Journal oflmrnunologicalMethods, 107 (1988) 41-46 41 Elsevier JIM04634 Establishment of a highly sensitive enzyme-linked immunosorbent assay for interleukin-la employing a fluorogenic substrate Naofumi Mukaida 1, Tadashi Kasahara 2, Yuechau Ko 1 and Tadashi Kawai 1 I Departments of Clinical Pathology, and 2 Medical Biology and Parasitology, Jichi Medical School, Minarnikawachi-machi, Tochigi-ken, 329-04, Japan (Received 13 July 1987, revised received 2 September 1987, accepted 21 September 1987) We have previously established a non-competitive solid-phase enzyme-linked immunosorbent assay (ELISA) specific for interleukin-la (IL-lc0 using a combination of polyclonal antibody as the immobilized antibody, biotinylated monoclonal antibody as the second antibody and avidin-peroxidase. The level of detection of that ELISA was 200-500 pg/ml. In order to improve its sensitivity, we have used streptavidin-/3-D-galactosidase and the fluorogenic substance 4-methylumbelliferyl-D-galactopyranosideas enzyme substrate. With this system IL-la could be detected at concentrations as low as 10-50 pg/ml, which was about 10-20 times more sensitive than conventional mouse thymocyte co-stimulator assays. Furthermore, the assay system was specific for IL-la in that neither IL-1/3 nor interleukin-2 (IL-2) interfered. Key words: Interleukin-la; Immunoassay; Fluorogenic substrate Introduction Interleukin-1 (IL-1) is produced by a variety of cells or cell lines and exerts multiple biological functions, including the stimulation of prolifera- tion of thymocytes, T cells and fibroblasts, en- hancement of IL-2 production by T cells or T cell lines, induction of acute-phase protein synthesis by hepatocytes and pyrogenic activity (Op- Correspondence to: N. Mukaida, Department of Clinical Pathology, Jichi Medical School, Minamikawachi-machi, Tochigi-ken, 329-04, Japan. Abbreviations: IL-1, interleukin-1; IL-2, interleukin-2; r, recombinant; ELISA, enzyme-linked immunosorbent assay; 4MU-Gal, 4-methylumbelliferyl-D-galactopyranoside; PHA, phytohaemagglutinin; ONPG, o-nitrophenyl-/3-D-galac- topyranoside; [3H]TdR, tritiated thymidine; PBS, phosphate- buffered saline; BSA, bovine serum albumin; FI, fluorescence intensity. penheim, 1986). Molecular cloning of IL-1 has revealed the presence of two distinct species of IL-1 molecules with acidic form of IL-la and neutral form of IL-1/3 (March, 1985). These two types of IL-1, however, exhibited the same biologi- cal functions (Wood, 1985). To date, several kinds of bioassays have been employed to detect IL-1 activity using mouse thymocyte (Mizel, 1978), D10.G4.1 cells (Kaye, 1984), LBRM-33A-1 cells (Conlon, 1983) and fibroblasts (Schmidt, 1983). Inhibitors interfere with these assays and the presence of interleukin-2 (IL-2) in samples gives rise to false positive re- sults. Furthermore, they are not able to dis- criminate between the two types of IL-1. We have previously raised polyclonal and monoclonal antibodies against IL-la and estab- lished a non-competitive, solid-phase enzyme-link- ed immunosorbent assay (ELISA) for IL-la using biotinylated antibody and avidin-peroxidase 0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)