However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system. Keywords:Phage-protein interaction, phage display, combinatorial libraries, hepatitis-C virus, monoclonal antibodies, fluorescence correlation spectroscopy Antibody engineering combined with combinatorial methods has become a new field for producing and selecting specific antibodies. a human monoclonal antibody to the hepatitis-C virus (HCV) envelope protein E2 and its cognate antigen (rE2 or rE1/E2). We could thus assess the specific interactions and determine the fraction of specific versus background phage (26% specific phage). Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross-correlation studies using the Mouse monoclonal to EP300 two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system. Keywords:Phage-protein interaction, phage display, combinatorial libraries, hepatitis-C virus, monoclonal antibodies, fluorescence correlation spectroscopy Antibody engineering combined with combinatorial methods has become a new field for producing and selecting specific antibodies. Molecular libraries, both of biological and chemical nature, based on other molecular scaffolds are also widespread. Quick and efficient screening of small quantities of single molecules with improved characteristics, such as increased binding affinity, altered specificity, or catalytic ability is therefore desired. The quantity of either receptor and ligand, such as antigen and antibody, for example, may be limiting in evolutionary strategies using enormous libraries of biological molecules as a source for detecting species with a new character. Large biological libraries of mutants or biologically diverse molecules, such as a repertoire of Ig specificities are available as presentation packages, linking phenotype and genotype in display technologies such as phage or ribosome display (Smith 1985;McCafferty et al. 1990;Hanes and Plckthun 1998). The extreme sensitivity of Fluorescence Correlation Spectroscopy (FCS) makes it Ibrutinib-biotin interesting to use for identification of rare clones in molecular libraries. Recent advances in laser techniques and microscopy have allowed this new technique to be utilized with full potential (Rigler and Widengren 1990;Rigler et al. 1993). It is based on the ability to measure single fluorescent molecules excited by monochromatic laser light in extremely small volumes. One records spatiotemporal correlations among intensity fluctuations belonging to the emission of discrete molecules. The sensitivity has been increased so that single molecules can be detected. This opens up the possibility of detecting rare events and allows working concentrations of fmoles or less to be used. FCS may thus provide substantially improved selection strategies for molecular libraries. To investigate this potential, we have used a model system comprised of a previously isolated Fab fragment displayed on the surface of filamentous M13 phage, and its cognate antigen, the Hepatitis-C Virus (HCV) Ibrutinib-biotin E2 protein (Allander et al. 2000). Two approaches for detecting phage were utilized (Fig. 1): Autocorrelated detection of specific phage with fluorescently labeled antigen, or detection of phage with labeled antiphage antibodies. == Fig. 1. == Principle for Fluorescence Correlation Spectroscopy -based detection utilizing two different colors in autocorrelation mode. == Results == == Phagestocks == The first phagestocks were produced with colony-forming units (cfu) titers varying between 10101012cfu/mL. These preparations were polyethylene glycol (PEG) precipitated once and they resulted in unreasonably high backgrounds. Testing all components we determined that the super broth (SB) medium contributed to the large signal (Table 1). Medium components were apparently co-precipitated with the phage during PEG precipitation. == Table 1. == Background values for different components used in phage preparations I is the mean fluorescence intensity. *The phage were resuspended in PBS buffer after treatment. The background does not depend on the concentration of phages but on phage medium components. New phagestocks prepared with one or two PEG precipitations as a means of purification were tested in FCS. The background was reduced for 2 PEG Ibrutinib-biotin precipitated material but the degree of aggregation had increased compared to 1 PEG precipitation. Aggregates of varying sizes are a problem in FCS measurements because identification.