Analytical Sciences

Measurement of Mouse Urinary Type IV Collagen Using Time-Resolved Fluoroimmunoassay

Michimasa KOBAYASHI,* Hiroko KIMURA,** Jie LIAO,* Masaaki ABE,*** Sachiko HIROSE,*** and Yasuhiko TOMINO* 

*Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo 113-8421, Japan
**Department of Forensic Medicine, Juntendo University School of Medicine, Tokyo 113-8421, Japan
***Department of Pathology, Juntendo University School of Medicine, Tokyo 113-8421, Japan

Urinary level of type IV collagen is an important indicator for early renal dysfunction, but there has been no practical system to measure mouse type IV collagen adaptable to extremely small amounts of urine samples. We developed a highly sensitive time-resolved fluoroimmunoassay (TR-FIA) to measure mouse urinary type IV collagen. Based on the structural features of type IV collagen molecule, dithiothreitol (DTT) was used for pretreatment of the samples. This assay permits measurement of 100 pg/ml type IV collagen in 5 ul urine samples. Urinary levels of type IV collagen derived from 12 samples of two different mouse strains (KK/Ta and BALB/c) were measured using this assay. The results demonstrated very clearly the difference in values of urinary type IV collagen between diabetic mice and nondiabetic mice. Compared with the conventional enzyme-linked immunosorbent assay (ELISA), this method requires far smaller volumes of samples, and is best suited to mouse models in future experiments.

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