Biosensors and Bioelectronics 21 (2006) 1880–1886 Multiplexed measurement of serum antibodies using an array biosensor Maria C. Moreno-Bondi a , Chris Rowe Taitt b , Lisa C. Shriver-Lake b , Frances S. Ligler b,∗ a Department Qu´ ımica Anal´ ıtica, Facultad de Qu´ ımica, Universidad Complutense, 28040 Madrid, Spain b Center for Bio/Molecular Science and Engineering, US Naval Research Laboratory, Washington, DC 20375-5348, USA Received 15 August 2005; received in revised form 12 December 2005; accepted 20 December 2005 Available online 24 January 2006 Abstract The array biosensor provides the capability for simultaneously measuring titers of antibody against multiple antigens. Human antibodies against four different targets, tetanus toxin, diphtheria toxin, staphylococcal enterotoxin B (SEB) and hepatitis B, were measured simultaneously in sera from eight different donors in a single assay and titers were determined. The assays could measure amounts of bound antibody as low as approximately 100fg. Each individual serum exhibited a different pattern of reactivity against the four target antigens. Applications of this biosensor capability include monitoring for exposure to pathogens and for efficacy of vaccination. © 2006 Elsevier B.V. All rights reserved. Keywords: Vaccination; Antibody production; Biosensor; Array; Immunization; Disease exposure; Seroconversion 1. Introduction With the fast-paced improvements in microarray technol- ogy, ever-increasing numbers of reports describe antigen- based microarrays for detection and characterization of antibody–antigen interactions. The goal of many of these stud- ies is vaccine development (Chen et al., 2004; Davies et al., 2005; Li et al., 2005; Lucas et al., 2005; Qui et al., 2005), char- acterization of autoimmune disorders or allergies (Joos et al., 2000; Wiltshire et al., 2000; Avseenko et al., 2002; Hiller et al., 2002; Robinson et al., 2002; Quintana et al., 2004; Shreffler et al., 2004; Feng et al., 2004; Duan et al., 2005), or screening of immunoglobulin libraries (De Wildt et al., 2000; Angenendt et al., 2003; Michaud et al., 2003). However, only a limited number of these studies have attempted to use these systems for serodi- agnosis of infectious diseases (Mezzasoma et al., 2002; Perrin et al., 2003; Bacarese-Hamilton et al., 2004a,b) or for determina- tion of efficacy of vaccines and vaccination protocols (Avseenko et al., 2002; Neuman De Vegvar et al., 2003; Robinson et al., 2003). While exquisitely elegant and sensitive, in many cases, ∗ Corresponding author. Tel.: +1 202 404 6002; fax: +1 202 404 8897. E-mail address: fligler@cbmse.nrl.navy.mil (F.S. Ligler). the technology is limited by complicated sample handling pro- cedures and the difficulty in performing analyses on multiple samples simultaneously. ELISA and challenge/neutralization studies are the most common technologies used to assess vaccine efficacy and are considered the gold standards for this latter purpose (for exam- ple, Mann et al., 2004; Warfield et al., 2004; Barman et al., 2005; Hope et al., 2005; Ni et al., 2005). However, these assays gen- erally require large quantities of reagents, significant time until results are available, a high degree of technical skill, and, in the case of neutralization studies, large numbers of animals and associated facilities. This report describes use of a facile, multiplexed assay pro- tocol for screening of human sera for antibodies directed against bacterial and viral antigens including Staphylococcus aureus enterotoxin B, tetanus toxin, diphtheria toxin and hepatitis B. Diluted sera from informed volunteers were analyzed using 75- min assays performed on a biosensor developed at the US Naval Research Laboratory (NRL). The NRL Array Biosensor uti- lizes a planar waveguide patterned with an array of immobilized recognition elements to capture targets of interest; bound targets are detected using labeled “tracer” antibodies and evanescent illumination (Ligler and Taitt, 2002; Taitt et al., 2005). The pat- tern of fluorescent spots is detected using a Peltier-cooled CCD 0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2005.12.018