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Preparation method and applications of olaquindox metabolite hapten

A technology for oquindox and metabolites, which is applied in the preparation methods of peptides, chemical instruments and methods, animal/human proteins, etc. The method is simple and feasible, the processing sample volume is large, and the cost is low.

Active Publication Date: 2013-09-18
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The current detection method of olaquindox is mostly chromatography, but it requires complex and expensive instruments, and the sample pretreatment process is cumbersome, which is not suitable for the screening of a large number of samples on site

Method used

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  • Preparation method and applications of olaquindox metabolite hapten
  • Preparation method and applications of olaquindox metabolite hapten
  • Preparation method and applications of olaquindox metabolite hapten

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, preparation and identification of olaquindox metabolite hapten

[0042] 1. Preparation of olaquindox metabolite hapten

[0043] (1) Add 0.38g MQCA, 0.20g GABA and a small amount of DMAP to 20mL dry DMF;

[0044] (2) Slowly add 0.8 g of DCC in 5 mL of dry DMF mixture at 0°C. After the addition, the temperature is naturally raised to room temperature, and the reaction is continued for 40 h;

[0045] (3) The solvent was evaporated, and the condensation product of MQCA and aminobutyric acid was obtained after purification by column chromatography, which was the product shown in formula (1).

[0046] Second, the identification of olaquindox metabolite hapten

[0047] The NMR identification of the olaquindox metabolite hapten prepared in step one.

[0048] NMR see figure 2 . The spectrum shows a carboxyl signal peak around 12.0 and an increased methylene signal peak between 1.5 and 3.5, indicating that the hapten is successfully synthesized.

Embodiment 2

[0049] Embodiment 2, preparation and identification of olaquindox metabolite artificial antigen

[0050] 1. Synthesis of olaquindox metabolite immune antigen

[0051] (1) Fully dissolve 30 mg of the olaquindox hapten in 1 mL of DMF;

[0052] (2) Weigh 50 mg of BSA, make it fully dissolved in 3 mL of PBS (pH 7.2), slowly add the olaquindox hapten solution drop by drop to the BSA solution to obtain solution A;

[0053] (3) Weigh 12.5mg of EDC, dissolve it in 1mL of water, slowly add it to A at room temperature, and stir for 24h;

[0054] (4) Dialyze with 0.01mol / L PBS for 3 days, and change the dialysate twice a day to remove unreacted small molecular substances. Centrifuge at 12,000 rpm for 30 min, collect the supernatant, aliquot, and store at -20°C for later use.

[0055] 2. Synthesis of olaquindox metabolite-coated antigen

[0056] (1) Dissolve 20 mg of olaquindox hapten in 1.0 mL of DMF, cool to 10°C, add 15 μL of isobutyl chloroformate, and stir for 30 minutes at 10°C ...

Embodiment 3

[0062] Example 3, Preparation and Specific Identification of Monoclonal Antibody

[0063] 1. Preparation of olaquindox metabolite monoclonal antibody

[0064] 1. Use 100 μg of the immunogen (MQCA-BSA) prepared above, dissolve the immunogen in normal saline and mix it with Freund’s complete adjuvant in an equal volume, and inject subcutaneously on the back of the neck to immunize Balb / c females aged 6-8 weeks. On the 7th, 14th, and 28th day after the initial immunization, mix the immunogen and Freund's incomplete adjuvant in equal volumes, and each additionally immunize once, and 3 days before the fusion, 100 μg of the immune complex / rat, without Freund's adjuvant Add another immunization.

[0065] 2. Proceed according to the conventional method, take the splenocytes of the immunized mice and mix them with the mouse myeloma cells (SP2 / 0) in the logarithmic growth phase, and then slowly add the preheated fusion agent (PEG4000) within 45 seconds for fusion , suspend evenly with...

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Abstract

The invention discloses olaquindox metabolite hapten, and corresponding artificial antigen and monoclonal antibody. In addition, the invention also discloses a preparation method and applications of the olaquindox metabolite hapten and corresponding artificial antigen and monoclonal antibody. The olaquindox metabolite antigen can be obtained by connecting the olaquindox metabolite hapten with a carrier protein. The olaquindox metabolite antigen can be applied to preparing olaquindox metabolite specific antibody. The preparation method is simple, convenient and feasible, and lower in cost, and the hapten yield is higher. The artificial olaquindox metabolite antigen can produce olaquindox metabolite-targeted specific antibody through immune animal, can be used for preparing an enzyme linked immunosorbent assay kit or a colloidal gold test paper card for detecting the residual olaquindox metabolite in food, and the olaquindox metabolite hapten has various advantages of being simple, fast, large in sample processing quantity, high in sensitivity, strong in specificity and the like.

Description

technical field [0001] The invention belongs to the technical field of food safety, and in particular relates to a method for preparing a olaquindox metabolite hapten, an antigen, and a monoclonal antibody and an application thereof. Background technique [0002] Olaquindox, also known as Kuaiyuling, Bayonox, Olaquin, Olaquindox, Fedan. Molecular formula: C 12 h 13 N 3 o 4 . It is light yellow crystalline powder formed by condensation of 2-methylquinoxaline-1,4-dioxide and ethanolamine by German Bayer company in 1965, slightly soluble in water. [0003] Because of its good broad-spectrum antibacterial effect, it has a good therapeutic effect on digestive tract diseases caused by Gram-negative pathogens such as Escherichia coli and Salmonella, and it can promote the digestion and utilization of feed by livestock and poultry, improve It is widely used in feed and feed additives because of its growth rate and other effects. In recent years, in aquatic feed, olaquindox wa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D241/44C07K14/765C07K14/77C07K1/113C07K16/44G01N33/577
Inventor 何方洋万宇平孙震冯静罗晓琴崔海峰余厚美韩京朋段盈盈
Owner BEIJING KWINBON BIOTECH
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