Interleukin-8 (IL-8) is a chemotactic and activating cytokine for neutrophils, which plays an important role in acute inflammatory responses. We aimed to develop a sensitive enzyme-linked immunosorbent assay (ELISA) for IL-8 and established 18 clones of anti-IL-8 monoclonal antibodies (mAbs). These mAbs were evaluated in terms of their antigen-binding affinities, and five clones were selected and used for the comparative study of various combinations of antibodies in sandwich ELISA. Affinity purified rabbit polyclonal antibody was also used in this study. One antibody pair, which showed relatively high sensitivity and which was not severely interfered with blood components, was selected and the assay conditions were optimized by choosing the appropriate buffer for sample dilution and by directly labeling the second antibody with enzyme. The finalized ELISA, using polyclonal antibody as first (coated) antibody and horseradish peroxidase-labeled mAb (clone EL139) Fab' fragment as second antibody, could detect as low as 2.5 pg/ml (0.125 pg/well) of IL-8 by in total 2 h incubation, without being affected by body fluid components. The ELISA was specific to IL-8, showing no cross-reactivity with other cytokines or various IL-8 family proteins which share some amino acid sequence homology with IL-8. As an example of its application to clinical specimens, plasma samples from patients with septic shock were measured. The results showed that sepsis patients contain significantly higher levels of plasma IL-8 compared to normal controls. When analyzed by gel-filtration chromatography, IL-8 in sepsis plasma was eluted in a molecular weight (M(r) region corresponding to the monomer form. The ELISA established here is expected to be effectively used for further investigations on the relationship between IL-8 and various diseases.