Furazolidone-metabolite-resistant monoclonal antibody and application thereof

A technology of monoclonal antibody and furazolidone, which is applied in the analysis of veterinary drug residues and the field of immunology, can solve the problems of high experimental equipment and technical requirements, cumbersome operation, and is not suitable for rapid detection of large batches of samples, and achieves strong controllability and repeatability sexual effect

Active Publication Date: 2017-02-08
BIOLOGY INST OF HEBEI ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently, the detection techniques for furazolidone residues mainly include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS) and immunoassay techniques. The three methods are sensitive and accurate, but the operation is cumbersome and requires high experimental equipment and technology, so it is not suitable for rapid detection of large quantities of samples

Method used

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  • Furazolidone-metabolite-resistant monoclonal antibody and application thereof
  • Furazolidone-metabolite-resistant monoclonal antibody and application thereof
  • Furazolidone-metabolite-resistant monoclonal antibody and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1 Preparation of furazolidone metabolite antigen of the present invention

[0041] (1) Derivatization of furazolidone metabolites: take 0.25g AOZ, dissolve it with about 2mL of methanol at room temperature; take 0.15g of 3-carboxybenzaldehyde, dissolve it with 2mL of methanol, slowly drop into the above In the AOZ solution, stir and react at room temperature for 5 hours, filter, and dry the filter residue to obtain the furazolidone metabolite derivative CPAOZ;

[0042] (2) Synthesis of immunogen: Weigh 50 mg of bovine serum albumin (BSA) and dissolve it in 1 mL of 0.1 mol / L PBS with a pH value of 7.4, add 1 mL of N,N-dimethylamide (DMF), stir to dissolve ; Dissolve 8 mg of CPAOZ obtained in step (a) in 1 mL of DMF solution, add 14 mg of dicyclohexylimide (DCC) and 6 mg of N-hydroxysuccinimide (NHS) under stirring, and stir overnight at 4° C., 8000r Centrifuge at 1 / min for 15 minutes, collect the supernatant and put it into a dialysis bag, dialyze with 0.01mo...

Embodiment 2

[0044] Example 2 Preparation of monoclonal antibody to furazolidone metabolites according to the present invention

[0045] (1) Animal immunization: immunize 6-8 week-old female Blab / c mice with the immunogen whose carrier protein is bovine serum albumin, and immunize once every 2 weeks. Determine the titer and inhibition rate, and select the mouse with the best result to prepare for fusion;

[0046] Table 1 Immunization flow chart

[0047]

[0048]

[0049] (2) Cell fusion: the fused mice were bleed from the eyeballs, and the serum was used as a positive control. After the neck was killed, the spleen was taken out under aseptic conditions to prepare spleen cells, which were fused with SP2 / 0 cells by PEG at a ratio of 5:1. The fused cell suspension was added to a 96-well plate covered with feeder cells, and cultured in an incubator at 37°C and 5% CO2;

[0050] (3) Screening of positive hybridoma cell lines: the fused cells were checked for contamination the next day, a...

Embodiment 3

[0052] Example 3 Characteristic Identification of Furazolidone Metabolite Monoclonal Antibody According to the Present Invention

[0053] (1) Potency determination

[0054] The indirect ELISA method is used, and the specific steps are as follows: Coating: Dilute the coating material with carbonate buffer solution to a concentration of 2 μg / mL, 100 μL / well in a 96-well microtiter plate, overnight at 4°C; washing: return the coated plate to room temperature , pour off the coating solution, add 300 μL of washing solution to each well, let stand for 1 min each time, wash 3 times, and pat dry for the last time; sealing: add 200 μL of washing solution containing 10% calf serum to each well, 37°C for 1 hour; Pour off the blocking solution, wash 3 times, and pat dry; add primary antibody: use the washing solution to dilute the monoclonal antibody at a ratio of 1:2000, add 100 μL to each well, and set a blank control well (PBS) and a negative control (negative control) Serum), placed ...

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Abstract

The invention relates to a furazolidone-metabolite-resistant monoclonal antibody and application thereof, and belongs to the technical field of immunology and the technical field of veterinary drug residue analysis. An anti-monoclonal antibody is generated through being secreted by hybridoma cell strains with the preservation number of CCTCC NO: C2016140; the valence of the antibody is 1 to 1024000; the subtype is IgG1; the affinity constant Ka is 1.6*10<9>L / mol; the inhibition rate IC50 on the furazolidone metabolites is 1.7 mu g / L; no cross reaction exists on other furazolidone metabolite analogues. The monoclonal antibody can be used for preparing an enzyme linked immunosorbent assay kit and a colloidal gold trail chromatography test paper strip for testing the furazolidone metabolites so as to achieve the goal of fast and sensitively detecting the furazolidone medicine metabolites in animal tissues, urine and milk.

Description

technical field [0001] The invention relates to an anti-furazolidone metabolite monoclonal antibody, a hybridoma cell line producing the monoclonal antibody, and the application of the antibody in detecting furazolidone metabolites, belonging to the technical field of immunology and the technical field of veterinary drug residue analysis. Background technique [0002] Furazolidone (FZD), also known as furazolidone, is a nitrofuran drug that has inhibitory effects on common Gram-negative and positive bacteria, acts on microbial enzyme systems, inhibits acetyl-CoA, and interferes with microbial Glucose metabolism, thereby playing an antibacterial role. Widely used in aquatic products and poultry to treat bacillary dysentery, enteritis, coccidiosis, etc., as well as scabies, red fin disease, ulcers, etc. of farmed fish. Because of its strong bactericidal ability, broad antibacterial spectrum, not easy to produce drug resistance, rapid oral absorption, and no cross-resistance w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44C12N5/20G01N33/577C12R1/91
CPCC07K16/44C07K2317/35C07K2317/92G01N33/577G01N2430/00
Inventor 李春生李玉静刘静静吴萌杜顺丰武孝利曹秀梅闫玉杰
Owner BIOLOGY INST OF HEBEI ACAD OF SCI
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