ELISA: COMPETITION ASSAY

1. Development of a disposable immunosensor for Aflatoxin M1 detection in milk S Micheli L.,Grecco R., Palleschi G., Moscone D., Dipartimento di Scienze e Tecnologie Chimiche, University of Rome “Tor Vergata,”, Via della Ricerca Scientifica, 00133 Rome, Italy. E-mail: <danila.moscone@uniroma2.it> P O O INTRODUCTION O OH O Milk is susceptible to contamination from external sources and one key analyte is aflatoxin M1 (AFM1). This hepatocarcinogenic mycotoxin could occur in milk of cows fed with aflatoxin B1-contamined feedstuffs. Acute effects by aflatoxin M1 ingestion or inhalation are primarily observed in liver damage, while chronic exposure to aflatoxin often leads to liver cancer. The maximum content of AFM1 in milk, according with EC directives, is so low as 50 ppt, and this level of AFM1 can be controlled only by accurate and sensitive methods of analysis. At present, methods available for aflatoxin detection are based on HPLC or TLC procedures, which require expensive instrumentation and hazardous reagents. O E E OCH3 AFM1 AIM The purpose of this work was the development of a disposable electrochemical immunosensor based on screen-printing technology for measurement of AFM1 in milk. This technology can combine the high selectivity of the immunoanalysis with the easiness of the electrochemical probes (screen-printed electrodes - SPEs -). The advantages of using this fabrication allow for cheap and reproducible production, decreasing the amount of reagents used and making disposability devices. The determination of AFM1 is based on direct competitive ELISA format, where an AFM1-Ab complex is formed. The concentration of enzymatic product is inversely proportional of amount analyte (sample or standard) to be analysed. Ab IMMOBIISATLION AFM1 and AFM1-enzyme ADDITION PRECOATING DETECTION Enzyme-antigen conjugate ELISA: COMPETITION ASSAY Solid support DEVELOPMENT OF WORK Antigen Electrochemical ELISA The electrochemical strips consisted of a silver pseudoreference and two carbon electrodes (working and counter), all screen-printed on a flexible film. The electrochemical immunosensors have been set up immobilising the antibodies directly on the surface of screen-printed electrodes (SPEs), and allowing the competition between free and conjugated AFM1 with HRP enzyme.  The electrochemical signal was generated by the electrochemical detection of enzymatic product.  The electrochemical technique used was the Chronoamperometry, with an applied potential of -100 mV  Competition in milk has been carried out Spectrophotometric ELISA Spectrophotometric Enzyme-Linked ImmunoSorbent Assay (ELISA) tes has been obtained set-up:  AFM1 labelled with Horseradish Peroxidase (HRP) has been used; 3, 3’, 5, 5’ tetramethylbenzidine and H202 as enzyme substrates have been utilised;  parameters such as buffer, pH and ionic strength, time, temperature, competition step, blocking reagent and matrix effect have been optimised;  competition in milk has been carried out. Anti-IgG (mouse) Counter electrode Reference electrode Working electrode Monoclonal PrimaryAb RESULTS Electrochemical ELISA PreCoating: Ab anti-IgG 10 µg/mL in carbonate buffer 50 mM pH 9.6, overnight at 4°C Blocking: 1% PVA in PBS, 30 min at RT Coating: Ab anti-AFM1 20 µg/mL in PBS, until dryness at RT Competition: AFM1 (ppt) 15 min after AFM1-HRP 1:8 v/v in PBS, total time : 40 min at RT Spectrophotometric ELISA PreCoating: Ab anti-IgG 10 µg/mL in carbonate buffer 50 mM pH 9.6 overnight at 4°C Blocking: 0.5% PVA in PBS, 1 h at 37°C Coating: Ab anti-AFM1 10 µg/mL in PBS, 2 h at 37°C Competition: AFM1 (ppt) 10 min after AFM1-HRP 1:40 v/v in PBS, total time : 2 h at RT Working range:30 - 480 ppt Working range:30 - 250 ppt CONCLUSION and FUTURE WORK •  Standard curves were obtained by the use of AFM1 standard solution prepared in PBS  spectrophometric and electrochemical results have been compared. • The matrix effect of blank samples was tested no matrix effect was observed • Calibration curves were constructed by adding AFM1 standard solutions to centrifuged milk  milk dilution was not necessary • The regulatory level of AFM1 in milk (50 ppt) can be measured by both systems • The future work will be focalised on the improvement of the assays and on the comparison of our results with the conventional methods (HPLC and TLC). Maximum level Maximum level