To ensure proper dosage of a drug, analytical quantification of it in biofluid is necessary. Liquid chromatography mass spectrometry (LC-MS) is the conventional method of choice as it permits accurate identification and quantification. However, it requires expensive instrumentation and is not appropriate for bedside use. Using soluble epoxide hydrolase (sEH) inhibitors (EC5026 and TPPU) as examples, we report development of a nanobody-based enzyme-linked immunosorbent assay (ELISA) for such small molecules and its use to accurately quantify the drug chemicals in human samples. Under optimized conditions, two nanobody-based ELISAs were successfully established for EC5026 and TPPU with low limits of detection of 0.085 ng/mL and 0.31 ng/mL, respectively, and two order of magnitude linear ranges with high precision and accuracy. The assay was designed to detect parent and two biologically active metabolites in the investigation of a new drug candidate EC5026. In addition, the ELISAs displayed excellent correlation with LC-MS analysis and evaluation of inhibitory potency. The results indicate that nanobody-based ELISA methods can efficiently analyze drug like compounds. These methods could be easily implemented by the bedside, in the field in remote areas or in veterinary practice. This work illustrates that nanobody based assays offer alternative and supplementary analytical tools to mass spectrometry for monitoring small molecule medicines during clinical development and therapy. Attributes of nanobody based pharmaceutical assays are discussed.
OBJECTIVE: To assess the phospholipase activity of endothelial (EL) and hepatic lipase (HL) in postheparin plasma of subjects with metabolic syndrome (MS)/obesity and their relationship with atherogenic and antiatherogenic lipoproteins. Additionally, to evaluate lipoprotein lipase (LPL) and HL activity as triglyceride (TG)-hydrolyses to complete the analyses of SN1 lipolytic enzymes in the same patient. APPROACH AND RESULTS: Plasma EL, HL, and LPL activities were evaluated in 59 patients with MS and 36 controls. A trend toward higher EL activity was observed in MS. EL activity was increased in obese compared with normal weight group (P=0.009) and was negatively associated with high-density lipoprotein-cholesterol (P=0.014 and P=0.005) and apolipoprotein A-I (P=0.045 and P=0.001) in control and MS group, respectively. HL activity, as TG-hydrolase, was increased in MS (P=0.025) as well as in obese group (P=0.017); directly correlated with low-density lipoprotein-cholesterol (P=0.005) and apolipoprotein B (P=0.003) and negatively with high-density lipoprotein-cholesterol (P=0.021) in control group. LPL was decreased in MS (P<0.001) as well as in overweight and obese compared with normal weight group (P=0.015 and P=0.004, respectively); inversely correlated %TG-very low-density lipoproteins (P=0.04) and TG/apolipoprotein B index (P=0.013) in control group. These associations were not found in MS. CONCLUSIONS: We describe for the first time EL and HL activity as phospholipases in MS/obesity, being both responsible for high-density lipoprotein catabolism. Our results elucidate part of the remaining controversies about SN1 lipases activity in MS and different grades of obesity. The impact of insulin resistance on the activity of the 3 enzymes determines the lipoprotein alterations observed in these states.