Enzyme-linked immunoassay kit for acid orange II

An enzyme-linked immunosorbent assay, acid orange technology, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of high price, difficult on-site operation, and time-consuming, and achieve the effect of high sensitivity

Inactive Publication Date: 2010-12-15
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the instrument method is expensive, time-consuming, requires large instruments and equipment, and requires specialized technicians, so it is difficult to use on-site operations

Method used

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  • Enzyme-linked immunoassay kit for acid orange II
  • Enzyme-linked immunoassay kit for acid orange II
  • Enzyme-linked immunoassay kit for acid orange II

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Components and preparation process of enzyme-linked immunosorbent assay kit for the detection of acid orange II

[0037] 1. Composition of enzyme-linked immunosorbent assay kit for the detection of acid orange II

[0038] A. A solid-phase carrier (ELISA plate) coated with a coated antigen (a conjugate of acid orange II and a carrier protein);

[0039] B. Standard solution of acid orange II: 0.1ng / mL, 0.5ng / mL, 1ng / mL, 5ng / mL, 10ng / mL.

[0040] C. Enzyme-labeled goat anti-rabbit antibody solution: The enzyme-labeled goat anti-rabbit antibody is horseradish peroxidase-goat anti-rabbit IgG stock solution, loaded, and used to prepare a working concentration of 1:2000 with washing solution.

[0041] D. Acid orange II antibody solution: the polyclonal antibody prepared by immunizing animals with artificial immunization antigen, and the obtained acid orange II antibody is diluted with washing solution to a working concentration of 1:40000.

[0042] E. Luminescence...

Embodiment 2

[0058] Embodiment 2, the establishment of ELISA detection method

[0059] (1) Optimal concentration of antibody and coating antigen (square matrix)

[0060] Coat the microtiter plate longitudinally with each coating antigen at serial dilutions of 10.0 μg / mL, 5.0 μg / mL, 1.0 μg / mL, 0.1 μg / mL, 0.01 μg / mL, 0.001 μg / mL, 100 μL / well , placed at 36°C for 2 hours, washed the plate three times with washing solution, each time patted dry; 250μL / well blocking solution was blocked, placed at 0-4°C overnight, washed three times, each time patted dry; added 100μL / well serially diluted Antibody (1:5000 to 1:640000), placed at 36°C for 0.5 hours, washed three times, and patted dry each time; added 100 μL / well of 1:2000 horseradish peroxidase-goat anti-rabbit IgG antibody, placed at 36°C After 0.5 hours, the plate was washed three times and patted dry each time; 100 μL / well of luminescence solution was added to measure the luminescence value. The specificity determination was carried out wit...

Embodiment 3

[0069] Example 3. Application of enzyme-linked immunosorbent assay kit for the detection of acid orange II

[0070] (1) Preparation of reagents

[0071] A. Sample Diluent: Dilute the concentrated phosphate buffer solution provided in the kit with distilled water 10 times before use.

[0072] B. Washing solution: Dilute the concentrated washing solution provided in the kit with distilled water 10 times before use.

[0073] C. Luminescence solution: 400ul TMB solution (10mgTMB+2ml DMSO)+10ml citric acid buffer (pH5.0)+2ul 30%H 2 O 2 solution

[0074] (2) Sample pretreatment

[0075] A. Orange Juice First, centrifuge the purchased orange juice sample at 4°C and 10,000 rpm for 15 minutes, and dilute the orange juice supernatant with washing solution to 1:10 to obtain the sample to be tested.

[0076] B. Weigh 5g samples of halogen products and chili noodles, grind and pulverize, add 20ml of anhydrous ethanol-ammonia water-water (volume ratio 70:1:29) solution, shake for 0.5h,...

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Abstract

The invention discloses an enzyme-linked immunoassay kit for acid orange II, which belongs to the technical field of enzyme-linked immunosorbent analysis. Reagents in the kit provided by the invention comprise an enzyme label plate coated by a coupled substance of the acid orange II serving as a coating antigen and a carrier protein, standard solution of the acid orange II, solution of enzyme labeled goat-anti-rabbit antigen, antibody solution of the acid orange II, luminous solution, washing solution, coating solution and closed solution. The carrier protein of the coupled substance is bovine serum albumin or egg albumin with a molecular weight of 6.7 to 6.8 KDa. The coating antigen of the coated enzyme label plate is prepared by coupling the acid orange II and egg albumin, and an artificial immunogen for preparing the angiten is prepared by coupling the acid orange II and the bovine serum albumin. The maximum acid orange II detection range of the kit is 0.1 to 10 ng/mL. The enzyme-linked immunoassay kit of the invention has the characteristics of simplicity, quickness, accuracy and high sensitivity and can play an important role in the acid orange II detection of synthetic non-edible pigment in foods (such as marinated products and cayenne pepper) and drinks.

Description

【Technical field】 [0001] The invention belongs to the technical field of enzyme-linked immunosorbent assay, relates to an enzyme-linked immunosorbent assay kit, and in particular relates to an enzyme-linked immunosorbent assay kit of acid orange II. 【Background technique】 [0002] Azo dyes are an important synthetic dye, and they are the most widely used synthetic dyes for textiles and garments in the printing and dyeing process. They are used in the dyeing and printing of a variety of natural and Coloring of plastic, rubber, etc. Azo dyes do not have any direct carcinogenic effects themselves, but they can be reductively decomposed in the body to form aromatic amine compounds. After the metabolic activities of aromatic amines in the body, they may interact with target cells to change DNA and become an inducing factor for human disease. Or animals are potentially carcinogenic and mutagenic. Due to their carcinogenicity and mutagenicity, many countries in the world have imp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/535
Inventor 郗日沫薛虎寅孟萌张太昌张元阳徐静王亚宾
Owner NANKAI UNIV
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