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Time-resolved immunofluorometric assay detection kit for enrofloxacin residue and detection method thereof

A detection kit, the technology of enrofloxacin, applied in the field of immunodetection, can solve the problem of no kit, etc., and achieve the effects of simple sample pretreatment, good effect, and short operation process

Inactive Publication Date: 2011-07-27
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] However, there is currently no relevant technical report on the detection of enrofloxacin by time-resolved immunoassay, and there is no kit suitable for time-resolved immunoassay detection of enrofloxacin, so as to achieve high sensitivity and simple operation in large quantities. Quick check

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  • Time-resolved immunofluorometric assay detection kit for enrofloxacin residue and detection method thereof
  • Time-resolved immunofluorometric assay detection kit for enrofloxacin residue and detection method thereof
  • Time-resolved immunofluorometric assay detection kit for enrofloxacin residue and detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0063] The preparation of embodiment 1 enrofloxacin antigen

[0064] a. Dissolve 50 μmol / L enrofloxacin in 1 mL of dimethylformamide (DMF), then add equimolar dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide to the solution Amine (NHS), react overnight at room temperature;

[0065] b. Centrifuge, take 800 μL of supernatant, slowly add to 4 mL of 15 mg / mL BSA or OVA carrier protein carbonate buffer solution, and then react for 4 hours under magnetic stirring;

[0066] c. After the reaction is completed, put it into a dialysis bag, first dialyze twice with distilled water, and then dialyze with 0.8% normal saline to obtain the product;

[0067] d. The binding ratio was measured by ultraviolet scanning (Chen Xinxin et al., 1998). Finally, the antigen was concentrated or lyophilized to obtain the enrofloxacin immunogen and the coating agent, which were stored in a refrigerator at -20°C.

Embodiment 2

[0068] The preparation of embodiment 2 antibody

[0069] (1) Enrofloxacin rabbit polyclonal antibody preparation

[0070] Animal immunization procedure: New Zealand white rabbits were used as immunized animals. Enrofloxacin and bovine serum albumin conjugates were used as the immunogen, and the immunization dose was 100 μg / monkey. At the first immunization, the immunogen was mixed with the same amount of Freund's complete adjuvant to make an emulsifier, and the intraperitoneal injection was performed at intervals of 3 Take the same dose of immunogen plus the same amount of Freund's incomplete adjuvant mixed and emulsified every week, and boost the immunization once, take rabbit blood, centrifuge, take the supernatant, use caprylic acid-ammonium sulfate method to purify the antibody, and freeze it in 100 μL / tube.

[0071] (2) Enrofloxacin rabbit monoclonal antibody preparation

[0072] Animal immunization: Balb / c mice were immunized at intervals with the conjugate of enroflox...

Embodiment 3

[0125] Example 3Eu 3+ Preparation and purification of labeled goat anti-rabbit

[0126] Take 1ml of 5mg / mL goat anti-rabbit IgG dissolved in 0.05mol / L PBS pH7.4, and switch the buffer salt condition through a PD-10 column, and the eluent is 50mmol / L pH 9.6NaCO 3 -NaHCO 3 buffer. The protein peaks were collected and quantified by UV absorption analysis (1.46A 280 -0.74A 260 ), dilute goat anti-rabbit to 2mg / mL with the eluent above. Take 500 μL of diluted goat anti-rabbit IgG and add 0.2 mg of Eu 3+ -N 2 -[p-isocyanic acid-benzyl]-diacetyltriaminetetraacetic acid (Eu 3+ -DTTA) in brown vials, placed in a constant temperature oven at 28°C to react for 48h, the reaction solution was chromatographed on Sepharose CL-6B (1×30cm) equilibrated with 50mmol / L Tris-HCl pH 7.8 buffer solution, and the fluorescence count of each tube was measured value, detect the first counting peak after dilution, and prepare for use after dilution.

[0127] Labeling rate = number of markers / num...

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Abstract

The invention provides a time-resolved immunofluorometric assay detection kit for enrofloxacin residues and a detection method thereof. The detection kit is coated with the elisa plate of an enrofloxacin antigen, and a lanthanide is marked with a goat anti rabbit or goat anti mouse antibody, an enrofloxacin antibody and the like. The invention also discloses a method applying the above detection kit to detect enrofloxacin residues. In the detection kit for enrofloxacin residues provided by the invention, the indirect competitive time immunofluorometric assay technology is adopted, so that the detection kit has the advantages of high sensitivity and good stability, is very suitable for screening a great quantity of samples and has an important practical meaning; and operation steps is greatly simplified, reaction time is greatly shortened, errors caused by complex operation are reduced, and cost is lowered.

Description

technical field [0001] The invention belongs to the technical field of immunodetection, and relates to a detection kit for chemical pollution residues and a preparation method thereof, in particular to a time-resolved immunoassay kit for detecting enrofloxacin residues and a preparation method thereof. Background technique [0002] Food safety is a major event related to the daily life of the broad masses of people. Food safety issues mainly include several factors such as physical hazards, chemical hazards and microbial hazards in food, among which chemical hazards mainly include veterinary drug residues, pesticide residues, heavy metals, and environmental hazards. , [0003] The United Nations Food and Agriculture Organization (FAO) and the World Health Organization (WHO) Joint Legislative Committee on Residues define veterinary drug residues as: parent compounds of veterinary drugs and their metabolites, and impurities related to veterinary drugs contained in any edible ...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/577G01N1/28G01N21/75G01J1/00
Inventor 孙远明雷红涛杨金易沈玉栋王弘肖治理
Owner SOUTH CHINA AGRI UNIV
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