Abstract
A heterologous indirect competitive enzyme linked immuno sorbent assay (icELISA) has been developed for the determination of enrofloxacin (ENR) residues in poultry. For this purpose, carbodiimide active ester method was employed to synthesize the artificial antigen of ENR- bovine serum albumin (BSA) while mixed-anhydride technique was used to synthesize the coating antigen of ENR- ovalbumin (OVA), to pursue the heterologous sensitivity. By square matrix titration, an icELISA method was developed, and the linear range was from 0.02 to 86.3 ng/mL, with limit of detection (LOD) and IC50 value of 0.8 and 0.01 ng/mL, respectively. After optimization, 5% of NaOH was used in the assay buffer and this ELISA system can tolerate methanol not higher than 30%. The correlation coefficients (R2) between concentration spiked and concentration determined were 0.9975 in chicken muscle and 0.9959 in duck muscle, respectively. Therefore, this assay has the potential to be incorporated into a quantitative monitoring program for the rapid screening of ENR residue in poultry muscles. Key words: Enrofloxacin, artificial antigen, polyclonal antibody, indirect competitive enzyme linked immuno sorbent assay (ELISA), heterologous, poultry.
Highlights
Enrofloxacin (ENR) is the first specific fluoroquinolone developed for veterinary application, which belongs to the second generation of quinolone antibiotics fluorinated in position 6 and bearing a piperazinyl moiety in position 7
The absorbance for ENR-bovine serum albumin (BSA) (279 and 321 nm) gave a significant shifted peak at 279 nm compared with the 269 nm peak for ENR (269, 321, and 333 nm), which indicated the ENR was successfully conjugated with BSA
The optimum concentration of coating antigen was 1.0 μg/mL and polyclonal antibody (pAb) was 1:10,000 dilutions. This assay allowed the detection of ENR (20-80% inhibition of color development) from 0.02 to 86.3 ng/mL, with an half-maximum inhibition concentration (IC50) value
Summary
Enrofloxacin (ENR) is the first specific fluoroquinolone developed for veterinary application, which belongs to the second generation of quinolone antibiotics fluorinated in position 6 and bearing a piperazinyl moiety in position 7. Awareness of residual antibiotics in animalderived food is growing as their application increases in both human and veterinary medicine (Yan et al, 2011). In order to monitor enrofloxacin residue levels in livestock and poultry products, simple and rapid analytical methods are required. Various analytical methods, such as high performance liquid chromatography (HPLC) (Christodoulou et al, 2008), liquid chromatography-mass spectrometry (LC-MS) (Delepine et al, 1998; San Martin et al, 2007), and LC-MS/MS
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