Antibody-profiling technologies for studying humoral responses to infectious agents

Abstract

Analyses of humoral responses against different infectious agents are critical for infectious disease diagnostics, understanding pathogenic mechanisms, and the development and monitoring of vaccines. While ELISAs are often used to measure antibody responses to one or several targets, new antibody-profiling technologies, such as protein microarrays, can now evaluate antibody responses to hundreds, or even thousands, of recombinant antigens at one time. These large-scale studies have uncovered new antigenic targets, provided new insights into vaccine research and yielded an overview of immunoreactivity against almost the entire proteome of certain pathogens. However, solid-phase antigen arrays also have drawbacks that limit the type of information obtained, including suboptimal detection of conformational epitopes, high backgrounds due to impure antigens and a narrow dynamic range of detection. We have developed a solution-phase antibody-profiling technology, luciferase immunoprecipitation systems (LIPS), which harnesses light-emitting recombinant antigen fusion proteins to quantitatively measure patient antibody titers. Owing to the highly linear light output of the luciferase reporter, some antibodies can be detected without serum dilution in a dynamic range of detection often spanning seven orders of magnitude. When LIPS is applied iteratively with multiple target antigens, a high-definition antibody profile is obtained. Here, we discuss the application of these different antibody-profiling technologies and their associated limitations with particular emphasis on protein microarrays. We also describe LIPS in detail and discuss several clinically relevant uses of the technology. Together, these new technologies offer new tools for understanding humoral responses to known and emerging infectious agents.

Immunodominant “Asymptomatic” Herpes Simplex Virus 1 and 2 Protein Antigens Identified by Probing Whole-ORFome Microarrays with Serum Antibodies from Seropositive Asymptomatic versus Symptomatic Individuals

“…Regardless of the number and nature of these antigens, it is logical that an efficient vaccine must include immunodominant asymptomatic antigens (ID-A-Ags) but not immunodominant symptomatic antigens (ID-S-Ags), to avoid inducing or exacerbating herpes disease. While an enzyme-linked immunosorbent assay (ELISA) is often used to identify one or several antigens targeted by antibodies (Abs), new promising antibodyprofiling technologies, such as protein microarrays, are emerging and can now be used to simultaneously identify hundreds, or even thousands, of pathogen-derived protein Ags (1,2,9).…”

Antibody Profiling of Borrelia burgdorferi Infection in Horses

“…In contrast to Western blot-based diagnostic testing, LIPS has the advantages of generating highly quantitative and robust antibody data against multiple, defined targets in a highthroughput capacity (6,10). Our approach of profiling three different B. burgdorferi antigens provides novel insight into equine Lyme disease.…”

“…The reason for this discrepancy may relate to horse immune differences as well as the likelihood that many of the horse serum samples represent late Lyme infection. Although not explored here, an antigen mixture of different B. burgdorferi strains by LIPS also might be useful for diagnostic screening (6,13,14). For example, based on the comparably low background signals of VOVO, DbpA, and DbpB, these antigens could be employed as a mixture, thereby simplifying data collection and analysis.…”

“…LIPS is based on employing light-emitting Renilla luciferase-fusion proteins in a liquid-phase immunoprecipitation assay. Serologic testing by LIPS for a variety of human pathogens provides the full exploitation of antibody profiles because of its robustness, multiplex capacity, and high sensitivity and specificity (6,11,12,14,15). Recently, a LIPS test employing a synthetic VOVO antigen consisting of two variants of immunodominant epitopes of the C6 peptide and OspC achieved 98% sensitivity and 100% specificity for the diagnosis of human Lyme disease, but the findings were not statistically different from those of the C6 ELISA (10).…”

Rhesus and Human Cytomegalovirus Glycoprotein L Are Required for Infection and Cell-to-Cell Spread of Virus but Cannot Complement Each Other

“…The luciferase immunoprecipitation system (LIPS) was used to measure antibody levels to RhCMV proteins (1). The open reading frames of RhCMV gB, gL, and pp65.2 proteins were each fused to a plasmid encoding Renilla luciferase (Ruc).…”