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: Sunao Imai, Shoichi Naito, Tatsuya Takahashi, Akira Yamauchi, Etsuo Nakamura, Masaaki Sato, Yuuichi Mitsuda, Hiroyuki Takagi, Yoshito Numata, Ikuo Fujii, Shoji Yamane
• Journal: Analytical biochemistry (Anal Biochem) Vol. 473 Pg. 72-9 (Mar 15 2015) ISSN: 1096-0309 [Electronic] United States
• PMID: 25524616 (Publication Type: Journal Article)
• Copyright: Copyright © 2015. Published by Elsevier Inc.

Chemical References

• Antibodies, Monoclonal
• Insulin

Topics

• Amino Acid Sequence
• Amino Acid Substitution
• Animals
• Antibodies, Monoclonal (immunology)
• Diabetes Mellitus, Experimental (blood)
• Enzyme-Linked Immunosorbent Assay (methods)
• Fasting (blood)
• Female
• Hybridomas (cytology)
• Insulin (blood, chemistry, genetics, immunology)
• Limit of Detection
• Mice
• Molecular Sequence Data
• Rats