Combined Gold Nanoparticle-Antibody Conjugate Enhances Sensitivity of Influenza A Antigen Detection in Lateral Flow Assay Natpapas Wiriyachaiporn, Nisachon Apiwat National Nanotechnology Center (NANOTEC) National Science and Technology Development Agency Pathumthani, Thailand Warangkana Chantima, Tararaj Dharakul Department of Immunology, Faculty of Medicine Siriraj Hospital Mahidol University Bangkok, Thailand Abstract-A combined gold nanoparticle (GNP)-monoclonal antibody (MAb) conjugate was generated for a direct influenza A antigen detection using lateral flow assay (LF) format. The combination of GNP-MAb conjugate targeting both nucleoprotein (NP) and matrix protein (M) allows the detection of multiple influenza A proteins. The LF system consisting of combined GNP- MAb conjugate showed an improved sensitivity, compared to the LF system containing single GNP-MAb conjugate targeting only one target protein, either NP or M. This was illustrated by the 2- to 260-fold lower limit of detection of the combined GNP-MAb conjugate LF than the single GNP-MAb conjugate LF. As such, the combined GNP-MAb conjugate LF could provide the basis for developing a direct influenza A antigen detection for rapid influenza A diagnosis. Index Terms-Combined gold nanoparticle-antibody conjugate, combined antigen detection, lateral flow assay, influenza A antigen detection. I. INTRODUCTION Over the past decade, nanoparticles have been used extensively for biomolecular detection. GNP, in particular, is commonly used as label in many diagnostic applications, including LF assay due to its intense color, ease in preparation and conjugation [1,2]. Within the LF system, GNP is conjugated to a targeting molecule, usually antibody (Ab) to detect the target antigen (Ag). The conjugated GNP-Ab-Ag complex is then captured by another capture Ab immobilized on the solid phase forming the conjugated GNP-Ab-Ag-Ab sandwich complex [3]. The accumulation of GNP from the complex enables the signal to be detected subsequently. In this study, GNP was conjugated to MAbs targeting different influenza A antigen. The use of combined GNP-MAb conjugate in developing LF assay to enhance sensitivity of influenza A antigen detection as a model was investigated. A combination of antigen detection allows the detection of the two most abundant influenza A proteins, the NP and M proteins, simultaneously [4,5]. Two types of the LF systems using single and combined GNP-MAb conjugate were fabricated. The performance of both LF systems in detecting influenza A antigen was evaluated and discussed. II. MATERIALS AND METHODS A. Source of antibodies and chemical reagents MAb specific to the NP protein (MAb NP) was supplied from a commercial source (Innova biotechnology, Thailand) and MAbs specific to the M protein (MAb M1 and MAb M2) were generated previously in this laboratory using standard hybridoma technique respectively [6]. GNP (40 nm) was supplied by BBI (UK). LF test strips were supplied by Innova biotechnology. All chemical reagents used in this study were of molecular grade and were supplied by Sigma-Alrich, UK unless stated otherwise. B. Source of virus isolates and viral infected allantoic fluid and cell lysate Influenza A virus isolates used in this study were provided by Prof. Pilaipan Puthavattana and Dr. Sontana Siritantikorn (Mahidol University). A total of 1 clinical human isolate of the seasonal influenza A H1N1 virus, 1 clinical human isolate of the seasonal influenza A H3N2 virus, and 1 isolate of the pandemic 2009 swine-origin influenza A H1N1 virus was used in this study. Allantoic fluid was harvested from 10-day-old embryonated specific-pathogen-free chicken eggs inoculated with sucrose gradient-purified influenza A virus isolates [7,8]. The infected allantoic fluid was inactivated using binary ethylenimine inactivation method [9]. Madin-Darby canine kidney (MDCK) cell cultures infected with other viruses were provided by Dr. Sontana Siritantikorn (Mahidol University). All viral manipulations were performed under appropriate biosafety level 2 plus laboratory conditions. C. Evaluation of MAb reactivity The reactivity of the MAbs was evaluated using Western blot analysis and ELISA. Western blot analysis was performed using the inactivated purified influenza A virus in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (15% (w/v) SDS-PAGE). To evaluate the reactivity of MAb NP, indirect ELISA was performed using the purified recombinant NP (rNP) protein (Kamolwan Watcharatanyatip and Sirikwan Boonmoh, Mahidol University). Color development of the immunoreaction in ELISA and Western blot analysis was performed using peroxidase reaction as described [6].    ,((( 36 Proceedings of the 6th IEEE International Conference on Nano/Molecular Medicine and Engineering November 4-7, 2012, Bangkok, Thailand