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Chemical luminescence ELISA detection kit for gatifloxacin

A chemiluminescent enzyme, gatifloxacin technology, applied in chemiluminescence/bioluminescence, analysis by making materials undergo chemical reactions, measurement devices, etc., can solve the time-consuming sample processing process, difficult to carry instruments, a large number of organic solvents, etc. problem, to achieve the effect of simple sample pretreatment, low detection limit and high sensitivity

Inactive Publication Date: 2009-04-08
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 are both expensive, bulky and difficult to carry instruments, and require a large amount of organic solvents and time-consuming sample processing

Method used

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  • Chemical luminescence ELISA detection kit for gatifloxacin
  • Chemical luminescence ELISA detection kit for gatifloxacin
  • Chemical luminescence ELISA detection kit for gatifloxacin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 Gatifloxacin immunogen, coating antigen, and preparation of polyclonal antibody

[0029] (1) Preparation of immunogen

[0030] A, weigh GAT (gatifloxacin) 15.0mg (40μmol), EDC (carbodiimide activated ester, 76.5mg, 400μmol), NHS (N-hydroxysuccinimide, 22.5mg, 200μmol) and dissolve in sequence In 5mL DMF (N-N dimethylformamide), avoid light, and stir at room temperature for 24 hours;

[0031] B, 90.0 mg (1.3 μmol) cBSA (activated bovine serum albumin) was dissolved in 10 mL PBS (phosphate buffered saline, 0.01 M, pH 7.4);

[0032] C. Slowly add the "A" reaction solution into the "B" reaction solution, and stir at room temperature for 3 hours; Change the dialysate every 4-6 hours; then dialyze with high-purity water for 3 days, during which the dialysate should be changed every 4-6 hours; after the dialysis, freeze-dry with a freeze dryer to obtain a white flocculent solid that is gatifloxacin Immunogen, stored at -20°C, for later use.

[0033] (2) Prepar...

Embodiment 2

[0039] Embodiment 2 The establishment of chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA)

[0040] (1) Optimal concentration of antibody and coated antigen (square matrix)

[0041] 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 with a gradient of 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, and place at room temperature Wash three times for 1 hour; add 100 μL / well of luminescence solution, and measure the luminescence v...

Embodiment 3

[0059] Embodiment 3, the assembly of the chemiluminescent ELISA kit for detecting gatifloxacin of the present invention

[0060] (1) The composition of the chemiluminescent ELISA kit for detecting gatifloxacin

[0061] 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);

[0062] b. Gatifloxacin antibody working solution (volume ratio concentration is 1:4000);

[0063] c. 6 bottles of gatifloxacin standard solution, the concentrations are 0.05ng / mL, 0.1ng / mL, 0.2ng / mL, 0.5ng / mL, 1ng / mL and 2ng / mL;

[0064] d, horseradish peroxidase-labeled goat anti-rabbit IgG antibody working solution (working concentration is 1: 1000);

[0065] e Concentrated phosphate buffer solution: it contains 80g of NaCl and KH per liter 2 PO 4 2.0g, Na 2 HPO 4 .12H 2 O 29.0g, KCl 2.0g aqueous solution;

[0066] f Concentrated washing solution: Tween 20 (Tween20) with a volume ratio ...

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Abstract

The invention discloses a chemical luminescence enzyme-linked immunoassay kit of gatifloxacin, comprising a kit body, an enzyme-marked plate and a reagent which are arranged in the kit body; wherein, all the holes of the enzyme-marked plate are coated with coating antigen; the coating antigen is prepared by the coupling of the gatifloxacin and ovalbumin; the reagent comprises a polyclonal antibody of the gatifloxacin, an enzyme-marked second antibody (namely, the antibody of horseradish peroxidase-marked goat anti-rabbit), a gatifloxacin series standard solution, a concentrated phosphate buffer, a concentrative cleaning solution and a luminescence solution. The kit has the advantages of high sensitiveness, good repeatability, simpleness, fastness and exactness; and compared with traditional colorimetric ELISA method, the sensitiveness can be improved by one magnitude and the method is expected to play an import role in the gatifloxacin residual detection of animal source foods (such as milk samples and animal tissue samples) and a urine sample.

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 quinolones, in particular to a chemiluminescence ELISA detection kit for gatifloxacin. Background technique [0002] Gatifloxacin (GAT) is a quinolone broad-spectrum antibiotic with low price and good bactericidal effect, and is widely used in the treatment of human and animal infections. However, gatifloxacin will accumulate residues in the food chain and cause abnormal blood glucose metabolism in patients, so more and more countries have set the highest detection limit for quinolones. In view of this, in order to protect the health of the people and ensure the quality of imported and exported animal-derived products, it is necessary to find a fast, sensitive and convenient detection method for gatifloxacin residues. [0003] The reported methods for the detection of gatifloxacin residues mainly include: high performance liqui...

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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