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Enzyme linked immunosorbent assay kit for detecting acid orange and application of enzyme linked immunosorbent assay kit

An enzyme-linked immunosorbent reagent and acid orange technology, which is applied in the detection field of acid orange content in marinated meat, bean paste and hot sauce, can solve the problem of low sensitivity of thin-layer chromatography scanning method, expensive equipment, unsuitable for popularization and application, etc. problem, to achieve the effect of efficient detection method, simple structure, and simple pretreatment process

Active Publication Date: 2016-02-03
BEIJING KWINBON BIOTECH
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among these methods, thin-layer chromatography scanning method has low sensitivity and is not suitable for trace acid orange analysis; fluorescence spectrometry has just been used for the detection of acid orange, and its application is seldom
The UPLC method has the fastest detection speed, but the equipment requirements are also the highest; HPLC and HPLC-MS methods are the most widely used methods, which are accurate, stable and reliable, and are also the current standard methods for the determination of acid oranges, but the equipment is expensive and the samples are pre-processed. The processing is cumbersome and time-consuming, requires large-scale equipment and professional technicians, is difficult to realize on-site operation, and is not suitable for popularization and application

Method used

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  • Enzyme linked immunosorbent assay kit for detecting acid orange and application of enzyme linked immunosorbent assay kit
  • Enzyme linked immunosorbent assay kit for detecting acid orange and application of enzyme linked immunosorbent assay kit
  • Enzyme linked immunosorbent assay kit for detecting acid orange and application of enzyme linked immunosorbent assay kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The preparation of embodiment 1 kit components

[0029] 1. Preparation of acid orange hapten

[0030] Dissolve 280mg of p-aminobenzoic acid into 20mL of 0.2mol / L HCl solution, and add 140mg of NaNO dropwise at 0-4°C 2 5mL aqueous solution, and react in the dark for 0.5~1h, then slowly dropwise add the mixture of 240mg acid orange II in 5mL0.5mol / L sodium acetate solution, and react in the dark for 1~3h; Wash with water, extract with ethyl acetate, and remove the solvent to obtain the Acid Orange II azo derivative recrystallized in ethanol-water, which is the Acid Orange hapten.

[0031] Get above-mentioned product and measure through proton nuclear magnetic resonance spectrum, such as figure 2 As shown, the increased carboxyl signal peak around 12.7ppm and the increased aromatic ring signal peak around 8.1-8.4ppm indicate that the hapten was successfully synthesized.

[0032] 2. Antigen preparation

[0033] Immunogen preparation—the acid orange hapten was coupled w...

Embodiment 2

[0050] Embodiment 2 detects the formation of the ELISA kit of acid orange

[0051] An enzyme-linked immunosorbent assay kit for acid orange detection was constructed to include the following components:

[0052] (1) A microtiter plate coated with acid orange conjugated antigen;

[0053] (2) 6 bottles of acid orange standard solution, the concentrations are 0μg / L, 0.5μg / L, 1.5μg / L, 4.5μg / L, 13.5μg / L, 40.5μg / L;

[0054] (3) Acid orange monoclonal antibody working solution;

[0055] (4) goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0056] (5) Substrate chromogenic solution is made up of A liquid and B liquid, and A liquid is carbamide peroxide, and B liquid is tetramethylbenzidine;

[0057] (6) The stop solution is 2mol / L sulfuric acid;

[0058] (7) The washing solution has a pH value of 7.4, contains 0.5% to 1.0% Tween-20, 0.01‰ to 0.03‰ sodium azide preservative, and 0.1 to 0.3mol / L phosphate buffer, and the percentage is mass volume percentage;

[...

Embodiment 3

[0060] The detection of sour orange in the sample of embodiment 3 salted stewed pork, bean paste and hot sauce

[0061] 1. Sample pretreatment

[0062] Marinated Pork and Chili Sauce Samples:

[0063] Weigh 2.0g±0.05g sample into a 50mL polystyrene centrifuge tube, add 4mL acetonitrile, then add 6mL deionized water, vortex with a vortexer until uniform, above 3000g, and centrifuge at room temperature (20-25°C) for 5min; Take 100 μL supernatant in a 2 mL polystyrene centrifuge tube, add 900 μL complex solution, then add 800 μL n-hexane, vortex for 30 s with a vortexer, centrifuge at room temperature (20-25 ° C) for 5 min; remove the upper organic phase , remove 50 μL of the layer for analysis.

[0064] Bean paste sample:

[0065] Weigh 2.0g±0.05g of sample into a 50mL polystyrene centrifuge tube, add 6mL of acetonitrile, then add 4mL of 0.1mol / L carbonate buffer (pH9.1), vortex with a vortexer until uniform, more than 3000g , centrifuge at room temperature (20-25°C) for 5 m...

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Abstract

The invention provides an enzyme linked immunosorbent assay kit for detecting acid orange. The enzyme linked immunosorbent assay kit comprises an elisa plate coated with coating antigen, an acid orange monoclonal antibody, an enzyme-labeled marker, an acid orange standard product solution, a substrate coloring solution, a stopping solution, a cleaning solution and a redissolved solution, wherein the coating antigen is an acid orange coupled antigen, and the enzyme-labeled marker is the enzyme-labeled antiantibody. The invention further discloses a method for detecting acid orange by applying the enzyme linked immunosorbent assay kit. The method comprises the following steps: firstly conducting sample pretreatment, then detecting with the assay kit, and finally analyzing the detection results. The provided enzyme linked immunosorbent assay kit can be used for quickly detecting the content of acid orange in pickled stewed meat, bean paste and chilli sauce, is simple and convenient in operation, low in cost and high in sensitivity, can realize on-site supervision and is suitable for mass sample screening.

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 acid orange, which is particularly suitable for detecting the content of acid orange in marinated pork, bean paste and hot sauce. Background technique [0002] Acid orange (Acidorange), also known as golden orange Ⅱ, orange Ⅱ, No. 2 orange, tangerine orange, acid golden or acid bright orange, commonly known as "golden powder", chemical name is 2-naphthol azo-p-benzenesulfonic acid sodium. Acid orange is a kind of chemical dye, which is mainly used for dyeing textiles such as leather, silk and wool. There are a lot of chemical additives in this dye. If it is left in that environment for a long time, it may affect future fertility, such as Infertile or deformed children may also have an impact on their own health. They are moderately toxic carcinogenic compounds and are prohibited from being used as food a...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/531
Inventor 万宇平罗晓琴何方洋冯静杨秀贤吴鹏刘春雪张芝
Owner BEIJING KWINBON BIOTECH
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