Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting furaltadone residue marker AMOZ

An enzyme-linked immunosorbent reagent, furaltadone technology, applied in the field of detection analysis and immunology, can solve the problems of low efficiency and inability to be widely used, and achieve the effect of high detection efficiency, good accuracy, and simple synthesis process

Active Publication Date: 2012-11-07
WUHAN SHANGCHENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method needs to react AMOZ with p-aldehyde benzoic acid before detecting, the efficiency of the reaction is low, and it cannot be widely used.

Method used

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  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting furaltadone residue marker AMOZ
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting furaltadone residue marker AMOZ
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting furaltadone residue marker AMOZ

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Synthesis of Antigens

[0031] 1.1 Synthesis of the hapten 3-p-formylbenzoyl-5morpholinomethyl-2-oxazolidinone (CPAMOZ)

[0032] Add 37.5 mg of p-aldehyde benzoic acid into a brown vial, slowly add methanol until completely dissolved, and stir magnetically. Add 50 mg of furaltadone residual marker AMOZ, and react with magnetic stirring at room temperature for 48 hours in the dark. Centrifuge at 8000 rpm for 10 minutes to remove the supernatant, wash with methanol three times, remove the supernatant, and dry the precipitate to obtain the white product CPAMOZ.

[0033]

[0034] 1.2 Synthesis of immunogen (CPAMOZ-BSA conjugate)

[0035] Take CPAMOZ 0.0167g and dissolve in 1ml N,N-dimethylformamide (DMF), add N,N'dicyclohexylcarbodiimide (DCC) 0.0137g and N-hydroxysuccinimide (NHS) 0.0072 g, stirred overnight at 4°C with magnetic force. Centrifuge at 5000rpm for 10min at 4°C and take the supernatant as solution A for later use. Weigh 0.070g of BSA and dis...

Embodiment 2

[0041] Embodiment 2: Preparation of monoclonal antibody

[0042] Preparation of hybridoma cell lines: refer to the method in Xue Qingshan's "Principles and Techniques of In Vitro Culture" Science Press, 2001 edition: immunize Balb / C mice with the CPAMOZ-BSA conjugate prepared in Example 1, and the immunization procedure is: basic Immunization After emulsifying the immunogen with an equal volume of complete Freund's adjuvant, inject it subcutaneously at multiple points on the back of the mouse, and then boost the immunization once every 2 weeks, replace it with incomplete adjuvant emulsification, and finally inject intraperitoneally three days before the fusion , Enhanced immunization, double the amount of antigen, no adjuvant. At the time of fusion, a Balb / C mouse that received the final booster immunization was taken, sacrificed by bloodletting from the orbit (serum was collected, it was positive serum), and sterilized by immersing in 75% alcohol by volume for 5 minutes.

[...

Embodiment 3

[0048] Embodiment 3: the establishment of ELISA detection method

[0049] 3.1 Optimization of ELISA conditions

[0050] Accurately pipette 100 μL of the diluted CPAMOZ-OVA coating original working solution into each well, pack one line for each concentration, and store in the refrigerator at 4°C overnight. Take out the ELISA plate, add 250 μL of washing solution (PBST) to each well, vibrate on the shaker for 1 min, shake off the washing solution, and pat dry on absorbent paper (or clean towel). Repeat the wash 2 times. Accurately pipette 250 μL of blocking solution (1% OVA) into each well, and incubate in a 37°C incubator for 2 hours. Shake off blocking solution and pat dry on absorbent paper. Accurately pipette 250 μL of washing solution into each well, vibrate on a shaker for 1 min, shake off the washing solution, and pat dry on absorbent paper. Repeat the wash 2 times.

[0051] Take the coated ELISA plate, add 50 μL of 20 mM PBS buffer solution with pH 7.4 to each well...

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Abstract

The invention discloses a monoclonal antibody capable of identifying a furaltadone residue marker 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ). The monoclonal antibody is secreted by a hybridoma cell AMOZ; and the hybridoma is preserved in China Center for Type Culture Collection, and has a preservation number of CCTCC NO:C201188. The invention also discloses an enzyme immunoassay method and a reagent kit for detecting furaltadone marker residue marker AMOZ, and application thereof to detection of the furanketone residue marker AMOZ. Compared with prior art, the monoclonal antibody prepared by the invention, the ELISA Kit and the ELISA method have high detection sensitivity, high precision and good accuracy; a hapten synthesis process is simple, and has high synthesis efficiency; and a derivatization reagent for sample pretreatment is benzene formaldehyde, which has advantage of small toxicity compared with other commonly used derivative reagent like o-nitrobenzaldehyde.

Description

technical field [0001] The invention belongs to the technical field of detection analysis and immunology, and specifically relates to a monoclonal antibody and an enzyme capable of detecting furaltadone residue marker 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) Immunization methods and kits. Background technique [0002] Furaltadone belongs to nitrofuran drugs and has the basic structure of 5-nitrofuran ring. It is a broad-spectrum antibacterial drug and is effective against gram-positive bacteria, gram-negative bacteria, fungi, protozoa, etc. It is widely used in aquaculture. Toxicology tests have proved that furaltadone and its metabolites have significant carcinogenic, mutagenic and other serious toxic effects. The European Union banned the use of furaltadone in the breeding industry in 1993, and my country's Announcement No. 193 "List of Veterinary Drugs and Other Compounds Prohibited for Food Animals" included nitrofuran drugs in the prohibited list. [0003] B...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44C12N5/20G01N33/577C12R1/91
Inventor 袁宗辉彭大鹏王玉莲郑丽陶燕飞陈冬梅刘振利
Owner WUHAN SHANGCHENG BIOTECH
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