CN109943535A - One plant of praziquantel monoclonal antibody hybridoma cell strain G and its application - Google Patents

Abstract

One plant of praziquantel monoclonal antibody hybridoma cell strain G and its application, belong to food safety field of immunodetection.The present invention provides one plant of praziquantel monoclonal antibody hybridoma cell strains G, it has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, classification naming is monoclonal cell strain, and deposit number is CGMCC No.17307, and the deposit date is on Mays 24th, 2018.The praziquantel monoclonal antibody of this cell strain secretion can be used for the analysis detection of praziquantel residue in food safety detection.The monoclonal antibody of cell strain secretion of the present invention has preferable specificity and detection sensitivity (IC to praziquantel₅₀It is worth for 7.4 ng/mL) detection, it can be achieved that praziquantel residue amount in aquatic products edible portion, there is practical application value.

Description

One plant of praziquantel monoclonal antibody hybridoma cell strain G and its application

Technical field

The present invention relates to one plant of praziquantel monoclonal antibody hybridoma cell strain G and its applications, belong to food safety and exempt from Epidemic disease detection field.

Background technique

Praziquantel (Praziquantel, PZQ) be it is a kind of for the mankind and the anthelmintic of animal, specifically treat tapeworm and suction Worm.It is especially effective for blood fluke, Chinese liver fluke, wide section diphyllobothrium.Praziquantel is World Health Organization's essential drugs mark Drug on quasi- inventory is one of drug most important for basic public health in the world.Praziquantel is also used for prevention and treatment aquatic products The parasitic infection of animal, praziquantel is widely used in production in recent years, and many helminths is not only made to produce drug resistance, And its residual in aquatic products causes potential threat to consumer.The instrument inspection of the bulletin regulation praziquantel of the Ministry of Agriculture 985 Rising limit is 10 μ g/kg；Standard GB/T/T 22956-2008 provides the inspection of the praziquantel residue amount in globe fish, sea eel, roast eel Rising limit is 5 μ g/kg.People take in various chronic diseases caused by residue of veterinary drug and induction pathogen to extended low level in recent years The problems such as generating drug resistance pays special attention to, and to ensure fish quality and consumer's health, promotes culture fishery Health, sustainable development, it is necessary to the medicament residue in aquatic products is monitored.

It is largely to study its pest-resistant mechanism and treatment method to the report of praziquantel both at home and abroad at present, to praziquantel Residues detection is mainly instrument detection, especially based on high performance liquid chromatography.However the preceding place of these instrument analytical methods It manages more complicated, is not suitable for the quick detection of a large amount of samples；Therefore the interests in order to safeguard the majority of consumers, it is necessary to establish A kind of efficient, quick detection method for praziquantel.

Enzyme-linked immunization (ELISA) is a kind of extremely efficient, sensitive, quick detection method, it can be achieved that a large amount of samples Quickly detection, and it is not high to the purity requirement of sample when detection.Therefore, it is necessary to establish efficient immunological detection method, And an important prerequisite for establishing the method need to filter out the high-specificity monoclonal monomer for praziquantel.

Summary of the invention

It is thin by this object of the present invention is to provide one plant of praziquantel monoclonal antibody hybridoma cell strain G and its application The antibody of born of the same parents' strain preparation has preferably specificity and detection sensitivity to praziquantel, can be used to establish the immunology inspection of praziquantel Survey method.

Technical solution of the present invention, one plant of praziquantel monoclonal antibody hybridoma cell strain G have been preserved in the micro- life of China Object culture presevation administration committee common micro-organisms center, abbreviation CGMCC, classification naming are monoclonal cell strain, deposit number For CGMCC No.17307, the deposit date is on Mays 24th, 2018.

Praziquantel monoclonal antibody, it is miscellaneous by the praziquantel monoclonal antibody that the deposit number is CGMCC No.17307 No. G secretion of tumor cell strain is handed over to generate.

The application of the praziquantel monoclonal antibody, the analysis detection for praziquantel residue in food safety detection.

The strain of praziquantel monoclonal antibody hybridoma cell G preparation basic step provided by the invention are as follows:

(1) synthesis of haptens: as shown in following reaction equation；

Weigh Compound SM (PZQ) 1.5g(4.8 mmol) it is placed in cooled on ice, c.H is added₂SO₄ 4.71 g (2.57 mL, 48 mmol), it is then slowly added into c.HNO₃1.52 g (1.06 mL, 24 mmol), reaction mixture is stirred at room temperature Mix 16h.Water (60mL) is added and mixture is placed in cooled on ice, is then slowly added into solid sodium bicarbonate up to water layer PH reaches 8.EtOAc(60mL is added into mixture), separate each layer and aqueous layer extracted.It is eluted with EtOAc(3 × 50mL).It will Combined organic layer is washed with salt water (50mL), and uses Na₂SO₄Dry, vacuum concentration obtains straw yellow solid, by quick Column chromatography is purified, and faint yellow solid (730mg, 2.04mmol, yield 42.54%), i.e. compound 1 are obtained.

By SnCl₂•2H₂999.4 mg of O (4.43 mmol, 2.8eq) is added to compound 1(565mg, 1.58mmol) Methanol solution in (20mL).Mixture is flowed back 8h at 65 DEG C.Methanol is removed in vacuum, uses 10%NaHCO₃Solution will be remaining Object is adjusted to pH 8, is extracted with ethyl acetate (3 × 30mL).Combined organic layer is washed with salt water (30mL), uses Na₂SO₄It is dry It is dry, it is filtered and concentrated in vacuo, obtains light yellow solid, passed through flash column chromatography.Obtain yellow solid MGD_1 (435mg, 1.33mmol, yield 84.04%), i.e. haptens PZQ-HS.

2) preparation of comlete antigen PZQ-HS-BSA: 5.5mg PZQ-HS(pyrrole quinoline ketone Yan Sheng Wu ︰ bovine serum albumin(BSA) is weighed (BSA) molar ratio is 60:1), it is dissolved in 600 μ L n,N-Dimethylformamide DMF, 50 μ L 1M HCl is added, ice bath is kept away After light reaction 30min, the NaNO of 5.56 μ L 30% is added₂Solution (ready-to-use) continues ice bath and is protected from light activation 30min, claims For A liquid；10 mg BSA are taken, are dissolved with pH=8.6,2mL 0.01M carbonate buffer solution CB, referred to as B liquid；Again dropwise by A liquid It is slowly added into B liquid, 4 DEG C of reaction 4-6h；Up to conjugate PZQ-HS-BSA mixed liquor；Then with 0.01M PBS(pH= 7.2) solution is dialysed three days, is removed unreacted small haptens, is obtained comlete antigen PZQ-HS-BSA, and pass through ultraviolet suction Scan method is received to be identified；

3) mouse is immune: after praziquantel comlete antigen and equivalent Freund's adjuvant mixing and emulsifying, carrying out neck to BALB/c mouse Dorsal sc multi-point injection is immune (except spurt is immune).First immunisation complete Freund's adjuvant, dosage are 100 μ g/；Repeatedly Booster immunization cannots be used up full Freund's adjuvant and dosage halves as 50 μ g/ only；Spurt is immune not to have to adjuvant, directly uses physiological saline It is injected intraperitoneally after dilution, dosage halves again as 25 μ g/ only.It is spaced one month between first immunisation and second of booster immunization, It is spaced 21 days between multiple booster immunization, spurt is immune to be spaced 18-21 days between last time booster immunization.By indirectly competing Strive potency and inhibition that enzyme-linked immunization (ic-ELISA) observation mouse immune effect detects mice serum；

4) cell fusion and cell strain are established: by polyethylene glycol (PEG 4000) method that mouse boosting cell and mouse myeloma is thin Born of the same parents are merged, and filter out hybridoma using selective medium (HAT culture medium), and carry out cell training with HT culture medium It supports.Fusion detects positive cell hole using ic-ELISA method after a week, and further measures positive cell using ic-ELISA method The inhibitory effect in hole is detected again after a week, is chosen by limiting dilution assay to inhibiting preferable positive cell hole to be subcloned Hole, subclone.The Monoclonal hybridomas of the hypersecretion specific antibody of praziquantel is obtained after being subcloned three times according to the above method Cell strain G；

5) identification of hybridoma cell strain property: pass through ic-ELISA measurement sensitivity and specificity.

Beneficial effects of the present invention: it is provided by the invention cell strain G secretion monoclonal antibody, to praziquantel have compared with Good specificity and detection sensitivity (IC₅₀Value is 7.4 ng/mL), it can be achieved that praziquantel residue in aquatic products edible portion The detection of amount has practical application value.

Biological material specimens preservation: one plant of praziquantel monoclonal antibody hybridoma cell strain G has been preserved in the micro- life of China Object culture presevation administration committee common micro-organisms center, abbreviation CGMCC, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3, Institute of Microorganism, Academia Sinica, the deposit date is on May 24th, 2018, deposit number was CGMCC No.17307.

Detailed description of the invention

The inhibition standard curve of Fig. 1 cell strain G 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.

Mouse is immunized by praziquantel comlete antigen in the present invention, and by cell fusion, HAT selective medium culture is led to Ic-ELISA screening cell conditioned medium is crossed, the hybridoma that there is hypersecretion specific antibody for praziquantel has been finally obtained Strain.

The preparation of 1 hybridoma cell strain of embodiment G

(1) synthesis of haptens:

Weigh Compound SM (PZQ) 1.5g(4.8 mmol) it is placed in cooled on ice, c.H is added₂SO₄ (4.71 g, 2.57 ML, 48 mmol), it is then slowly added into c.HNO₃(1.52 g, 1.06 mL, 24 mmol), by reaction mixture in room temperature Lower stirring 16h.Water (60mL) is added and mixture is placed in cooled on ice, is then slowly added into solid sodium bicarbonate until water The pH of layer reaches 8.EtOAc(60mL is added into mixture), separate each layer and aqueous layer extracted.It is washed with EtOAc(3 × 50mL) It is de-.Combined organic layer is washed with salt water (50mL), and uses Na₂SO₄Dry, vacuum concentration obtains straw yellow solid, leads to It crosses flash column chromatography to be purified, obtains faint yellow solid (730mg, 2.04mmol, yield 42.54%), i.e. compound 1.

By SnCl₂•2H₂O (999.4 mg, 4.43 mmol, 2.8eq) is added to compound 1(565mg, In methanol solution 1.58mmol) (20mL).Mixture is flowed back 8h at 65 DEG C.Methanol is removed in vacuum, uses 10%NaHCO₃ Residue is adjusted to pH 8 by solution, is extracted with ethyl acetate (3 × 30mL).Combined organic layer is washed with salt water (30mL) It washs, uses Na₂SO₄It dries, filters and is concentrated in vacuo, obtain light yellow solid, passed through flash column chromatography.Obtain Huang Color solid MGD_1(435mg, 1.33mmol, yield 84.04%), i.e. haptens PZQ-HS.

(2) 5.5mgPZQ-HS(praziquantel derivative and bovine serum albumin the preparation of comlete antigen PZQ-HS-BSA: are weighed White (BSA) molar ratio is 60:1), it is dissolved in 600 μ L n,N-Dimethylformamide DMF, 50 μ L 1M HCl, ice bath is added After being protected from light 30min, the NaNO of 5.56 μ L 30% is added₂Solution (ready-to-use) continues ice bath and is protected from light activation 30min, Referred to as A liquid；10 mg BSA are taken, are dissolved with pH=8.6,2mL 0.01M carbonate buffer solution CB, referred to as B liquid；Again dropwise by A Liquid is slowly added into B liquid, 4 DEG C of reaction 4-6h；Up to conjugate PZQ-HS-BSA mixed liquor；Then with 0.01M PBS(pH= 7.2) solution is dialysed three days, is removed unreacted small haptens, is obtained comlete antigen PZQ-HS-BSA, and pass through ultraviolet suction Scan method is received to be identified；

(3) after praziquantel comlete antigen and equivalent Freund's adjuvant mixing and emulsifying, nape animal immune: is carried out to BALB/c mouse The subcutaneous multi-point injection in portion is immune (except spurt is immune).First immunisation complete Freund's adjuvant, dosage are 100 μ g/；Repeatedly add It is strong immune to cannot be used up full Freund's adjuvant and dosage halves as 50 μ g/ only；Spurt is immune not to have to adjuvant, directly dilute with physiological saline It is injected intraperitoneally after releasing, dosage halves again as 25 μ g/ only.It is spaced one month between first immunisation and second of booster immunization, it is more It is spaced 21 days between secondary booster immunization, spurt is immune to be spaced 18-21 days between last time booster immunization.Pass through indirect competition It is to detect the potency and inhibition of mice serum that enzyme-linked immunization (ic-ELISA), which observes mouse immune effect,；

(4) cell fusion: after spurt is three days immune, according to conventional PEG(polyethylene glycol, molecular weight 4000) method progress is carefully Born of the same parents' fusion, the specific steps are as follows:

A, mouse plucks eyeball and takes blood, after cervical dislocation puts to death mouse, is immediately placed in 75% alcohol and sterilizes, and impregnates 5 min The spleen of mouse is taken out in left and right, sterile working, is moderately ground with syringe rubber head and obtains splenocyte by 200 mesh cell screen clothes Suspension is collected, and is centrifuged (1200rpm, 8 min), is washed splenocyte three times with RPMI-1640 culture medium, after last time is centrifuged, Splenocyte is diluted to certain volume, is counted, it is spare；

B, collect SP2/0 cell: 7-10 days before fusion, SP2/0 oncocyte being used and contains 10% FBS(fetal calf serum) RPMI- 1640 culture mediums are in 5% CO₂It is cultivated in incubator.SP2/0 oncocyte quantity is required to reach (1-4) × 10 before fusion⁷, guarantee SP2/0 oncocyte is in logarithmic growth phase before merging.When fusion, oncocyte is collected, RPMI-1640 basic culture solution is suspended in In, carry out cell count；

C, the PEG 4000 of 1mL is added drop-wise in cell by the 7min: the 1min of fusion process from slow to fast；2min is stood. 1mL RPMI-1640 culture medium is added dropwise in 3min and 4min in 1min；5min and 6min is added dropwise in 1min 2mL RPMI-1640 culture medium；The RPMI-1640 culture medium of 1mL is added dropwise in 7min, every 10s.Then 37 DEG C of warm bath 5 min.It being centrifuged (800 rpm, 10 min), abandons supernatant, cell gently strikes scattered, and it is added into it and contains 20% fetal calf serum, 2% 50 The RPMI-1640 selective medium (HAT culture medium) of × HAT is added to 96 porocyte plates according to 200 holes μ L/, is placed in 37 DEG C, 5% CO₂It is cultivated in incubator；

(5) cell screening and cell strain are established: partly being changed with HAT culture medium fused cell within the 3rd day after cell fusion Liquid；It is changed entirely with the RPMI-1640 transition culture solution (HT culture medium) containing 20% fetal calf serum, 1% 100 × HT within 5th day Liquid；Cell conditioned medium is taken to be screened within 7th day.Screen in two steps: the first step first filters out positive cell hole with ic-ELISA method, the It is standard items that two steps, which select praziquantel, carries out inhibitory effect measurement to positive cell with ic-ELISA method.Selection is to praziquantel mark Quasi- product have the cell hole preferably inhibited, are subcloned using limiting dilution assay, are detected after seven days with same method.It presses The above method is subcloned three times, final to obtain praziquantel cell strain of monoclonal antibody G；

(6) preparation and identification of monoclonal antibody: taking 8-10 week old BALB/c mouse, and every mouse peritoneal injects sterile paraffin oil 1mL；Every mouse peritoneal injection 1 × 10 after 7 days⁶ Praziquantel hybridoma collects ascites since the 7th day, ascites is led to It crosses octanoic acid-saturated ammonium sulfate method and carries out antibody purification.Under the conditions of meta-acid, caprylic acid can be precipitated in ascites except ball is immunized in IgG Other foreign proteins outside albumen, are then centrifuged for, and abandon precipitating；Again with the Dan Ke of the ammonium sulfate precipitating IgG type of equivalent saturation degree Supernatant is abandoned in grand antibody, centrifugation, and after the dissolution of 0.01 M PBS solution (pH7.4), dialysis desalting finally obtains list after purification Clonal antibody is placed in -20 DEG C of preservations.

6.1 coating: by coating antigen praziquantel-OVA with 0.05M pH9.6 carbonate buffer solution 3 multiple proportions since 1 μ g/mL Dilution, 100 holes μ L/, 37 DEG C of reaction 2h；

6.2 washings: solution in plate is inclined, and is washed 3 times with cleaning solution, each 3min；

6.3 closings: after patting dry, 200 hole μ L/ confining liquids, 37 DEG C of reaction 2h are added.It is dried for standby after washing；

6.4 sample-addings: by antiserum since 1:1000 doubling dilution, and be added in the coating hole of each dilution, 100 holes μ L/, 37 DEG C of reaction 30min；Sufficiently after washing, the diluted HRP- sheep anti-mouse igg of 1:3000,100 holes μ L/, 37 DEG C of reactions are added 30min；

6.5 colour developings: ELISA Plate is taken out, and sufficiently after washing, the TMB developing solution of 100 μ L is added in every hole, and 37 DEG C are protected from light 15min；

6.6 terminate and measure: 50 μ L terminate liquids are added to terminate reaction in every hole, and the OD in each hole is then measured with microplate reader₄₅₀Value.

With the IC of ic-ELISA measurement monoclonal antibody praziquantel₅₀Are as follows: 7.4ng/mL illustrates there is good spirit to praziquantel Sensitivity can be used for the detection of praziquantel immunoassay.

The configuration of solution:

Carbonate buffer solution (CBS): Na is weighed₂CO₃1.59 g, NaHCO₃2.93 g are mixed after being dissolved in a small amount of distilled water respectively It closes, adds distilled water to mix to about 800mL, adjust 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, 0.2g KH₂PO₄, 2.9g Na₂HPO₄·12 H₂O, it is molten In 800mL pure water, with NaOH or HCl tune pH to 7.2~7.4, it is settled to 1000mL；

PBST: the PBS containing 0.05 % polysorbas20；

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

Claims (3)

1. one plant of praziquantel monoclonal antibody hybridoma cell strain G, has been preserved in Chinese microorganism strain preservation conservator Meeting common micro-organisms center, abbreviation CGMCC, classification naming are monoclonal cell strain, and deposit number is CGMCC No.17307, are protected The hiding date is on May 24th, 2018.

2. praziquantel monoclonal antibody, it is characterised in that: its deposit number as described in claim 1 is CGMCC No.17307's No. G secretion of praziquantel monoclonal antibody hybridoma cell strain generates.

3. the application of praziquantel monoclonal antibody described in claim 2, it is characterised in that: for praziquantel in food safety detection Remaining analysis detection.