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Indirect competition enzyme linked immunosorbent assay kit for detecting toluidine fast red

An enzyme-linked immunosorbent reagent, toluidine red technology, applied in measuring devices, color/spectral characteristic measurement, instruments, etc., can solve the difficulties of rapid detection and supervision of toluidine red, expensive equipment and detection costs, and inability to realize on-site detection, etc. problems, to achieve the effect of no radioactive isotope pollution, long storage time of reagents, and high accuracy

Inactive Publication Date: 2010-12-15
INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have a series of advantages such as high detection precision, sensitivity, accuracy, stability, etc., but the equipment and detection costs are expensive, the operation is complicated, and the analysis time is long, which cannot realize on-site detection, which limits its promotion and application, and also limits the rapid development of toluidine red. Detection and regulation pose difficulties
At present, there is no rapid immunoassay method report specifically for toluidine red at home and abroad, so it is urgent to establish a high-sensitivity rapid detection technology for toluidine red

Method used

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  • Indirect competition enzyme linked immunosorbent assay kit for detecting toluidine fast red
  • Indirect competition enzyme linked immunosorbent assay kit for detecting toluidine fast red
  • Indirect competition enzyme linked immunosorbent assay kit for detecting toluidine fast red

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Synthesis of Immunogen and Preparation of Polyclonal and Monoclonal Antibodies

[0025] 1.1 Reagents and instruments

[0026] Toluidine red (Germany Dr), succinic anhydride (Sigma), pyridine (Sigma), EDC (Shanghai Covalent Chemical Reagent Company), bovine serum albumin (BSA), key-hole limpet hemocyanin (KLH, Sigma) ), horseradish peroxidase (HRP) (Shanghai Kaiyang Biotechnology Co., Ltd.), and other reagents were of analytical grade.

[0027] Double-beam ultraviolet-visible spectrophotometer (TU-1909, Beijing Puxi General Instrument Co., Ltd.), chromatography device (3057 portable recorder, Chongqing Chuanyi No. 4 Factory; SBS series numerical control drop counter, constant flow pump, automatic part Collector, chromatography column, Shanghai Huxi Analytical Instrument Factory), magnetic stirrer (Shanghai Dongrongfeng Scientific Instrument Co., Ltd.), desktop centrifuge (Minispin maximum speed 13400rpm maximum centrifugal force 12100rcf, 2ml×12).

[0028] 1....

Embodiment 2

[0054] Example 2 Establishment of Indirect Competitive ELISA Method

[0055] 2.1 ELISA checkerboard method to determine the optimal working concentration of coated antigen and antibody

[0056] Coat the microtiter plate with 100 μl per well of toluidine red-OVA with serial concentrations of 100 μg / ml, 10 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, and 0.125 μg / ml, and coat at 4°C for 24 hours , wash 4 times with washing solution, pat dry on absorbent paper, block each well with 200 μl blocking solution at 4°C for 12 h, wash 3 times, and pat dry on absorbent paper. Add 100 μl of antibody solution diluted 1:1000, 1:2000, 1:4000, 1:6000, 1:8000, 1:10000, react at room temperature for 1 hour, wash 4 times, immediately add 100 μl enzyme-labeled goat anti-rabbit (or goat anti-rabbit) Mouse) antibody, react at room temperature for 30 minutes, wash three times, add 50 μl of chromogenic reagent A solution and 50 μl of chromogenic reagent B solution, protect from light at room temperature f...

Embodiment 3

[0061] Example 3 Toluidine Red Indirect Competitive ELISA Kit Formation

[0062] Set up an enzyme-linked immunosorbent assay kit for detecting toluidine red, so that it includes the following components:

[0063] (1) ELISA plate coated with toluidine red antigen;

[0064] (2) Anti-toluidine red monoclonal or polyclonal antibody working solution;

[0065] (3) goat anti-rabbit or goat anti-mouse antibody labeled with horseradish peroxidase;

[0066] (4) 6 bottles of toluidine red standard solution, the concentrations are: 0μg / L, 1μg / L, 3μg / L, 9μg / L, 27μg / L, 81μg / L;

[0067] (5) Substrate chromogenic solution A is carbamide peroxide or hydrogen peroxide, and substrate chromogenic solution B is tetramethylbenzidine or o-phenylenediamine;

[0068] (6) The concentrated washing solution is 10 times phosphate buffer containing 0.5% Tween 20;

[0069] (7) The concentrated sample diluent is 10 times phosphate buffered saline of 0.1% Tween-20;

[0070] (8) The stop solution is 2mol / ...

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Abstract

The invention discloses an indirect competition enzyme linked immunosorbent assay kit for detecting toluidine fast red, belonging to the field of biological detection. The enzyme linked immunosorbent assay kit for toluidine fast red comprises a toluidine fast red specific monoclonal or polyclonal antibody, a coupler of toluidine fast red and carrier protein and an enzyme-labeled secondary antibody. The enzyme linked immunosorbent assay kit for detecting toluidine fast red is sensitive, quick and accurate and is mainly used for screening a large quantity of samples. Main reagents in the kit are provided in the form of working solutions, the operation is convenient, and the kit has the characteristics of high sensitivity, high precision, high accuracy and the like and can quickly detect para red remained in food.

Description

technical field [0001] The invention belongs to the technical field of detection and analysis of prohibited additives in the field of ELISA and food safety. Specifically, the invention relates to an indirect competition ELISA detection kit for detecting parared. technical background [0002] Toluidine red, like para red, belongs to the azo series chemical synthetic dyeing agent, which is mainly used in the coloring of oil paint and rubber products, and is solid at room temperature. Because its structure (see the picture below) is similar to the carcinogen Sudan Red, azobenzene can be degraded during metabolism to produce aniline, which can directly act on liver cells and cause toxic liver disease. Long-term intake of aniline can cause human body Nervous system damage and is potentially carcinogenic, so it is banned for use in food production. [0003] [0004] Since the addition of toluidine red can make chili products, tomato products and sausages and other foods mainta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/558G01N33/543G01N21/31
Inventor 杨曙明邱静于洪侠陈刚陈爱亮张妍程玛丽张薇薇
Owner INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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