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Azithromycin enzyme-linked immunoassay kit and application thereof

A technology of azithromycin enzyme and azithromycin, which is applied in the field of biotechnology detection, can solve the problems of high instrument cost, high requirements for test personnel, and long test time, and achieve the effect of high detection sensitivity, good technical parameters and low detection cost

Active Publication Date: 2021-03-19
北京元恩生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In August 2005, the Ministry of Agriculture issued Announcement No. 560. Azithromycin is an antibacterial drug used in human clinical control. Using it in food animals will cause drug resistance problems, affect animal disease control, food safety and human health, and do not meet the requirements of veterinary drugs. Safe and Effective Approval Principles, Azithromycin Drugs Are Listed as Prohibited Veterinary Drugs
Although the instrumental analysis method has the characteristics of accurate test results, more reliable results, and good repeatability, its pretreatment procedures are cumbersome, the measurement time is long, the cost of the instrument is high, and the requirements for testing personnel are high, which is not conducive to rapid on-site detection.

Method used

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  • Azithromycin enzyme-linked immunoassay kit and application thereof
  • Azithromycin enzyme-linked immunoassay kit and application thereof
  • Azithromycin enzyme-linked immunoassay kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] 1. Azithromycin enzyme-linked immunoassay kit contains components:

[0027] (1) 96-well microtiter plate coated with azithromycin hapten-OVA conjugate;

[0028] (2) Azithromycin monoclonal antibody labeled with horseradish peroxidase, 7mL / bottle;

[0029] (3) Azithromycin standard 0μg / L, 0.02μg / L, 0.06μg / L, 0.18μg / L, 0.54μg / L, 1.62μg / L, 1mL / bottle.

[0030] (4) 20× concentrated washing solution, the composition contains 2% Tween 20, 0.2mol / L phosphate buffer, pH 7.0, the percentage is mass volume ratio, 30mL / bottle, use deionized water at a ratio of 1:19 The ratio is diluted.

[0031] (5) The complex solution working solution, the composition is 2% sodium chloride solution, the percentage is mass-volume ratio, 50mL / bottle.

[0032] (6) The substrate A solution is hydrogen peroxide, and the substrate B solution is TMB, both 7mL / bottle.

[0033] (7) The stop solution is 2mol / L H 2 SO 4 , 7mL / bottle.

[0034] 2. Preparation of main components:

[0035] 2.1. Synthes...

Embodiment 2

[0072] The difference from Example 1 is that the enzyme-labeled antibody is a horseradish peroxidase-labeled azithromycin polyclonal antibody. Preparation of polyclonal antibody to azithromycin: New Zealand white rabbits were immunized with azithromycin hapten-BSA conjugate mixed with Freund's complete adjuvant in a ratio of 1:1. For the first immunization, second immunization and third immunization, Freund's complete adjuvant was replaced with Freund's incomplete adjuvant, and the dose was the same as the first time. After the third immunization, the interval between each immunization was 2 weeks. A total of 5 immunizations were performed. Blood was collected from the carotid artery 7 days after the last immunization, and purified polyclonal antibody to azithromycin was obtained by fractional precipitation with ammonium sulfate.

Embodiment 3

[0073] The detection of azithromycin in embodiment 3 milk, chicken sample

[0074] 1. Milk sample pretreatment method

[0075] Pipette 1 mL of milk sample into a 2 mL polystyrene centrifuge tube, add 1 mL of 1% trichloroacetic acid, vortex for 3 min, and centrifuge at 4000 rpm for 5 min at room temperature; pipette 50 μL of the supernatant into a 2 mL polystyrene centrifuge tube, add 450 μL for reconstitution solution, vortexed for 1 min, and took 50 μL for analysis. The working solution for reconstitution is 2% sodium chloride solution, and the percentages are mass-volume ratios.

[0076] 2. Chicken sample pretreatment method

[0077] Weigh 1.0±0.05g tissue sample into a 50mL polystyrene centrifuge tube, add 4mL of 0.025M sodium hydroxide solution, vortex for 5min, and centrifuge at 4000rpm for 5min at room temperature; take 100μL supernatant and add 400μL reconstitution working solution, and vortex Vortex for 1 min to fully mix; take 50 μL for analysis.

[0078] 2. Stand...

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Abstract

The invention provides an azithromycin enzyme-linked immunoassay kit which comprises a 96-hole elisa plate, an enzyme-labeled antibody, an azithromycin standard substance, a 20 x concentrated washingsolution, a reconstitution fluid working solution, a substrate solution A, a substrate solution B and a stop solution, wherein the 96-hole elisa plate is coated with azithromycin hapten carrier protein; the enzyme marker concentrated solution is an azithromycin specific antibody marked by horse radish peroxidase. The invention also provides application of the azithromycin enzyme-linked immunoassaykit in detection of milk and honey samples. The azithromycin enzyme-linked immunoassay kit disclosed by the invention is simple in structure, convenient to use, low in requirement on detection personnel, high in detection sensitivity, good in technical data such as accuracy and precision of a detection result and the like, and is a very ideal azithromycin residue detection product.

Description

technical field [0001] The invention relates to the field of biotechnology detection, and specifically relates to a rapid detection kit for azithromycin ELISA and its application. Background technique [0002] Azithromycin (AZM) is a semi-synthetic fifteen-membered macrolide antibiotic developed in the 1980s. Azithromycin has a good broad-spectrum antibacterial effect, and is widely used in the prevention and treatment of human respiratory tract, urinary tract and soft tissue infections and sexually transmitted diseases, and has also been used in the clinical treatment of livestock and poultry diseases. In August 2005, the Ministry of Agriculture issued Announcement No. 560. Azithromycin is an antibacterial drug used in human clinical control. Using it in food animals will cause drug resistance problems, affect animal disease control, food safety and human health, and do not meet the requirements of veterinary drugs. According to the principle of safe and effective approval...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/58G01N33/543
CPCG01N33/5308G01N33/581G01N33/543
Inventor 杨昌松王世恩郝士元
Owner 北京元恩生物技术有限公司
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