Chemical luminescence ELISA detection reagent kit for furazolidone

A technology of chemiluminescence enzyme and furazolidone, which is applied in the fields of chemiluminescence/bioluminescence, analysis by causing chemical reactions of materials, and measuring devices, etc., which can solve the problems of cumbersome derivatization, time-consuming sample processing process, expensive and bulky instruments that are difficult to carry, etc. , to achieve the effect of simple sample pretreatment, low detection limit and high sensitivity

Inactive Publication Date: 2009-06-24
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both HPLC and LC-MS have the characteristics of high sensitivity and high specificity, but they require expensive, bulky and difficult to carry instruments, a large amount of organic solvents, tedious derivatization and time-consuming sample processing

Method used

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  • Chemical luminescence ELISA detection reagent kit for furazolidone
  • Chemical luminescence ELISA detection reagent kit for furazolidone
  • Chemical luminescence ELISA detection reagent kit for furazolidone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 13

[0028] Example 13- Derivatization of amino-2-oxazolone (AOZ), preparation of immunogen, coated antigen, and antibody

[0029] (1) Derivatization of AOZ

[0030] The preparation method of derivative CPAOZ of AOZ is as follows:

[0031] Dissolve 75mg (0.5mmol) of 3-carboxybenzaldehyde in 5mL of methanol to obtain liquid A. Dissolve 51 mg of AOZ (0.5 mmol) in 15 mL of methanol to obtain liquid B. Mix liquids A and B, stir, and reflux at 65°C. Track by thin chromatography, and the reaction is completed in about 9 hours. After rotary evaporation to dryness, about 20 mL of ethanol was added to wash and filter with suction. The derivative CPAOZ of AOZ is obtained.

[0032] (2) Preparation of immunogen

[0033] The mixed acid anhydride method prepares furazolidone and its metabolite (AOZ) immunogen CPAOZ-cBSA steps as follows:

[0034] Weigh 50 mg (0.21 mmol) of CPAOZ and dissolve in 10 mL of anhydrous DMF (anhydrous N-N dimethylformamide) and stir to dissolve, add 77 μL (0.32 m...

Embodiment 2

[0041] Embodiment 2 The establishment of chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA)

[0042] (1) Optimal concentration of antibody and coated antigen (square matrix)

[0043]Use 100μL / well in the longitudinal direction of the microplate plate, and the concentration gradient is 80.0μg / mL, 40.0μg / mL, 20.0μg / mL, 10.0μg / mL, 5.0μg / mL, 2.5μg / mL, 1.25μg / mL, and 0.625 Coat with μg / mL coating antigen solution, place overnight at 4°C, wash the plate three times with 280 μL / well washing solution, then block with 250 μL / well blocking solution, place at room temperature for 2.5 hours, wash three times; add 100 μL / well horizontally, Dilute the antibody solution at 1:100, 1:200~1:51200, place at room temperature for 2 hours, wash three times; add 100 μL / well of 1:1000 horseradish peroxidase-labeled goat anti-rabbit antibody, place at room temperature Wash three times for 1 hour; add 100 μL / well of luminescence solution, and measure the luminescence value. Specificity det...

Embodiment 3

[0053] Embodiment 3, the assembly of the chemiluminescent ELISA kit for detecting furazolidone of the present invention

[0054] (1) The composition of the chemiluminescent ELISA kit for detecting furazolidone

[0055] a. A solid-phase carrier (milky white opaque polystyrene 96-well chemiluminescence microplate plate) coated with a coated antigen (a conjugate of AOZ and ovalbumin);

[0056] b. Furazolidone antibody working solution (volume ratio concentration is 1:4000);

[0057] c. 6 bottles of furazolidone standard solution, the concentrations are 0.1ng / mL, 0.5ng / mL, 0.8ng / mL, 1ng / mL, 5ng / mL and 10ng / mL;

[0058] d. Horseradish peroxidase-labeled goat anti-rabbit IgG antibody working solution (working concentration is 1:1000);

[0059] e Concentrated Phosphate Buffer Solution:: NaCl 80g, KH 2 PO 4 2.0g, Na 2 HPO 4 .12H 2 o 2 29.0g, KCl 2.0g dissolved in 1000mL distilled water

[0060] f Concentrated washing solution: Tween 20 (Tween20) with a volume ratio concentr...

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Abstract

The invention discloses a reagent kit for the chemiluminescence enzyme-linked immunoassay of furazolidone, which comprises a kit body, an enzyme label plate arranged in the kit body and a reagent arranged in the kit body, wherein, holes of the enzyme label plate are coated by an envelope antigen which is manufactured through coupling between 3-azyl-2-oxazolone that is a metabolin of furazolidone and ovalbumin; and the reagent contains a polyclonal antibody of furazolidone, an enzyme label secondary antibody that is a goat anti-rabbit antibody marked by horseradish peroxidase, a furazolidone-series standard solution, a concentrated phosphate buffer solution, a concentrated cleaning solution and a luminescent liquid. The reagent kit has the characteristics of high sensitivity, good reproducibility, simplicity, convenience, speediness and accuracy; compared with the traditional color comparison ELISA method, the sensitivity can be improved by one order higher, and the reagent is expected to play an important role in furazolidone residue detection of water used in stock raising, feeds and animal-derived foods (such as milk samples, animal tissue samples and urine samples).

Description

technical field [0001] The invention belongs to the technical field of drug residue analysis and immunity, and relates to an ELISA detection kit for nitrofurans, in particular to a chemiluminescence ELISA detection kit for furazolidone. Background technique [0002] Furazolidone (FZ) is a broad-spectrum antibiotic of nitrofurans, which is cheap and has good bactericidal effect. It can effectively treat a variety of infections caused by Gram-positive and negative bacteria, and has certain effects on some protozoa and fungi. , which are widely used in livestock and poultry and aquaculture, and are used as therapeutic drugs and feed additives. However, furazolidone is rapidly metabolized in the body, and the metabolite is 3-amino-2-oxazolone (AOZ). This molecule binds to cell membrane proteins and forms a bound state, which can remain stable for a long time, thereby delaying the clearance rate of the original drug in the body. However, ordinary food processing methods (such as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/545G01N21/76
Inventor 郗日沫丁锴刘中秋李伟华尹伟伟刘伟
Owner SHANDONG UNIV
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