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Development of a novel immunoassay specific for mouse intact proinsulin.

Analytical biochemistry (2015-06-01)
Sunao Imai, Tatsuya Takahashi, Shoichi Naito, Akira Yamauchi, Chihiro Okada, Yoshihide Notsu, Ikue Sakikawa, Michiyoshi Hatanaka, Takanori Iwasaki, Atsushi Morita, Ikuo Fujii, Shoji Yamane
ABSTRACT

The blood concentration of intact proinsulin, but not total proinsulin, has been suggested to be a diagnostic marker for type 2 diabetes mellitus (T2DM), but a sensitive assay specific for rodent intact proinsulin is lacking. Here, a novel enzyme-linked immunosorbent assay (ELISA) for mouse intact proinsulin was developed. The developed ELISA detected mouse intact proinsulin with the working range of 8.3 to 2700pg/ml. Cross-reactivity with mouse split-32,33 proinsulin was approximately 100times lower than the reactivity with mouse intact proinsulin, and no cross-reactivity with mouse insulin was detected. The developed ELISA was sufficiently sensitive to detect low levels of intact proinsulin in normal mouse plasma. The measurement by the developed ELISA revealed that intact proinsulin was elevated in the plasma of type 2 diabetic db/db mice as mice aged, and the ratio of intact proinsulin/insulin in plasma was correlated with levels of glycated hemoglobin A1c as seen in T2DM patients. These results suggest that the plasma level of intact proinsulin, but not total proinsulin, is a sensitive marker for pancreatic dysfunction and the ensuring diabetic disease progression of db/db mice. This ELISA could aid nonclinical evaluation of therapeutic interventions in T2DM.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
3,3′,5,5′-Tetramethylbenzidine, ≥99%
Sigma-Aldrich
3,3′,5,5′-Tetramethylbenzidine, ≥98% (TLC)
Sigma-Aldrich
3,3′,5,5′-Tetramethylbenzidine, ≥98.0% (NT)
Sigma-Aldrich
3,3′,5,5′-Tetramethylbenzidine, tablet, 1 mg substrate per tablet
Sigma-Aldrich
Edelfosine, ≥95% (HPLC)