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Light induced chemiluminescence immunoassay method for detecting enrofloxacin and kit thereof
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A light-induced chemiluminescence and enrofloxacin technology, which is applied in the field of light-induced chemiluminescence immunoassay, can solve the problems of expensive equipment, easy inactivation of marker enzymes, and limited sensitivity, and achieve short detection time, simple operation, and high sensitivity. high effect
Inactive Publication Date: 2010-11-24
JIANGSU INST OF NUCLEAR MEDICINE
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[0003] At present, physical and chemical detection methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) are mostly used for the detection of enrofloxacin, but these methods require expensive equipment, cumbersome operations and limited sensitivity.
In recent years, there have been reports on immuno
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Embodiment 1
[0017] Embodiment 1 preparation kit and detection eel sample
[0018] Preparation of luminescent particles coated with ENR-OVA artificial antigen:
[0019] Add 1 mg of luminescent particles to a centrifuge tube, add 12.5 μL of 1% Tween-20, 0.05 mg of ENR-OVA artificial antigen, 10 μL of 25 mg / mL sodium borohydride solution, and use 0.1M, pH6.0 2-(N- The volume of morpholine)ethanesulfonic acid (MES) buffer was added to 200 μL, and the reaction was shaken at 37° C. in the dark for 48 hours. Add 10 μL of 0.3 M pH 5.0 carboxymethoxylamine hemihydrochloride (CMO) solution to block the unbound sites, incubate at 37°C in the dark for 1 hour, and then centrifuge to separate the luminescent particles linked to ENR-OVA, and dilute them for later use.
[0020] Preparation of reagents:
[0021] Preparation of standard ENR reagents: standard ENR: 0ng / mL, 0.1ng / mL, 1ng / mL, 10ng / mL, 50ng / mL, 100ng / mL, obtained by dilution from pure ENR, diluent is distilled water.
[0022] The compositio...
Embodiment 2
[0037] Embodiment 2 chicken sample determination
[0038] The reagent provided by the kit is the same as in Example 1, and is used to detect chicken samples.
[0039]The specific detection steps are as follows: Weigh 3g±0.03g chicken sample into a 50mL centrifuge tube, add 9mL of acetonitrile-0.1mol / L sodium hydroxide solution (84+16, v / v), shake and mix for 10min, and centrifuge at 4000r / min 10min; pipette 4mL of the supernatant into a 50mL centrifuge tube, add 4mL of 0.02ml / L phosphate buffer, add 8mL of dichloromethane, shake for 10min, centrifuge at 4000r / min for 10min, take 6mL of the lower organic phase into a 10mL test tube , blow dry with nitrogen in a water bath at 50°C; add 0.02ml / L phosphate buffer 0.5mL, vortex for 2min to dissolve the residue, add n-hexane 1mL, vortex for 2min, centrifuge at 4000r / min for 5min; take 50μL of the supernatant for analysis .
[0040] Add 20 μL of luminescent particles coated with ENR-OVA to a white opaque microwell plate; add 20 μL ...
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Abstract
The invention discloses a light induced chemiluminescence immunoassay method for detecting enrofloxacin and a kit thereof, and belongs to the technical field of light induced chemiluminescence immunoassay (LICLIA). The detection basis of the kit is homogeneous phase labeled immunoreaction. The method comprises the following steps of: adding ENR-OVA-coated luminous particles into a microplate, sequentially adding an ENR standard or sample, a rabbit anti-ENR antibody, a biotinylated goat anti-rabbit antibody for lucifugal reaction, adding streptavidin-coated sensitive particles for incubation, and performing detection; competitively connecting the ENR-OVA on the luminous particles and the ENR to the ENR antibody, and forming a complex with the biotinylated goat anti-rabbit antibody and the streptavidin-coated sensitive particles; under light induction, due to the generation and transmission of singlet state ionized oxygen, transmitting the energy to the luminous particles to generate fluorescence, detecting optical signals by using a light excited chemiluminescent detector, wherein the intensity of the optical signals and the concentration of ENR in the sample are in inverse proportion, and contrasting a standard curve to measure the content of the ENR in the sample. The kit for detecting the ENR has the advantages of simple structure, simple and convenient operation, short detection time and high sensitivity.
Description
technical field [0001] A kit for detecting enrofloxacin (ENR) and a detection method thereof belong to the technical field of light-induced chemiluminescence immunoassay (LICLIA), and are used for detecting the ENR content in fish, meat and their products. Background technique [0002] Enrofloxacin (enrofloxacin, ENR), also known as enrofloxacin carboxylic acid, is a new class of synthetic antibacterial drugs, and its metabolite ciprofloxacin (ciprofloxacin, CIP) has emerged since the 1980s It is one of the third-generation fluoroquinolones (FQNs) with a wider antibacterial spectrum and stronger antibacterial effect. This type of drug can kill bacteria by inhibiting topoisomerase II and topoisomerase IV to inhibit DNA replication in bacteria, and it will soon be widely used in medicine and veterinary medicine. However, adverse reactions caused by long-term medication, residues in livestock and poultry products, increase in drug resistance, and ecological effects in the envi...
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