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Enzyme-linked immunosorbent inspect kit for inspecting sulfa drugs and method thereof

A technology of enzyme-linked immunosorbent reagents and sulfonamides, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of complex instrument equipment pretreatment process, unsatisfactory specificity of sulfonamides, etc.

Active Publication Date: 2009-11-04
BEIJING WANGER BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the complicated equipment and cumbersome pre-treatment process, the instrumental analysis method is not suitable for on-site monitoring and screening of a large number of samples. Among them, the ELISA method has been used as a new screening method for sulfonamide drug residues in animal source foods, such as the Chinese patent Application (200510086777.5) and Chinese patent (200710056432.4) disclose two kinds of ELISA methods for detecting sulfonamide drug residues, but these two method kits are unsatisfactory to the specificity of detecting sulfa drug, all can only detect sulfa drug wherein several drugs

Method used

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  • Enzyme-linked immunosorbent inspect kit for inspecting sulfa drugs and method thereof
  • Enzyme-linked immunosorbent inspect kit for inspecting sulfa drugs and method thereof
  • Enzyme-linked immunosorbent inspect kit for inspecting sulfa drugs and method thereof

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Experimental program
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Effect test

Embodiment 1

[0078] Embodiment 1 Preparation of kit components

[0079] 1. Antigen Synthesis

[0080] a, sulfonamide drug hapten synthesis (synthetic route such as figure 2 )

[0081] 1) Put 10g of para-acetaminobenzenesulfonyl chloride into a 100ml round bottom flask, add 25ml of pyridine to dissolve, add 55ml of pyridine solution containing 0.52g of p-aminobenzoic acid dropwise under vigorous stirring, heat up the oil bath, and react at 44°C 1h. After the reaction stopped, cool to room temperature, spin evaporate, remove pyridine, dissolve with 0.01mol / L sodium hydroxide, add ethyl acetate for extraction, separate the organic phase, wash with water to remove sodium hydroxide, and evaporate to dryness to obtain a yellow solid substance of 0.72 g;

[0082]2) Dissolve 6g of the yellow product in 30ml of 0.1mol / L NaOH aqueous solution, raise the temperature in the oil bath, and reflux for 3 hours. After the reaction, cool to room temperature, adjust the pH value to weak acidity with 1mol...

Embodiment 2

[0115] Example 2 Construction of an enzyme-linked immunosorbent assay kit for detecting sulfonamides

[0116] The enzyme-linked immunosorbent assay kit for detecting sulfonamides was set up to include the following components:

[0117] (1) Enzyme plates coated with sulfa drug-conjugated antigens;

[0118] (2) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0119] (3) Monoclonal antibody working solution of sulfa drugs;

[0120] (4) 6 bottles of sulfamethoxazole standard solution, the concentrations are 0 μg / L, 1 μg / L, 3 μg / L, 9 μg / L, 27 μg / L, 81 μg / L;

[0121] (5) The substrate chromogenic solution is composed of A liquid and B liquid, the substrate chromogenic liquid A liquid is carbamide peroxide, and the substrate chromogenic liquid B liquid tetramethylbenzidine;

[0122] (6) The stop solution is 2mol / L hydrochloric acid;

[0123] (7) The concentrated washing solution is a carbonate buffer solution with a pH value of 7.2, containing 0.7-1.0% Tween-2...

Embodiment 3

[0125] The detection of sulfa drugs in the sample of embodiment 3

[0126] 1. Sample pretreatment

[0127] (1), chicken, pork, fish, shrimp sample pretreatment method

[0128] Weigh 2.0±0.05g of the homogeneous substance into a 50ml polystyrene centrifuge tube, add 8ml of ethyl acetate, shake immediately with a shaker for 5min, and centrifuge at 3000g or more for 5min; take 4ml of the supernatant and put it in a clean glass test tube of 10ml. Blow dry in a water bath at 50-60°C under nitrogen flow; add 1ml of n-hexane, vortex for 30s with a vortexer to dissolve the dry residue, then add 1ml of complex solution working solution, vortex for 30s with a vortexer, then transfer to a 2ml centrifuge tube, Above 3000g, centrifuge at room temperature (20-25°C) for 5min; remove the upper n-hexane phase, and take 50μl of the lower aqueous phase for analysis.

[0129] (2) Honey sample pretreatment method

[0130] Weigh 1.0±0.05g honey sample into a 50ml polystyrene centrifuge tube; add...

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Abstract

The invention provides an enzyme-linked immunosorbent kit for inspecting sulfa drugs, comprising an ELISA plate which is coated with coating antigen, an enzyme label, sulfa drug specific antibody working liquid (being contained when the antigen is coated on the ELISA plate and the enzyme label is enzyme labeling antibody or antibody is coated on the ELISA plate and the enzyme label is enzyme labeling antigen), sulfamethoxy-isoxazole standard product solution, substrate color development solution, stop solution, concentrated washing liquid and concentrated complex solution. The invention further discloses a method which applies the enzyme-linked immunosorbent kit for inspecting the sulfa drugs, and the method comprises the steps of firstly carrying out the pre-treatment on a sample, then using the kit for inspecting and finally analyzing the inspection result. The provided enzyme-linked immunosorbent kit can be used for inspecting the residual amount of the sulfa drugs in animal tissues(chicken, pork, fish and shrimp), honey, eggs, milk, feeds and other samples, the operation is simple, the cost is low, the sensitivity is high and the enzyme-linked immunosorbent kit can be monitore d on-site and is applicable in screening mass samples.

Description

technical field [0001] The present invention relates to enzyme-linked immunoassay detection technology, in particular to an enzyme-linked immunosorbent assay kit for detecting sulfonamides, which is especially suitable for sulfonamides in chicken, pork, fish, shrimp tissues and honey, eggs, milk, and feed samples Drug residue detection. technical background [0002] Sulfa drugs are widely used antibiotics, which play an important role in the control and treatment of livestock and poultry diseases, but they also have serious side effects. Sulfonamides are potentially carcinogenic. The No. 235 document of the Ministry of Agriculture of my country stipulates that the residue limit is 100μg / kg. [0003] At present, the detection methods for sulfa veterinary drug residues mainly include thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (HPLC / MS), gas chromatography (GC), and enzyme-linked immunosorbent assay....

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/566G01N33/531G01N33/577
Inventor 何方洋张建军万宇平冯才伟冯才茂冯静崔海峰徐念琴赵正苗李勇刘福林刘平朱亮亮陈炜琳罗贵昆
Owner BEIJING WANGER BIOTECH
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