Chemical luminescence ELISA detection reagent kit of ciprofloxacin

A technology of chemiluminescence enzyme and ciprofloxacin, which is applied in chemiluminescence/bioluminescence, analysis by causing chemical reactions of materials, and measurement devices, can solve the problems of complex processing, long time, high detection cost, etc., and achieve sensitivity high effect

Inactive Publication Date: 2009-02-18
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of microbiological methods are: time-consuming and lack of specificity
The disadvantages of thin-layer chromatography are: the operation process is complicated and takes a long time; the operators need to undergo professional training; there are many interference factors affecting the analysis, and ...

Method used

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  • Chemical luminescence ELISA detection reagent kit of ciprofloxacin
  • Chemical luminescence ELISA detection reagent kit of ciprofloxacin
  • Chemical luminescence ELISA detection reagent kit of ciprofloxacin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1. Preparation of immunogen, coated antigen and antibody

[0022] (1) Synthesis of immunogen

[0023] The immunogen was obtained by coupling ciprofloxacin and bovine serum albumin (BSA) by EDC method. It includes the following steps:

[0024] A. Weigh 20.0mg (54.4μmol) of ciprofloxacin, 104.2mg (544μmol) of water-soluble carbodiimide (EDC), 31.3mg (272μmol) of N-hydroxysuccinimide (NHS) and dissolve in 5mL. In DMF, keep away from light and react with magnetic stirring at room temperature for 24 hours;

[0025] B. Dissolve 123.3mg (1.8μmol) BSA (molecular weight range of 6.7KDa~6.8KDa) in 10mL phosphate buffer solution (PBS) (0.01M, pH7.4);

[0026] C, slowly add the "product of step A" to the "product of step B", and react with magnetic stirring at room temperature for 3 hours; after the reaction is completed, transfer the reaction solution to a semi-permeable membrane, and use PBS (0.01 M, pH7.4) dialysis for 3 days, during which the dialysate was changed every 4-6...

Embodiment 2

[0030] Example 2. Establishment of CL-ELISA detection method

[0031] 2.1 The optimal concentration of antibody and coating antigen (square matrix)

[0032] For each coating antigen in the longitudinal direction, serial dilutions of 40.0μg / mL, 20.0μg / mL, 10.0μg / mL, 5.0μg / mL, 2.5μg / mL, 1.25μg / mL, 0.625μg / mL, 0.3125μg / mL Degree-coated microtiter plate, 100μL / well, placed overnight at 0-4°C, wash the plate three times with washing solution, and pat dry each time; block with 250μL / well blocking solution, place at room temperature for 3 hours, wash the plate three times, pat dry each time ; Add 100μL / well of a series of diluted antibodies (1:100 to 1:1024000), place at room temperature for 2 hours, wash the plate three times, and pat dry each time; add 100μL / well of 1:1000 horseradish peroxidase labeled Goat anti-rabbit antibody, place at room temperature for 1 hour, wash the plate three times, and pat dry each time; add 100 μL / well of luminescence solution to determine the luminescenc...

Embodiment 3

[0042] Example 3. Chemiluminescence enzyme-linked immunoassay kit for detecting ciprofloxacin

[0043] (1) The composition of a chemiluminescence enzyme-linked immunosorbent assay kit for the detection of ciprofloxacin

[0044] a. Solid-phase carrier (enzyme-labeled plate) coated with coated antigen (conjugate of ciprofloxacin and carrier protein);

[0045] b. 6 bottles of ciprofloxacin standard solution with concentrations of 0.1ng / mL, 0.2ng / mL, 0.5ng / mL, 1ng / mL, 2ng / mL, 5ng / mL;

[0046] c. Ciprofloxacin antibody working solution (working concentration is 1:8000);

[0047]d. Horseradish peroxidase labeled goat anti-rabbit IgG antibody working solution (working concentration is 1:1000);

[0048] e. Concentrated phosphate buffer solution: NaCl 80.0g, KH 2 PO 4 2.0g, Na 2 HPO 4 . 12H 2 O 2 29.0g, KCl 2.0g, add double distilled water to 1000mL, pH 7.5;

[0049] f. Concentrated washing liquid: NaCl 80.0g, KH 2 PO 4 2.0g, Na 2 HPO 4 .12H 2 O 2 29.0g, KCl 2.0g, tween-200.5mL add doub...

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Abstract

The invention discloses a chemiluminescence enzyme-linked immunosorbent test kit of ciprofloxacin, which includes a test kit box body, an enzyme label plate and reagent arranged in the test kit. The test kit is characterized in that holes of the enzyme label plate are coated with envelope antigen which is namely the conjugate of ciprofloxacin and carrier protein; the reagent includes polyclonal antibodies of the ciprofloxacin, enzyme-labeled second antibody which is namely the goat anti rabbit antibody marked by horseradish peroxidase, standard liquid of the ciprofloxacin, concentrated washing liquid and luminescence liquid. With simplicity, celerity and accuracy, the chemiluminescence enzyme-linked immunosorbent test kit has the advantages that the sensitivity is high, and the sensitivity can be enhanced by one order of magnitude compared with traditional colour comparison ELISA; thus playing an important role in ciprofloxacin residue detection of animal food (such as milk, animal tissue and urine samples).

Description

Technical field [0001] The invention relates to an enzyme-linked immunoassay kit, in particular to a chemiluminescent enzyme-linked immunoassay kit for ciprofloxacin. Background technique [0002] Ciprofloxacin (CIP) is a synthetic third-generation quinolone antibacterial drug with broad-spectrum antibacterial activity and good bactericidal effect. The antibacterial activity against almost all bacteria is 2~4 stronger than Norfloxacin and Enoxacin Times, ciprofloxacin is widely used in urinary and respiratory system infections. However, its excessive use has also caused serious pollution to organisms and the environment. Therefore, detection, especially rapid, simple and sensitive detection of ciprofloxacin, has become more and more important. [0003] At present, the main methods for detecting ciprofloxacin are: microbiological method, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), color / mass combined analysis (LC-MS), liquid / mass combined use An...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N21/76
Inventor 郗日沫李伟华丁锴刘中秋尹伟伟刘伟王亚宾
Owner SHANDONG UNIV
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