Hybridoma cell strain for secreting anti 1-amino-hydantoin monoclonal antibody and application of hybridoma cell strain

Abstract

The invention discloses a hybridoma cell strain for secreting an anti 1-amino-hydantoin monoclonal antibody and application of the hybridoma cell strain, and belongs to the technical field of biologics. The hybridoma cell strain is named a hybridoma cell strain 2G9A3-35H11, and the preservation number is CCTCC NO: C2015141. According to the hybridoma cell strain, a conjugate formed by AHD-CP (1-Aminohydantoin Hydrochloride-4-Carboxyphenyl Oxime) and bovine serum albumin is adopted as antigen to immunize BALB / c mice, spleen cells obtained after immunization are fused with rejuvenated SP2 / 0 myeloma cells, cell culture is performed with a culture medium without antibiotics, and thus the hybridoma cell strain is prepared through multiple times of screening and cloning; in in-vitro culture, the hybridoma cell strain can secrete a great number of specific antibodies of an anti-AHD derivatization product, and thus the hybridoma cell strain can be applied to rapid and accurate immunodetection and immunoassay on macrodantin metabolin.

Description

technical field

[0001] The invention relates to the field of biotechnology, in particular to a hybridoma cell strain secreting anti-1-amino-hydantoin monoclonal antibody and application thereof. Background technique

[0002] Nitrofurantoin (Nitrofurantoin, CAS No.67-20-9), belonging to nitrofuran drugs, is a synthetic antibacterial drug, often used as a broad-spectrum antibiotic for the prevention and treatment of diseases caused by Salmonella and Escherichia Gastrointestinal diseases of pigs, cattle, poultry and bees. However, studies in recent years have shown that nitrofuran drugs and their metabolites are highly toxic, have teratogenic side effects, and can induce cancer. Feed use.

[0003] Due to the thermal and chemical instability of nitrofuran drugs, the detection of its metabolite 1-amino-hydantoin (AHD) is often used as a residue marker to determine whether nitrofuran is used in animal breeding.

[0004] At present, the detection methods of AHD residue mainly in...

Examples

Embodiment 1

[0039] The hybridoma cell line 2G9A3-35H11 was delivered to the China Center for Type Culture Collection (Address: China. Wuhan. Wuhan University) on August 26, 2015. The viability of the cell line was tested on September 10, 2015. The result was survival , deposit number CCTCC No: C2015141.

[0040] The hybridoma cell line uses the conjugate (AHD-CP-BSA) of derivatives of nitrofurantoin metabolites (AHD-CP) and bovine serum albumin (BSA) as an antigen to immunize BALB / c mice, and the mice after immunization The splenocytes of the splenocytes were fused with the rejuvenated SP2 / 0 myeloma cells, cultured in medium without adding antibiotics, and obtained after screening and 6 times of cloning. The specific process is as follows:

[0041] 1. Hapten synthesis

[0042]Weigh 300 mg of p-4-carboxybenzaldehyde (4-Carboxybenzaldehyde, 4-CBA) in 1 mL of distilled water, add dropwise dimethyl sulfoxide (DMF) until 4-CBA is completely dissolved, add 100 mg of nitrofurantoin metabolite ...

Embodiment 2

[0083] F of 2G9A3-35H11 cell line 3 Substitute for the production of monoclonal antibodies, antibodies can be prepared in vivo or in vitro.

[0084] 1. In vivo preparation of AHD monoclonal antibody

[0085] 6-week-old male Balb / c mice were intraperitoneally injected with 0.5 mL of liquid paraffin 2 weeks in advance → take 2×10 6 the F 3 Inject hybridoma cells into the abdominal cavity of mice → 7-12 days later, when the mice are slightly enlarged, inject 1×10 6 hybridoma cells → 2 to 5 days later, when the abdomen of the mouse is enlarged, draw the ascites with a large needle → kill the mouse by guiding the spine, wash the peritoneal cavity with PBS, collect the washing fluid, merge it into the ascites → purify the antibody, each mouse Mice can obtain 5-15 mg of antibody.

[0086] 2. In vitro → preparation of AHD monoclonal antibody

[0087] The 2G9A3-35H11 cell line was inserted into a glass cell bottle, added with 1640 medium (containing 10% fetal bovine serum), at 37°...

Embodiment 3E

[0094] Example 3 ELISA method detects nitrofurantoin metabolites in chicken (application of primary purified antibody)

[0095] (1) Coating: Dilute AHD-CP-OVA to 300ng / mL with carbonate buffer solution of pH=9.6, add 100μL / well into the microwell strip of the microplate, overnight at 4°C, add 200μL to each well after washing the plate 10% skimmed milk powder, sealed at 37°C for 2 hours, washed and dried in the shade for later use.

[0096] (2) Sample pretreatment: Prepare 3 Snow Mountain Ma chicken breast muscle samples, weigh 1±0.05g of homogeneous samples, weigh 12 copies of each sample, and separate them with 1-amino-2-lactoin (AHD) standard solution Do 0, 0.2, 0.5, 1.0 μg / kg addition recovery test, and do 3 parallels for each concentration. Add 4mL deionized water, 0.5mL 1mol / L hydrochloric acid and 100μL 0.01mol / L 2-nitrobenzaldehyde (2-NP) solution to each sample, place in a 56°C water bath for 2h, add 5mL 0.1mol / L K 2 HPO 4 , 0.4mL 1mol / L NaOH and 5mL ethyl acetate, ...

technical field

[0001] The invention relates to the field of biotechnology, in particular to a hybridoma cell strain secreting anti-1-amino-hydantoin monoclonal antibody and application thereof. Background technique

[0002] Nitrofurantoin (Nitrofurantoin, CAS No.67-20-9), belonging to nitrofuran drugs, is a synthetic antibacterial drug, often used as a broad-spectrum antibiotic for the prevention and treatment of diseases caused by Salmonella and Escherichia Gastrointestinal diseases of pigs, cattle, poultry and bees. However, studies in recent years have shown that nitrofuran drugs and their metabolites are highly toxic, have teratogenic side effects, and can induce cancer. Feed use.

[0003] Due to the thermal and chemical instability of nitrofuran drugs, the detection of its metabolite 1-amino-hydantoin (AHD) is often used as a residue marker to determine whether nitrofuran is used in animal breeding.

[0004] At present, the detection methods of AHD residue mainly in...

Examples

Embodiment 1

[0039] The hybridoma cell line 2G9A3-35H11 was delivered to the China Center for Type Culture Collection (Address: China. Wuhan. Wuhan University) on August 26, 2015. The viability of the cell line was tested on September 10, 2015. The result was survival , deposit number CCTCC No: C2015141.

[0040] The hybridoma cell line uses the conjugate (AHD-CP-BSA) of derivatives of nitrofurantoin metabolites (AHD-CP) and bovine serum albumin (BSA) as an antigen to immunize BALB / c mice, and the mice after immunization The splenocytes of the splenocytes were fused with the rejuvenated SP2 / 0 myeloma cells, cultured in medium without adding antibiotics, and obtained after screening and 6 times of cloning. The specific process is as follows:

[0041] 1. Hapten synthesis

[0042]Weigh 300 mg of p-4-carboxybenzaldehyde (4-Carboxybenzaldehyde, 4-CBA) in 1 mL of distilled water, add dropwise dimethyl sulfoxide (DMF) until 4-CBA is completely dissolved, add 100 mg of nitrofurantoin metabolite ...

Embodiment 2

[0083] F of 2G9A3-35H11 cell line 3 Substitute for the production of monoclonal antibodies, antibodies can be prepared in vivo or in vitro.

[0084] 1. In vivo preparation of AHD monoclonal antibody

[0085] 6-week-old male Balb / c mice were intraperitoneally injected with 0.5 mL of liquid paraffin 2 weeks in advance → take 2×10 6 the F 3 Inject hybridoma cells into the abdominal cavity of mice → 7-12 days later, when the mice are slightly enlarged, inject 1×10 6 hybridoma cells → 2 to 5 days later, when the abdomen of the mouse is enlarged, draw the ascites with a large needle → kill the mouse by guiding the spine, wash the peritoneal cavity with PBS, collect the washing fluid, merge it into the ascites → purify the antibody, each mouse Mice can obtain 5-15 mg of antibody.

[0086] 2. In vitro → preparation of AHD monoclonal antibody

[0087] The 2G9A3-35H11 cell line was inserted into a glass cell bottle, added with 1640 medium (containing 10% fetal bovine serum), at 37°...

Embodiment 3E

[0094] Example 3 ELISA method detects nitrofurantoin metabolites in chicken (application of primary purified antibody)

[0095] (1) Coating: Dilute AHD-CP-OVA to 300ng / mL with carbonate buffer solution of pH=9.6, add 100μL / well into the microwell strip of the microplate, overnight at 4°C, add 200μL to each well after washing the plate 10% skimmed milk powder, sealed at 37°C for 2 hours, washed and dried in the shade for later use.

[0096] (2) Sample pretreatment: Prepare 3 Snow Mountain Ma chicken breast muscle samples, weigh 1±0.05g of homogeneous samples, weigh 12 copies of each sample, and separate them with 1-amino-2-lactoin (AHD) standard solution Do 0, 0.2, 0.5, 1.0 μg / kg addition recovery test, and do 3 parallels for each concentration. Add 4mL deionized water, 0.5mL 1mol / L hydrochloric acid and 100μL 0.01mol / L 2-nitrobenzaldehyde (2-NP) solution to each sample, place in a 56°C water bath for 2h, add 5mL 0.1mol / L K 2 HPO 4 , 0.4mL 1mol / L NaOH and 5mL ethyl acetate, ...

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