Evaluation of affinity-tagged protein expression strategies using local and global isotope ratio measurements.

Elucidation of protein-protein interactions can provide new knowledge on protein function. Enrichments of affinity-tagged (or "bait") proteins with interaction partners generally include background, nonspecific protein artifacts. Furthermore, in vivo bait expression may introduce additional artifacts arising from altered physiology or metabolism.

A general system for studying protein-protein interactions in Gram-negative bacteria.

One of the most promising methods for large-scale studies of protein interactions is isolation of an affinity-tagged protein with its in vivo interaction partners, followed by mass spectrometric identification of the copurified proteins. Previous studies have generated affinity-tagged proteins using genetic tools or cloning systems that are specific to a particular organism. To enable protein-protein interaction studies across a wider range of Gram-negative bacteria, we have developed a methodology based on expression of affinity-tagged "bait" proteins from a medium copy-number plasmid.

Identification of an antilaminin-1 scFv that preferentially homes to vascular solid tumors.

The tumor vasculature and extracellular matrix make attractive targets for distinguishing solid tumors from normal cells. In solid tumors, the processes of angiogenesis and metastasis potentially give rise to unique epitopes not usually accessible in homeostatic organs. Specific targeting of solid tumors for radioimmunotherapy requires that the targeting agent accumulate rapidly and at high levels at the tumor site.

Interleukin-4 produces a breakdown of tolerance in vivo with autoantibody formation and tissue damage.

B cell susceptibility to Fas-mediated apoptosis is downmodulated by engagement of IL-4 and sIg receptors. IL-4 produces Fas-resistance in both normal and tolerant B lymphocytes and has been associated with autoantibody production in mice expressing heterogeneous B cell receptors. To study the in vivo effects of IL-4 on autoreactive B cells in a more well-defined system, mice triply transgenic for IL-4, soluble HEL and anti-HEL B cell receptors were generated.

A diabody that dissociates to monomer forms at low concentration: effects on binding activity and tumor targeting.

A human scFv, 15-9, was selected from a phage display library for binding to murine laminin-1. A diabody was made from the scFv by shortening the linker from 15 to 5 amino acids between the VH and VL sequence. Radioiodinated scFv and diabody were analyzed for size, binding to laminin, and biodistribution in tumor bearing mice.

Phage display selection of scFv to murine endothelial cell membranes.

The diversity of endothelial cells is becoming more apparent and more important in defining vessel systems that supply blood to normal organs and to tumors. Reagents that identify expression of cell surface determinants on these cells are crucial for differentiating among different vessel types. As a first step in this process we have selected a panel of 25 scFvs from a phage display library that bind to the endothelial cell line LEII.

Genetic analysis of Pseudomonas aeruginosa by enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR) and arbitrarily primed PCR: gel analysis compared with microchip gel electrophoresis.

Objectives: To assess the applicability of a newly emerging microchip gel electrophoresis for rapid strain differentiation among clinical isolates of Pseudomonas aeruginosa, and to compare this technique with the traditional gel method for DNA separation.

Methods: One hundred clinical strains of P. aeruginosa obtained from a hospital in northwestern Ohio were tested for reactivity to 3 serotype-specific monoclonal antibodies by enzyme-linked immunosorbent assay.

A monoclonal antibody to a carbohydrate epitope expressed on glycolipid and on alpha3beta1 integrin on human esophageal carcinoma.

A mouse monoclonal antibody (MAb-9) produced by immunization with a human esophageal carcinoma cell line, TE-2 (derived from undifferentiated squamous cell carcinoma) reacted specifically with about 30% of esophageal carcinoma cell lines and tissue sections from clinical samples. MAb-9 showed minimal reactivity with normal esophageal tissue. (125)I, fluorescent or gold particle labeled MAb-9 bound to TE-2 cell surfaces.