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Enzyme linked immunosorbent assay kit for detecting nitroimidazole drugs and application thereof

An enzyme-linked immunosorbent reagent and nitroimidazole technology, applied in the field of enzyme-linked immunoassay detection, can solve the problems of cumbersome operation of the instrument detection method, limitation of popularization and application, endangering food safety, etc. Sensitive effect

Active Publication Date: 2013-10-23
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Nitroimidazoles are antiprotozoal and antibacterial drugs commonly used in livestock and poultry feed, mainly used for the prevention and treatment of histomonas and parasites in poultry, trichomonas in cattle, hemorrhagic enteritis in pigs, parasitic infections in fish, bees Microsporidiosis, etc., and have growth-promoting effects, but such drugs and some metabolites have carcinogenic, teratogenic, mutagenic and genotoxic effects on mammals, so the unreasonable use of nitroimidazoles has caused possible Drug residues in carnivorous animal tissues endanger human food safety. my country has banned the use of metronidazole, demetonidazole, and lonidazole in meat foods, and tinidazole has been prohibited from being used in veterinary drugs. Therefore, there is a need for a Sensitive and precise method for simultaneous determination of nitroimidazoles in honey
[0003] At present, the methods for detecting nitroimidazole drugs mainly include: high performance liquid chromatography-ultraviolet chromatography, high performance liquid chromatography diode array detector method, gas chromatography nitrogen phosphorus detector method, high performance liquid chromatography-electrospray mass spectrometry, gas chromatography method, etc., the instrument detection method is cumbersome and expensive, and its popularization and application are limited

Method used

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  • Enzyme linked immunosorbent assay kit for detecting nitroimidazole drugs and application thereof
  • Enzyme linked immunosorbent assay kit for detecting nitroimidazole drugs and application thereof
  • Enzyme linked immunosorbent assay kit for detecting nitroimidazole drugs and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Preparation of embodiment 1 kit components and kit assembly

[0036] Coating Antigen Preparation

[0037] Metronidazole hapten preparation

[0038] 0.85g metronidazole and 1.0g succinic anhydride, add 10ml pyridine, stir at room temperature for 12-20 hours, evaporate the solvent to dryness, dissolve in ethyl acetate, wash with saturated ammonium chloride solution, wash with water, dry, remove the solvent and place in cyclohexane -Recrystallization from ethyl acetate gave a white solid, which was the metronidazole hapten.

[0039] Conjugation of metronidazole hapten with ovalbumin to obtain coated antigen

[0040] 1) Dissolve 15mg of metronidazole hapten in 1.5ml of DMF and cool to 10°C;

[0041] 2) Take 10 μl of isobutyl chloroformate and add it to 1), stir and react at 10°C for 30 minutes;

[0042] 3) Take 25mg ovalbumin with 2.2ml 50mmol / L Na 2 CO 3 Dissolve and react at 10°C for 4h, then overnight at 4°C;

[0043] 4) 0.01mol / l PBS was dialyzed at 4°C for 3 day...

Embodiment 2

[0079] The detection of nitroimidazoles in the sample of embodiment 2

[0080] 1. Sample pretreatment

[0081] The pretreatment of the sample is mainly to obtain a nitroimidazole drug solution from the sample for subsequent detection. The following is the sample pretreatment method:

[0082] Weigh 3.0±0.05g of honey into a 50ml polystyrene centrifuge tube; add 3ml of 0.1M carbonate buffer to fully dissolve the honey; add 9ml of ethyl acetate, shake for 5min, above 3000g, centrifuge at room temperature for 5min; take 6ml of the upper organic phase To a 50ml polystyrene centrifuge tube, add 1ml ethyl acetate, 1ml methanol, 1ml n-hexane, then add 2ml 2M sodium hydroxide solution, vortex for 5min; above 3000g, centrifuge at room temperature for 5min; pipette 4ml of the upper organic phase to In a 10ml clean glass test tube, dry it in a water bath at 50-60°C under nitrogen flow; add 1ml of n-hexane, vortex for 30s, add 0.5ml reconstituted working solution, vortex for 1min, centri...

experiment example 1

[0087] Experimental example 1 standard product precision test:

[0088] A batch of ELISA plates were extracted from the ELISA plates prepared in three different time periods, 5 kits were extracted from each batch, 20 microwells were extracted from each plate, and the absorbance value of the 0.6 μg / L standard solution was measured , to calculate the coefficient of variation.

[0089] Table 1 Standard repeatability test (CV%)

[0090]

[0091] From the above test results, it can be concluded that the coefficient of variation of each batch of 5 standard products is between 2.4% and 9.2%.

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Abstract

The invention provides an enzyme linked immunosorbent assay kit for detecting nitroimidazole drugs. The kit comprises an enzyme labeling plate coated with metronidazole coupling antigen, enzyme labeling antiantibody working solution, specific antibody working solution of the nitroimidazole drugs, metronidazole standard substance solution, substrate developing solution, stop solution, concentrated washing solution and redissolving solution. The invention further discloses a method for detecting the nitroimidazole drugs by utilizing the enzyme linked immunosorbent assay kit. The method comprises the following steps of: firstly, carrying out sample preprocessing; secondly, carrying out detection by utilizing the kit; and thirdly, analyzing a detection result. The enzyme linked immunosorbent assay kit provided by the invention can be used for detecting the content of the nitroimidazole drugs in a honey sample, is simple and convenient to operate, is low in cost, can realize on-site monitoring and is suitable for screening of a large number of samples.

Description

technical field [0001] The invention relates to an ELISA detection technology, in particular to an ELISA reagent kit for detecting nitroimidazoles, which is suitable for the determination of nitroimidazoles in honey. technical background [0002] Nitroimidazoles are antiprotozoal and antibacterial drugs commonly used in livestock and poultry feed, mainly used for the prevention and treatment of histomonas and parasites in poultry, trichomonas in cattle, hemorrhagic enteritis in pigs, parasitic infections in fish, bees Microsporidiosis, etc., and have growth-promoting effects, but such drugs and some metabolites have carcinogenic, teratogenic, mutagenic and genotoxic effects on mammals, so the unreasonable use of nitroimidazoles has caused possible Drug residues in carnivorous animal tissues endanger human food safety. my country has banned the use of metronidazole, demetonidazole, and lonidazole in meat foods, and tinidazole has been prohibited from being used in veterinary dr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531C07D233/94
Inventor 万宇平吴鹏罗晓琴孙震冯静余厚美赵正苗段盈盈何丽霞李晓芳
Owner BEIJING KWINBON BIOTECH
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