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Chloramphenicol chemiluminescence enzyme-linked immunodetection kit

A chemiluminescence enzyme and immunodetection 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 immunoassay 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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  • Chloramphenicol chemiluminescence enzyme-linked immunodetection kit
  • Chloramphenicol chemiluminescence enzyme-linked immunodetection kit
  • Chloramphenicol chemiluminescence enzyme-linked immunodetection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1: Preparation of immunogen, coating agent and enzyme-labeled monoclonal antibody

[0043] (1) Chloramphenicol hapten preparation

[0044] A. Dissolve chloramphenicol in methanol, add 5% Pd / C, pass in hydrogen, keep a certain pressure, react at room temperature for 2 hours, filter to remove Pd / C, evaporate the solvent to obtain a light yellow viscous liquid, obtained chloramphenicol hapten. The molar ratio of Pd / C to chloramphenicol was 1:10. Adjust the pH value of the target substance obtained above to 1-2 with hydrochloric acid at 0-5°C, and add 0.1-1mol / L NaNO dropwise under stirring. 2 solution to make the starch potassium iodide test paper turn blue, then add 0.1-1mol / L urea solution dropwise to make the starch potassium iodide test paper light blue, then add 0.1-1mol / L NaOH solution to adjust the pH value to 7-9, and obtain the clear liquid as Solution A is ready for use.

[0045] B. Measure 21.8 mL of solution A and add it to 3 mL of DMF, pre-cool at ...

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 enzyme-labeled chloramphenicol monoclonal antibodies (1:1000 to 1:512000), incubate at room temperature (20-25°C) for 15 minutes, wash the plate five times, and pat dry for the last time; add 100 μL / well of chemiluminescence solution , to measure the luminescence value. Specificity determination was carried out with the coating antigen concentration and antibody dilution having obvious gradient changes in luminescenc...

Embodiment 3

[0069] Embodiment 3: Chemiluminescent ELISA kit for detecting chloramphenicol

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

[0071] A, the solid phase carrier (elisa plate) that is coated with coating former (CAP-OVA);

[0072] B, Chloramphenicol standard solution: 0pg / mL, 20pg / mL, 60pg / mL, 180pg / mL, 540pg / mL and 1620pg / mL.

[0073] C, enzyme-labeled chloramphenicol antibody solution: the monoclonal antibody prepared by immunizing animals with artificial immune antigen (CAP-BSA), and the obtained chloramphenicol antibody is labeled with horseradish peroxidase and diluted to 1: 16000 working concentration.

[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. Mix liquid A and liquid...

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Abstract

The present invention discloses a chloramphenicol 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 chloramphenicol and carrier protein conjugate, and the reagents comprise horseradish peroxidase-labeled chloramphenicol monoclonal antibody, a series of chloramphenicol standard solutions, a concentrated phosphate buffer, a concentrated washing solution and a chemiluminescence solution. The chloramphenicol chemiluminescence enzyme-linked immunodetection kit has characteristics of high sensitivity, simple and rapid detection, and high accuracy, provides a substantially reduced operation time compared to the conventional colorimetric ELISA method, and can be used for detection of chloramphenicol residues in animal tissues (pork, chicken, pork liver and chicken liver), aquatic products (fish and shrimp) and milk.

Description

technical field [0001] The invention relates to a chemiluminescent immunoassay kit for detecting chloramphenicol, which is used for detecting chloramphenicol in animal tissues (pork, chicken, pig liver, chicken liver), aquatic products (fish, shrimp), honey and milk content or residue. It belongs to the field of immunological detection. Background technique [0002] Chloramphenicol (Chloramphenicol, CAP) is a cheap and efficient broad-spectrum antibiotic, which has a good inhibitory effect on Gram-positive and negative bacteria, so it was once widely used in agriculture and animal husbandry. But animal-derived Food is ingested by the human body along the food chain for a long time, which can cause a variety of diseases. The light ones destroy the balance of the normal flora in the human body, and the flora is out of balance, causing the human body to produce drug-resistant bacteria beads, which will bring adverse effects to the use of antibiotics in the future. Impact: Peo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N21/76
Inventor 冯才伟吴鹏杨昌松王鑫冯静杜亚菲蒲小容韩京朋
Owner BEIJING KWINBON BIOTECH
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