Human serum albumin detection

FIGURE 47.7 Layout of the microfluidic chip including running buffer reservoir (1), eluent buffer reservoir (2), sample reservoir (3), sample waste reservoir (4), washing reservoirs (5,6) and waste reservoir (7) and electropherograms of human serum albumin detected above (a) and below the affinity monolith column with immobilized Pisum sativum agglutinin (b). (Reprinted with permission from Mao, X., et al.. Anal. Chem., 76, 6941, 2004. Copyright 2004 American Chemical Society.)... 

Consequently, the research work of Hara s group continued focusing on the improvement of protein determination using CE combined with online CL detection. By replacing EY by the Rhodamine B isothiocyanate (RITC) dye in the binary complexes formed with the proteins BSA or human serum albumin (HSA) and using a different imidazole buffer solution of pH 6, the sensitivity was increased [72], However, best detection limits for these determinations were found employing the tetramethylrhodamine isothiocyanate isomer (TRITC) dye, left for 4 h with a standard solution of BSA in acetonitrile followed by introduction into the capillary. For BSA, a detection limit of 6 nM was reached [73],... 

Recently, sulfur mustard has been shown to alkylate a cysteine residue in human serum albumin (10). The site of alkylation was identified in a tryptic digest of albumin from blood exposed to [14C]sulfur mustard. A sensitive method for its analysis was developed based on Pronase digestion of alkylated albumin to the tripeptide S-[2-[(hydroxyethyl)thio]ethyl-Cys-Pro-Phe, and detection using micro-LC-MS-MS. In vitro exposure of human blood to > 10 nM sulfur mustard could be detected employing this method. The analytical procedure was successfully applied to albumin samples from Iranian casualties of the Iraq-Iran war. 

In this section, use of the IO YI as an immunosensor is demonstrated, being relevant, e.g., for identification of biomarkers for early disease detection. The sensor is used to monitor the anti-human serum albumin - human serum albumin (a-HSA-HSA) immunoreaction. 

A similar approach for detecting the presence of specific proteins has also been reported by Nilsson and coworkers [26]. In this study, a complex between a calmodulin, a small calcium-binding protein, and the zwitter-ionic polythiophene POWT was used to detect the presence of calcineurin. The interaction between the POWT-camodulin complex and calcineurin changed the emission profile from POWT, and no observable changes were observed upon exposure of the complex human serum albumin, suggesting that the complex could be used for the specific detection of calcineurin. 

A reversible, direct fluoroimmunosensor for human serum albumin (HS A) measurement has been described by Bright et al.(m> Antibody Fab fragments are first immobilized on small quartz plates by hinge-region thiols, and then dansylated. The immunosensor is formed by attaching the quartz plates with bound Fab to the distal end of a bifurcated fiber-optic probe, which transmits both the excitation and emission. Binding of ffSA to the immunosensor results in a three- to five-fold enhancement of dansyl fluorescence. The sensor can be reused up to 50 times, with a detection limit of about 1.8 x 10-8 M, and a somewhat limited dynamic range. 

K. L. Bell and H. C. Brenner, Phosphorescence and optically detected magnetic resonance study of the tryptophan residue in human serum albumin, Biochemistry 21, 799-804 (1982). 

Braun and Alsenz6 used an ELISA to detect aggregates in interferon-alpha (IFN-a) formulations. They analyzed IFN-a formulations for possible aggregate formation because all marketed interferons are reported to induce antibodies to some extent. Because of its stabilizing effects, human serum albumin (HSA) is used in the formulation of marketed IFN-a at a great excess over IFN-a itself. HSA can also interact with other proteins. Braun and Alsenz developed an ELISA for the detection of both IFN-a-IFN-a and HSA-IFN-a aggregates. A MAb was used for the capture and detection of the IFN-a and a polyclonal for the detection of HSA. The assay is shown schematically in Figure 11.4. 

ED Moody, PJ Viskari, CL Colyer. Noncovalent labeling of human serum albumin with indocyanine green a study by capillary electrophoresis with diode laser-induced fluorescence detection. J Chromatogr B 729 55-64, 1999. 

LC-MS/MS has also been used to detect adducts to human serum albumin following pronase E digestion and analysis of the resulting Cys-Pro-Phe tripeptide. Modification of this tripeptide was observed for paracetamol and additionally demonstrated using albumin isolated from blood from patients [42]. 

Damsten, M.C., Commandeur, J.N.M., Fidder, A., Hulst, A.G.,Touw, D., Noort, D. and Vermeulen, N.P.E. (2007) Liquid chromatography/tandem mass spectrometry detection of covalent binding of acetaminophen to human serum albumin. Drug Metabolism and Disposition The Biological Fate of Chemicals, 35 (8), 1408-1417. 

Heineman s group [13,14] proposed the method of heterogeneous immunoassay to detect human serum albumin (HSA) labeled with In... 

Mohamadi et al. [96] reported online preconcentration of human serum albumin (HSA) and its immunocomplex with a monoclonal antibody on-chip coupled to isotachophoresis. The sample injection, preconcentration, and separation were carried out continuously and controlled by a sequential voltage switching program. Preconcentration was carried out with on-chip nondenaturing gel electrophoresis in methylcellulose solution. Furthermore, the authors applied this method for immunoassay of HSA. The separation of HSA and its immunocomplex was achieved in 25 seconds in 1 cm of the microchannel with induced fluorescence detection at 7.5 pM. 

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