Simultaneous Detection of Antibodies to Six Nonhuman-Primate Viruses by Multiplex Microbead Immunoassay

Abstract

To maintain healthy nonhuman primates for use in biomedical research, animals are routinely screened for several infectious agents at most facilities. Commonly, monkey serum samples are tested by conventional immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) or Western blotting, for antibodies to specific infectious agents. For testing for antibodies against multiple agents in each sample, conventional immunoassays are laborious and time-consuming. More efficient immunoassays are needed. Accordingly, we have developed a novel multiplex serodiagnostic system based on individually identifiable, fluorescent microbead sets, where each bead set is coated with antigens from a purified preparation of a specific virus. The coated bead sets are mixed to enable the detection of antibodies to multiple viruses in one serum or plasma sample. These viruses include four agents that are routinely tested for maintenance of specific-pathogen-free monkeys, namely, simian immunodeficiency virus, simian type D retrovirus, simian T-cell lymphotropic virus, and herpes B virus, as well as simian foamy virus and rhesus cytomegalovirus, both of which are commonly found in nonhuman primates. This multiplex microbead immunoassay (MMIA) enabled the simultaneous detection of antibodies to all six viruses in single serum samples as small as 1 microliter. The results obtained by MMIA analysis correlated with results of conventional ELISAs, which detect antibodies to single agents. Thus, this multiplex microbead detection system is an efficient diagnostic modality for serosurveillance of nonhuman primates.

Development of a custom pentaplex sandwich immunoassay using Protein-G coupled beads for the Luminex® xMAP® platform

“…The soluble pattern recognition receptor PTX3 and immunoglobulin-like receptor TREM-1 were eliminated from the panel of interest in this study due to antibody cross-reactivity issues detected by xMAP® but not by ELISA. This indicates that the presentation of antigenic epitopes on microspheres is different from that in ELISA, as has been previously reported (Khan et al, 2006). Expansion of the array to a heptaplex might be possible if screening of alternative PTX3 and TREM-1 antibody pairs (i.e.…”

“…Further differences between ELISA and xMAP® used in this study were highlighted by the slightly decreased sensitivity of xMAP® analysis for IP-10 compared to ELISA; this scenario is not unusual. Khan et al suggest that the availability and accessibility of the antigenic epitopes may be limiting due to the smaller surface area of xMAP® microspheres, whilst the concentration of antigenic molecules may not be limiting due to the larger available surface areas in ELISA microtitre plate wells (Khan et al, 2006).…”

Plasma Antibody Profiles as Diagnostic Biomarkers for Tuberculosis

“…Microbead sets (Luminex Corp, Austin, TX) were coupled through carbodiimide linkages with each of the M. tuberculosis recombinant antigens as previously described (14)(15)(16).…”

Multiplexed Immunoassays

“…At present, the most successful and robust multiplexing technology is Luminex xMAP platform, which combines advanced fluidics, optics, and digital signal processing with proprietary microsphere technology to deliver multiplexed assay capabilities. Importantly, Luminex is an open technology and numerous companies have marketed the Luminex system, including Bio-Rad, Qiagen, Invitrogen, and Millipore ect.. And a steadily growing list of ready-to-use multiplexed immunoassays have also been provided by these companies for applications in biomarker discovery , autoimmune disease diagnostics [77][78][79][80] , infectious disease diagnostics [81][82][83][84][85][86][87][88][89][90][91][92][93] , neurological diseases [94][95][96][97][98][99][100][101] , HLA testing [102][103][104] , and drug discovery 105,106 ( Table 4).…”

“…Moreover, encoded microsphere based multiplexed immunoassay technology could improve diagnostics of infectious diseases by enabling the simultaneous detection of antibodies or antigens to multiple infectious pathogens, such as human immunodeficiency virus (HIV), the Hepatitis A, B, C virus, Mycobacterium tuberculosis, as well as a large number of other viral, bacterial and parasitic pathogens. [81][82][83][84][85][86][87][88][89][90][91][92][93] FDA-approved assays for infectious diseases (e.g. EBV, HSV, MMRV, Syphilis, and ToRC assays on BioPlex TM 2200 or Multi-Lyte TM ) are already available on market.…”