Simultaneous Measurements of Auto-Immune and Infectious Disease Specific Antibodies Using a High Throughput Multiplexing Tool Atul Asati, Olga Kachurina, Anatoly Kachurin* Sanofi Pasteur, VaxDesign Campus, Orlando, Florida, United States of America Abstract Considering importance of ganglioside antibodies as biomarkers in various immune-mediated neuropathies and neurological disorders, we developed a high throughput multiplexing tool for the assessment of gangliosides-specific antibodies based on Biolpex/Luminex platform. In this report, we demonstrate that the ganglioside high throughput multiplexing tool is robust, highly specific and demonstrating ,100-fold higher concentration sensitivity for IgG detection than ELISA. In addition to the ganglioside-coated array, the high throughput multiplexing tool contains beads coated with influenza hemagglutinins derived from H1N1 A/Brisbane/59/07 and H1N1 A/California/07/09 strains. Influenza beads provided an added advantage of simultaneous detection of ganglioside- and influenza-specific antibodies, a capacity important for the assay of both infectious antigen-specific and autoimmune antibodies following vaccination or disease. Taken together, these results support the potential adoption of the ganglioside high throughput multiplexing tool for measuring ganglioside antibodies in various neuropathic and neurological disorders. Citation: Asati A, Kachurina O, Kachurin A (2012) Simultaneous Measurements of Auto-Immune and Infectious Disease Specific Antibodies Using a High Throughput Multiplexing Tool. PLoS ONE 7(8): e42681. doi:10.1371/journal.pone.0042681 Editor: Sylviane Pied, Lile 2 University, France Received March 5, 2012; Accepted July 11, 2012; Published August 30, 2012 Copyright: 2012 Asati et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project has been funded in whole or in part with Federal funds from Biomedical Advanced Research and Development Authority, Department of Health and Human Services, under Contract No. HHSO100201000035C. The funders had no role in study design, data collection and analysis, or preparation of the manuscript. Competing Interests: The authors declare competing financial interest. All authors are employed by Sanofi Pasteur. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: Anatoly.Kachurin@sanofipasteur.com Introduction Monitoring antibodies against neuronal ganglioside antigens is necessary for the diagnosis and therapy of various immune disorders. Ganglioside-specific antibodies are known to participate in various immune mediated neuropathies such as Guillain-Barre syndrome (GBS), multifocal motor neuropathy (MMN), Miller Fisher syndrome (MFS), acute and chronic form of inflammatory demyelinating polyradiculoneuropathy (AIDP; CIDP) [1–9]. Moreover, ganglioside antibodies were found to have a role in the pathogenesis of the Alzheimer disease, and are suggested as peripheral blood biomarkers for Alzhiemer disease progression [10]. Various forms of multiple sclerosis (MS) have shown an increased level of circulating ganglioside antibodies that can serve as potential markers of axonal damage in MS [11]. Also, there are evidences connecting ganglioside antibodies with epilepsy, Syden- ham chorea, autoimmune CNS inflammation and celiac disease [12–17]. Very recently, an elevated levels of GM1-ganglioside antibodies have been recently reported in mice after immunization against many influenza strains (1976, 1991–1992 and 2004–2005 vaccines) [18], [19]. Although conventional ELISA has been widely used for the detection of ganglioside antibodies [20–22], it has certain limitations such as considerable assay time, limited concentration sensitivity and lack of the multiplexing capacity that allows simultaneous detection of ganglioside and infectious antigen specific antibodies in a single sample volume. Alaedini et al [23], [24] reported an elegant express method to assess the presence of antibodies specific to the whole pool of neuronal gangliosides. The assay is based on agglutination of latex beads coated with the extract of human gangliosides with the antibodies. While being robust and time-saving, the method of Alaedini et al detects ganglioside antibodies at concentration 100–1000 times larger than the ELISA assays [24], lacks multiplexing capacity and is not able to discriminate antibodies specific to various gangliosides [23], [24]. Gangliosides are known as very labile compounds which make development of immunoassays complicated and may lead to false positive results [25]. Consequently, we reasoned that a more robust, specific, sensitive and multiplexing detection tool would be desirable for measuring ganglioside specific antibodies to help discern their roles in autoimmune disease and their usefulness as disease biomarkers. Considering a possible alternative between using multiplexing microarray ELISA-like technique and bead array BioPlex/Luminex platform, we decided in favor of the latter, due to the above mentioned instability of gangliosides [25]. We hypothesized that a reliable multiplexing system using Bioplex/ Luminex beads can be designed to detect the presence of various ganglioside- and infectious disease-specific antibodies in a single sample volume. PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e42681