Currently, high-performance liquid chromatographic (HPLC) methods are mainly used to measure antiretroviral plasma concentrations in HIV-infected patients. Although the utility of routine therapeutic drug monitoring (TDM) as an additional tool to optimize long-term antiretroviral therapy is unclear, if TDM is to be widely used, the availability of simple, cheap and reliable methods for the measurement of antiretroviral drug levels are needed, particularly in resource-limited settings. In this study, an immunochromatograhic (IC) strip test to detect the presence of nevirapine (NVP) in body fluids has been developed. Antiserum to NVP was first raised in rabbits by immunization against NVP chemically conjugated with bovine serum albumin, and subsequently validated by Western immunoblotting and competitive indirect ELISA. The partially purified anti-NVP antibodies were conjugated with colloidal gold particles. The conjugation of the colloidal gold and polyclonal antibodies was monitored by UV-vis spectroscopy, while transmission electron microscopy images were used to characterize the particle size and shape of the conjugates. The resulting colloidal gold conjugates were used for the production of an IC strip test to detect nevirapine in human plasma. Preliminary assessment suggests no-cross reactivity of the NVP polyclonal antibodies but assessment of plasma samples from HIV-infected patients receiving HAART needs to be conducted. This assay could potentially be used for drug monitoring as part of the clinical care of HIV infected patients. 相似文献
Relative quantification of N-glycoproteomes shows great promise for the discovery of candidate biomarkers and therapeutic targets. The traditional protocol for quantitative analysis of glycoproteomes is usually off-line performed, and suffers from long sample preparation time, and the risk of sample loss or contamination due to manual manipulation. In this study, a novel integrated sample preparation platform for quantitative N-glycoproteome analysis was established, with combination of online N-glycopeptide capture by a HILIC column, sample buffer exchange by a N2-assisted HILIC–RPLC interface, deglycosylation by a hydrophilic PNGase F immobilized enzymatic reactor (hIMER) and solid dimethyl labeling on a C18 precolumn. To evaluate the performance of such a platform, two equal aliquots of immunoglobulin G (IgG) digests were sequentially pretreated, followed by MALDI-TOF MS analysis. The signal intensity ratio of heavy/light (H/L) labeled deglycosylated peptides with the equal aliquots was 1.00 (RSD = 6.2%, n = 3), much better than those obtained by the offline protocol, with H/L ratio as 0.76 (RSD = 11.6%, n = 3). Additionally, the total on-line sample preparation time was greatly shortened to 160 min, much faster than that of offline approach (24 h). Furthermore, such an integrated pretreatment platform was successfully applied to analyze the two kinds of hepatocarcinoma ascites syngeneic cell lines with high (Hca-F) and low (Hca-P) lymph node metastasis rates. For H/L labeled Hca-P lysates with the equal aliquots, 99.6% of log 2 ratios (H/L) of quantified glycopeptides ranged from −1 to 1, demonstrating high accuracy of the developed sample preparation strategy. By triplicated analysis of glycopeptides and non-glycopeptides of Hca-F and Hca-P lysates, 43 up-regulated and 30 down-regulated (Hca-F/P) N-glycosylation sites, and 11 significantly changed N-glycoproteins were successfully quantified, and most of them were related to tumorigenesis and tumor metastasis. All these results demonstrate the developed integrated N-glycoprotein pretreatment platform is of great power for the accurate, precise and high-throughput analysis of N-glycoproteomes. 相似文献
The monomer 3‐ethyl‐1‐vinyl‐2‐pyrrolidone ( 3 ) and the homopolymer poly(3‐ethyl‐1‐vinyl‐2‐pyrrolidone) ( 5 ) have been synthesized. Polymer 5 is soluble in water and shows a critical temperature (Tc) of 27 °C. The presence of cyclodextrin causes a slight shift of the Tc. The lower critical solution temperature (LCST) could be varied between 27 and 40 °C by copolymerization with N‐vinyl‐2‐pyrrolidone. A linear correlation between the Tc and the copolymer composition is observed.
