Chemiluminescence ELISA detection kit of furadantin

A chemiluminescent enzyme, nitrofurantoin technology, applied in chemiluminescence/bioluminescence, analysis by chemical reaction of materials, measurement devices, etc., can solve the problems of cumbersome derivatization, expensive and bulky instruments that are difficult to carry, and time-consuming sample processing processes. , to achieve the effect of simple sample pretreatment, low detection limit and high sensitivity

Inactive Publication Date: 2009-03-25
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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  • Chemiluminescence ELISA detection kit of furadantin
  • Chemiluminescence ELISA detection kit of furadantin
  • Chemiluminescence ELISA detection kit of furadantin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 11

[0030] Example 11-Derivatization of aminohydantoin (AHD), preparation of immunogen, coating antigen, and antibody

[0031] (1) Derivatization of AHD

[0032] Dissolve 75mg (0.5mmol) of 3-carboxybenzaldehyde (CBA) in 5mL of methanol to obtain liquid A. Dissolve 76mg of AHD (0.5mmol) in 15mL of methanol to obtain liquid B. Mix liquids A and B, stir, and reflux at 65°C. Tracked by TLC, the reaction is completed in about 18 hours. After rotary evaporation to dryness, about 20 mL of ethanol was added to wash and filter with suction. The AHD derivative 1-(4-carboxybenzylidene)-aminohydantoin (CPAHD) is obtained.

[0033] (2) Preparation of immunogen

[0034] The mixed acid anhydride method prepares nitrofurantoin and its metabolite (AHD) immunogen CPAHD-cBSA steps as follows:

[0035] Slowly drop 18 mg (300 μmol) of ethylenediamine (EDA) into 20 mL of ice-cold (0.01M pH7.4) phosphate buffer saline PBS, stir at 4°C, add concentrated hydrochloric acid dropwise to the solution, an...

Embodiment 2

[0042] Embodiment 2 The establishment of chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA)

[0043] 2.1 Optimal concentration of antibody and coated antigen (square matrix)

[0044] 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.

[0045] Speci...

Embodiment 3

[0065] Embodiment 3, the assembly of the chemiluminescent ELISA kit for detection of nitrofurantoin of the present invention

[0066] (1) The composition of the chemiluminescent ELISA kit for detecting nitrofurantoin

[0067] a. A solid phase carrier (milky white opaque polystyrene 96-well chemiluminescent microplate plate) coated with antigen (conjugate of AHD and carrier protein);

[0068] b, nitrofurantoin antibody working solution (volume ratio concentration is 1:5000);

[0069] c. 6 bottles of nitrofurantoin standard solution, the concentrations are 0.1ng / mL, 0.5ng / mL, 1ng / mL, 5ng / mL and 10ng / mL;

[0070] d. Horseradish peroxidase-labeled goat anti-rabbit IgG antibody working solution (working concentration is 1:1000);

[0071] e Concentrated phosphate buffer solution: 80g of NaCl per liter, KH 2 PO 4 2.0g, Na 2 HPO 4 .12H 2 o 2 29.0g, KCl2.0g aqueous solution.

[0072] f Concentrated washing solution: a concentrated phosphate buffer solution of pH 7.5 and 0.1 ...

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Abstract

The invention discloses a chemiluminescence enzyme immunoassay detection reagent kit for nitrofurantion, which comprises a kit body, an ELIAS plate arranged in the kit body and a reagent in the kit body, wherein each hole of the ELIAS plate is enveloped with an envelope antigen, and the envelope antigen is produced by coupling a metabolite 1-aminohydantoin (AHD) of the nitrofurantion and carrier protein; and the reagent comprises a polyclonal antibody of the nitrofurantion, a goat anti-rabbit antibody labeled by an ELIAS secondary antibody (horseradish peroxidase), a nitrofurantion series standard solution, a condensed phosphate buffer liquid, a concentrated cleaning solution, and a luminescent solution. The reagent kit has the characteristics of high sensitivity, good repetitiveness, simplicity, convenience, quickness, and accuracy; compared with the prior colorimetric ELISA method, the sensitivity can be improved by one order of magnitude; and the reagent kit is expected to play an important role in residue detection of the nitrofurantion of water for livestock raising, feedstuff, and animal derived food, 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 a nitrofuran ELISA detection kit, in particular to a nitrofurantoin chemiluminescent ELISA detection kit. Background technique [0002] Nitrofurantoin belongs to the class of Nitrofurans antibiotics. Nitrofuran drugs are broad-spectrum antibiotics. They have very good antibacterial and pharmacokinetic properties. Because of their low price and good effect, they are widely used as growth-promoting additives for pigs, poultry and aquatic products. It is mainly used to treat enteritis, boils, red fin disease, ulcer disease, etc. caused by Escherichia coli or Salmonella. It has a stable drug effect and can inhibit or kill a variety of Gram-positive and negative bacteria. Fungi play a role. After nitrofuran drugs are taken by animals, the original drug molecules are rapidly metabolized in the animal body, and its stability in the body does not exceed several...

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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