Quantitative Bead-Based Multiplex Immunoassays for Mouse Anti-Virus Immune Response

Abstract

Abstract In response to pathogens, especially viruses, cells release interferons and other cytokines to combat infection. Interferons are typically divided into three types: 1 (e.g., IFN-α, IFN-β), 2 (e.g., IFN-γ), and 3 (e.g., IFN-λ1, IFN-λ2). All inter-ferons are important for fighting viral infections and for regulating the immune system. In addition, interferons are critically involved in cancer and autoim-mune diseases such as psoriasis, systemic lupus erythematosus, and multiple sclerosis. Studying the expression profile of interferons and other related cyto-kines is essential to understanding the immune response against viral pathogens and other related disease processes. We have developed a multiplex panel, using fluorescence-encoded beads, which is suitable for use on commonly available flow cytometers. The panel simultaneously quantifies 13 mouse anti-viral immune proteins, including IFN-α, IFN-β, IFN-γ, CXCL1 (KC), TNF-α, CCL2 (MCP-1), IL-12p70, CCL5 (RANTES), IL-1β, CXCL10 (IP-10), GM-CSF, IL-10, and IL-6. The assay was validated for specificity, linearity, cross-reactivity, and inter and intra-assay precision. The assay was further validated using biological samples to quantify analyte concentration changes in response to poly (I:C) stimulation compared to untreated controls. This multiplex panel is a robust tool for measuring the concentration of anti-viral inflammatory mediators in murine biological samples, and offers greater efficiency and broader dynamic ranges compared to conventional ELISA assays.