J Vet Sci.  2015 Dec;16(4):431-437. 10.4142/jvs.2015.16.4.431.

Magnetic nanoparticle based purification and enzyme-linked immunosorbent assay using monoclonal antibody against enrofloxacin

• Kim NG
• Kim MA
• Park YI
• Jung TS
• Son SW
• So B
• Kang HG

• KMID: 2133625
• DOI: http://doi.org/10.4142/jvs.2015.16.4.431

Abstract

Monoclonal anti-enrofloxacin antibody was prepared for a direct competitive enzyme-linked immunosorbent assay (ELISA) and purification system using monoclonal antibody (mAb) coupled magnetic nanoparticles (MNPs). The IC50 values of the developed mAb for enrofloxacin (ENR), ciprofloxacin, difloxacin, sarafloxacin, pefloxacin, and norfloxacin were 5.0, 8.3, 9.7, 21.7, 36.0, and 63.7 ng/mL, respectively. The lowest detectable level of ENR was 0.7 ng/mL in the prepared ELISA system. To validate the developed ELISA in the food matrix, known amounts of ENR were spiked in meat and egg samples at 10, 20 and 30 ng/mL. Recoveries for ENR ranged from 72.9 to 113.16% with a coefficient of variation (CV) of 2.42 to 10.11%. The applicability of the mAb-MNP system was verified by testing the recoveries for ENR residue in three different matrices. Recoveries for ENR ranged from 75.16 to 86.36%, while the CV ranged from 5.08 to 11.53%. Overall, ENR-specific monoclonal antibody was prepared and developed for use in competitive to ELISAs for the detection of ENR in animal meat samples. Furthermore, we suggest that a purification system for ENR using mAb-coupled MNPs could be useful for determination of ENR residue in food.

Keyword

enrofloxacin; enzyme-linked immunosorbent assay; magnetic nanoparticle; monoclonal antibody

MeSH Terms

Animals
Ciprofloxacin
Enzyme-Linked Immunosorbent Assay*
Inhibitory Concentration 50
Meat
Nanoparticles*
Norfloxacin
Ovum
Pefloxacin
Ciprofloxacin
Norfloxacin
Pefloxacin

Figure

• 

  Fig. 1 Standard curve of direct competitive enzyme-linked immunosorbent assay (ELISA) using coating antigen enrofloxacin (ENR)-bovine serum albumin (BSA) (100 ng/mL) and ENR monoclonal antibody (mAb)-HRP (diluted 1/1,000, final dilution in the well). (A) Standard curves of chicken muscle samples. (B) Standard curves of egg samples. (C) Standard curves of cattle samples. logC, ENR standard concentration in extract solution (40, 20, 10, 5, 2.5, and 0 ng/mL).

• 

  Fig. 2 Binding efficiency of ENR mAb to different amounts of magnetic nanoparticles (MNPs). ENR mAb (130 µg) was coupled with each amount of MNP (n = 3). Data shown represent the mean ± SE (n = 3).

• 

  Fig. 3 High-performance liquid chromatography chromatogram of ENR. (A) Standard sample: MeOH in distilled water (DW) containing 25 ng/mL ENR. (B) Sample: chicken muscle spiked with 25 ng/mL ENR (dilution). (C) MeOH in DW sample.

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