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Enzyme-linked immunosorbent assay kit for detecting diclazuril and application thereof

An enzyme-linked immunosorbent reagent, diclazuril technology, applied in the direction of biological testing, measuring devices, material inspection products, etc., can solve the problems that it is difficult to meet the rapid detection of a large number of samples and on-site samples, the pretreatment process is complicated, and the cost is high. The inspection method is convenient and easy, the pretreatment process is simple, and the effect of high accuracy is

Active Publication Date: 2018-12-21
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods can simultaneously carry out the multi-residue qualitative and quantitative analysis of diclazuril and other similar drugs, but require expensive instruments and specialized technicians. testing needs

Method used

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  • Enzyme-linked immunosorbent assay kit for detecting diclazuril and application thereof
  • Enzyme-linked immunosorbent assay kit for detecting diclazuril and application thereof
  • Enzyme-linked immunosorbent assay kit for detecting diclazuril and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The preparation of embodiment 1 kit components

[0028] 1. The synthesis of diclazuril hapten (see the attached figure 1 )

[0029] Take 1 g of diclazuril, add 5 mL of glacial acetic acid to dissolve and clarify, add 20 mL of aqueous hydrobromic acid, heat at 100 ° C, and stir for 2 h. Stop the reaction, add water 100mL, add 1mol / L NaOH to neutralize to pH 6, add ethyl acetate 200mL×3, extract three times, combine organic phases, dry with 20g of anhydrous sodium sulfate, evaporate to dryness, put on a silica gel column, ethyl acetate / Petroleum ether (v / v, 1 / 1) was eluted and separated to obtain 0.91 g of carboxyclazuril hapten product with a yield of 87.5%.

[0030] 2. Synthesis and identification of diclazuril conjugated antigen

[0031] Immunogen preparation—diclazuril hapten was coupled with bovine serum albumin (BSA) to obtain immunogen.

[0032] Take 16mg of carboxyclazuril hapten, add N,N-dimethylformamide 0.5mL to dissolve, add N-hydroxysuccinimide (NHS) 6mg...

Embodiment 2

[0061] Embodiment 2 detects the formation of the ELISA kit of diclazuril

[0062] An enzyme-linked immunosorbent assay kit for detecting diclazuril was constructed to include the following components:

[0063] (1) A microtiter plate coated with a diclazuril-coupled antigen;

[0064] (2) 5 bottles of diclazuril standard solution, the concentrations are 0 μg / L, 1 μg / L, 3 μg / L, 9 μg / L, 27 μg / L;

[0065] (3) Diclazuril monoclonal antibody working solution;

[0066] (4) goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0067] (5) Substrate chromogenic solution is made up of A liquid and B liquid, and A liquid is carbamide peroxide, and B liquid is tetramethylbenzidine;

[0068] (6) The stop solution is 2mol / L sulfuric acid;

[0069] (7) The washing solution has a pH value of 7.4 and contains 0.5% to 1.0% Tween-20, 0.01‰ to 0.03‰ sodium azide, and 0.1 to 0.3mol / L phosphate buffer;

[0070] (8) The complex solution is a phosphate buffer solution with a pH valu...

Embodiment 3

[0071] The detection of diclazuril in the sample of embodiment 3

[0072] 1. Sample pretreatment

[0073] Weigh 1.0g ± 0.05g tissue sample into a 50mL centrifuge tube, add 1mL 0.1mol / L sodium hydroxide, vortex for 1min with a vortex, then add 7mL acetonitrile, vortex for 2min with a vortex, 3000g at room temperature (20 ~25℃) for 5min; pipette 1mL of the upper organic phase into a 10mL clean and dry glass tube, and dry it in a water bath at 50~60℃ under nitrogen flow; add 1mL of complex solution and vortex for 30s with a vortexer; take 50μL for analysis .

[0074] 2. Detection with kit

[0075] Add 50 μL / well of diclazuril standard solution or pretreated sample solution to the microwells of the microtiter plate coated with diclazuril-conjugated antigen, and then add horseradish peroxidase-labeled goat anti-mouse Add 50 μL / well of anti-antibody, then add 50 μL / well of diclazuril monoclonal antibody working solution, shake gently to mix, cover the plate with a cover film and ...

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Abstract

The invention provides an enzyme-linked immunosorbent assay kit for detecting diclazuril. The enzyme-linked immunosorbent assay kit comprises an enzyme linked immunosorbent assay plate which is coatedby diclazuril coupling antigens, diclazuril monoclonal antibodies, enzyme labeled anti-antibodies, diclazuril standard substance solution, substrate developing solution, stop solution, cleaning solution and compound solution. The invention also discloses a method for detecting the diclazuril through application of the enzyme-linked immunosorbent assay kit. The method comprises the steps of carrying out sample pretreatment; carrying out detection through utilization of the kit; and analyzing a detection result. The enzyme-linked immunosorbent assay kit provided by the invention can be used fordetecting content of the diclazuril in animal tissues. The enzyme-linked immunosorbent assay kit is simple and convenient in operation, low in cost and high in sensitivity. Field monitoring can be carried out. The enzyme-linked immunosorbent assay kit is applicable to screening of a large number of samples.

Description

technical field [0001] The invention relates to an enzyme-linked immunoassay technology, in particular to an enzyme-linked immunoassay kit for detecting diclazuril, which is particularly suitable for detecting the content of diclazuril in animal tissues. Background technique [0002] Diclazuril is a non-polyether chemically synthesized anticoccidial drug developed by the Belgian Janssen Pharmaceutical Company in the 1980s. It belongs to the triazine benzyl acetonitrile compound. It has the characteristics of high efficiency, low toxicity and broad spectrum. It is often used in Prevention and treatment of anticoccidiosis in the poultry and rabbit industries. Diclazuril can inhibit or kill sporozoites in aquatic animals, and it is mainly used in aquaculture to prevent and control sporozoites such as myxospores, iodine worms, cercospores, tetrapolar worms, and monopolar worms in aquaculture. sick. Because diclazuril has the characteristics of low toxicity, broad spectrum, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/58G01N33/543G01N33/535
CPCG01N33/535G01N33/543G01N33/581
Inventor 万宇平何方洋崔海峰王琳琛韩深王兆芹韩光耀冯才伟杨春艳杨烁
Owner BEIJING KWINBON BIOTECH
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