ELISA kit for detecting roxarsone, and application thereof

An enzyme-linked immunoassay detection and kit technology, which is applied in measurement devices, instruments, scientific instruments, etc., can solve problems such as environmental pollution, and achieve the effects of convenient use, high sensitivity, and rapid detection methods.

Inactive Publication Date: 2016-05-11
JIANGSU WISE SCI & TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A large amount of animal excrement containing arsine enters the environment, causing pollution to the environment

Method used

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  • ELISA kit for detecting roxarsone, and application thereof
  • ELISA kit for detecting roxarsone, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The preparation of embodiment 1 kit components

[0036] 1. Roxarsone hapten preparation

[0037] 1) Dissolve 20mg of roxarsone, 10ul, 1,3-propylenediamine, and a catalytic amount of 4-dimethylaminopyridine in 2ml of N,N'-dimethylformamide to obtain liquid I;

[0038] 2) Dissolve 20mg of N,N'-dicyclohexylcarbodiimide in 0.5ml of DMF to obtain liquid II;

[0039] 3) Slowly add liquid II to liquid I dropwise at 0°C, and continue to react for 20 hours after returning to room temperature;

[0040] 4) Evaporation of solvent, column chromatography (eluent: dichloromethane / methanol, volume ratio 20:1), to obtain roxarsone hapten, such as figure 1 .

[0041] 2. Antigen preparation

[0042] Immunogen preparation—Roxarsone hapten was coupled with bovine serum albumin (BSA) to obtain immunogen.

[0043] 1) Dissolve 5.3 mg of roxarsone hapten in 0.5 ml of DMF to obtain liquid I;

[0044] 2) Dissolve 30 mg of BSA in 2.0 ml, pH 7.0, 0.1 mol / L phosphate buffer solution to obtain ...

Embodiment 2

[0065] Example 2 The formation of the enzyme-linked immunosorbent assay kit for detecting roxarsone

[0066] An enzyme-linked immunosorbent assay kit for detecting roxarsone was constructed to include the following components:

[0067] (1) a microtiter plate coated with roxarsone-coupled antigen;

[0068] (2) 6 bottles of roxarsone standard solution, the concentrations were 0ug / L, 1ug / L, 3ug / L, 9ug / L, 27ug / L, 81ug / L.

[0069] (3) concentrated enzyme conjugate;

[0070] (4) enzyme conjugate dilution;

[0071] (5) Substrate chromogenic solution is made up of substrate liquid A liquid and substrate liquid B liquid, and substrate liquid A liquid is carbamide peroxide, and substrate liquid B liquid is tetramethylbenzidine;

[0072] (6) The stop solution is 2mol / L sulfuric acid.

Embodiment 3

[0073] The detection of roxarsone in the sample of embodiment 3

[0074] 1. Sample pretreatment

[0075] Use a homogenizer to homogenize the feed sample; weigh 5.0g±0.05g of the homogenized feed sample into a 50ml polystyrene centrifuge tube, add 25m150% methanol, vibrate vigorously with an oscillator for 5min, over 3000r, room temperature (20 Centrifuge at -25°C / 68-77°F) for 5min; take 500ul of the supernatant to a 2ml polystyrene centrifuge tube, add 500ul of 10% sodium chloride aqueous solution and shake for 1min with a shaker, mix well; take 20ul for analysis.

[0076] 2. Detection with kit

[0077] Add ochratoxin A standard solution / sample 20ul to the microwells of the microtiter plate coated with roxarsone-conjugated antigen, and then add 100ul of enzyme conjugate working solution (dilute the concentrated enzyme conjugate with the enzyme conjugate diluent according to l: 20 volume for dilution), seal the plate with a cover film, react in the dark at 25°C for 10 minut...

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Abstract

The invention provides an ELISA kit for detecting roxarsone. The ELISA kit for detecting roxarsone comprises an ELISA plate coated with a roxarsone coupled antigen, a roxarsone standard substance solution, a concentrated enzyme conjugate, a enzyme conjugate operating fluid, a substrate colorimetric solution and a stopping solution. The invention also discloses a method using the ELISA kit to detect roxarsone in a sample. The method comprises the following steps: preprocessing the sample, detecting roxarsone by using the kit, and analyzing a detection result. The ELISA kit can be used to detect the residual quality of roxarsone in the feed (raw material, batch and concentrate) sample, and has the advantages of simple operation, low cost, high sensitivity, realization of onsite monitoring, and suitableness for screening a large amount of samples.

Description

technical field [0001] The invention relates to an enzyme-linked immunosorbent detection technology, in particular to an enzyme-linked immunosorbent assay kit for detecting roxarsone, which is suitable for the determination of roxarsone in feed (raw materials, batch materials, concentrated materials). technical background [0002] As a feed additive, organic arsine preparations are widely used in the world to promote the growth of poultry, and have achieved considerable economic benefits in poultry breeding. At present, the organic arsine preparations approved by my country and the U.S. Drug and Food Administration (FDA) mainly include roxarsone and arsanic acid. Roxarsone is more commonly used. They all have a broad-spectrum bactericidal effect, and also inhibit and kill intestinal parasites. It can also enhance the permeability of capillaries, mainly acting on the intestinal tract, and improving the physical and chemical environment of the intestinal tract. Has a simila...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531
Inventor 洪霞刘静
Owner JIANGSU WISE SCI & TECH DEV
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