Simultaneous Determination of Penicillin G and Chloramphenicol in Milk by a Magnetic Nanoparticle-Based Fluorescent Immunoassay

Background: Antibiotic residues are a problem of increasing importance and have direct consequences for human and animal health. The frequent use of antibiotics in veterinary practice causes their excretion in milk in dairy cattle. This way, they can easily enter the human body through the consumption of milk and dairy products. Objectives: This induces the need for accurate and sensitive methods to monitor antibiotic levels in milk. The aim of this study was to develop a rapid and sensitive magnetic nanoparticle-based fluorescence immunoassay for the simultaneous detection of chloramphenicol and penicillin G in milk. Methods: Magnetic nanoparticles were synthesized and functionalized with (3-aminopropyl)triethoxysilane. Chloramphenicol-Ovalbumin and Chloramphenicol-Ovalbumin-Fluorescein-5-isothiocyanate conjugates were prepared. Penicillin G – ATTO 633 fluorescent conjugate was synthesized. Antibodies against chloramphenicol and penicillin G were immobilized onto the magnetic nanoparticles. The competitive fluorescent immunoassay was developed. The optimal concentration of the antibody-magnetic nanoparticles and the fluorescent conjugates for the assay was determined. The calibration curves for the antibiotics in buffer and milk were plotted. Fluorescent immunoassay for the simultaneous determination of chloramphenicol and penicillin G in milk was developed. Results: The limit of detection by the simultaneous immunoassay of chloramphenicol and penicillin G in milk was 0.85 ng/mL and 1.6 ng/mL, respectively. The recovery of different concentrations of chloramphenicol and penicillin G in milk samples varied from 98% to 106%. Conclusions: A rapid and sensitive magnetic nanoparticle-based immunofluorescent assay for the simultaneous determination of chloramphenicol and penicillin G in milk was developed. The magnetic nanoparticles ensured rapid and easy procedure.

Development of a new biochip array for APOE4 classification from plasma samples using immunoassay-based methods

Background:Apolipoprotein E (APOE) is a key player in lipid transport and metabolism and exists in three common isoforms: APOE2, APOE3 and APOE4. The presence of theE4allelic variant is recognized as a major genetic risk factor for dementia and other chronic (neuro)degenerative diseases. The availability of a validated assay for rapid and reliable APOE4 classification is therefore advantageous.Methods:Biochip array technology (BAT) was successfully applied to identify directly the APOE4 status from plasma within 3 h, through simultaneous immunoassay-based detection of both specific APOE4 and total APOE levels.Results:Samples (n=432) were first genotyped by polymerase chain reaction (PCR), and thereafter, using BAT, the corresponding plasma was identified as null, heterozygous or homozygous for theE4allele by calculating the ratio of APOE4 to total APOE protein. Two centers based in Austria and Ireland correctly classified 170 and 262 samples, respectively, and achieved 100% sensitivity and specificity.Conclusions:This chemiluminescent biochip-based sandwich immunoarray provides a novel platform to detect rapidly and accurately an individual’sAPOE4status directly from plasma. TheE4genotype of individuals has been shown previously to affect presymptomatic risk, prognosis and treatment response for a variety of diseases, including Alzheimer’s disease. The biochip’s potential for being incorporated in quantitative protein biomarker arrays capable of analyzing disease stages makes it a superior alternative to PCR-basedAPOEgenotyping and may deliver additional protein-specific information on a variety of diseases in the future.