Enzyme-Linked Immunosorbent Assay for T-Cell Dependent Immunogenicity Assessment of Therapeutic Peptides
Immunogenicity assessment of therapeutic peptides, proteins, oligonucleotides, and hybrid molecules, such as nucleopeptides, is a major aspect in understanding their safety and efficacy. Both T-cell independent and dependent immune reactions contribute to an immunogenic response against antigen, including secretion of cytokines and production of an antigen-specific antibody. Various assays exist for detecting and quantifying such immunogenic responses by human T-cells ex vivo or in mouse serum, which primarily include enzyme-linked immunosorbent assay (ELISA, direct and indirect), flow-cytometry and surface plasmon resonance (SPR). ELISA is a popular choice due to its robustness, reliability, sensitivity, ease of automation, and the requirement of simple equipment commonly available in most molecular biology and biochemistry laboratories. The chapter describes the detailed protocol of cytokine analysis by an ELISA method and highlights few crucial steps to be considered while performing the assay for successful immunogenicity studies.
Key wordsImmunogenicity Bioactive peptides Cytokines
This work was supported in part by the Lee Kong Chian School of Medicine, Nanyang Technological University Singapore Start-Up Grant to N.K.V., Singapore Eye Research Institute (SERI) Pilot Grant (Project No. R1229/35/2015), and the Singapore Ministry of Education (MOE) under its Singapore MOE Academic Research Fund (AcRF) Tier 2 Grant (MOE2017-T2-2-004). R.L. acknowledges support from the Singapore National Research Foundation under its Translational and Clinical Research Flagship Program (NMRC/TCR/008-SERI/2013) administered by the Singapore Ministry of Health’s National Medical Research Council (NMRC) and NMRC under its Centre Grant Programme- Optimization of core platform Technologies for Ocular Research (INCEPTOR)(NMRC/CG/M010/2017_SERI).
- 8.Hornbeck P (2001) Enzyme-linked immunosorbent assays. Curr Protoc Immunol, Chapter 2: Unit 2.1Google Scholar