Hybridoma cell line and chloramphenicol-resistant monoclonal antibody produced by same

A hybridoma cell line and monoclonal antibody technology, applied in the field of immunochemistry, to achieve high sensitivity and affinity, rapid and sensitive detection, and strong specificity

Active Publication Date: 2011-01-12
JIANGSU HUACHUANG MEDICINE RES & DEV PLATFORM MANAGEMENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low price of chloramphenicol and its remarkable curative effect, the use of chloramphenicol in clinical and breeding has been repeatedly prohibited. In order to strengthen the detection and supervision of chloramphenicol residues in animal food

Method used

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  • Hybridoma cell line and chloramphenicol-resistant monoclonal antibody produced by same
  • Hybridoma cell line and chloramphenicol-resistant monoclonal antibody produced by same
  • Hybridoma cell line and chloramphenicol-resistant monoclonal antibody produced by same

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Reagent and material preparation

[0033] Chloramphenicol sodium succinate (sigma, C3787); bovine serum albumin (BSA) (Amresco, 0332); human serum albumin (HSA) (Sigma, A9511); ovalbumin (OVA) (Sigma, A5503); Carbodiimide hydrochloride (EDC) (Sigma, E6383); N-hydroxysuccinimide (NHS) (Sigma, 130672); N, N-dimethylformamide (DMF) (Sigma, D4551); Freund's complete adjuvant (Sigma, F5881); Freund's incomplete adjuvant (Sigma, F5506); Tetramethylbenzidine (TMB) (Amresco, 0759); HAT (Sigma, H0262) and HT (Sigma, H0137) ; sodium chloride (Amresco, 0241); potassium chloride (Amresco, 0395); potassium dihydrogen phosphate (Sigma, P9791); disodium hydrogen phosphate (Sigma, 71639); 035-044); Dimethyl sulfoxide (DMSO) (Applichem, 0231); Polyethylene glycol 4000 (PEG4000) (Sigma, P7306); DMEM high glucose medium (Gibco, 11995); Fetal bovine serum (Gibco, C2027050).

[0034] Experimental animals and cells: Balb / c mice (6-8 weeks old, female) were purchased from the Animal Center...

Embodiment 2

[0114] Embodiment 2 drug cross reaction test

[0115] Carry out by the monoclonal antibody screening indirect competition ELISA method established in embodiment one, the monoclonal antibody of chloramphenicol and chloramphenicol hapten structural analog such as chloramphenicol base, chloramphenicol sodium succinate, thiamphenicol chloramphenicol , tetracycline, gentamicin, ampicillin, and florfenicol for competition experiments, dilute these standards into different concentrations for indirect competition ELISA, draw the inhibition standard curve, and calculate the IC of the competitors 50 value and cross-reactivity rate, the cross-reaction rate of this monoclonal antibody and chloramphenicol sodium succinate reaches 120%, and the cross-reaction rate with other similar drug reactions is below 5%, therefore, the anti-chloramphenicol prepared in the present invention The monoclonal antibody can be used to detect chloramphenicol and chloramphenicol sodium succinate residues at th...

Embodiment 3

[0118] Example 3 Application of Monoclonal Antibody 7C10' in the ELISA Method for Detecting Chloramphenicol Residues

[0119] The monoclonal antibody 7C10' can be applied to the ELISA method for detecting chloramphenicol residues, for example, it can be used for the detection of chloramphenicol residues in eel, shrimp, and chicken. Taking eel (purchased in the market, river eel) as an example, the main steps for detecting chloramphenicol residues in eel are as follows:

[0120] 1. Sample pretreatment: Use 20% ethanol and eel samples to homogenize according to the volume to mass ratio of 3:1, then centrifuge the homogenate at 5000G at 4 degrees for 10 minutes, take the middle layer liquid, and dilute it with PBS solution 2 times before proceeding detection. 2. The detection principle of the kit in the present invention is the indirect competition ELISA method, the former OVA-CAP of coating is coated on the microporous plate, adds the chloramphenicol standard substance or sampl...

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Abstract

The invention relates to a chloramphenicol-resistant monoclonal antibody, which is mainly used for detecting chloramphenicol residue in livestock products and milk products. The chloramphenicol-resistant monoclonal antibody is secreted from a hybridoma cell line 7C10; and the preparation method comprises the following steps of: coupling EDC-activated chloramphenicol and carrier protein to obtain immunogen and coating antigen; performing intraperitoneal injection of mouse immunogen and enhancing the immunity; screening out a mouse who is subjected to immunity enhancement and has high serum titer; fusing spleen cells and SP2/0 myeloma cells; performing sub-clone to obtain the monoclonal antibody hybridoma cell line 7C10 capable of stably secreting the chloramphenicol; performing intraperitoneal injection on the mouse with the hybridoma cell line 7C10; collecting ascitic fluid; and performing liquid-phase chromatographic purification to obtain the chloramphenicol-resistant monoclonal antibody 7C10'. The chloramphenicol-resistant monoclonal antibody has the advantages of high specificity, high flexibility and affinity, large-scale production and effect of quickly and sensitively detecting the chloramphenicol residue in foods such as milk, meats, aquatic products and the like.

Description

technical field [0001] The invention relates to an anti-chloramphenicol specific monoclonal antibody, a hybridoma cell line producing an anti-chloramphenicol monoclonal antibody, a preparation method of an anti-chloramphenicol specific monoclonal antibody and its application in detecting chloramphenicol drug residues The application belongs to the technical field of immunochemistry. Background technique [0002] Chloramphenicol is a broad-spectrum bacteriostatic agent. It can diffuse into bacterial cells through fat solubility, mainly acts on the 50s subunit of bacterial 70s ribosomes, inhibits transpeptidase, hinders the growth of peptide chains, and inhibits the formation of peptide chains, thereby preventing protein synthesis. Gram-negative bacteria are more potent than Gram-positive bacteria. Chloramphenicol has been widely used to treat infections of various sensitive bacteria, but its clinical application has been strictly controlled because of its serious adverse re...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44G01N33/577
Inventor 唐宏王晓艳崔迎利王文夏文静林纪昀戴威韩晓娟杨利尹丽梅倪同浩郁靓汪纪宗
Owner JIANGSU HUACHUANG MEDICINE RES & DEV PLATFORM MANAGEMENT CO LTD
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