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Time resolution immunoassay test kit and method of ciprofloxacin residual

A detection kit and technology of ciprofloxacin, which is applied in the field of immunoassay, can solve the problems such as the absence of ciprofloxacin time-resolved immunoassay kit, and achieve the effect of simple sample pretreatment, short operation process and good effect

Inactive Publication Date: 2011-03-30
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] However, there is no relevant technical report on the detection of ciprofloxacin by time-resolved immunoassay, and there is no kit suitable for time-resolved immunoassay detection of ciprofloxacin, so as to achieve high sensitivity and simple operation in large quantities. Quick check

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  • Time resolution immunoassay test kit and method of ciprofloxacin residual
  • Time resolution immunoassay test kit and method of ciprofloxacin residual
  • Time resolution immunoassay test kit and method of ciprofloxacin residual

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Embodiment 1

[0062] The preparation of embodiment 1 antigen

[0063] Preparation of ciprofloxacin antigen:

[0064] a. Dissolve 50 μmol / L ciprofloxacin in 1 mL of DMF, then add equimolar DCC and NHS to the solution, and let it 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 carbonic acid 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 determined by ultraviolet scanning (Chen Xinjian et al., 1998). Finally, the antigen was concentrated or lyophilized to obtain the ciprofloxacin immunogen and the coating agent, which were stored in a refrigerator at -20°C.

Embodiment 2

[0068] The preparation of embodiment 2 antibody

[0069] Preparation of ciprofloxacin rabbit monoclonal antibody:

[0070] Animal immunization: Balb / c mice were immunized at intervals with the conjugate of ciprofloxacin and carrier protein as the immunogen, and the spleens of mice containing ciprofloxacin-specific antibodies in the blood were detected by indirect immunoassay.

[0071] Cell fusion and cloning: the splenocytes of Balb / c mice producing specific antibodies were fused with myeloma cells SP20, the cell supernatant was measured by indirect competitive enzyme-linked immunosorbent method, and positive wells were screened. The positive wells are cloned by limiting dilution method or microcloning method, and hybridoma cell lines producing monoclonal antibodies are obtained and established.

[0072] Cell cryopreservation and recovery: take the hybridoma cells in the logarithmic growth phase and use the cryopreservation medium to make a cell suspension, aliquot them into ...

Embodiment 3

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

[0126] Take 1ml of 5mg / mL goat anti-mouse IgG dissolved in 0.05mol / L PBS pH7.4, switch the buffer salt condition through the 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-mouse to 1 mg / mL with the eluent above. Take 500 μL of diluted goat anti-mouse 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 / number of...

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Abstract

The invention provides a time resolution immunoassay test kit and method of ciprofloxacin residual. The test kit of the invention is coated with an elisa plate of a ciprofloxacin antigen, a lanthanide-labeled goat anti-rabbit or goat anti-mouse antibody, a ciprofloxacin antibody and the like. The invention also discloses the method for testing the ciprofloxacin residual by utilizing the test kit. The test kit for testing the ciprofloxacin provided by the invention has the advantages that an indirect competition time resolution immunoassay technique is adopted, the sensitivity is high, the stability is good, the operation step and the reaction time are greatly simplified, errors caused by the complex operation are reduced, and the cost is lowered, thereby being extremely suitable for screening a large number of samples, and having important realistic significance.

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 ciprofloxacin 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 pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531G01N21/64C07K16/44C07K16/06C12N15/12C12N15/70
Inventor 孙远明王弘徐振林雷红涛沈玉栋杨金易
Owner SOUTH CHINA AGRI UNIV
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