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A kind of ELISA kit for detecting safrole and its application

An enzyme-linked immunosorbent reagent, safrole technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of high professional requirements for operators, expensive detection costs, cumbersome steps, etc., and achieve simple pretreatment process and detection speed Fast, sensitive results

Active Publication Date: 2021-03-09
ZHENGZHOU TOBACCO RES INST OF CNTC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the above methods require advanced detection equipment, expensive detection costs, cumbersome steps, time-consuming, and high professional requirements for operators, they are not suitable for large-throughput rapid screening detection in grassroots enterprises and institutions.

Method used

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  • A kind of ELISA kit for detecting safrole and its application
  • A kind of ELISA kit for detecting safrole and its application
  • A kind of ELISA kit for detecting safrole and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Preparation of each component of the ELISA kit for detecting safrole

[0029] 1. Preparation of safrole hapten

[0030] Take 1.0 g of 5-(2-bromo-2-propen-1-yl)-1,3-benzodioxolane, add 50 mL of anhydrous DMSO to dissolve, stir to clarify, add KOH 0.48 g, add anhydrous iodine Add 0.2 g of sodium chloride, stir vigorously, add 0.41 g of allanine, and react at 100°C for 4 hours. Stop the reaction, add 50 mL of water, add 1mol / L hydrochloric acid to adjust the pH value to 6, add 80 mL of ethyl acetate, extract three times, combine the organic phases, evaporate to dryness by rotary evaporation, put on a silica gel column, ethyl acetate / petroleum ether (v / v, 1 / 5) was eluted and separated to obtain 0.91 g of the hapten product of safrole propionate, with a yield of 87.37%.

[0031] The above haptens were identified by H NMR spectroscopy, 1 H-NMR (CDCl 3 , 300MHz) δ: 11.0 (1H, -COOH), 6.90 (1H, ArH, J=6.864), 6.68 (1H, ArH), 6.76 (1H, ArH), 6.07 (2H, s, -CH 2 -),...

Embodiment 2

[0058] Example 2 The formation of an enzyme-linked immunosorbent assay kit for detecting safrole

[0059] Set up the enzyme-linked immunosorbent assay kit that detects safrole so that it includes the following components:

[0060] (1) Enzyme plate coated with safrole-conjugated antigen;

[0061] (2) 6 bottles of safrole standard solution, the concentrations are 0 µg / L, 10 µg / L, 30 µg / L, 90 µg / L, 270 µg / L, 810 µg / L;

[0062] (3) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0063] (4) The substrate chromogenic solution is composed of the substrate chromogenic solution A and the substrate chromogenic solution B, the A solution is carbamide peroxide, and the B solution is tetramethylbenzidine;

[0064] (5) The stop solution is 2 mol / L sulfuric acid;

[0065] (6) The washing solution has a pH value of 7.4 and contains 0.5%~1.0% Tween-20, 0.01‰~0.03‰ sodium azide, and 0.1~0.3 mol / L phosphate buffer;

[0066] (7) The complex solution is a phosphate buffer ...

Embodiment 3

[0067] Example 3 Detection of safrole in tobacco

[0068] 1. Sample pretreatment

[0069] Weigh 1.0±0.05 g of pulverized tobacco samples into a 50 mL polystyrene centrifuge tube, add 5 mL of methanol, vortex for 3 min with a vortexer, and mix well; centrifuge at 3000 rpm for 5 min; take 100 μL of supernatant, Add 150 μL of complex solution, vortex for 30 s with a vortexer, and mix well; take 50 μL for analysis.

[0070] 2. Detection with kit

[0071] Add 50 μL / well of safrole standard solution or pre-treated sample solution to the microwells of the enzyme-labeled plate coated with safrole-conjugated antigen, then add 50 μL / well of safrole-specific antibody working solution, shake gently to mix, and cover with a cap After the cover plate was placed in a light-proof environment at 25°C, the reaction was carried out for 30 minutes; the cover film was carefully peeled off, the liquid in the well was dried, and the washing solution was used to wash 250 μL / well for 4-5 times with ...

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Abstract

The invention discloses an ELISA kit for detecting safrole, which comprises: an enzyme label plate coated with a coating source, a safrole standard solution, a safrole specific antibody, an enzyme-labeled secondary antibody, a substrate chromogenic solution, a termination solution, washing solution, and reconstitution solution; the original coating is safrole-conjugated antigen, the enzyme-labeled secondary antibody is enzyme-labeled goat anti-mouse anti-antibody, and the safrole-specific antibody is prepared by using safrole-conjugated antigen as an immunogen; the safrole The coupled antigen is obtained by coupling a safrole hapten with a carrier protein, and the safrole hapten is composed of 5‑(2‑bromo‑2‑propene‑1‑yl)‑1,3‑benzodioxolane and amino Propionic acid reaction to get. The invention also discloses a method for detecting safrole using the enzyme-linked immunosorbent assay kit. The ELISA kit provided by the invention can be used to detect the content of safrole in samples, and has the advantages of simple operation, low cost, high sensitivity, on-site monitoring and 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 safrole, which can qualitatively and quantitatively detect the residual amount of safrole in tobacco flavors and fragrances, tobacco and tobacco products. Background technique [0002] Safrole, also known as safrole, safrole, is a colorless or slightly yellow liquid, insoluble in water, easily soluble in chloroform, ether and other non-polar organic solvents, with camphor wood smell, naturally occurring in cinnamon, nutmeg, black Plants such as pepper and perilla are the main constituents of many edible natural essential oils such as sassafras, star anise and camphor. Because of its pleasant smell, sassafras oil was once used as a flavoring and fragrance in food, detergents, and cosmetics. Safrole compounds mainly include safrole, isosafrole, dihydrosafrole and so on. Safrole is also an important compon...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/535G01N33/558
CPCG01N33/535G01N33/558
Inventor 陈黎刘惠民鲁亚辉李旭赵乐崔华鹏樊美娟王晓瑜贾芳芳余晶晶
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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