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Tylosin hapten, artificial antigen and antibody as well as preparation methods and application thereof

A technology of tylosin and artificial antigen, which is applied in chemical instruments and methods, animal/human protein, sugar derivatives, etc., to achieve the effects of high sensitivity and accuracy, low cost and simple method

Active Publication Date: 2018-06-19
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved in the present invention is to overcome the defects and deficiencies of the above-mentioned existing tylosin residue detection technology, and to provide a new tylosin hapten structure, specifically the macrolide side of the tylosin molecule The acetaldehyde group of the chain is directly oxidized into a carboxyl group as a hapten, and the oxidized acetic acid arm is of moderate length, not suitable for folding, and has a high degree of overlap with the skeleton structure, which maintains the three-dimensional structure of telosin to the greatest extent, and is conducive to the stable immune induction of antibodies , and then the hapten is coupled to the carrier protein, its specific structure protrudes from the surface of the carrier protein, and an antigenic epitope as the carrier is exposed to the animal immune system, and specific antibodies can be obtained, laying the foundation for the establishment of an immune detection method

Method used

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  • Tylosin hapten, artificial antigen and antibody as well as preparation methods and application thereof
  • Tylosin hapten, artificial antigen and antibody as well as preparation methods and application thereof
  • Tylosin hapten, artificial antigen and antibody as well as preparation methods and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The preparation of embodiment 1 tylosin hapten

[0048] (1) Dissolve 1 g of Tylosin tartrate in a mixture of 50 mL of tert-butanol and 30 mL of water, and add 0.62 g of NaH 2 PO 4 , 1mL dimethyl sulfoxide, 0.2g NaClO 2 , react for 30min;

[0049] (2) Ethyl acetate (50 mL) and water (10 mL) were added to the reaction solution for extraction three times, and the organic phase was collected. The aqueous phase was extracted three more times with ethyl acetate (50 mL). The combined organic phases were washed with water (20 mL) and brine (20 mL), washed with anhydrous MgSO 4 Dry and filter. After the filtrate was rotary evaporated under reduced pressure, it was purified by silica gel column chromatography (methanol:chloroform=1:10) to obtain a white solid, which was the hapten.

[0050] The mass spectrum of tylosin hapten is as follows figure 2 As shown, an ion peak with m / z of 932.5 appeared, indicating that the molecular weight of the product after deprotonation was...

Embodiment 2

[0052] The preparation of embodiment 2 tylosin artificial antigen

[0053] (1) Weigh 93.2mg (0.1mmoL) of the hapten prepared in Example 1 and dissolve it in 0.5mL DMF, stir and add 51.2mg (0.2mmol) DCC and 23mg (0.2mmoL) NHS, and react overnight with magnetic stirring at 4°C , and the supernatant after centrifugation was liquid A.

[0054] (2) Weigh 15 mg of carrier protein (BSA, OVA) and dissolve it in 5 mL of 0.1 mol / L phosphate buffer solution (pH=8.0), stir and dissolve to prepare solution B. Under magnetic stirring, solution A was gradually dropped into solution B, and reacted at 4°C for 12 hours.

[0055] (3) Put the reaction solution into a dialysis bag, dialyze with a phosphate buffer solution of pH=8.0 at 4° C. for 3 days, and change the dialysis solution 2 to 3 times a day to obtain the tylosin artificial antigen.

[0056] The artificial antigen structural formula of tylosin is shown in formula (II):

[0057]

Embodiment 3

[0058] The preparation of embodiment 3 tylosin antibody

[0059] The antibody preparation method described in this example refers to conventional methods in the art.

[0060] Mix the tylosin artificial antigen with an equal dose of immune adjuvant (Freund's complete adjuvant for the first immunization, and Freund's incomplete adjuvant for subsequent booster immunizations), emulsify completely with a stirrer, and place on the back. Healthy New Zealand white rabbits were immunized subcutaneously, and boosted immunization every three weeks thereafter. Use indirect ELISA to measure the serum titer and inhibition rate. After the titer is stable, boost the immunization again. Blood was collected from the heart one week after the last immunization, and the serum was retained by centrifugation. The serum was purified by octanoic acid-ammonium sulfate salting-out method to obtain highly specific antibodies.

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Abstract

The invention discloses tylosin hapten, artificial antigen and antibody as well as preparation methods and application thereof. Specifically, a glyoxyl functional group of a large ring side chain of tylosin is used as a derived coupling site; formyl is converted into carboxyl through a one-step oxidization method, so as to obtain the tylosin hapten with a carboxymethylated coupling arm. The obtained hapten and a framework structure have a high overlapping degree and a foundation is laid for exposure of a characteristic structure of the tylosin and production of a high-specificity antibody. Furthermore, the hapten and carrier protein are coupled to obtain the artificial antigen and an animal is immunized to obtain the tylosin high-specificity antibody; an established enzyme linked immunosorbent assay has high accuracy and good sensitivity; the detection limit reaches 0.051ng / mL and the half inhibitory concentration is 1.39ng / mL; the enzyme linked immunosorbent assay is simple to operateand low in cost, is very suitable for field detection, can be used for rapidly and effectively detecting the tylosin in environment and food samples and has a wide application prospect and economic value.

Description

technical field [0001] The invention belongs to the technical field of drug residue detection. More specifically, it relates to a tylosin hapten, artificial antigen and antibody, a preparation method and application thereof. Background technique [0002] Tylosin (TYL), also known as Tylosin, is a macrolide antibiotic obtained by Hamill et al. in 1959 from the culture medium of Streptomyces freundii. TYL has obvious inhibitory effect on Gram-positive bacteria, and also has inhibitory effect on some Gram-negative bacteria. It has a good therapeutic effect on mycoplasma diseases in livestock and poultry, and is the first choice drug for treating mycoplasma diseases. It has a certain inhibitory and killing effect on most spirochetes, viruses, etc., and can also promote animal growth and improve feed efficiency, so it is widely used in animal husbandry. However, the residues of these drugs will cause allergic reactions (rash, anaphylactic shock, etc.) in the human body, inhibit...

Claims

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Application Information

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IPC IPC(8): C07H17/08C07K14/765C07K14/77C07K14/795C07K16/44G01N33/58
CPCC07H17/08C07K14/765C07K14/77C07K14/795C07K16/44G01N33/581
Inventor 沈玉栋姚婵媛杨金易王弘徐振林孙远明韦晓群
Owner SOUTH CHINA AGRI UNIV
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