One-step immune detecting method for enrofloxacin residual in aquatic product
An immunoassay method, enrofloxacin technology, applied in the field of detection of drug residues in aquatic products, can solve the problems of long detection time, cumbersome operation, poor anti-interference ability, etc., achieve simple and fast operation, save cumbersome operations, and shorten analysis the effect of time
Inactive Publication Date: 2013-04-24
OCEAN UNIV OF CHINA
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Problems solved by technology
However, the existing immunoassay method is mainly based on two-step enzyme-linked immunosorbent assay (ELISA), which has problems such as long detection time, cumbersome operation, and poor anti-interference ability, which limits its practical application as a rapid screening method. application
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Embodiment 1
[0017] Example 1: Rapid detection of enrofloxacin residues in eels
[0018] 1. Antibody preparation and purification
[0019] Antibodies to enrofloxacin were prepared by immunizing mice with biological immunization techniques. Enrofloxacin antiserum was separated after immunization of mice, precipitated with 30-50% saturated ammonium sulfate, and then purified by superdexTM75 column.
[0020] 2. Preparation of enzyme-labeled antibody
[0021] Weigh 5mg of horseradish peroxidase and dissolve in 1ml of distilled water, add 0.2mL of freshly prepared 0.1M NaIO 4 The solution was stirred at room temperature in the dark for 20 minutes; the solution was put into a dialysis bag, and dialyzed against 1mM pH4.4 sodium acetate buffer at 4°C overnight; after taking it out, add 20μL of 0.2M pH9.6 carbonate buffer to The pH of the above-formylated horseradish peroxidase was raised to 9.0-9.6, then immediately added 10 mg of mouse serum antibody dissolved in 1 mL of 0.01 M carbonate buffer...
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The invention discloses a one-step immunodetection method for enrofloxacin residue in an aquatic product. In the method, a specific polyclonal antibody is prepared by a biological immune technology; the prepared specific antibody is combined with horseradish peroxidase to form an enzyme-labeled antibody; a 96-well polystyrene plate is taken as a solid phase carrier, a corresponding complete antigen are used for coating in advance and carrier protein is blocked; a negative control is prepared from extracting solution without a substance to be detected in detection, and a standard curve is established according to gradient solution of an enrofloxacin standard substance; an absorbance value of the standard curve is detected and corresponding inhibition ratio is computed by the conventional ELISA, thus further determining the concentration of the substance to be detected in a sample according to the standard curve. The immunodetection method has the advantages of high sensitivity, good accuracy and being capable of meeting the detection requirements of corresponding standards, simple, convenient and fast detection and effectively shortened analysis time, thus the method can be applicable to field rapid screening and detection of the enrofloxacin residue.
Description
technical field [0001] The invention relates to a method for detecting drug residues in aquatic products, in particular to a method for detecting enrofloxacin residues. The invention is funded by the National Natural Science Foundation of China (No. 30400336) and the National Agricultural Industry Research Project (NYHYZX 07-046). Background technique [0002] Enrofloxacin (Enrofloxacin) is a chemically synthesized broad-spectrum antibacterial drug, which belongs to the representative product of fluoroquinolones, and is currently widely used in the treatment and control of bacterial infectious animal diseases in the field of aquaculture. However, more and more studies have shown that enrofloxacin has potential hazards to human health, and the relevant testing and monitoring requirements are becoming more stringent. The detection methods of enrofloxacin include liquid chromatography, mass spectrometry-liquid chromatography and immunological detection methods. However, the e...
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IPC IPC(8): G01N33/543
Inventor 曹立民林洪李宗妍隋建新许旭
Owner OCEAN UNIV OF CHINA