scan method identification；

(3) mouse is immune: the BALB/c mouse for choosing 6-8 week old is immunized.Immunogene and freund adjuvant emulsification is complete Afterwards, mouse is immunized by subcutaneous multi-point injection, first immunisation uses Freund's complete adjuvant, and booster immunization is not exclusively helped using Fu Shi Agent, immunizing dose is the half of a preceding immunizing dose when spurt is immune, directly carries out abdominal cavity after mixing with physiological saline Injection；Each secondary immunization interval is three weeks.After third time is immune, interval blood sampling in one week detection serum titer and inhibition；

(4) cell fusion and cell strain are established: by polyethylene glycol (PEG 2000) method, making mouse boosting cell and mouse myeloma Cell fusion detects positive cell hole using indirect ELISA, and further utilize indirect competition by HAT culture medium culture ELISA method measure positive cell hole inhibitory effect, by limiting dilution assay to have the positive cell hole preferably inhibited carry out three Secondary subclone finally screens acquisition hybridoma cell strain D；

(5) it the identification of hybridoma cell strain property: is set with and is measured with ELIAS secondary antibody using mouse monoclonal Ig class/subgroup identification； IC₅₀The measurement of value, cross reacting rate and affinity passes through ELISA method.

Beneficial effects of the present invention: (1) the anti-chlorine promazine monoclonal antibody that the present invention obtains has preferable inspection to chlorpromazine Survey sensitivity and affinity；(2) a kind of method of new synthesis chlorpromazine immunogene, synthesis step more simplifies, effectively, for the present The research of people provides the thinking and method of synthetic immunogen afterwards.

Biological material specimens preservation: one plant anti-chlorine promazine monoclonal antibody hybridoma cell strain D, it is micro- China has been preserved in Biological inoculum preservation administration committee common micro-organisms center CGMCC, in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 of address Institute of microbiology, the academy of sciences, state, classification naming are monoclonal cell strain, preservation date on May 24th, 2018, deposit number CGMCC No.17304。

Detailed description of the invention

Fig. 1 is the ultra-violet absorption spectrum characterization of immunogene.

Fig. 2 is the standard suppression curve of chlorpromazine monoclonal antibody.

Specific embodiment

The following examples of the present invention are only used as the further explanation of the content of present invention, not as a limitation of the present invention interior Perhaps range.Below by embodiment, the invention will be further described.

The present invention is by being immunized mouse for chlorpromazine comlete antigen, by cell fusion, HAT selective medium culture, Cell conditioned medium is screened by indirect ELISA and indirect competitive ELISA, having finally obtained has preferable affinity and sensitive to chlorpromazine The monoclonal antibody hybridoma cell strain of degree.

Embodiment 1: the preparation that anti-chlorine promazine monoclonal antibody hybridoma cell is strain D

1, the synthesis of haptens: acetylpromazine maleate (APZ, 45 mg, 0.01 mmol) and half hydrochloride of carboxylic methoxamine (CMO, 25 mg, 0.02 mmol) is dissolved in deionized water (5 mL), and sodium acetate is added dropwise in the case where rotor stirring, Adjustment pH is 8-9, is 2-3 with 0.1 M hydrochloric acid adjustment pH, then by 15mL then by mixture in 40 DEG C of 10 h of back flow reaction Ethyl acetate is added.Ethyl acetate layer is collected, it is dry with Rotary Evaporators, obtain chlorpromazine haptens (CPZ-hapen).

2, the synthesis of comlete antigen: taking 2.2mg chlorpromazine haptens to be dissolved in 2mL dimethylformamide, and 4.5mg is added EDC(1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride) and 3.6mg NHS(N- HOSu NHS), room Temperature stirring, activates 4h；Separately take 10mg KLH(keyhole limpet hemocyanin) it is dissolved in the CB solution (carbonate of 3mL, 0.05M, pH9.6 Buffer solution) in, above-mentioned activating solution is added dropwise in KLH solution, is stirred at room temperature after reaction overnight, 4 DEG C are dialysed three days, and -20 DEG C packing save.

3, animal immune: the BALB/c mouse of the 6-8 week old of health is selected to be immunized.Take chlorpromazine comlete antigen After the emulsification uniformly of (1mg/mL) and equivalent freund adjuvant, BALB/c mouse, every 100 μ L are immunized by subcutaneous multi-point injection.It is first It is secondary it is immune use Freund's complete adjuvant, booster immunization uses freund 's incomplete adjuvant, and immunizing dose is preceding primary when spurt is immune The half of immunizing dose is directly injected intraperitoneally after mixing with physiological saline；Each secondary immunization interval is three weeks.For the third time After immune, interval blood sampling in one week detection serum titer and inhibition；Selection inhibits best mouse, and spurt in 18 days is exempted from after exempting from five Epidemic disease prepares fusion.

4, cell fusion: after spurt immune three days, according to conventional PEG(polyethylene glycol, molecular weight 2000) method into Row cell fusion, the specific steps are as follows:

(1) sterile to take mouse spleen, it grinds and obtains splenocyte suspension by 200 mesh cell screen clothes, and carry out cell count；

(2) SP2/0 cell is collected, is suspended in RPMI-1640 basic culture solution, cell count is carried out；

(3) splenocyte and SP2/0 cell are mixed according to the counting ratio of 2-10:1, are merged after centrifugation with PEG, 1 min of time, Later according to from slowly to fast, RPMI-1640 basic culture solution is added, be suspended in after centrifugation containing 20% fetal calf serum, 2% 50 × In the RPMI-1640 screening and culturing liquid of HAT, 96 porocyte culture plates are added to, are placed in 37 DEG C, 5% CO₂Incubator in cultivate.

5, cell screening and cell strain are established: carrying out RPMI-1640 screening training to fused cell within the 3rd day in cell fusion Nutrient solution partly changes liquid, carries out within the 5th day being changed entirely with the RPMI-1640 transition culture solution of 100 × HT containing 20% fetal calf serum, 1% Liquid took cell conditioned medium to be screened at the 7th day.Screen in two steps: the first step first filters out positive cell hole with indirect ELISA, It is standard items that second step, which selects chlorpromazine, carries out inhibitory effect measurement to positive cell with indirect competitive ELISA.Selection is to chlorine third Piperazine has the cell hole preferably inhibited, is subcloned using limiting dilution assay, is detected with same method.In triplicate, Obtain anti-chlorine promazine monoclonal antibody hybridoma cell strain D.

6, the preparation and identification of monoclonal antibody: taking 8-10 week old BALB/c mouse, and every mouse peritoneal injects paraffin oil 1mL；Every mouse peritoneal injection 1 × 10 after 7 days⁶Hybridoma collected ascites since the 7th day, and ascites is passed through octanoic acid- The purifying of saturated ammonium sulfate method, the monoclonal antibody of acquisition are placed in -20 DEG C of preservations.

It is sub- that immunoglobulin is carried out using the monoclonal antibody that mouse monoclonal subtype identification kit obtains ascites purifying Type identification, hypotype are IgG2a type, specific as shown in table 1.

The subtype identification of 1 chlorpromazine monoclonal antibody of table

Using indirect competitive ELISA method, monoclonal antibody is measured to the IC of chlorpromazine₅₀For 3.61 μ g/L, and it is demonstrated to him The IC of Da Lafei etc.₅₀And cross reacting rate, it is specific as shown in table 2.

2 chlorpromazine monoclonal antibody of table to chlorpromazine, fenazil, put forth energy to be close, Chlorprothixene IC₅₀And cross reacting rate

7, antibody application: anti-chlorine promazine monoclonal antibody hybridoma cell strain D is resisted by monoclonal prepared by internal ascites Body is applied to chlorpromazine ELISA and adds recovery test, the specific steps are as follows:

(1) chlorpromazine for the 0.1 μ g/mL for using carbonate buffer solution (CBS) to dilute is coated with as coating 96 hole enzyme mark of primordial covering Plate, after every hole 100 μ L, 37 DEG C of 2 h of coating, three times with PBST washing lotion board-washing, each every 200 μ L of hole, 3 min, is patted dry every time；

(2) it is closed with the CBS containing 0.2% gelatin, every hole 200 μ L, 37 DEG C of 2 h of closing, three times with PBST washing lotion board-washing, often 200 μ L of secondary every hole, each 3min are patted dry；

(3) the chlorpromazine standard of 0,0.02,0.05,0.1,0.2,0.5,1,2 μ g/L is respectively configured with phosphate buffer (PBS) Solution.Standard solution and sample to be tested extracting solution are added separately in the ELISA Plate closed, every 50 μ L of hole, Each sample repeats 3 holes, then the 50 diluted anti-chlorine promazine monoclonal antibodies of μ L 1:16000,37 DEG C of reaction 0.5h are added in every hole Afterwards, board-washing pats dry；

(4) 100 μ L of the every hole addition sheep anti-mouse igg secondary antibody of the diluted HRP label of the PBS 1:3000 containing 0.1% gelatin, 37 DEG C reaction 0.5h after, board-washing pats dry；

(5) every hole is added 100 μ L TMB developing solutions, after 37 DEG C of colour developing 15min, 50 μ L 2M H of every hole addition₂SO₄Terminate liquid, 450nm surveys light absorption value；

(6) addition recycling and sample pre-treatments: taking fresh or the milk 5g of (stored refrigerated) of rising again, and adds three various doses Chlorpromazine standard items, respectively 1ng, 5ng, 10ng.It places it in 50mL centrifuge tube, is slowly dropped into 50% potassium hydroxide solution 1mL sufficiently vibrates on vortex mixer, is slowly dropped into ethyl acetate 20mL, 10min is vibrated on vortex mixer, then It is put into centrifuge and 5min is centrifuged with 3000r/min.4mL supernatant is pipetted in another centrifuge tube, is dried with nitrogen, is added The PBS that 1mL contains 10% methanol redissolves, and takes 50 μ L for detecting.Recovery test is added using indirect competitive ELISA, Its rate of recovery is respectively 98%, 92%, 105%.

The configuration of solution:

Carbonate buffer solution (CBS): Na is weighed₂CO₃1.59g NaHCO₃2.93g is mixed after being dissolved in a small amount of distilled water respectively, Add distilled water to mix to about 800mL, adjusts pH value to 9.6, add distilled water to be settled to 1000mL, 4 DEG C of storages are spare；

Phosphate buffer (PBS): 8.00g NaCl, 0.2g KCl, 0.24g KH₂PO₄, 3.62g Na₂HPO₄·12 H₂O, It is dissolved in 800mL pure water, with NaOH or HCl tune pH to 7.2-7.4, is settled to 1000mL；

PBST: the PBS containing 0.05% Tween20；

TMB developing solution: A liquid: Na₂HPO₄·12H₂O 18.43g, citric acid 9.33g, pure water are settled to 1000mL；B liquid: 60 Mg TMB is dissolved in 100mL ethylene glycol.A, B liquid 1:5 mixing by volume is TMB developing solution, current existing mixed.

It is in summary only presently preferred embodiments of the present invention, practical range not for the purpose of limiting the invention.It is i.e. all Equivalent changes and modifications made by content according to the present patent application range all should be technology scope of the invention.

Claims (3)

1. hybridoma cell strain D of one plant of secretion anti-chlorine promazine monoclonal antibody, being preserved in Chinese microorganism strain preservation Administration committee common micro-organisms center CGMCC, the micro- life of the address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences Object research institute, classification naming are monoclonal cell strain, preservation date on May 24th, 2018, deposit number CGMCC No.17304.

2. anti-chlorine promazine monoclonal antibody, it is characterised in that: it is by deposit number CGMCC No.17304 hybridoma cell strain D Number secretion generate.

3. the application of anti-chlorine promazine monoclonal antibody described in claim 2, it is characterised in that: remaining for chlorpromazine in food Detection.