title: Development of an ultrasensitive immunoassay using affinity maturated antibodies for the measurement of rodent insulin.
abstract: The measurement of plasma insulin is important for clinical diagnosis of diabetes and for preclinical research of metabolic diseases, especially in rodent models used in drug discovery research for type 2 diabetes. Fasting immunoreactive insulin (F-IRI) concentrations are used to calculate the homeostasis model assessment ratio (HOMA-R), an index of insulin sensitivity. However, even the most sensitive commercially available enzyme-linked immunosorbent assay (ELISA) kits cannot measure the very low F-IRI concentrations in normal rats and mice. Therefore, we sought to develop a new rodent insulin ELISA with greater sensitivity for low F-IRI concentrations. Despite repeated efforts, high-affinity antibodies could not be generated by immunizing mice with mouse insulin (self-antigen). Therefore, we generated two weak monoclonal antibodies (13G4 and 26B2) that were affinity maturated and used to develop a highly sensitive ELISA. The measurement range of the sandwich ELISA with the affinity maturated antibodies (13G4m1 and 26B2m1) was 1.5 to 30,000 pg/ml, and its detection limit was at least 10 times lower than those of commercially available kits. In conclusion, we describe the development of a new ultrasensitive ELISA suitable for measuring very low plasma insulin concentrations in rodents. This ELISA might be very useful in drug discovery research in diabetes.
authors.affiliation: Shionogi Pharmaceutical Research Center, Shionogi & Company, Toyonaka, Osaka 561-0825, Japan.
authors.forename: Sunao
authors.initials: S
authors.lastname: Imai
authors.affiliation: Shionogi Pharmaceutical Research Center, Shionogi & Company, Toyonaka, Osaka 561-0825, Japan.
authors.forename: Shoichi
authors.initials: S
authors.lastname: Naito
authors.affiliation: Shionogi Pharmaceutical Research Center, Shionogi & Company, Toyonaka, Osaka 561-0825, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Takahashi
authors.affiliation: Shionogi Pharmaceutical Research Center, Shionogi & Company, Toyonaka, Osaka 561-0825, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Yamauchi
authors.affiliation: Shionogi Pharmaceutical Research Center, Shionogi & Company, Toyonaka, Osaka 561-0825, Japan.
authors.forename: Etsuo
authors.initials: E
authors.lastname: Nakamura
authors.affiliation: Shionogi Pharmaceutical Research Center, Shionogi & Company, Toyonaka, Osaka 561-0825, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Sato
authors.affiliation: Shionogi Pharmaceutical Research Center, Shionogi & Company, Toyonaka, Osaka 561-0825, Japan.
authors.forename: Yuuichi
authors.initials: Y
authors.lastname: Mitsuda
authors.affiliation: Shionogi Pharmaceutical Research Center, Shionogi & Company, Toyonaka, Osaka 561-0825, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Takagi
authors.affiliation: Shionogi Pharmaceutical Research Center, Shionogi & Company, Toyonaka, Osaka 561-0825, Japan.
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Numata
authors.affiliation: School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
authors.forename: Ikuo
authors.initials: I
authors.lastname: Fujii
authors.affiliation: Shionogi Pharmaceutical Research Center, Shionogi & Company, Toyonaka, Osaka 561-0825, Japan. Electronic address: shoji.yamane@shionogi.co.jp.
authors.forename: Shoji
authors.initials: S
authors.lastname: Yamane
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D003921:Diabetes Mellitus, Experimental / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005215:Fasting / Q000097:blood; D005260:Female; D006825:Hybridomas / Q000166:cytology; D007328:Insulin / Q000097:blood* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D057230:Limit of Detection; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats
chemical_list: D000911:Antibodies, Monoclonal; D007328:Insulin
keywords: Affinity maturation; Monoclonal antibody; Phage display; Rodent insulin; Sandwich enzyme immunoassay