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Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting mequindox and olaquindox metabolites

An enzyme-linked immunosorbent reagent and monoclonal antibody technology, which is applied in the field of veterinary drug residue analysis and immunology, to achieve the effects of increasing affinity, saving time and cost, and causing less harm to health

Inactive Publication Date: 2015-04-29
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, at present, there are few reports on detection methods for quinoxaline metabolites. Therefore, a monoclonal antibody with good specificity and high sensitivity is established to detect the metabolism of acemetquine and olaquindox in animals or meat foods. Product residues appear to be necessary

Method used

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  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting mequindox and olaquindox metabolites
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting mequindox and olaquindox metabolites
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting mequindox and olaquindox metabolites

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The preparation of embodiment 1 immunogen and coating former

[0034] 1.1 Preparation of carboxyl-dedioxoquine cydodol (COOH-DCYX) and human serum albumin (HSA) conjugate (COOH-DCYX-HSA)

[0035] Take 29.3mg of COOH-DCYX, dissolve in 1mL DMSO, add 15.0mg of NHS and 20.2mg of DCC, activate the reaction at room temperature for 12h, centrifuge to remove the precipitate, and obtain liquid A; take 66.0mg of HSA, dissolve in 6mL of PBS (pH=7.4 ), this is liquid B; add liquid A dropwise to liquid B, and react in the dark at room temperature for 12 hours. After the reaction is completed, transfer the reaction solution into a dialysis bag, dialyze in PBS at 4°C for 5 days, replace the dialysate every 4 to 6 hours, and freeze-dry the sample after the dialysate to obtain the conjugate COOH-DCYX-HSA. Store at 20°C. The reaction formula is as follows:

[0036]

[0037] 1.2 Preparation of desdiomethaquine-carboxymethylhydroxylamine and ovalbumin conjugates (DMEQ-AOAA) and (DMEQ...

Embodiment 2

[0042] The preparation of embodiment 2 monoclonal antibody

[0043]Preparation of hybridoma cells: with reference to Yang Hanchun's "Animal Immunology", the immunogen carboxyl-dedioxycedol (COOH-DCYX)-human serum albumin conjugate (COOH-DCYX-HSA) prepared in Example 1 Immunize Balb / C mice (purchased from Experimental Animal Center of Hubei Academy of Medical Sciences). The immunization procedure is as follows: for basic immunization, a protein emulsion containing 100 μg of immunogen emulsified with an equal volume of Freund’s complete adjuvant is injected subcutaneously at the back of the neck of the mouse; Immunogen protein emulsion for booster immunization. From the third immunization, the tail blood was collected on the 8th day after each immunization, the serum was separated, and the serum antibody titer was detected by indirect ELISA method. Immunization qualified mice (high titer, good sensitivity) stop immunization in preparation for fusion. At the time of fusion, ta...

Embodiment 3

[0048] Example 3 Establishment of desdioxymethoquine and desdioxoquinethanol indirect competition ELISA detection method

[0049] 3.1 Preparation of reagents (the reagents used in this example were prepared by the following methods unless otherwise specified)

[0050] 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;

[0051] 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;

[0052] Washing liquid: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, KCl 0.2g, Tween20 0.5mL, add double distilled water to 1000mL, adjust pH to 7.4;

[0053] Blocking solution: Ovalbumin 1g dissolved in 100mL phosphate buffer;

[0054] Substrate solution A: 3,3',5',5-tetramethylbenzidinediamine (TMB) 200mg, absolute ethanol 100mL, add double distilled water to 1000mL;

[0055] Substrate B: Na 2 HPO 4 14.6g, citric acid 9....

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Abstract

The invention discloses a specific monoclonal antibody capable of distinguishing mequindox and olaquindox metabolites, and an enzyme-linked immunosorbent assay method and kit for detecting the mequindox and olaquindox metabolites. According to the invention, the monoclonal antibody is secreted by a hybridoma cell strain DMEQ1A3 of which the preservation number is CCTCC N0.C201496. Compared with the prior art, the monoclonal antibody prepared by the invention can distinguish desoxy mequindox and desoxy olaquindox at the same time, and has higher sensitivity and specificity. The ELISA method and the kit, disclosed by the invention, can detect the residue of the mequindox and olaquindox metabolites in meat food at the same time, and have the advantages of high detection efficiency, high sensitivity, high precision, high accuracy and the like.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue analysis and immunology, and relates to a monoclonal antibody capable of recognizing metabolites of methaquine and olaquindox and an enzyme-linked immunosorbent method (ELISA) for detecting metabolites of methaquine and olaquindox ) and kits. Background technique [0002] Desdioximethaquine and desdioxoquinethanol belong to quinoxaline drugs, which are the products of the deoxygenation metabolism of the prototype drugs acetylmethaquine and olaquindox in animals, and have the basic Structure, acemethaquine and olaquindox have a broad antibacterial spectrum, and their anti-gram-negative bacteria are stronger than positive bacteria. They have been widely used in veterinary clinics and aquaculture. People abuse drugs in pursuit of economic benefits, resulting in a large amount of drug residues in animal food. Quinoxaline drugs can cause toxic and side effects such as tertiary effects...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44C12N5/20G01N33/577G01N33/543
Inventor 袁宗辉何秀平彭大鹏潘源虎王玉莲陈冬梅陶燕飞刘振利
Owner HUAZHONG AGRI UNIV
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