Hapten, artificial antigen and monoclonal antibody for tylosin and preparation methods and applications thereof

A technology of monoclonal antibody and tylosin, which is applied in the field of food safety, can solve the problems of unrecognized antibodies, etc., and achieve the effect of shortening the detection time, improving the detection efficiency and reducing the detection cost

Inactive Publication Date: 2012-12-19
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the immunoassay techniques for these kinds of animal-specific macrolide drugs at home and abroad all use the hydroxyl group in the tylosin or tilmicosin molecule to directly link with the carrier protein as an immunogen to prepare antibodies, and the obtained Antibodies do not recognize other drugs of the same class

Method used

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  • Hapten, artificial antigen and monoclonal antibody for tylosin and preparation methods and applications thereof
  • Hapten, artificial antigen and monoclonal antibody for tylosin and preparation methods and applications thereof
  • Hapten, artificial antigen and monoclonal antibody for tylosin and preparation methods and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Experiment 1: Synthesis example of tylosin hapten with structure of formula 1

[0033] (a) Add 20 mL of distilled water and 0.5 mL of glacial acetic acid to a three-neck flask, then add 1 mmol of tylosin tartrate and 1 mmol of 6-aminocaproic acid, stir to dissolve, and then heat to reflux until the reaction solution turns yellow.

[0034] (b) Then, the yellow solution was concentrated and evaporated at 40°C to obtain a yellow solid. The yellow solid was dissolved in 10 mL of ethanol, and 20 mL of ethyl acetate was added to the ethanol solution to precipitate a solid product. The precipitate was dried at 60°C to obtain the tylosin hapten. Its structural formula is as shown in formula 1:

[0035]

[0036] Formula 1.

[0037] The product has a melting point of 156°C; infrared spectrum (IR) characterization data: (KBr)V max 3350, 3399, 3200, 2973, 2929, 2830, 1619, 1530, 1500, 1390, 1110, 1066, 921, 611cm -1 . Compared with the infrared data of tylosin, there are mo...

Embodiment 2

[0060] Experiment 1: Antibody Preparation

[0061] (1) Using the tylosin artificial antigen prepared in the above-mentioned Example 1 as the immunogen, immunize 5 Balb / C mice with an immunization dose of 100-300 μg / only, and the immunization method is as follows: mix the immunogen with an equal amount of The complete adjuvant was fully emulsified and injected subcutaneously at multiple points on the back of the neck. At intervals of 2-3 weeks, after the immunogen was emulsified with an equal amount of Freund's incomplete adjuvant, a booster immunization was given, and a total of 6 booster immunizations were given.

[0062] (2) Seven days after the last immunization, the mouse with the highest serum titer in the above step 1) was selected and sacrificed by dislocation of the cervical spine. Under sterile conditions, the spleen was removed, splenocytes were isolated, and fused with mouse myeloma SP2 / 0 cells at a ratio of 10:1. Positive hybridoma cells were screened using tylos...

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Abstract

The invention discloses a hapten, an artificial antigen and a monoclonal antibody for tylosin and preparation methods and applications thereof. The hapten contains a common structural part of tylosin, tilmicosin, acetyl-isovaleryl tylosin and a tylosin metabolin, i.e., cladinose-removed tylosin; and in the preparation process of a tylosin artificial antigen, introduced carboxyl is connected with a carrier protein, i.e., the common structural part is kept far away from the carrier protein and is fully exposed to an immunological recognition of animals. A broad-spectrum specific antibody which can be used for recognizing the tylosin, tilmicosin, the acetyl-isovaleryl tylosin and the tylosin metabolin, i.e., cladinose-removed tylosin simultaneously can be obtained by taking the artificial antigen as an immunogen immune animal. When the broad-spectrum specific antibody is used for rapidly immunologically analyzing and sieving, the detection efficiency is increased, the detection time is shortened, and the detection cost is lowered.

Description

technical field [0001] The invention relates to the field of food safety, in particular to a hapten, an artificial antigen and a monoclonal antibody of tylosin and a preparation method and application thereof. Background technique [0002] Macrolides are a class of antibiotics with a 14- to 16-membered ring lactone structure. Since the discovery of the representative variety erythromycin in 1952, many varieties have come out. Veterinary clinical applications mainly include erythromycin, tylosin, tilmicosin, tyvanectin, kitasamycin, spiramycin, oleandomycin, etc., of which tylosin, tilmicosin and Tivalamectin is a special drug for animals. Tylosin against mycoplasma and various G + Bacteria have strong antibacterial effect. Tilmicosin has broad-spectrum antibacterial activity similar to tylosin, and has inhibitory effect on Gram-positive bacteria and some Gram-negative bacteria, mycoplasma, spirochetes, etc. Tyvalosin has a stronger antibacterial effect than tylosin. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H17/08C07H1/00C07K14/765C07K14/77C07K1/113C07K16/44G01N33/577
Inventor 刘聚祥王建平王丽莉刘静张会彩
Owner HEBEI AGRICULTURAL UNIV.
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