A Highly Sensitive and Specific SARS-CoV-2 Spike- and Nucleoprotein-Based Fluorescent Multiplex Immunoassay (FMIA) to Measure IgG, IgA, and IgM Class Antibodies

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays with excellent clinical performance are essential for reliable estimation of the persistence of immunity after infection or vaccination. In this paper we present a thoroughly validated SARS-CoV-2 serological assay with excellent clinical performance and good comparability to neutralizing antibody titers.

937 Advanced understanding of the tumor microenvironment with multiplex analysis: an automated 7-color multiplex assay using Akoya’s opal technology

BackgroundImmunotherapy and precision medicine are rapidly developing approaches to cancer therapy. Biomarkers that detect the tumor and tumor microenvironment allow for the development of strategies that accelerate the development of treatments that enhance a patient‘s immune system. Akoya’s MOTiF™ PD-1/PD-L1 Panel is a validated, multiplex immunoassay enabling detection of the 6 most clinically relevant immuno-oncology and spatial markers: PD-1, PD-L1, FoxP3, CD8, CD68, and PanCK. This panel provides unparalleled quantitative data for pre-clinical and translational IO research.MethodsThe MOTiF™ PD-1/PD-L1 Panel was used to stain normal and tumor lung tissues. Stained slides were analyzed using the InForm® and Visiopharm® image analysis platforms.ResultsWe introduce the workflow and image analysis solutions using InForm® and Visiopharm® software to provide robust, quantifiable data.ConclusionsThis data provides insight into the innate and adaptive immune environment for targeted design of new immunotherapies. These new targeted immunotherapies could potentially improve efficacy and reduce toxicity.

A novel high performing multiplex immunoassay Multi-HTLV for serological confirmation and typing of HTLV infections

Background Human T-Cell Lymphotropic Viruses (HTLV) type 1 and type 2 account for an estimated 5 to 10 million infections worldwide and are transmitted through breast feeding, sexual contacts and contaminated cellular blood components. HTLV-associated syndromes are considered as neglected diseases for which there are no vaccines or therapies available, making it particularly important to ensure the best possible diagnosis to enable proper counselling of infected persons and avoid secondary transmission. Although high quality antibody screening assays are available, currently available confirmatory tests are costly and have variable performance, with high rates of indeterminate and non-typable results reported in many regions of the world. The objective of this project was to develop and validate a new high-performance multiplex immunoassay for confirmation and discrimination of HTLV-1 and HTLV-2 strains. Methodology/Principal findings The multiplex platform was used first as a tool to identify suitable antigens and in a second step for assay development. With data generated on over 400 HTLV-positive blood donors sourced from USA and French blood banks, we developed and validated a high-precision interpretation algorithm. The Multi-HTLV assay demonstrated very high performance for confirmation and strain discrimination with 100% sensitivity, 98.1% specificity and 100% of typing accuracy in validation samples. The assay can be interpreted either visually or automatically with a colorimetric image reader and custom algorithm, providing highly reliable results. Conclusions/Significance The newly developed Multi-HTLV is very competitive with currently used confirmatory assays and reduces considerably the number of indeterminate results. The multiparametric nature of the assay opens new avenues to study specific serological signatures of each patient, follow the evolution of infection, and explore utility for HTLV disease prognosis. Improving HTLV diagnostic testing will be critical to reduce transmission and to improve monitoring of seropositive patients.

Seropositivity to Nucleoprotein to detect SARS-CoV-2 infections: a tool to detect breakthrough infections after COVID-19 vaccination

Background With COVID-19 vaccine roll-out ongoing in many countries globally, monitoring of breakthrough infections is of great importance. Antibodies persist in the blood after a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Since COVID-19 vaccines induce immune response to the Spike protein of the virus, which is the main serosurveillance target to date, alternative targets should be explored to distinguish infection from vaccination. Methods Multiplex immunoassay data from 1,513 SARS-CoV-2 RT-qPCR-tested individuals (352 positive and 1,161 negative) with a primary infection and no vaccination history were used to determine the accuracy of Nucleoprotein-specific immunoglobulin G (IgG) in detecting past SARS-CoV-2 infection. We also described Spike S1 and Nucleoprotein-specific IgG responses in 230 COVID-19 vaccinated individuals (Pfizer/BioNTech). Results The sensitivity of Nucleoprotein seropositivity was 85% (95% confidence interval: 80-90%) for mild COVID-19 in the first two months following symptom onset. Sensitivity was lower in asymptomatic individuals (67%, 50-81%). Participants who had experienced a SARS-CoV-2 infection up to 11 months preceding vaccination, as assessed by Spike S1 seropositivity or RT-qPCR, produced 2.7-fold higher median levels of IgG to Spike S1 ≥14 days after the first dose as compared to those unexposed to SARS-CoV-2 at ≥7 days after the second dose (p=0.011). Nucleoprotein-specific IgG concentrations were not affected by vaccination in naïve participants. Conclusions Serological responses to Nucleoprotein may prove helpful in identifying SARS-CoV-2 infections after vaccination. Furthermore, it can help interpret IgG to Spike S1 after COVID-19 vaccination as particularly high responses shortly after vaccination could be explained by prior exposure history.

A Multiplex Noninvasive Salivary Antibody Assay for SARS-CoV-2 Infection and Its Application in a Population-Based Survey by Mail

Given the enormous impacts of the COVID-19 pandemic, developing tools for population surveillance of infection is of paramount importance. This article describes the development of a multiplex immunoassay on a Luminex platform to measure salivary immunoglobulin G responses to the spike protein, its two subunits and receptor binding domain, and the nucleocapsid protein of SARS-CoV-2.

Extracellular Vesicles as Biological Indicators and Potential Sources of Autologous Therapeutics in Osteoarthritis

Along with cytokines, extracellular vesicles (EVs) released by immune cells in the joint contribute to osteoarthritis (OA) pathogenesis. By high-resolution flow cytometry, we characterized 18 surface markers and 4 proinflammatory cytokines carried by EVs of various sizes in plasma and synovial fluid (SF) from individuals with knee OA, with a primary focus on immune cells that play a major role in OA pathogenesis. By multiplex immunoassay, we also measured concentrations of cytokines within (endo) and outside (exo) EVs. EVs carrying HLA-DR, -DP and -DQ were the most enriched subpopulations in SF relative to plasma (25–50-fold higher depending on size), suggesting a major contribution to the SF EV pool from infiltrating immune cells in OA joints. In contrast, the CD34+ medium and small EVs, reflecting hematopoietic stem cells, progenitor cells, and endothelial cells, were the most significantly enriched subpopulations in plasma relative to SF (7.3- and 7.7-fold higher). Ratios of EVs derived from neutrophils and lymphocytes were highly correlated between SF and plasma, indicating that plasma EVs could reflect OA severity and serve as systemic biomarkers of OA joint pathogenesis. Select subsets of plasma EVs might also provide next generation autologous biological products for intra-articular therapy of OA joints.

A highly sensitive and specific SARS-CoV-2 spike- and nucleoprotein-based fluorescent multiplex immunoassay (FMIA) to measure IgG, IgA and IgM class antibodies

Background. Validation and standardization of accurate serological assays are crucial for the surveillance of the coronavirus disease 2019 (COVID-19) pandemic and population immunity. Methods. We describe the analytical and clinical performance of an in-house fluorescent multiplex immunoassay (FMIA) for simultaneous quantification of antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein and spike glycoprotein. Furthermore, we calibrated IgG-FMIA against World Health Organisation (WHO) International Standard and compared FMIA results to an in-house enzyme immunoassay (EIA) and a microneutralisation test (MNT). We also compared the MNT results of two laboratories. Results. IgG-FMIA displayed 100% specificity and sensitivity for samples collected 13-150 days post-onset of symptoms (DPO). For IgA- and IgM-FMIA 100% specificity and sensitivity were obtained for a shorter time window (13-36 and 13-28 DPO for IgA- and IgM-FMIA, respectively). FMIA and EIA results displayed moderate to strong correlation, but FMIA was overall more specific and sensitive. IgG-FMIA identified 100% of samples with neutralising antibodies (NAbs). Anti-spike IgG concentrations correlated strongly (ρ=0.77-0.84, P<2.2x10-16) with NAb titers. The NAb titers of the two laboratories displayed a very strong correlation (ρ=0.95, P<2.2x10-16). Discussion. Our results indicate good correlation and concordance of antibody concentrations measured with different types of in-house SARS-CoV-2 antibody assays. Calibration against WHO international standard did not, however, improve the comparability of FMIA and EIA results. keywords: SARS-CoV-2, COVID-19, antibody, immunoassay, nucleoprotein, spike glycoprotein, WHO international standard, neutralising antibodies, microneutralisation, receptor-binding domain