Diclazuril ELISA (enzyme linked immunosorbent assay) kit as well as construction and detection method

An enzyme-linked immunosorbent reagent and diclazuril technology, which is applied to measurement devices, instruments, scientific instruments, etc., can solve the problems of complicated operation process, limited application scope, long detection time, etc. Effects with low pre-processing requirements

Inactive Publication Date: 2017-04-26
ZHENJIANG XIANCHUANG BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, there are few foreign detections of diclazuril residues in chicken tissues: the methods generally include gas chromatography, liquid chromatography, and liquid chromatography-mass spectrometry, and most of them detect multiple drugs at the same time; but commonly used physical and chemical analysis The method requires expensive instruments and equipment, skilled professionals and other conditions, and the operation process is complicated and the detection time is long, so its application range is limited, and it is difficult to popularize and apply in actual production

Method used

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  • Diclazuril ELISA (enzyme linked immunosorbent assay) kit as well as construction and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The synthesis of embodiment 1 immunogen and the preparation of monoclonal antibody

[0045] 1.1 Reagents and instruments

[0046] Diclazuril was purchased from Sigma, N, N-dicyclohexylcarbodiimide and N-hydroxysuccinimide were purchased from Shanghai Chemical Reagent Company, bovine serum albumin, chicken ovalbumin, Freund's complete adjuvant and Freund's incomplete adjuvant from Pierce products.

[0047] Bio-Rad imark 680 microplate reader, AE260 electronic balance, purchased from Germany METTLER company; HI9321 acidity meter, American HANNA company; hand-held homogenizer, purchased from Germany IKA company; 93-3 time constant temperature two-way magnetic stirrer, purchased from Shanghai Yarong Biochemical Instrument Factory.

[0048] 1.2 Synthesis of Diclazuril Artificial Antigen

[0049] (1) Weigh 3 mg of diclazuril, 3 mg of N-hydroxyxaporil and 3 mg of N, N-dicyclohexylcarbodiimide and dissolve them in 1 mL of DMF solvent, react at room temperature for 3 hours, Ge...

Embodiment 2

[0061] Embodiment 2 The establishment of immunoassay method

[0062] 2.1 ELISA method to determine the optimal coating concentration

[0063] Coat the enzyme with ovalbumin-diclazuril conjugate at a concentration of 10 μg / mL, 5 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL in an amount of 50 μL per well The target plate was coated at 4°C for 2 hours, washed 4 times, patted dry, added blocking solution, blocked at 4°C for 24 hours, washed 4 times, and patted dry. Join 1:8×10 4 50 μL / well of diluted antiserum was incubated at room temperature for 30 min, washed 4 times, and 50 μL / well of enzyme-labeled goat anti-mouse antibody was immediately added. React at room temperature for 30 minutes, wash 4 times, add chromogenic solution, 50 μL / well, chromogenic reaction at room temperature for 15 minutes, add 50 μL / well stop solution to terminate the reaction, and detect the A value with a microplate reader (450 nm). At the same time, set blank control wells (no antibody, only a...

Embodiment 3

[0075] Embodiment 3 detects the formation of the ELISA kit of diclazuril

[0076] The ELISA kit of diclazuril is set up so that it comprises the following components:

[0077] (1) A microtiter plate coated with diclazuril artificially synthesized antigen;

[0078] (2) 2mL, 8×10 4 times the anti-diclazuril monoclonal antibody;

[0079] (3) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0080] (4) 6 bottles of diclazuril standard solution, the concentrations are 0μg / L, 10μg / L, 20μg / L, 30μg / L, 50μg / L, 100μg / L;

[0081] (5) Substrate chromogenic solution A is carbamide peroxide, and substrate chromogenic solution B is tetramethylbenzidine; chromogenic agent A and chromogenic agent B should be mixed in equal proportions.

[0082] (6) The washing solution is a phosphate buffer containing 0.05% Tween-20;

[0083] (7) The concentrated sample diluent is a phosphate buffered saline solution of 0.1% Tween-20;

[0084] (8) The stop solution is 2mol / L hydrochlor...

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Abstract

The invention discloses an ELISA (enzyme linked immunosorbent assay) kit for detecting diclazuril, which comprises a kit body, wherein a diclazuril specific antibody, a conjugate and a conjugate reagent of diclazuril and carrier protein, an ELISA plate coated with a diclazuril antigen or specific antibody, a diclazuril standard solution, a substrate developing solution, a cleaning solution and a stop solution are arranged in the kit body; the diclazuril specific antibody is a diclazuril monoclonal antibody. In the diclazuril ELISA kit, main reagents are provided in the form of working solution, the use is convenient and the price is low; during the assay, the pretreatment requirement on a sample is low, the treatment process is simple, and a large batch of samples can be rapidly screened at the same time by the ELISA kit; compared with instrumental analysis techniques, the ELISA kit has the characteristics of high speed, great convenience, high accuracy, high sensitivity and the like.

Description

technical field [0001] The invention relates to the fields of ELISA and veterinary drug residue detection. In particular, it relates to an enzyme-linked immunoimmunoassay kit for detecting diclazuril residues in feed and its composition and detection method. Background technique [0002] Diclazuril was developed by the Belgian Janssen company in 1986. Under the trade name of Clinicox, it is listed in 43 countries including the UK and Germany. In 1995, it applied to the Ministry of Agriculture of my country for registration of premixes with specifications of 0.2% and 0.5%. It was approved for import in 1997. Diclazuril is insoluble in water, and has very low solubility in organic solvents such as acetonitrile and methanol, and is only slightly soluble in dimethylformamide, tetrahydrofuran and dimethyl sulfoxide. [0003] The original drug of diclazuril has poor solubility in water, and is generally used as a feed additive, and the usual dosage is lppm. In the case of large...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/545
CPCG01N33/545G01N33/577G01N2430/10
Inventor 洪霞刘静张淑雅
Owner ZHENJIANG XIANCHUANG BIOTECH CO LTD
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