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Development of a Generic Enzyme-Immunoassay for the Detection of Fluoro(quinolone)-Residues in Foodstuffs Based on a Highly Sensitive Monoclonal Antibody

Abstract

An enzyme immunoassay (EIA) was developed based on a generic and highly sensitive monoclonal antibody (mAb) enabling simultaneous detection of all fluoroquinolone/quinolone antibiotics (FQs) approved for the treatment of food-producing animals. To generate the group-specific antibody, norfloxacin was conjugated to glucose oxidase (GlcOX) by one-step carbodiimide method and used as immunogen. For the establishment and optimization of a direct, competitive EIA, three different FQ-horseradish peroxidase (HRP) conjugates were generated. Best results were obtained by using a hapten-heterologous conjugate (clinafloxacin-HRP) prepared by applying a simple and highly efficient periodate method. Under optimized conditions, the assay showed a very high sensitivity, e.g., the detection limit for norfloxacin was 0.02 ng/mL. Furthermore, due to the high affinity of the employed mAb, all FQs approved within the EU for the treatment of food-producing animals can be detected well below their maximum residue levels (MRLs) with IC50 values ranging from 0.16 ng/mL (marbofloxacin) to 3.82 ng/mL (sarafloxacin). Additionally, the established EIA showed cross-reactivity with 23 other FQs and IC50 values ranged from 0.05 ng/mL (rufloxacin) to 73.2 ng/mL (pradofloxacin). Thus, the established EIA could be potentially applied both for the detection of approved and illegally used quinolones in food. To demonstrate the applicability of the assay, artificially contaminated milk, meat, fish, and shrimp samples were analyzed; mean recovery rates were 88.9%, 76.2%, 74.4%, and 77.9%, respectively. These data demonstrate that the developed EIA is suited for the rapid and sensitive monitoring of FQs residues in animal-derived food.

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Abbreviations

DMF:

dimethylformamide

EDC:

N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide

EIA:

Enzyme immunoassay

EU:

European Union

GlcOX:

Glucose oxidase

FQs:

Fluoroquinolones/quinolones

HRP:

Horseradish peroxidase

LOD:

Limit of detection

mAb:

Monoclonal antibody

MRL:

Maximum residue limit

PBS:

Phosphate buffered saline

IFA:

Incomplete Freund’s adjuvants

TMB:

3,3′,5,5′-tetramethyl-benzidine

BES:

Besifloxacin

CIN:

Cinoxacin

CIP:

Ciprofloxacin

CLN:

Clinafloxacin

DAN:

Danofloxacin

DEL:

Delafloxacin

DIF:

Difloxacin

ENO:

Enoxacin

ENR:

Enrofloxacin

FLR:

Fleroxacin

FLU:

Flumequine

GAT:

Gatifloxacin

IBA:

Ibafloxacin

LEV:

Levofloxacin

LOM:

Lomefloxacin

MAR:

Marbofloxacin

MOX:

Moxifloxacin

NAD:

Nadifloxacin

NA:

Nalidixic acid

NOR:

Norfloxacin

OFL:

Ofloxacin

ORB:

Orbifloxacin

OA:

Oxolinic acid

PRA:

Pradofloxacin

PAZ:

Pazufloxacin

PEF:

Pefloxacin

PA:

Pipemidic acid

PRU:

Prulifloxacin

RUF:

Rufloxacin

SAR:

Sarafloxacin

SPR:

Sparfloxacin

TOS:

Tosufloxacin

TRO:

Trovafloxacin

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Acknowledgments

We thank Ms. Brunhilde Minich and Mr. Mostefa Djeffal for the excellent technical support. This study was summarized from the doctoral thesis of Ulas Acaroz.

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Correspondence to Ulas Acaroz.

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Conflict of Interest

Ulas Acaroz declares that he has no conflict of interest. Richard Dietrich declares that he has no conflict of interest. Maria Knauer declares that she has no conflict of interest. Erwin Märtlbauer declares that he has no conflict of interest.

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All institutional and national guidelines for the care and use of laboratory animals were followed.

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Electronic supplementary material

Maximum residue limits (MRLs) for fluoroquinolone/quinolone (FQ) antibiotics in food of animal origin, in accordance with Regulation (EU) 37/2010; The summary of generic immunoassays for the detection of FQs; Schematic representation of the immunogen synthesis via carbodiimide method; UV spectra of Norfloxacin-GlcOX and Clinafloxacin-HRP conjugates with unconjugated starting materials; Standard curves of other FQs used in human medicine or veterinary medicine; Comparative presentation of standard curves for danofloxacin and marbofloxacin in PBS or reconstituted skimmed milk powder; Characteristics of quinolone standard curves generated in reconstituted milk powder which are employed for the recovery analysis; Characteristics of quinolone standard curves generated in PBS which are employed for the recovery analysis of beef, salmon and shrimp samples.

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Acaroz, U., Dietrich, R., Knauer, M. et al. Development of a Generic Enzyme-Immunoassay for the Detection of Fluoro(quinolone)-Residues in Foodstuffs Based on a Highly Sensitive Monoclonal Antibody. Food Anal. Methods 13, 780–792 (2020). https://doi.org/10.1007/s12161-019-01695-1

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Keywords

  • (Fluoro)quinolones
  • Monoclonal antibody
  • Enzyme immunoassay
  • Foodstuffs