A rapid, simple measurement of human albumin in whole blood using a fluorescence immunoassay (I).

BACKGROUND: Human serum albumin (HSA) is the most abundant plasma protein and plays key a role in metabolism. The variation in albumin concentration provides valuable information related to metabolic diseases and diagnostic application. METHODS: We constructed two assay systems to quantify the albumin concentration. The immunoassay used a fluorescence (FL) dye to detect albumin in samples and employed the conventional chromatography as a separation system. The assay system consists of an anti-HSA-mAb or an HSA immobilized test strip in a disposable cartridge, a fluorescence-labeled detector buffer and a laser-fluorescence scanner. We mixed the sample with detector, loaded it onto a cartridge, incubated it for 10 min and measured the concentration of albumin in a laser-fluorescence scanner. We examined the comparability of assay with an automated BCG dye binding method using a Hitachi 747 biochemical analyzer. RESULTS: The correlation of coefficient between AT/AC as converted from the relative fluorescence units (RFU) and albumin concentration displayed reasonable reliability in both the competition and the inhibition assay systems (r = 0.998). Using the Bland-Altman difference plot analysis, we observed an acceptable agreement between two methods, the fluorescence immunochromatography assay (FL-ICA) and the automated BCG dye-binding method of a Hitachi biochemical analyzer, over the clinical relevant range of HSA concentrations. The coefficient of variation (CV) of within- and between-run variation in the immunoassay system was < 8% and the recovery fell within 5% in each control sample. In addition to its reliable analytical performance, the assay with whole blood can be completed in 12 min using a one-step operation without any pretreatment. CONCLUSION: The developed immunoassay system using fluorescence dye and lateral-flow chromatography is a simple, fast and reliable method for quantifying the albumin concentration in whole blood.

ProteoChip: a highly sensitive protein microarray prepared by a novel method of protein immobilization for application of protein-protein interaction studies.

We have developed a highly sensitive microarray protein chip, ProteoChip, coated with ProLinker, novel calixcrown derivatives with a bifunctional coupling property that permits efficient immobilization of capture proteins on solid matrixes and makes high-throughput analysis of protein-protein interactions possible. The analysis of quartz crystal microbalance showed that both monoclonal antibody (mAb) and antigen (Ag) bound to the gold film of the sensor surface coated with ProLinker B and that it is useful for studies of Ab-Ag interactions. ProteoChip, aminated glass slide coated with ProLinker A, was also demonstrated to be useful for preparation of high-density array spots by using a microarrayer and for analysis of analyte Ags either by direct or sandwich methods of fluorescence immunoassay. The detection sensitivity of ProteoChip was as low as 1-10 femtogram/mL of analyte protein, useful for detection of tumor markers. ProteoChip was also useful for studies of direct protein-protein interactions as demonstrated by analysis of integrin-extracellular matrix protein interaction. These experimental results suggest that ProteoChip is a powerful tool for development of chip-based lead screening microarrays to monitor protein-protein interactions (i.e. drug target) as well as for biomarker assays which require high detection sensitivity.