ELISAs and similar immunoassays are a backbone of biomedical research and clinical practice. Here we review the major factors to consider in the development and application of ultrasensitive ELISAs for analysis of human immune responses in plasma, serum, urine, or tissue culture supernatants. We focus on cytokine and chemokine biomarkers of health and chronic inflammatory diseases including allergy, asthma, autoimmunity, and cardiovascular disease. Detailed protocols for ELISA and Meso Scale Discovery assays (an improved variant of ELISA) are provided for 15 cytokines and 11 chemokines that play immune-regulatory roles in human innate and adaptive immunity. Protocols have been individually optimized to yield ultrasensitive limits of detection and quantification. Major factors enhancing immunoassay sensitivity, precision, and reproducibility, as well as key pitfalls in assay design and execution, are critically reviewed.
Keywords: Adaptive; Allergy; Antigen-specific; Asthma; Cytokine; ELISA; Human; Innate; Meso Scale discovery (MSD); Pattern recognition receptor; Primary culture.