Monoclonal antibody and enzyme-linked immunosorbent assay kit for detecting amantadine and rimantadine

An enzyme-linked immunosorbent reagent and monoclonal antibody technology, which is applied in the field of veterinary drug residue analysis and immunology, can solve the problems of no simultaneous detection of amantadine ELISA method, etc., and achieve the effect of easy operation, good precision, and low operator requirements

Active Publication Date: 2017-04-19
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is currently no report on an ELISA method for th

Method used

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  • Monoclonal antibody and enzyme-linked immunosorbent assay kit for detecting amantadine and rimantadine
  • Monoclonal antibody and enzyme-linked immunosorbent assay kit for detecting amantadine and rimantadine
  • Monoclonal antibody and enzyme-linked immunosorbent assay kit for detecting amantadine and rimantadine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation of Immunogen and Coating Original

[0030] 1.1 Preparation of immunogen RMA-HS-EDC-KLH

[0031] Weigh 1 g of rimantadine (RMA) and 2 g of succinic anhydride (HS) in a round bottom flask, dissolve them in 8 ml of pyridine, and reflux for 2 days in an oil bath at 90°C with magnetic stirring. After the reaction was completed, the solvent pyridine was rotary evaporated, redissolved with methanol and then rotary evaporated, and repeated twice. Dissolve the product with sodium hydroxide solution and adjust the pH to alkaline (PH=9-10). Wash the lye with ethyl acetate, discard the organic phase, and repeat three times. Finally, adjust the pH of the obtained aqueous phase to neutral (PH=6-7) with hydrochloric acid, wash it three times with ethyl acetate, discard the organic phase and dry it to obtain a yellow-brown product, which is the hapten rimantadine hemisuccinic acid Esters (RMA-HS).

[0032] Weigh 18 mg of hapten (RMA-HS), 15 mg of water-soluble...

Embodiment 2

[0035] Example 2 Preparation of Monoclonal Antibody

[0036] 2.1 Animal immunity

[0037] The immunogen (RMA-HS-EDC-KLH) prepared by the inventor's National Laboratory for Veterinary Drug Residues was used to immunize Balb / C mice (purchased from the Experimental Animal Center of Hubei Provincial Center for Disease Control and Prevention). The immunization procedure is to take the immunogen RMA-HS-EDC-KLH, mix them with equal amounts of adjuvant and inject them into the mice to produce specific serum.

[0038] 2.2 Cell fusion and cloning

[0039] With reference to Yang Hanchun's "Animal Immunology", the RMA-HS-EDC-KLH immunogen prepared by the inventor's National Laboratory for Veterinary Drug Residues was used to immunize Balb / C mice (purchased from the Experimental Animal Center of the Hubei Provincial Center for Disease Control and Prevention). The procedure is as follows: after the basic immunization, the immunogen was emulsified with an equal volume of complete Freund's ...

Embodiment 3

[0044] Example 3 Establishment of Indirect Competitive ELISA Detection Method

[0045] 3.1 Preparation of reagents (the reagents used in this example were prepared by the following methods unless otherwise specified)

[0046] Phosphate buffer: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 .12H 2 O 2.9g, KCl 0.2g, add double distilled water to 1000mL, adjust pH to 7.4;

[0047] Coating solution: Take Na 2 CO 3 1.59g, NaHCO 3 2.93g, add triple distilled water to 1000mL, adjust the pH value to 9.6;

[0048] Washing liquid: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 .12H 2 O 2.9g, KCl 0.2g, Tween 200.5mL, add double distilled water to 1000mL, adjust pH to 7.4;

[0049] Blocking solution: 1.5g skimmed milk powder dissolved in 100mL phosphate buffer;

[0050] Substrate solution A: 3,3',5',5-tetramethylbenzidinediamine (TMB) 200mg, absolute ethanol 100mL, add double distilled water to 1000mL;

[0051] Substrate B: Na 2 HPO 4 14.6g, citric acid 9.3g, 0.75% urea hydrogen pero...

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Abstract

The present invention discloses a specific monoclonal antibody capable of recognizing amantadine and rimantadine, wherein the specific monoclonal antibody is secreted by a hybridoma cell line 2G3, and the hybridoma cell line 2G3 is preserved in China Center for Type Culture Collection, and has the preservation number of CCTCC NO:C201625. The invention further discloses an enzyme-linked immunosorbent assay method for detecting the amantadine residue and the rimantadine residue in edible animal tissues, and a kit thereof. According to the present invention, the prepared monoclonal antibody has advantages of high sensitivity, high detection accuracy, good precision, gradual change, rapidness, easy operation and the like, and overcomes the gap in the ELISA method on the market.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue analysis and immunology, and in particular relates to a monoclonal antibody capable of recognizing amantadine (AMA) and rimantadine (RMA), an enzyme-linked immunosorbent method (ELISA) and a kit. Background technique [0002] Amantadine is the most effective human antiviral drug against influenza virus, but it is also widely used in animal husbandry, mainly using amantadine hydrochloride and rimantadine hydrochloride to treat and prevent economic animals such as chickens and pigs Influenza and other common clinical viral diseases. [0003] In addition to affecting the weight gain of laying hens, amantadines have side effects such as neurotoxicity and gastrointestinal reactions, and the problem of drug resistance is becoming more and more serious. Drug-resistant viruses have stronger pathogenicity, transmissibility and Cross-resistance between different drugs. In order to avoid th...

Claims

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Application Information

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IPC IPC(8): C07K16/44C12N5/20G01N33/94G01N33/577C12R1/91
CPCC07K16/44G01N33/577G01N33/94
Inventor 袁宗辉彭大鹏魏薇戴梦红王玉莲潘源虎陈冬梅周琪冯亮
Owner HUAZHONG AGRI UNIV
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