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Background: A rapid and sensitive time-resolved fluoroimmunoassay (TRFIA) based on the biotin-streptavidin amplification system was developed for the determination of anticyclic citrullinated peptide (anti-CCP)., Methods: Europium-labeled streptavidin derivatives combined with europium and anhydride of diethylene triamine pentaacetic acid were used to label streptavidin, biotin was coupled with rabbit anti-human IgG to form a biotin-anti-human IgG bridge between streptavidin-europium and the anti-CCP antibody in the immunoassay. The anti-CCP assay was carried out by measuring the fluorescence of Eu(3+) -streptavidin at 615 nm., Results: The presented method produced a wide linear range from 0.58 to 9,463 U/ml, while it was only 591.4-18.48 U/ml when using an ELISA kit, and featured a detection limit up to 0.5 U/ml for anti-CCP. The values determined by the biotin-streptavidin-TRFIA and ELISA correlated well (R(2) = 0.8927). The method was applied to determine anti-CCP in serum samples with satisfied recoveries of 96.45-104.63%., Conclusion: The assay results obtained by the present method showed that biotin-streptavidin-amplified TRFIA improve the traditional ELISA kit for anti-CCP detection. Therefore, it offers a better alternative immunoassay in rheumatoid arthritis management., (© 2014 Wiley Periodicals, Inc.)

For developing a magnetic bioassay system, an investigation to determine the presence of a specific biomolecular interaction between biotin and streptavidin was done using magnetic nanoparticles and a silicon substrate with a self-assembled monolayer. Streptavidin was immobilized on the magnetic particles, and biotin was attached to the monolayer-modified substrate. The reaction of streptavidin-modified magnetic particles on the biotin-modified substrate was clearly observed under an optical microscope. The magnetic signals from the particles were detected using a magnetic force microscope. The results of this study demonstrate that the combination of a monolayer-modified substrate with biomolecule-modified magnetic particles is useful for detecting biomolecular interactions in medical and diagnostic analyses., ((c) 2004 Wiley Periodicals, Inc)

Screening of peptide ligands for affinity chromatography usually involves incubation with the target protein in a batch system. In an additional step, peptides with fast binding kinetics have to be selected in respect to satisfactory performance under flow conditions on a support ensuring optimal three-dimensional presentation of the peptide. We have developed a rapid screening system based on peptide synthesis and screening on CIM((R)) disks. The disk size was minimized to fit into microplates usually applied for solid-phase extraction. In combination with a vacuum manifold, semi-automated peptide synthesis and screening for binding to a target protein under simulated chromatography conditions are possible. Various analytical methods can be applied for parallel and automated determination of the quantity, integrity, or activity of the target protein in the flow through or bound to the affinity support. This system also allows parallel screening for suitable chromatographic conditions like running buffer, washing, and elution conditions., (Copyright 2002 Wiley Periodicals, Inc.)

A number of potential matrix candidates were investigated with regard to the importance of the pH in the matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) analysis of non-covalently bound protein complexes. The matrices examined were 2,5-dihydroxybenzoic acid (DHB), 4-hydroxy-alpha-cyanocinnamic acid (HCCA), 2-aminonicotinic acid (ANA), 4-nitroaniline (NA), 2-amino-4-methyl-5-nitropyridine (AMNP) and 3-hydroxypicolinic acid (HPA). In solution these matrix compounds permitted the preparation of MALDI samples at pH in the range 2-7. Among the matrices tested, complex formation, by specific non-covalent interactions, could only be observed when HPA (pH 3.8) was used as the matrix for the MALDI analysis. Under these conditions, specific non-covalent complex formation of recombinant streptavidin and glutathione-S-transferases were observed but not for human hemoglobin. The MALDI spectra obtained with the neutral compounds ANA (pH 4.4), NA (pH 6.4) and AMNP (pH 7.1) as matrices contain only peaks of the subunit with no signal of the non-covalent bound complexes present. Considering the results obtained in this study with basic and acidified matrix materials, there does not appear to be a strong correlation between the pH of the matrix solution and the utility of a matrix for the analysis of non-covalently bound complexes.