LIPS arrays for simultaneous detection of antibodies against partial and whole proteomes of HCV, HIV and EBV

Abstract

For many infectious agents, the detection of antibodies is critical for diagnosis, monitoring and understanding vaccine responses. To facilitate the highly quantitative and simultaneous analysis of antibodies against multiple proteins from infectious agents, we have developed Luciferase Immunoprecipitation Systems (LIPS) arrays. By configuring microtiter plates with multiple antigens and testing control and infected serum samples at one time in solution, LIPS arrays provided highly reproducible antibody titers to panels of antigens with a wide dynamic range of detection. While all serum samples showed similar positive and negative immunoreactivity with internal control antigens derived from Influenza and Renilla luciferase-alone protein, respectively, antibody titers to many HCV and HIV antigens were generally 10 to over 400-fold higher in the infected versus uninfected samples. Additional screening of 18 proteins from the EBV proteome with serum samples from healthy EBV-infected individuals showed statistically significant antibody titers to 50% of the proteins tested. Antibody titers for the different EBV antigens in the healthy EBV-infected individuals were markedly heterogeneous highlighting the complexity of host humoral responses. These results suggest that LIPS arrays offer a highly discriminating platform for simultaneously profiling a wide spectrum of antibodies associated with many infectious agents.

Quantitative differences in HTLV-I antibody responses: classification and relative risk assessment for asymptomatic carriers and ATL and HAM/TSP patients from Jamaica

“…In contrast, the LIPS assay used in this study provides quantitative measures of Ab responses for multiple virus proteins that can be used to efficiently evaluate humoral responses in patients and controls. 42,43 Based on these quantitative measures of Ab responses for HTLV-I Gag, Env, and Tax by the LIPS assay, we modeled and classified the HTLV-I Ab responses in conjunction with subject information, sex, race, and age, between HAM/TSP patients and ACs (Model 1), between HAM/TSP patients and ATL patients (Model 2), and between ATL patients and ACs (Model 3). Under training modeling conditions using classification criteria based on cross-validation modeling conditions, Model 1 distinguished HAM/TSP patients from ACs at a true-positive rate of 85.42% and a false-positive rate of 29.32% (Table 3 and Figure 2A right graph).…”

Serology-Enabled Discovery of Genetically Diverse Hepaciviruses in a New Host

“…Briefly, animal sera were processed in a 96-well format at room temperature as previously described (6,8,9). Serum samples were first diluted 1:10 in assay buffer A (50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM MgCl 2 , 1% Triton X-100) using a 96-well polypropylene microtiter plate.…”

“…Given the high genetic diversity observed in other RNA viruses, including HCV, we used the evolutionarily conserved CHV helicase protein as the target antigen. The CHV serine protease/helicase (NS3) coding region corresponding to the highly immunogenic region of HCV helicase protein (11) was cloned into pREN2 eukaryotic expression vector, for recombinant expression of an NS3-Renilla luciferase fusion protein in COS1 cells (6)(7)(8)(9)(10). CHV-luciferase fusion proteins specifically bound to antibodies immobilized on protein A/G beads and were measured by a standard luciferase assay (Fig.…”

Kinetics of Epstein-Barr Virus (EBV) Neutralizing and Virus-Specific Antibodies after Primary Infection with EBV

“…The LIPS assay to quantify the level of antibody to EBV proteins has been described previously (11). Antibodies to 8 EBV proteins were chosen based on a previous study of antibodies to 18 viral proteins; antibodies to these 8 proteins showed the highest signal-to-noise ratios in healthy EBV-seropositive blood donors (11).…”

“…The LIPS assay to quantify the level of antibody to EBV proteins has been described previously (11). Antibodies to 8 EBV proteins were chosen based on a previous study of antibodies to 18 viral proteins; antibodies to these 8 proteins showed the highest signal-to-noise ratios in healthy EBV-seropositive blood donors (11). Briefly, each EBV antigen was cloned as a fusion protein with Renilla luciferase in a plasmid, Cos-1 cells were transfected with each plasmid, and lysates from cells were incubated with human sera and immunoprecipitated with protein A/G beads.…”