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Enzyme-linked immunosorbent assay kit for detecting morphine and codeine, and applications thereof

An enzyme-linked immunosorbent reagent and kit technology, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of high detection cost, inability to operate on-site, complicated pretreatment, etc., and achieve high accuracy, high precision, Simple pre-processing

Active Publication Date: 2017-10-03
BEIJING KWINBON BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have their own characteristics. Currently, high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry are the mainstream methods. These two methods are sensitive, accurate, specific, and well-separated. They can simultaneously determine multiple drugs, but require expensive The pretreatment of advanced instruments and samples is complex, cumbersome and time-consuming, the cost of detection is high, and it cannot be operated on site, and it needs to be operated by professionals, so its application is limited.

Method used

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  • Enzyme-linked immunosorbent assay kit for detecting morphine and codeine, and applications thereof
  • Enzyme-linked immunosorbent assay kit for detecting morphine and codeine, and applications thereof
  • Enzyme-linked immunosorbent assay kit for detecting morphine and codeine, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 Preparation of kit components

[0029] 1. Synthesis of morphine hapten (see appendix for synthetic route) figure 1 ) and identification

[0030] Take 20 mg of 3-fluoro-4 nitroaniline, add 20 μL of dilute sulfuric acid, add water, stir to dissolve, add 18 mg of sodium nitrite at 5°C, and stir for 1 hour to obtain a diazonium salt solution; take 15 mg of morphine, add sodium hydroxide ethanol solution to dissolve , added to the diazonium salt, reacted for 2h, stopped the reaction, centrifuged to obtain a red solid compound, added ethanol and rotary evaporated, evaporated to dryness to remove water, added dichloromethane:petroleum ether=1:20 to wash and crystallize to obtain a hapten product.

[0031] The above haptens were identified by H NMR spectroscopy, the results are shown in the attached figure 2 . 1 H-NMR (CDCl 3 ,300MHz) δ: 8.40(s,1H,ArH), 7.90(s,1H,ArH), 7.62(s,1H,ArH), 6.82(s,1H,ArH), 5.90(s,1H,-CH- ), 5.59(s,1H,-CH-), 5.35(s,1H,-OH), 4.30(d,1...

Embodiment 2

[0063] Example 2 Construction of ELISA kits for detecting morphine and codeine

[0064] An ELISA kit for detecting morphine and codeine was constructed to include the following components:

[0065] (1) ELISA plate coated with morphine-conjugated antigen;

[0066] (2) 6 bottles of morphine standard solution, the concentrations were 0 μg / L, 0.3 μg / L, 0.9 μg / L, 2.7 μg / L, 8.1 μg / L, 24.3 μg / L;

[0067] (3) Morphine monoclonal antibody working solution;

[0068] (4) goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0069] (5) Substrate chromogenic solution is made up of A liquid and B liquid, and A liquid is carbamide peroxide, and B liquid is tetramethylbenzidine;

[0070] (6) The stop solution is 2mol / L sulfuric acid;

[0071] (7) The washing liquid is pH 7.6, containing 0.2-0.4mol / L phosphate buffer solution of 0.8%-1.2% Tween-20 and 0.01‰-0.02‰ sodium azide;

[0072] (8) The complex solution is a 0.1-0.3 mol / L phosphate buffer solution with a pH of 7.6 an...

Embodiment 3

[0073] Example 3 Detection of Morphine and Codeine in Hot Pot Base Material and Chili Sauce Samples

[0074] 1. Sample pretreatment

[0075] Weigh 1.0g±0.05g of the homogenized sample into a 50mL polystyrene centrifuge tube, add 10mL of ethanol respectively, vortex for 5min with a vortexer, centrifuge at 3000g for 5min at room temperature (20-25°C); pipette 1mL of the upper organic layer Put the phase (if there is suspended matter in the upper layer, avoid it) into a 10mL clean and dry glass tube, and blow it to dryness with nitrogen in a water bath at 50-60°C; add 1mL of n-hexane, vortex for 2min with a vortexer until the oil is dissolved, and then add 1mL of compound Solution, continue to vortex for 3 minutes until uniform; transfer the mixed liquid into a 2mL centrifuge tube, centrifuge at room temperature (20-25°C) at 4500g for 2 minutes; remove the upper grease layer and emulsified layer, and take 50 μL of the lower layer for analysis.

[0076] 2. Detection with kit

[...

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Abstract

The present invention provides an enzyme-linked immunosorbent assay kit for detecting morphine and codeine. The enzyme-linked immunosorbent assay kit comprises an enzyme label plate coated with a morphine-conjugated antigen, a morphine monoclonal antibody, an enzyme-labeled anti-antibody, a morphine standard substance solution, a substrate coloring solution, a stopping solution, a washing liquid and a reconstituted solution. The invention further discloses a method for using the enzyme-linked immunosorbent assay kit to detect morphine and codeine, wherein the method comprises: pre-treating a sample, detecting with the kit, and analyzing the detection result. According to the present invention, the enzyme-linked immunosorbent assay kit can be used for detecting the residual morphine and the residual codeine in the hot pot bottom material and the chilli sauce, has characteristics of simple operation, low cost, high sensitivity and on-site monitoring, and is suitable for screening of a large number 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 morphine and codeine, which is particularly suitable for detecting residual amounts of morphine and codeine in hot pot bottoms and chili sauce. Background technique [0002] Poppy husks, commonly known as smoke husks, are the dried and mature husks of poppy plants in the family Papaveraceae after cutting the juice. They contain more than 20 kinds of alkaloids, among which the most abundant alkaloids are morphine, codeine, thebaine, and opium poppy. Alkaline, narcotine. Although the content of alkaloids in poppy shells is small, if you eat foods containing such drugs for a long time, you will experience symptoms such as chills, sweating, fatigue, sallow complexion, and drowsiness. In severe cases, it may cause damage to the nervous system and digestive system. Symptoms such as endocrine disorders occur. ...

Claims

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Application Information

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IPC IPC(8): G01N33/543
CPCG01N33/543
Inventor 张林田何方洋梁希扬万宇平黄学泓冯才伟张冬辉罗晓琴杨晞王琳琛崔海峰
Owner BEIJING KWINBON BIOTECH
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