Establishment of mouse Mac-2 binding protein enzyme-linked immunosorbent assay and its application for mouse chronic liver disease models.
OBJECTIVE
We identified Mac-2 (galectin-3) binding protein (Mac-2bp) as a novel diagnostic and liver fibrosis predicting biomarker for nonalcoholic steatohepatitis in humans. In mouse models, there are no serum biomarkers predicting liver disease severity. In this study, we developed a mouse Mac-2bp enzyme-linked immunosorbent assay (ELISA) system and determined its efficacy for predicting the severity of liver disease in mouse models, especially in non-alcoholic fatty liver disease (NAFLD) models.


METHODS
We established several rat monoclonal antibodies against mouse Mac-2bp, selected two clones for the ELISA, and checked the accuracy and reproducibility of the ELISA, especially for NAFLD models and liver fibrosis models. We also investigated the relationships between serum levels and hepatic gene expression of Mac-2bp in mouse models.


RESULTS
Our ELISA system had high accuracy and reproducibility (R2= 0.999). The intra-assay and inter-assay results for the coefficient of variation were 2.0-3.7% and 1.7-6.9%, respectively. The levels of bilirubin, hemoglobin, and chyle did not affect the Mac-2bp serum levels detected by our ELISA kit. In the mouse models, serum Mac-2bp levels increased with liver disease progression (F0/F1/F2/F3, 239.1 ± 36.7 / 259.1 ± 43.0 / 457.5 ± 162.0 / 643.7 ± 116.0 ng/mL; P < 0.0001), and were significantly correlated with hepatic gene expression of Mac-2bp (R = 0.42, P < 0.0001).


CONCLUSIONS
Our mouse Mac-2bp ELISA system effectively predicts severity of NAFLD and liver fibrosis in mouse models.
Department of Molecular Biochemistry and Clinical Investigation, Osaka University, Graduate School of Medicine, Suita, Japan.
Ayumi
A
Iwata
Department of Molecular Biochemistry and Clinical Investigation, Osaka University, Graduate School of Medicine, Suita, Japan.|Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Suita, Japan.
Yoshihiro
Y
Kamada
Department of Molecular Biochemistry and Clinical Investigation, Osaka University, Graduate School of Medicine, Suita, Japan.
Yusuke
Y
Ebisutani
Department of Molecular Biochemistry and Clinical Investigation, Osaka University, Graduate School of Medicine, Suita, Japan.
Akiko
A
Yamamoto
Department of Molecular Biochemistry and Clinical Investigation, Osaka University, Graduate School of Medicine, Suita, Japan.
Yui
Y
Ueda
Department of Molecular Biochemistry and Clinical Investigation, Osaka University, Graduate School of Medicine, Suita, Japan.
Hitomi
H
Arai
Department of Molecular Biochemistry and Clinical Investigation, Osaka University, Graduate School of Medicine, Suita, Japan.
Hironobu
H
Fujii
Department of Molecular Biochemistry and Clinical Investigation, Osaka University, Graduate School of Medicine, Suita, Japan.
Shinji
S
Takamatsu
Immuno-Biological Laboratories Co., Ltd., Fujioka, Japan.
Nobuhiro
N
Maruyama
Immuno-Biological Laboratories Co., Ltd., Fujioka, Japan.
Masahiro
M
Maeda
Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Suita, Japan.
Tetsuo
T
Takehara
Department of Molecular Biochemistry and Clinical Investigation, Osaka University, Graduate School of Medicine, Suita, Japan.
Eiji
E
Miyoshi
Mac-2bp; NAFLD; biomarker; liver fibrosis