Multiplexed measurement of serum antibodies using an array biosensor

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 100 fg. 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.

Introduction

With the fast-paced improvements in microarray technology, ever-increasing numbers of reports describe antigen-based microarrays for detection and characterization of antibody–antigen interactions. The goal of many of these studies is vaccine development (Chen et al., 2004, Davies et al., 2005, Li et al., 2005, Lucas et al., 2005, Qui et al., 2005), characterization 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 serodiagnosis of infectious diseases (Mezzasoma et al., 2002, Perrin et al., 2003, Bacarese-Hamilton et al., 2004a, Bacarese-Hamilton et al., 2004b) or for determination 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, the technology is limited by complicated sample handling procedures 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 example, Mann et al., 2004, Warfield et al., 2004, Barman et al., 2005, Hope et al., 2005, Ni et al., 2005). However, these assays generally 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 protocol 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 utilizes 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 pattern of fluorescent spots is detected using a Peltier-cooled CCD camera, and the extracted data are used to determine the identity and concentration of target. A critical aspect of this system is the ability to analyze multiple samples in parallel on the same sensor substrate; the two-dimensional nature of the arrayed substrate further tests each sample for the presence of multiple analytes. Therefore, multiple standards and controls can be analyzed for multiple targets at the same time as unknowns, enabling a quantitative assessment of matrix effects. For purposes of the current study, the parallel processing capability was used to analyze serum samples from multiple donors simultaneously, as well as to include a series of dilutions to determine antibody titers more accurately.