Mitogen inducible gene-6 (Mig-6) is a feedback inhibitor of epidermal growth factor receptor (EGFR) signaling pathway. The liver-specific knockout mice of the Mig-6 gene (Mig-6d/d) showed hepatomegaly and increased hypercholesterolemia. In this study, the biomarkers of insulin resistance and the effects of high-fat diets in the wild (Mig-6f/f) and Mig-6d/d mice were analyzed. The fasting plasma concentrations of glucose, triglyceride, cholesterols, free fatty acids, and HOMA-IR were measured and the glucose tolerance and insulin resistance tests were performed in the 25-week-old Mig-6f/f and the Mig-6d/d mice. The protein levels of active insulin receptor, glucose 6-phosphatase, and phosphoenolpyruvate carboxykinase were analyzed in the liver and fat. The fasting plasma cholesterol and glucose concentration were higher in the Mig-6d/d mice than the Mig-6f/f mice with increased fat deposition in the liver. But the Mig-6d/d mice had the improved glucose intolerance and insulin resistance without increased amount of phosphoinsulin receptor after insulin infusion in the liver. The hepatic concentration of phosphoenolpyruvate carboxykinase was increased in fasting Mig-6d/d mice. The feeding of high-fat diet accelerated the plasma lipids profiles and HOMA-IR in the Mig-6d/d mice but had no differential effects in oral glucose tolerance test and insulin tolerance test in both genotypes. These results suggest that the activated EGFR signaling might increase the fasting plasma glucose concentration through inducing the hepatic steatosis and the improved whole-body insulin resistance in the KO mice be caused by decreased adipogenesis in fat tissues.
Cross-reactive material 197 (CRM 197 ) is a non-toxic mutant of diphtheria toxin containing a single amino acid substitution of glycine 52 with glutamic acid. CRM 197 has been used as a carrier protein for poorly immunogenic polysaccharide antigens to improve immune responses. In this study, to develop a sandwich ELISA that can detect CRM 197 and CRM 197 conjugate vaccines, we generated a human anti-CRM 197 monoclonal antibody (mAb) 3F9 using a phage-displayed human synthetic Fab library and produced mouse anti-CRM 197 polyclonal antibody. The affinity (K D ) of 3F9 for CRM 197 was 3.55 nM, based on Bio-Layer interferometry, and it bound specifically to the B fragment of CRM 197 . The sandwich ELISA was carried out using 3F9 as a capture antibody and the mouse polyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA was <1 ng/ml CRM 197 . In addition, the 3F9 antibody bound to the CRM 197 -polysaccharide conjugates tested in a dose-dependent manner. This ELISA system will be useful for the quantification and characterization of CRM 197 and CRM 197 conjugate vaccines. To our knowledge, this study is the first to generate a human monoclonal antibody against CRM 197 and to develop a sandwich ELISA for CRM 197 conjugate vaccines.
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