Monoclonal antibody for detecting furazolidone metabolites, ELISA method, and kit

A technology of furazolidone and its metabolites, which is applied in the field of monoclonal antibodies, can solve the problems of animal death and easy toxic reactions, and achieve the effects of strong specificity, high affinity, good accuracy and precision

Active Publication Date: 2015-12-09
WUHAN SHANGCHENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This type of drug has certain toxicity to livestock and poultry, large dose or long-term continuous use, prone to toxic reactions, manifested as anor

Method used

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  • Monoclonal antibody for detecting furazolidone metabolites, ELISA method, and kit
  • Monoclonal antibody for detecting furazolidone metabolites, ELISA method, and kit
  • Monoclonal antibody for detecting furazolidone metabolites, ELISA method, and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The preparation of embodiment 1 immunogen and coating former

[0031] Take 1g of furazolidone metabolite (AOZ) and 1.65g of 5-hydroxy-2nitro-benzaldehyde, dissolve them in absolute ethanol, stir and react at room temperature for 5h, let stand overnight in a refrigerator at 4°C, filter, and use N,N-2 Dissolve methylformamide (DMF), add 100mg of NaOH, slowly dropwise add 600mg of ethyl 4-bromobutyrate; after the reaction is complete, add water, extract with ethyl acetate, combine organic phases, filter, and evaporate the filtrate to dryness After adding a small amount of DMF to dissolve it, add 40 mL of 1NNaOH, stir and react at room temperature for 2 hours, adjust the pH of the reaction solution to 3 with HCL, and filter to obtain the hapten CNPAOZ, whose structural formula is as follows:

[0032]

[0033] Take 0.2g of AOZ, stir it with about 2mL of methanol at room temperature to dissolve it; take 0.15g of 3-carboxybenzaldehyde, dissolve it with 2mL of methanol, slow...

Embodiment 2

[0037] The preparation of embodiment 2 monoclonal antibody

[0038] 2.1 Immunization of mice

[0039] The CNPAOZ-KLH conjugate prepared in Example 1 was used as the immunogen to immunize Balb / C female mice (purchased from Hubei Provincial Center for Disease Control and Prevention). For the first immunization, a protein emulsion containing 50 μg of immunogen emulsified with an equal volume of Freund’s complete adjuvant was injected subcutaneously on the back of the neck of the mouse for basic immunization, and then every 15 days, protein emulsion containing 50 μg of immunogen emulsified with Freund’s incomplete adjuvant was used for basic immunization. The protein emulsion of the immunogen strengthens the immunity. From the third immunization, the tail blood of the mice was collected on the 7th day after each immunization, the serum was separated, and the serum antibody titer was detected by the commonly used indirect ELISA method (refer to the method reported by Dong Guowei a...

Embodiment 3E

[0044] The establishment of embodiment 3ELISA detection method

[0045] 3.1 Determination of the best reaction conditions

[0046] By square matrix titration (results are shown in Table 1), the coating concentration increases along with the dilution of antibody, and its OD value declines not obviously, illustrates that the affinity of coating former of the present invention and antibody is not strong, and this is to the competition of medicine. It is more advantageous, which is also one of the reasons why the present invention chooses CPAOZ-BSA as the coating agent. Therefore, the original coating concentration of 1ppm CPAOZ-BSA was finally selected as the optimal coating concentration.

[0047] Table 1 Determination of the best ELISA reaction conditions

[0048]

[0049]

[0050] 3.2 Establishment of standard curve

[0051] Prepare AOZ standard products into 6 concentration gradients of 0, 0.05, 0.1, 0.2, 0.4, and 0.8 μg / L, with 2 parallel wells for each concentratio...

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Abstract

The invention belongs to the technical field of veterinary drug residue analysis and immuno-analysis, and specifically relates to a monoclonal antibody for detecting furazolidone metabolites, an ELISA method, and a kit. The invention mainly comprises the following steps: synthesizing semiantigen, immunogen, and coating antigen, preparing a monoclonal antibody, and establishing an ELISA method. The provided kit is mainly composed of an monoclonal antibody for preventing furazolidone metabolites (AOZ), horse radish peroxidase labeled goat-anti-mouse IgG, and an ELISA plate, in which a coating antigen specifically bonded with the AOZ monoclonal antibody is coated. The provided ELISA kit has the advantages of high sensitivity and high performance/price ratio and can be used to detect the residues of furazolidone metabolites (AOZ) in aquatic products.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue analysis, in particular to a monoclonal antibody (monoclonal antibody) for detecting furazolidone metabolites, an ELISA (enzyme-linked immunosorbent) method and a kit. Background technique [0002] Furazolidone belongs to nitrofuran drugs. Because it is difficult to absorb after oral administration, its concentration in the intestine is high and the concentration in the blood is low, so it is suitable for the treatment of various intestinal infections. This type of drug has certain toxicity to livestock and poultry, large dose or long-term continuous use, prone to toxic reactions, manifested as anorexia, diarrhea, gastrointestinal bleeding, peripheral neuritis, excitement, convulsions, paralysis, etc., and severe poisoning can cause animal death . Furazolidone is rapidly metabolized in the body, and its metabolite 3-amino-2-oxazolidinone (AOZ) can bind to proteins to form a more s...

Claims

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Application Information

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IPC IPC(8): C07K16/44C12N5/20G01N33/577C12R1/91
Inventor 冯亮周琪陈建军
Owner WUHAN SHANGCHENG BIOTECH
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