Development of a multiplex microbead-based immunoassay for quantitative analysis of mouse immune checkpoint proteins

Abstract

Abstract Immune checkpoint protein research plays a significant role in improving antitumor immune response as well as achieving a deeper understanding of the immune system and the disease states of inflammation, sepsis, and autoimmunity. Various mouse models have contributed to the clinical successes of immune checkpoint inhibitor immunotherapies and the understanding of checkpoint molecule-related inflammatory processes. Here we report the assay verifications and applications of a newly developed Luminex bead-based mouse immune-oncology checkpoint protein multiplex immunoassay for the quantitative profiling of 28 mouse immune checkpoint molecules and immune regulators (PD-1, PD-L1, PD-L2, CTLA-4/CD152, TIM-3, LAG-3, B7-H2/ICOSL, B7-H3/CD276, BTLA, GITR, HVEM, CD27, CD40, CD80/B7-1, CD226/DNAM-1, 4-1BBL/CD137L/TNFSF9, CD137/4-1BB, 5’-NT/CD73, BCA-1/CXCL-13, CD25/IL-2Rα, Granzyme B, E-cadherin, Galectin-1, Galectin-3, IFNγ, IL-10, TLR-2, and TNFα). We present quantitative protein biomarker profiling of mouse serum/plasma samples and conditioned media and lysates from mouse tumor cell lines, including LLC, B16-F10, MCA205, ID8, MB49, AT3, CT-2A, and YUMM1.7, as well as DC2.4 (dendritic cell line), C2C12 (myoblast cell line), and bEnd.3 (endothelial cell line). Our results demonstrate that this mouse immune checkpoint protein multiplex immunoassay is a powerful research tool for the quantitation of key immune regulator proteins and has potential application in biomarker discovery and translational research.