Rabbit monoclonal antibody based ciprofloxacin residue analysis enzyme-linked immune adsorption kit

An enzyme-linked immunosorbent, monoclonal antibody technology, applied in the field of kits, can solve problems such as no reports, and achieve the effect of reducing detection costs

Inactive Publication Date: 2010-04-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This kit uses hybridoma technology to obtain ciprofloxacin rabbit monoclonal antibody, and develops a ciprofloxacin indirect ELISA kit based on rabbit monoclonal antibody, which has not been reported at home and abroad

Method used

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  • Rabbit monoclonal antibody based ciprofloxacin residue analysis enzyme-linked immune adsorption kit
  • Rabbit monoclonal antibody based ciprofloxacin residue analysis enzyme-linked immune adsorption kit
  • Rabbit monoclonal antibody based ciprofloxacin residue analysis enzyme-linked immune adsorption kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Synthesis of Ciprofloxacin Immunization Antigen and Coating Antigen

[0027] The specific operation steps are as follows: add 5.76mg NHS, 20.64mg EDC, 3.86mg CPFX to 0.23ml DMF, incubate at room temperature, shading, for 24h (solution I); add 26.4mg BSA to 2.3ml 0.01MpH7.4 PBS (solution II). Then, the incubated solution I was added dropwise to solution II, shaken while adding, and stirred magnetically for 3 hours at room temperature. The reaction mixture was transferred into a dialysis bag and dialyzed against 0.01M pH7.4 PBS to remove free CPFX and other small molecular substances. The coating antigen OVA-CPFX was prepared in the same way and used to coat the microtiter plate.

[0028] The coupling principle of ciprofloxacin and carrier protein is as follows:

[0029]

[0030] Note: R-COOH is ciprofloxacin: For the NHS: R-CO-NH-R 1 for coupling products

Embodiment 2

[0031] Example 2: Production of Ciprofloxacin Rabbit Monoclonal Antibody

[0032] 1. Immunization of rabbits

[0033] For primary immunization, mix 500 μg of immune antigen with an equal amount of Freund’s complete adjuvant, and after complete emulsification, inject white rabbits (1 mL / only) subcutaneously at 5 points on the back; three weeks later, use 200 μg of immune antigen with Freund’s incomplete adjuvant After mixing, carry out the second immunization in the same way; after that, immunize once every two weeks. After a total of 3 to 7 immunizations, when the serum titer reaches 64,000 or more, inject 500 μg of immune antigen into the ear vein, and collect whole blood from the carotid artery 4 days later. , take the spleen aseptically and prepare for cell fusion.

[0034] 2. Preparation of rabbit monoclonal antibody

[0035]Rabbit spleen cells were fused with 240E in the logarithmic growth phase, with 50% PEG as the fusion agent and HAT as the selective medium, and spre...

Embodiment 3

[0036] Embodiment 3: the coating of microtiter plate

[0037] Ciprofloxacin-coated antigen was diluted to 4 μg / mL with pH9.6, 0.05mol / L carbonate buffer (containing 2.93g sodium bicarbonate and 1.59g sodium carbonate, dissolved in double distilled water or ultrapure water 1L) , add 100 μL to each well of the ELISA plate, coat at 4°C for 12 hours or 37°C for 2 hours, pour off the coating liquid, wash with PBST for 3 to 5 times, pat dry, then add 200 μL of 5% glycine to each well, Seal at 37°C for 1 hour, wash 3 to 5 times after taking it out, dry and seal the film for storage.

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Abstract

The invention discloses a rabbit monoclonal antibody based ciprofloxacin residue analysis enzyme-linked immune adsorption kit. In the ciprofloxacin detection, the IC50 value of the kit is 1.41ng/mL, the linear fitting equation of an inhibition ratio curve is y = 0.0239x - 1.3159, the detection range is between 0.19 and 10ng/mL, and the lowest detection limit is 0.095ng/mL. The ciprofloxacin antibody in the kit has higher specificity, and has lower crossing-over rate with other quinolone micromolecules; and the cross reaction rates between the ciprofloxacin antibody and enrofloxacin, ofloxacin, norfloxacin, fleroxacin or pefloxacin are 28.8 percent, 13.1 percent, 11 percent, 22.6 percent and 20.4 percent respectively. The ciprofloxacin antibody has no cross reaction with other antibiotics and sulfanilamide medicaments. The kit is suitable for detecting ciprofloxacin residue in samples of milk, urine and the like. The kit can detect samples on a large scale at the same time, is convenient and quick, has quite important realistic significance for on-site supervision and trace analysis, meanwhile greatly reduces the detection cost, and has potential economic value.

Description

technical field [0001] The invention relates to a kit, in particular to an enzyme-linked immunosorbent assay kit for ciprofloxacin residue analysis based on a rabbit monoclonal antibody. Background technique [0002] Ciprofloxacin (ciprofloxacin, ciprofloxacin, CPFX or CIP), chemical name 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7(1-piperazinyl) -3-quinolinecarboxylic acid. It is a broad-spectrum high-efficiency drug, and it is effective for G - Bacillus has strong lethality and is also effective against severe infections caused by drug-resistant bacteria. It is one of the third-generation fluoroquinolone antibacterial drugs with higher antibacterial activity. This type of drug absorbs rapidly, has high bioavailability and is widely distributed in the body, and has no cross-resistance reaction with other antibiotics. It is an effective drug for preventing and treating bacterial infection and mycoplasma disease in livestock and poultry, and its MIC is below 1 μg / ml. Howeve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/52
Inventor 郑晓冬黄斌刘娜陆蕾倪庚
Owner ZHEJIANG UNIV
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