Development of an ultrasensitive immunoassay using affinity maturated antibodies for the measurement of rodent insulin |
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Authors: | Sunao Imai Shoichi Naito Tatsuya Takahashi Akira Yamauchi Etsuo Nakamura Masaaki Sato Yuuichi Mitsuda Hiroyuki Takagi Yoshito Numata Ikuo Fujii Shoji Yamane |
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Affiliation: | 1. Shionogi Pharmaceutical Research Center, Shionogi & Company, Toyonaka, Osaka 561-0825, Japan;2. School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan |
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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. |
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Keywords: | Monoclonal antibody Rodent insulin Affinity maturation Sandwich enzyme immunoassay Phage display |
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