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Enzyme linked immunosorbent assay kit for detecting trimethoprim medicament and application thereof

A technology of trimethoprim and trimethoprim, applied in the field of enzyme-linked immunoassay, can solve the problems of complex instruments and equipment, unsuitable for on-site monitoring and screening of a large number of samples, and cumbersome processes

Inactive Publication Date: 2013-03-13
贵州勤邦食品安全科学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Conventional detection methods for trimethoprim residues mainly include high performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), etc. High skill requirements, not suitable for on-site monitoring and screening of a large number of samples

Method used

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  • Enzyme linked immunosorbent assay kit for detecting trimethoprim medicament and application thereof
  • Enzyme linked immunosorbent assay kit for detecting trimethoprim medicament and application thereof
  • Enzyme linked immunosorbent assay kit for detecting trimethoprim medicament and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The preparation of embodiment 1 kit components

[0063] 1. Antigen Synthesis

[0064] a. Synthesis of hapten

[0065] A hapten with a carboxyl functional group was obtained from trimethoprim by the succinic anhydride method.

[0066] Specific steps for hapten:

[0067] 1) Dissolve 0.5g of trimethoprim and 0.24g of succinic anhydride in 15ml of pyridine,

[0068] 2) Heating and stirring with a magnetic heating stirrer, and reacting at 70° C. for 24 hours.

[0069] 3) Use a rotary evaporator to evaporate the liquid, add 15ml of acetone to dissolve, then evaporate to dryness, repeat 3 times.

[0070] 4) Pass through a silica gel column, the developing solvent is methanol:hexyl hexanoate = 1:1, collect the point whose polarity is higher than that of the raw material, and evaporate to dryness to obtain the hapten.

[0071] b. Immunogen synthesis

[0072] 1) Fully dissolve 10 mg of trimethoprim hapten, 10 mg of NHS, and 12.5 mg of EDC in 1 mL of DMF, and stir at room te...

Embodiment 2

[0102] Embodiment 2 detects the formation of the ELISA kit of trimethoprim

[0103] An enzyme-linked immunosorbent assay kit for detecting trimethoprim was assembled to include the following components:

[0104] (1) A microtiter plate coated with trimethoprim-coupled antigen;

[0105] (2) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0106] (3) Trimethoprim monoclonal antibody working solution;

[0107] (4) 6 bottles of trimethoprim standard solution, the concentrations are 0 μg / L, 2 μg / L, 6 μg / L, 18 μg / L, 54 μg / L, 162 μg / L;

[0108] (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;

[0109] (6) The stop solution is 2mol / L hydrochloric acid;

[0110] (7) The concentrated washing solution is a phosphate buffered saline solution of 0.8-1.2% Tween-20 and 0.05-0.01‰ sodium azide preservative, and ...

Embodiment 3

[0112] The detection of trimethoprim in the sample of embodiment 3

[0113] 1. Sample pretreatment

[0114] Weigh 3.0±0.05g of honey sample into a 50ml polystyrene centrifuge tube, add 3ml of pH6.0 phosphate buffer, vortex for 5min until fully dissolved, add 2.0g of basic alumina, vortex for 10s, add chloroform 6ml, shake for 5min, centrifuge at 3000g or more for 5min; take 3ml of supernatant to another polystyrene centrifuge tube, add 0.75ml of pH 10.6 carbonate buffer solution, vortex for 5s, add 4ml of ethyl acetate, 4ml of n-hexane, shake 5min, centrifuge at 3000g or more for 5min; take 4ml of the upper organic phase into a 10ml clean glass test tube, and dry it in a water bath at 50-60°C under nitrogen flow; add 1ml of n-hexane, vortex for 30s to dissolve the dry residue, add reconstitution working solution 0.45 ml, vortexed for 2 min, and centrifuged at 3000 g for 5 min; remove the upper n-hexane phase, and take 50 μl of the lower aqueous phase for analysis.

[0115] 2...

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Abstract

The invention provides an enzyme linked immunosorbent assay kit for detecting a trimethoprim medicament and application thereof. The enzyme linked immunosorbent assay kit comprises an enzyme label plate coated with envelope antigen, an enzyme marker, trimethoprim specificity antibody working solution (when the enzyme label plate is coated with the envelope antigen and the enzyme marker is enzyme-labeled anti-antibody or the enzyme label plate is coated with the envelop antigen and the enzyme marker is the enzyme-labeled antigen), trimethoprim standard solution, substrate developing solution, stop solution, concentrated washing solution and composite solution. The invention also discloses a method for detecting the trimethoprim by applying the enzyme linked immunosorbent assay kit. The method comprises the following steps of: performing sample pretreatment; detecting by the kit; and analyzing a detection result. The enzyme linked immunosorbent assay kit provided by the invention can be used for detecting the residual amount of the trimethoprim in a honey sample, has the advantages of simple operation, low expense and high sensitivity, can be monitored in field, and is suitable for screening mass 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 trimethoprim drug, which is particularly suitable for detecting trimethoprim drug residues in honey. technical background [0002] Trimethoprim (trimethoprim, TMP) is a kind of antibacterial agent, structural formula sees figure 1 , The antibacterial range is similar to that of sulfa drugs, and when used in combination with sulfa drugs, the curative effect can be enhanced several times to dozens of times. It is thus a commonly used sulfonamide synergist, which is effective against a wide variety of Gram-positive and negative bacteria. But it is a restricted drug in animal food. [0003] Conventional detection methods for trimethoprim residues mainly include high performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), etc. The high skill requirements are not ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543G01N33/577G01N33/68
Inventor 何方洋万宇平冯才伟赵正苗汪善良冯才茂陈伟玲刘平崔海峰冯静刘玉梅郝士元杨昌松段盈盈
Owner 贵州勤邦食品安全科学技术有限公司
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