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Tildipirosin hapten derivative, and preparation method and detection kit thereof

A technology of tediloxine and hapten, applied in the field of immunology, can solve the problems of complex veterinary drug structure of immune response, and achieve the effect of good potency

Active Publication Date: 2018-06-22
WUHAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, a lot of work has been done on the synthesis of artificial antigens at home and abroad, and there are many experiences that can be used for reference. However, due to the complexity of the immune response and the diversity of veterinary drug structures

Method used

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  • Tildipirosin hapten derivative, and preparation method and detection kit thereof
  • Tildipirosin hapten derivative, and preparation method and detection kit thereof
  • Tildipirosin hapten derivative, and preparation method and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Preparation of Tedirosine Hapten Derivatives

[0031] Weigh 750 mg of tedirosine and dissolve it in 10 mL of dichloromethane, then add 1.1 times the molar equivalent of tedirosine TMSCl and 10 mg of imidazole, stir and react at 25°C for 4 hours, wash the reacted solution with water, and keep the lower layer two Methylene chloride organic phase, after drying it with anhydrous sodium sulfate, distill off the solvent dichloromethane; dissolve all the products after distilling off the solvent in 10mL of dichloromethane, and then add 1.5 times the molar equivalent of tedirosine Succinic anhydride and 10mL pyridine, stirred and reacted overnight at 25°C, monitored with thin-layer chromatography TLC, stopped the reaction after the reaction raw materials had been reacted, washed the solution after the reaction with a small amount of water, kept the lower methylene chloride organic phase, and After it was dried with anhydrous sodium sulfate, it was deprotected by addi...

Embodiment 2

[0034] Example 2: Synthesis of Tedirosine Complete Antigen

[0035] Get 10mg of the tedirosine hapten derivative (0.012mmol) obtained in Example 1, 3mg N-hydroxysuccinimide NHS (0.024mmol) and 5mg DCC (0.024mmol) and add in 0.5mL solvent (DMF and 1, 4 Dioxane (prepared according to the volume ratio of 1:1) dissolved, at room temperature 25 ° C, magnetic stirring reaction overnight, the solution after the reaction is A solution (if there is precipitation in A solution, filter to get the supernatant Then proceed to the next step); Take 16mg of BSA (0.0002mmol), dissolve it with 5mL of pure water to form B solution; drop A solution into B solution, and then react at room temperature for 8h at 25°C; after the reaction, in Dialyze with 0.01mol / L, pH=7.4 PBS buffer as the dialysate at 4°C for 3 days, change the dialysate twice a day, and centrifuge after the dialysis to obtain the Tedirosin-BSA conjugate, which is Taidi Roxin-BSA complete antigen.

[0036] The above-mentioned BSA ...

Embodiment 3

[0037] Embodiment three: the preparation of monoclonal antibody, comprises the following steps:

[0038] 1. Antigen immunization animals

[0039] Antigen quality control of Tedirosin-OVA complete antigen and Tedirosin-BSA complete antigen prepared in Example 2, such as image 3 shown.

[0040] way of immunization

[0041] Subcutaneous injection, Freund's immunization and rapid immunization were used to immunize 22 SPF Balb / c mice in 6 batches, 8 weeks old, female, from the Chinese Center for Disease Control and Prevention.

[0042] Experimental operation

[0043] Freund's immunity

[0044] Mice were immunized with 50 μg of Tedirosin-BSA complete antigen prepared in Example 1 for each dose, and the total injection volume was 500 μL, including 250 μL of complete Freund’s adjuvant, complete antigen and 0.01mol / L, pH=7.4 The total amount of PBS buffer was 250 μL. After the preparation was completed, it was emulsified with an emulsifying mixer for 40 minutes, and then injected s...

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Abstract

The invention relates to the field of immunology, in particular to a tildipirosin hapten derivative, and a preparation method and a detection kit thereof. The preparation method specifically comprisesthe following steps: first, reacting TMSCl (Trimethylchlorosilane) and tildipirosin to obtain C-2 site-OH TMS protected tildipirosin; then, reacting the C-2' site-OH TMS protected tildipirosin with succinic anhydride so as to esterify C-3 site-OH; finally, deprotecting to obtain C-3 site esterification product, namely, the tildipirosin hapten derivative; coupling with carrier protein to completethe synthesizing of a complete antigen; immunizing a mouse by taking the tildipirosin complete antigen as an artificial immunogen to obtain serum, taking tildipirosin-VOA complete antigen as a coatingantigen to complete the preparation of a monoclonal antibody, subcloning screening, preparation of a tildipirosin enzyme linked immunosorbent assay kit and the like. A main reagent of the kit is provided in the form of working fluid; a detecting method is convenient and feasible, and has the characteristics of high specificity, high sensitivity, high precision, high accuracy and the like.

Description

technical field [0001] The invention relates to the field of immunology, in particular to a tedirosin hapten derivative, a preparation method thereof, and an enzyme-linked immunoassay kit for detecting tedirosin residues in animal foods. Background technique [0002] In the immunochemical residue analysis of veterinary drugs, whether a stable artificial antigen with good immunogenicity can be synthesized is the most critical step in the preparation of antibodies and the establishment of immunoassay methods. At present, a lot of work has been done on the synthesis of artificial antigens at home and abroad, and there are many experiences that can be used for reference. However, due to the complexity of the immune response and the diversity of veterinary drug structures. For a new and specific veterinary drug to be tested, the synthesis of its artificial antigen should be based on the structural characteristics, and specific issues need to be treated specifically. The most ide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H17/08C07H1/00G01N33/577G01N33/535
CPCC07H1/00C07H17/08G01N33/535G01N33/577
Inventor 何斌陈洁刘武姚艳丰杨文海邵志勇吴利军陈夏冰金尔光冉志平陶弼菲邓蓉蓉周华
Owner WUHAN ACADEMY OF AGRI SCI
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