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

An enzyme-linked immunosorbent reagent, diazepam technology, applied in the detection of diazepam content in animal tissue, feed, water quality, urine, enzyme-linked immunosorbent kits, can solve the problem of rapid detection of a large number of samples and on-site samples , Unable to meet the requirements of residue analysis and detection, and the processing process is complicated, etc., to achieve the effect of simple structure, efficient detection method, and low pre-treatment requirements

Active Publication Date: 2017-05-10
BEIJING KWINBON BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

HPLC, TLC and other methods are limited by their sensitivity and cannot meet the current requirements for residue analysis and detection; HPLC-MS / MS, as the most reported method at present, can simultaneously perform qualitative and quantitative analysis of diazepam and its metabolites, but requires expensive instruments and specialized technicians, the sample pretreatment process is complex, costly, and time-consuming, and it is difficult to meet the needs of a large number of samples and rapid detection of on-site samples

Method used

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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The preparation of embodiment 1 kit components

[0028] 1, the synthesis of diazepam hapten (synthetic route sees attached figure 1 )

[0029] A. Take 1 g of maleimidobenzoic acid, add dichloromethane to dissolve, add 1.09 g of oxalyl chloride, stir at room temperature for 2 h; add ice water to shake, separate layers, take the lower organic phase, dry it with anhydrous sodium sulfate, and evaporate to dryness , to obtain an oily product, recrystallized from chloroform / n-hexane (1 / 1, v / v) to obtain 0.93 g of maleimidobenzoyl chloride product, with a yield of 86.92%;

[0030] B. Dissolve 0.93g of maleimidobenzoyl chloride with pyridine, add 1.0g of nordiazepam, heat in an oil bath, reflux for 4 hours, stop the reaction, and detect by TLC that there are almost no remaining raw materials; rotary steaming, remove pyridine , a red oil was obtained, and recrystallized from 1,2-dichloroethane to obtain 1.2 g of diazepam maleimide, a hapten product, with a yield of 66.67%.

...

Embodiment 2

[0062] Embodiment 2 detects the formation of the ELISA kit of diazepam

[0063] The ELISA kit for detecting diazepam was set up to include the following components:

[0064] (1) A microtiter plate coated with diazepam-conjugated antigen;

[0065] (2) 6 bottles of diazepam standard solution, the concentrations are 0 μg / L, 0.1 μg / L, 0.2 μg / L, 0.4 μg / L, 0.8 μg / L, 1.6 μg / L;

[0066] (3) Diazepam monoclonal antibody working solution;

[0067] (4) goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0068] (5) Substrate chromogenic solution is made up of A liquid and B liquid, and A liquid is carbamide peroxide, and B liquid is tetramethylbenzidine;

[0069] (6) The stop solution is 2mol / L sulfuric acid;

[0070] (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;

[0071] (8) The complex solution is a phosphate buffer solution with a pH value of 7.0 and 0.1 mol / L. ...

Embodiment 3

[0072] The detection of diazepam in the sample of embodiment 3

[0073] 1. Sample pretreatment

[0074] Tissue sample:

[0075] Weigh 2.0g±0.05g of the homogenized tissue sample into a 50mL polystyrene centrifuge tube, add 8mL of 0.1mol / L sodium hydroxide solution, oscillate with an oscillator for 5min, and centrifuge at 3000g at room temperature (20-25°C) for 10min; Pipette 1mL of supernatant into a 50mL polystyrene centrifuge tube, add 10mL of n-hexane, shake with a shaker for 5min, centrifuge at 3000g at room temperature (20-25°C) for 10min; pipette 5mL of the upper organic phase into a 10mL clean and dry glass tube , blow dry in a water bath at 50-60°C under nitrogen flow; add 1mL of complex solution, vortex for 2min with a vortexer; take 50μL for analysis.

[0076] Urine, water samples:

[0077] Pipette 1mL of the clear sample into a 50mL polystyrene centrifuge tube, add 4mL of 0.1mol / L sodium hydroxide solution, shake with a shaker for 2min; pipette 1mL of the mixed s...

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Abstract

The invention provides an enzyme-linked immunosorbent assay kit for detecting diazepam. The enzyme-linked immunosorbent assay kit comprises an ELISA (Enzyme Linked Immunosorbent Assay) plate coated with a diazepam coupled antigen, a diazepam monoclonal antibody, an enzyme-labeled anti-antibody, a diazepam standard product solution, a substrate color developing solution, a stopping solution, a washing solution and a dissolution solution. The invention further discloses a method for utilizing the enzyme-linked immunosorbent assay kit to detect the diazepam. The method comprises the following steps: firstly, pre-treating a sample; secondly, detecting by utilizing the kit; finally, analyzing a detection result. The enzyme-linked immunosorbent assay kit provided by the invention can be used for detecting the content of the diazepam in animal tissues, urine, feed and water, is simple and convenient to operate, low in cost and high in sensitivity, can be monitored in field and is suitable for screening a lot of samples.

Description

technical field [0001] The invention relates to an ELISA detection technology, in particular to an ELISA kit for detecting diazepam, which is particularly suitable for detecting the content of diazepam in animal tissues, urine, feed and water quality. Background technique [0002] Diazepam, also known as Valium, is a central nervous system depressant with the chemical name 1-methyl-5-phenyl-7-chloro-1,3-dihydro-1,4-benzodiazepine -2-ketone, which is mainly used in the treatment of human and animal sedation, hypnosis, and epilepsy; in animal husbandry, it is used as an anti-transportation stress drug for animals, and it is also illegally used as an animal weight gain promoter in large quantities. When the human body ingests food containing diazepam residues, the burden on the liver will increase, the mind will be dizzy, the memory will be impaired, and individual people will have rashes, leukopenia, and motor nerve and muscle functions are inhibited. For this reason, the Mini...

Claims

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Application Information

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IPC IPC(8): G01N33/94G01N33/531
CPCG01N33/531G01N33/948
Inventor 万宇平韩深冯才伟崔廷婷吴小胜崔海峰罗晓琴杨春燕
Owner BEIJING KWINBON BIOTECH
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