Sulfanilamide drug chemiluminescence enzyme-linked immunodetection kit

A chemiluminescence enzyme and sulfonamide drug technology, applied in the field of immunological detection, can solve the problems of the use of imported reagents, the high price of chemiluminescence instruments, and the inability to popularize the chemiluminescence immune method, and achieve the effect of high sensitivity

Active Publication Date: 2013-04-03
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The performance of imported chemiluminescence instruments and reagents is better, but foreign technology monopoly leads to high prices of chemiluminescence instruments, luminescent substrate solutions and kits, and the reagents or kits match the instruments. Imported reagents are often not available in domestic instruments. The use of chemiluminescent immunization methods cannot be popularized at the grassroots level

Method used

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  • Sulfanilamide drug chemiluminescence enzyme-linked immunodetection kit
  • Sulfanilamide drug chemiluminescence enzyme-linked immunodetection kit
  • Sulfanilamide drug chemiluminescence enzyme-linked immunodetection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Preparation of maternal nuclear hapten, immunogen, coating source and monoclonal antibody

[0044] (1) Synthesis of sulfonamide nuclear hapten:

[0045] A. Add 0.87g of 6-aminonicotinic acid, 20ml of ethanol, 3.7ml of hydrochloric acid (12mol / L) into a 25ml single-mouth bottle, heat to reflux, react for 8h, monitor by TLC, after the reaction is complete, remove the solvent under reduced pressure, dissolve in saturated NaHCO3 The aqueous solution was extracted with ethyl acetate, dried over anhydrous NaS2O4, and purified by column chromatography (ethyl acetate / petroleum ether, 2 / 1, v / v), with a yield of 85%.

[0046] B. Add 0.89g of ethyl 6-aminonicotinate, 1.6ml of Et3N, and 210ml of CH2Cl into a 25ml single-necked bottle. After stirring and dissolving, add a catalytic amount of DMAP. Slowly drip into 2ml of dichloromethane solution of 4-acetamidobenzenesulfonyl chloride, TLC monitoring, 5h, the reaction is complete, aftertreatment, drying, column chromatogr...

Embodiment 2

[0057] Embodiment 2: the establishment of ELISA detection method

[0058] (1) Optimization of antibody and coated antigen concentration (square matrix)

[0059] Serially dilute each coated antigen longitudinally at 160.0 μg / mL, 80.0 μg / mL, 40.0 μg / mL, 20.0 μg / mL, 10.0 μg / mL, 5.0 μg / mL, 2.5 μg / mL, 1.25 μg / mL Coat the ELISA plate with 100 μL / well, place it in a 37°C incubator for 2 hours, and then pat it dry; seal it with 150 μL / well blocking solution, place it in a 37°C incubator for 2 hours, wash the plate once, and pat it dry; add 50 μL / well A series of diluted sulfonamide monoclonal antibodies (1:1000 to 1:512000), and then add 50 μL / well washing solution to prepare the enzyme-labeled goat anti-mouse antibody working concentration of 1:2000. Incubate at room temperature (20-25°C) for 15 minutes, wash the plate five times, and pat dry the last time; add 100 μL / well of chemiluminescence solution, and measure the luminescence value. Specificity determination was carried out w...

Embodiment 3

[0069] Example 3: Chemiluminescence enzyme-linked immunosorbent assay kit for detection of sulfonamides

[0070] (1) The composition of the chemiluminescent ELISA kit for detecting sulfonamides

[0071] A, Solid phase carrier (elisa plate) coated with coating agent (SAs-OVA).

[0072] B, Standard solutions of sulfonamides: 0ng / mL, 1.0ng / mL, 3.0ng / mL, 9.0ng / mL, 27.0ng / mL and 81.0ng / mL.

[0073] C, sulfa antibody solution: the monoclonal antibody prepared by immunizing animals with artificial immune antigen (SAs-BSA), and dilute the obtained sulfa antibody with washing solution to a working concentration of 1:64000.

[0074] D, luminescent solution: A solution is tris(hydroxymethyl)aminomethane solution with luminol content of 0.01M and p-cresol content of 0.001M pH=8.8, B solution is 100mL solution containing 2.1g of citric acid, anhydrous Na 2 HPO 4 2.82g, a solution of 0.75% urea hydrogen peroxide in 0.64mL.

[0075] E, concentrated phosphate buffer is NaH per liter 2 P...

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Abstract

The present invention discloses a sulfanilamide drug chemiluminescence enzyme-linked immunodetection kit, which comprises a kit body, an enzyme label plate placed inside the kit body, and reagents placed inside the kit body, and is characterized in that every hole of the enzyme label plate is coated with coating antigen, the coating antigen is a sulfanilamide mother nucleus and carrier protein conjugate, and the reagents comprise sulfanilamide monoclonal antibody, horseradish peroxidase-labeled goat anti-mouse antibody, a series of sulfanilamide standard solutions, a concentrated phosphate buffer, a concentrated washing solution and a chemiluminescence solution. The sulfanilamide drug chemiluminescence enzyme-linked immunodetection kit has characteristics of high sensitivity, simple and rapid detection, high accuracy, and more drug detection types, provides a substantially reduced operation time compared to the conventional colorimetric ELISA method, and can be used for detection of residues of the 17 sulfanilamide drugs in animal tissues (pork, chicken, pork liver and chicken liver), aquatic products (fish and shrimp), eggs, milk and milk powder.

Description

technical field [0001] The invention relates to a chemiluminescent immunoassay kit for detecting sulfonamides, which is used for detecting the content or residual amount of sulfonamides in animal tissues (muscle, liver), aquatic products (fish, shrimp), eggs, milk and milk powder. It belongs to the field of immunological detection. Background technique [0002] The basic structure of sulfonamides is p-aminobenzenesulfonamide, which has aromatic amino groups and sulfonamide groups. Most of them are amphoteric compounds, and the drugs have certain acidity. The antibacterial effect of sulfonamides is due to the fact that sulfanilamide can be decomposed from sulfa drugs, and its molecular size and shape are similar to those of p-aminobenzoic acid in folic acid, and its chemical properties are also similar. Due to the lack of selectivity of bacteria to the two, a large amount of sulfanilamide is absorbed by bacteria instead of p-aminobenzoic acid, which interferes with the synth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N21/76
Inventor 何方洋万宇平吴鹏冯月君扶胜岳新荣段盈盈韩京朋
Owner BEIJING KWINBON BIOTECH
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