Development and Validation of High Sensitivity SimpleStep Ignite® ELISAs for Quantitative Measurement of Human Immune Checkpoint Proteins 2333298

Abstract Introduction Immune checkpoint pathways play critical roles in regulating T cell activation, tumor immune evasion, and responses to immunotherapy. Quantitative assessment of checkpoint related proteins–such as Fibrinogen Like Protein 1 (FGL1), Programmed Death Ligand 1 (PD L1), Programmed Death 1 Ligand 2 (PD L2), Programmed Death 1 (PD 1), and Lymphocyte Activation Gene 3 (LAG 3)–is essential for mechanistic studies, biomarker discovery, and preclinical immuno-oncology research. To address the need for rapid, highly sensitive, and user-friendly immunoassays, we developed a suite of ELISA kits optimized for detection of these key checkpoint molecules in biological matrices. Methods Antibody pairs were screened to identify optimal sensitivity and dynamic range. Biological validation included precision, spike and recovery performance, and dilutional linearity to ensure minimal matrix effect across serum, plasma, cell culture supernatant, and relevant immune cell lysate matrices. Cross reactivity and interference testing ensured assay specificity. Results The resulting ELISAs demonstrated low pg/mL range LOD, with broad dynamic ranges supporting quantification in diverse sample types. Spike and recovery values ranged from 80—120%, and parallelism studies confirmed reliable quantification across diluted matrices. Each assay exhibited high specificity, with no detectable cross reactivity to related family members. The streamlined single wash protocol enabled completion in under 55 minutes while extending sensitivity compared to traditional sandwich ELISAs. Conclusion These newly developed SimpleStep Ignite® ELISA kits for human FGL1, PD L1, PD-L2, PD 1, and LAG 3 provide rapid, sensitive, and reproducible tools for quantifying key immune checkpoint proteins. Their strong analytical performance and simplified workflow support high throughput immuno-oncology research, enabling deeper characterization of checkpoint pathways and accelerating biomarker driven discovery. Funding Source Abcam Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)