A Multiplex Microsphere-Based Immunoassay Increases the Sensitivity of SIV-Specific Antibody Detection in Serum Samples and Mucosal Specimens Collected from Rhesus Macaques Infected with SIVmac239

Abstract

Results from recent HIV-1 vaccine studies have indicated that high serum antibody (Ab) titers may not be necessary for Ab-mediated protection, and that Abs localized to mucosal sites might be critical for preventing infection. Enzyme-linked immunosorbent assay (ELISA) has been used for decades as the gold standard for Ab measurement, though recently, highly sensitive microsphere-based assays have become available, with potential utility for improved detection of Abs. In this study, we assessed the Bio-Plex® Suspension Array System for the detection of simian immunodeficiency virus (SIV)-specific Abs in rhesus macaques (RMs) chronically infected with SIV, whose serum or mucosal SIV-specific Ab titers were negative by ELISA. We developed a SIVmac239-specific 4-plex bead array for the simultaneous detection of Abs binding to Env, Gag, Pol, and Nef. The 4-plex assay was used to quantify SIV-specific serum IgG and rectal swab IgA titers from control (SIV-naive) and SIVmac239-infected RMs. The Bio-Plex assay specifically detected anti-SIV Abs in specimens from SIV-infected animals for all four analytes when compared to SIV-naive control samples (p≤0.04). Furthermore, in 70% of Env and 79% of Gag ELISA-negative serum samples, specific Ab was detected using the Bio-Plex assay. Similarly, 71% of Env and 48% of Gag ELISA-negative rectal swab samples were identified as positive using the Bio-Plex assay. Importantly, assay specificity (i.e., probability of true positives) was comparable to ELISA (94%–100%). The results reported here indicate that microsphere-based methods provide a substantial improvement over ELISA for the detection of Ab responses, aid in detecting specific Abs when analyzing samples containing low levels of Abs, such as during the early stages of a vaccine trial, and may be valuable in attempts to link protective efficacy of vaccines with induced Ab responses.

A betacoronavirus multiplex microsphere immunoassay detects early SARS-CoV-2 seroconversion and antibody cross reactions

“…We hypothesize that the high sensitivity and speci city of this assay for SARS-CoV-2 serology is due both to the physics of the Luminex xMAP-based platform, which enables a high dynamic range of measurement, as well as its multiplexing design with CoV S glycoproteins. Multiplex microsphere-based immunoassays have been shown to be more sensitive than standard ELISA for SARS-CoV-2 antibody detection (25) and several other virus infections, including Lassa virus, Ebola virus, and simian immunode ciency virus (11)(12)(13). Additionally, and perhaps more importantly, simultaneously incubating serum against spike proteins of seasonal HCoVs appears to enable the establishment of a lower threshold of positivity for detection of SARS-CoV-2 speci c antibodies.…”

“…We hypothesized that this approach would result in a highly sensitive and speci c assay for detecting SARS-CoV-2 speci c antibodies through two mechanisms. First, the Luminex xMAP-based platform has a large dynamic range and has been shown to be more sensitive than ELISA for the detection of antibodies to other viral infections (11)(12)(13). Second, given the high seroprevalence of the common human β-CoVs (14)(15)(16), cross-reactive antibodies present in subject samples (17,18) could be concurrently measured and accounted for in a multiplex approach.…”

Detection of Salmonid IgM Specific to the Piscine Orthoreovirus Outer Capsid Spike Protein Sigma 1 Using Lipid-Modified Antigens in a Bead-Based Antibody Detection Assay

“…Bead-based multiplex immunoassays, such as the Luminex xMAP technology, have been successfully used to detect mammalian antibodies for more than a decade (3–5). This method has the potential to detect specific antibodies against several antigens simultaneously, and can be used to identify antibodies directed against a wide range of antigens in one sample using small amounts of antigens and sample material.…”

“…The possibility to measure multiple analytes in the same sample further decrease the cost of each analysis. In addition to this, the xMAP assay is time-saving, can be used with much smaller sample volumes, uses around 1/50 the amount of capture antigen and offers broader dynamic range and higher sensitivity (3, 6, 7). The first bead-based multiplex immunoassays made to detect virus-specific antibodies in farmed Atlantic salmon were created and published in 2017 (8).…”

Testes fluorimétricos na sorologia da toxoplasmose humana: detecção simultânea de anticorpos de IgG e IgM específicos

“…os NHs-ésteres reagem preferencialmente com o grupo hidroxila de tirosinas, podendo também resultar em um acoplamento altamente eficiente (Kalkhof e Sinzs, 2008 (Lew, 1984 (Kunita et al, 2011, Powell et al, 2013, Zhang et al, 2011.…”