A Novel Method for Antibiotic Detection in Milk Based on Competitive Magnetic Immunodetection

Abstract

The misuse of antibiotics as well as incorrect dosage or insufficient time for detoxification can result in the presence of pharmacologically active molecules in fresh milk. Hence, in many countries, commercially available milk has to be tested with immunological, chromatographic or microbiological analytical methods to avoid consumption of antibiotic residues. Here a novel, sensitive and portable assay setup for the detection and quantification of penicillin and kanamycin in whole fat milk (WFM) based on competitive magnetic immunodetection (cMID) is described and assay accuracy determined. For this, penicillin G and kanamycin-conjugates were generated and coated onto a matrix of immunofiltration columns (IFC). Biotinylated penicillin G or kanamycin-specific antibodies were pre-incubated with antibiotics-containing samples and subsequently applied onto IFC to determine the concentration of antibiotics through the competition of antibody-binding to the antibiotic-conjugate molecules. Bound antibodies were labeled with streptavidin-coated magnetic particles and quantified using frequency magnetic mixing technology. Based on calibration measurements in WFM with detection limits of 1.33 ng·mL−1 for penicillin G and 1.0 ng·mL−1 for kanamycin, spiked WFM samples were analyzed, revealing highly accurate recovery rates and assay precision. Our results demonstrate the suitability of cMID-based competition assay for reliable and easy on-site testing of milk.

Development of Fast and Portable Frequency Magnetic Mixing-Based Serological SARS-CoV-2-Specific Antibody Detection Assay

“…Afterward, IFCs were washed as described above, and 500 μl of 60 μg ml –1 streptavidin-functionalized superparamagnetic particles with a hydrodynamic diameter of 70 nm (synomag ® -D, article number 104-19-701; micromod Partikeltechnologie GmbH, Rostock, Germany) was added to columns and incubated for additional 5 min at RT. After final washing with 750 μl PBS, measuring signal correlating to enriched S1-specific antibodies was obtained by inserting the columns into the handheld and portable frequency mixing magnetic detection device ( Rettcher et al, 2015 ; Pietschmann et al, 2020a , b ).…”

“…Here, magnetic immunodetection (MID) based on sensing superparamagnetic particles by frequency magnetic mixing detection (FMMD) technology might be a powerful tool. The implementation of MID-based assays for the detection of various antigens like bacterial and fungal toxins, plant and human pathogens, as well as peptide-specific antibodies was previously demonstrated ( Meyer et al, 2007a , b , c ; Rettcher et al, 2015 ; Achtsnicht et al, 2019a ; Pietschmann et al, 2020a , b , c . The basic principle of magnetic immunodetection is the specific enrichment of an analyte within an immunofiltration column (IFC) and its subsequent labeling with functionalized magnetic nanoparticles, resulting in an analyte dose-dependent retention of particles.…”

“…The basic principle of magnetic immunodetection is the specific enrichment of an analyte within an immunofiltration column (IFC) and its subsequent labeling with functionalized magnetic nanoparticles, resulting in an analyte dose-dependent retention of particles. Subsequently a particle-dose-depending measuring signal in millivolt (mV) is obtained, using a portable measuring FMMD device ( Krause et al, 2007 ; Achtsnicht et al, 2019a , c ; Pietschmann et al, 2020a , b , c . With this, a correlation between antigen and measuring signal can be seen, resulting not only in a qualitative but also quantitative data.…”

Comparative Modeling of Frequency Mixing Measurements of Magnetic Nanoparticles Using Micromagnetic Simulations and Langevin Theory

“…Their signal can usually be picked up using a receive coil; however, other magnetic detectors can also be employed [ 6 ]. Due to its high sensitivity, FMMD has been successfully applied to realize magnetic immunoassays for the detection of a multitude of different analytes, for instance, viruses [ 7 ], antibiotics [ 8 ], or bacterial toxins [ 9 ]. Furthermore, FMMD enables the distinction of different types of MNP based on their different frequency mixing response spectrum [ 10 , 11 , 12 ], which opens the potential for multi-analyte detection.…”

Multiplex Detection of Magnetic Beads Using Offset Field Dependent Frequency Mixing Magnetic Detection

“…Frequency mixing magnetic detection (FMMD) has proven to be very selective for superparamagnetic particles and thus it has been successfully used for detection of a variety of biological targets. For example, recently, the quantification of Aflatoxin B1 [ 14 ] and of different antibiotics in milk [ 15 ] has been shown using a competitive magnetic immunoassay. Furthermore, by employing a noncompetitive sandwich immunoassay method, the detection of cholera toxin subunit B [ 16 ], Francisella tularensis [ 17 ], C-reactive protein [ 18 ], plant viruses [ 19 ], and influenza viruses [ 18 , 19 , 20 ] has been shown.…”