BEADYPLEX™: A novel multi-antibiotic flow cytometric screening method for food commodities

C. Suárez-Pantaleón1, A. Gallo1, V. Chabottaux1, O. Heynen1, B. Granier1, A. Braseur2, A.-C. Huet2, Ph. Delahaut2 and N. Gillard2

1Unisensor S.A., Allée de la Cense Rouge 98, 4102 Liège (Ougrée) - BELGIUM (celia.suarez@unisensor.be) 2CER Groupe, Health Department, Rue du Point du Jour 8, 6900 Marloie-BELGIUM

Introduction

Methodology and Assay principle BEADYPLEX™ combines in the same single reaction several competitive immunoassays in a bead suspension format. A simple and rapid buffer-based sample extraction is followed by the FCIA, which consists on a first incubation of the extract with encoded antibiotic-conjugated beads (assay competitors) and broad-range antibiotic primary binders, followed by an additional incubation with secondary fluorescent binders. Finally, the resulting “bead-binders” complexes are characterized using the Flow Cytometer, including bead identification (according to their size and internal fluorescence) and the final assay signal. The presence of residues from an antibiotic family is identified by the fluorescence decrease of the corresponding bead.

The misuse of antibiotics in animal farming may lead to the presence of residues in edible products, thus compromising consumer health (toxicological and allergic reactions, as well as bacterial antibiotic resistance development) and industrial food processes. Rapid and efficient screening analytical methods for the early detection of antibiotic residues are therefore essential to guarantee both food safety and to reduce the negative impact on industrial fermentation processes . BEADYPLEX™ is a competitive Flow Cytometric ImmunoAssay (FCIA) for the simultaneous multi-residue analysis of more than 80 antibiotics from 10 families in food commodities, providing the family identification in a single test. Herein, we present the validation results (detection capabilities) in porcine, bovine and poultry muscle following 2010 CRL Guidelines supplementing European Decision 2002/657/EC. Furthermore, preliminary results show the potential applicability of BEADYPLEX™ to other matrices, such as fish, seafood, milk or eggs.

Sample Preparation Flow Cytometric Immunoassay

Results The validation of BEADYPLEX™ in porcine, bovine and poultry muscle was conducted by CER Groupe following CRL Guidelines for the validation of screening methods for residues of veterinary medicines (20/01/2010, 1-18; supplement of Commission Decision 2002/657/EC). The table below contains the CCβ values (detection capabilities) for the whole set of antibiotics detected in the three matrices. For most compounds, the detection capabilities are at or below the corresponding European MRL value (Commission Regulation EU N°37/2010).

The most relevant veterinary antimicrobial compounds are detected at or below the corresponding regulatory limits : more than 80 antibiotics from 10 families .

The main features of BEADYPLEX ™ comprise: broad-range detection profile, broad-applicability, identification of the antibiotic family in one single test per sample, and a high throughput capability. Both analysis time and global cost may be significantly reduced, as the choice of the required confirmatory method is straightforward.

The work herein presented illustrates the potential of Flow Cytometry for food analysis. In order to expand the application of this versatile and flexible technology to other type of contaminants, new FCIA kits are under development.

Conclusions and Perspectives

A fortification study with a multi-antibiotic mixture was conducted in different food commodities.

The normalized response (B/B0*100) was calculated with respect to the corresponding blank matrix for every individual bead (BL = bead level).

All of the evaluated antibiotics were detected by the method in all the matrices, as shown by the positive response (signal close to or below the corresponding cut-off), thus demonstrating the broad-applicability of BEADYPLEX™.

Acknowledgment • This work has received funding from Eureka and the Belgian Walloon Region.

Porcine muscle Bovine muscle Poultry muscle

Family An5bio5c MRL CCβ MRL CCβ MRL CCβ

Aminoglycosides

Streptomycin 500 250 500 250 - 250

Dihydrostreptomycin 500 125 500 125 - 125

Gentamicin 50 50 50 50 - 50

Neomycin B 500 100 500 100 500 100

Kanamycin A 100 100 100 100 100 100

Paromomycin 500 250 500 500 500 500

β-Lactams

Cefquinome 50 50 50 50 - 50

CeLiofur 1000 50 1000 50 1000 50

DesfuroylceLiofur 1000 2000 1000 2000 1000 2000

Cefoperazone - 3 - 3 - 3

Cefalexin - 3500 200 3500 - 3500

Cefalonium - 5 - 5 - 5

Cefapirin - 75 50 50 - 50

Desacetylcefapirin - 100 - 100 - 100

Cefazolin - 100 - 100 - 100

Cefacetrile - 50 - 50 - 50

Penicillin V 25 12,5 - 12,5 25 12,5

Penicillin G 50 5 50 5 50 5

Ampicillin 50 25 50 25 50 25

Amoxicillin 50 25 50 25 50 25

Oxacillin 300 50 300 50 300 50

Cloxacillin 300 50 300 50 300 50

Dicloxacillin 300 30 300 30 300 30

Nafcillin - 300 300 300 300 300

Piperacillin - 5 - 5 - 5

Porcine muscle Bovine muscle Poultry muscle

Family An5bio5c MRL CCβ MRL CCβ MRL CCβ

Sulfonamides

Sulfadimidine

Combined sulfonamides

= 100

100

Combined sulfonamides

= 100

100

Combined sulfonamides

= 100

100

Sulfadiazine 50 50 50

Sulfamethoxazole 10 10 10

Sulfadimethoxine 50 50 50

Sulfadoxine 10 10 10

Sulfachloropyridazine 1 1 1

Sulfapyridine 0,5 0,5 0,5

Sulfaguanidine 1 1 1

Sulfathiazole 0,5 0,5 0,5

Sulfamethoxypyridazine 0,25 0,25 0,25

Sulfamerazine 20 20 20

Sulfaclozine 500 500 1000

Sulfamethizole 1 1 1

Sulfamonomethoxine 10 20 10

Sulfaquinoxaline 50 50 50

Sulfameter 5 5 5

Sulfanilamide 50 50 50

Dapsone 0,2 0,2 0,2

Macrolides

Tilmicosin 50 50 50 50 75 50

Tylosin A 100 10 100 10 100 10

Tylvalosin 50 125 50 125 - 125

Tildipirosin 1200 1200 400 1200 - 1200

Spiramycin 250 250 200b 200 200b 200

Lincosamides Lincomycin 100 25 100 25 100 25

Clindamycin - 150 500 - 300

Polymyxin Colis5n 150 150 150 150 150 150

Porcine muscle Bovine muscle Poultry muscle

Family An5bio5c MRL CCβ MRL CCβ MRL CCβ

Tetracyclines

Chlortetracycline 100 25 100 100 100 100

Doxycycline 100 25 100 25 100 25

Oxytetracycline 100 50 100 100 100 100

Tetracycline 100 50 100 50 100 50

Demeclocycline - 50 - 100 - 100

Methacycline - 25 - 50 - 50

(F)Quinolones

Marbofloxacin 150 20 150 20 - 20

Flumequine 200 800 200 2000 400 2000

Enrofloxacin Sum = 100

5 Sum = 100

5 Sum = 100

5

Ciprofloxacin 10 20 20

Danofloxacin 100 250 200 200 200 200

Oxolinic acid 100 800 100 1000 100 1000

Difloxacin 400 50 400 50 300 50

Norfloxacin - 2,5 - 10 - 10

Sarafloxacin - 125 - 125 - 125

Pefloxacin - 10 - 20 - 20

Enoxacin - 50 - 50 - 50

Lomefloxacin - 15 - 15 - 15

Ofloxacin - 15 - 15 - 15

Cinoxacin - 800 - 800 - 800

Nalidixic acid - 2000 - 2000 - 2000

Phenicols

Chloramphenicol 0,3 0,45 0,3 0,45 0,3 0,45

Florfenicol 300 2100 200 2100 100 2100

Thiamphenicol 50 100 50 100 50 100

Pleuromu5lins Valnemulin 50 50 - 50 - 50

Tiamulin 100 25 - 25 100 25

Incubate 30 min + Wash Incubate 15 min + Wash

Detection Capabilities (µg/kg) for BEADYPLEX™ in muscle (non-exhaustive list)