Untitled - MendelNet 2013 - Mendelova zemědělská a lesnická ...

MENDELNET 2013INTRODUCTIONInfluenza is probably the most powerful member of the group of potential pandemic agents,because of the high speed of constant mutational changes in surface antigens, hemagglutinin (HA)and neuraminidase (NA). HA is a trimeric glycoprotein expressed on the membrane of influenzavirus (Suenaga, Mizuno et al. 2012). It binds to SA receptors on the surface of the host cell andsubsequently mediates fusion of the viral and host membranes (McCullough, Wang et al. 2012).For this reason, HA is considered to be the main target for antibodies upon vaccination as well asinfection (Lingwood, McTamney et al. 2012). For this reason HA is utilized for vaccine preparation(Wang, Ni et al. 2012). Quantitative and qualitative analysis of vaccine antigens is a turning pointbefore the vaccine is placed on the pharmaceutical market and used for immunization (Williams,Pirkle et al. 2012).Nowadays, new sensors based on magnetic beads separation are coming to the foreground (Gijs2004). Paramagnetic particles (MPs) are the excellent tool with many advantageous features, suchas easy handling and possibility for separation by the magnetic field. The easily modified surface isthe next great advantage. Because of these reasons, these particles have found many applications toenhance the selectivity, sensitivity, and speed of isolation methods (Krejcova, Dospivova et al.2012). In current study, the microfluidic assay based on MPs isolation and HA indirect detectionby QDs is described. Microfluidic device for isolation and detection of HA-QDs complex werefabricated using three-dimensional (3D) printing, which is an example of additive manufacturing orof solid freeform fabrication technology (Polzin, Spath et al. 2013).EXPERIMENTAL SECTIONChemicalsTris(2-carboxyethyl)phosphine (TCEP) was purchased from Molecular Probes (Oregon, USA).Co(NH 3 ) 6 Cl 3 and other chemicals were purchased from Sigma Aldrich (Sigma-Aldrich, USA)unless indicated otherwise. Deionised water was used for rinsing, washing, and preparation ofbuffers. Stock solutions were prepared from ACS water.HemagglutininAs the sample of Influenza hemagglutinin was used vaccine Vaxigrip (Sanofi Pasteur, France),which contained three strands (A/California/7/2009 (H1N1), A/Victoria/361/2011 (H3N2) andB/Wisconsin/1/2010) of inactivated and splitted influenza virions. Vaxigrip contains 15 µg of allthree HA per 0.5 mL.CdS QDsCdS QDs were prepared by a slightly modified method published by Li and co-workers (Li, Shih etal. 2007). The obtained yellow solution was stirred for 1 h. Prepared CdS QDs were stored in thedark at 4 ºC and were used for labeling of vaccine HA.Labeling of vaccine HA by QDsVaxigrip (500 µL) was reduced and washed with water (5 x 400 µL) on an centrifugal filter device- Amicon 3k (Millipore, Massachusetts, USA) and mixed with a QDs solution (500 µL), mixturewas shaken (24 h, room temperature) on a Biosan Orbital Shaker OS-10 (Biosan Ltd. Riga, Latvia).Volume of solution was reduced to 100 µL (Amicon Ultra 3k), washed, diluted to 1 mL and usedfor isolation.754 | P age