ABSTRACT. A novel sandwich enzyme-linked immunosorbent assay (ELISA) was established to determine the serum insulin concentrations in domestic cats. By using a solid-phase mouse anti-bovine insulin monoclonal antibody and a peroxidase-conjugated guinea pig antirat insulin polyclonal antibody, feline serum insulin concentrations in the range of 0.1 to 3.6 ng/ml could be measured. The intraassay CV and interassay CV were less than 6% and less than 10%, respectively. The present insulin assay will strongly help studies on feline diabetes mellitus. Diabetes mellitus is one of the most common endocrine diseases in cats, with peak occurence in middle aged and older cats. Determination of serum insulin is very helpful for the diagnosis and classification of diabetes mellitus. Previous reports showed that several radioimmunoassay (RIA) kits designed to measure human insulin concentrations could be applied to feline samples [2,4]. However, only relative values of serum insulin were provided since those RIA kits were calibrated with human insulin but not with feline insulin. There has been no commercially available non-radiometric assay designed for and calibrated with feline insulin. In the present study, we established a novel sandwich enzyme-linked immunosorbent assay (ELISA) to determine the serum insulin concentrations in cats.To establish the ELISA system, we newly produced a monoclonal antibody against bovine insulin (BAN25433). Balb/c mice were immunized with purified bovine insulin (Sigma, St. Louis, MO, U.S.A.). Splenocytes from the immunized mice were fused to P3X53 mouse myeloma cells. Antigen-producing hybridoma clones were selected by the reactivity to bovine insulin. Purified BAN 25433 antibody was used as a solid phase antibody. Plastic ELISA plates (Sumilon MS-8896F, Tokyo) were coated with 50 l/ well of BAN25433 antibody (8 g/ml) in bicarbonate buffer (pH 9.6) at 4C overnight and then blocked with 200 ml/ well of ApplieDuo (1:10, Seikagaku Co., Tokyo) at room temperature (RT) for 1 hr.To prepare the standard feline insulin, an approximately 8 g of frozen feline pancreatic tissue was kindly gifted from Dr. Tsujimoto, Laboratory of Veterinary Internal Medicine, The University of Tokyo. Feline insulin in the pancreatic tissue was extracted by ethanol-HCl [1], partially purified with an affinity column (HiTrap, Amersham Pharmacia Biotech, Uppsala, Sweden) with BAN25433 antibody, and then thoroughly purified by a reverse-phase high-performance liquid chromatography as previously described [1]. The protein concentration was determined (Protein Assay Kit, Bio-Rad, Hercules, CA, U.S.A.) and the feline insulin standard solution was prepared. The other ELISA reagents used in this study including peroxidase-conjugated anti-rat insulin antibody, antibody diluting solution, washing buffer, substrate solution containing 3,3',5,5'-tetramethylbenzidine (TMB) and stop solution (1N sulfuric acid) were the parts of a commercial ELISA kit for rat insulin (Morinaga Institute of Biological Science, Inc., Yokohama).The ELIS...