SummaryVarious protocols were used in the development of enzyme‐linked immunosorbent assays (ELISA) to improve the sensitivity and range of detection of human tumour necrosis factor‐α (TNF‐α). ELISA can provide a specific, sensitive and rapid method for detection of TNF‐α in patient's sera, and it is important that the assay used should be sufficiently sensitive to detect low levels of TNF‐α. The double sandwich ELISA proved to be the most sensitive, detecting<0080 ng/mL TNF. Of eight different protocols, one assay using a purified monoclonal antibody to human TNF‐α and rabbit polyclonal anti‐TNF‐α antibody had the greatest sensitivity and range of detection. The study illustrates methods for the development of sensitive immunoassays which may have applications in many assay systems.