Search PubMed⌕ Search

Biomedical subjects

Ian M Tomlinson

Publications and source records attributed to Ian M Tomlinson.

5 recordsLinked to original sources
PubMed2005

Engineering high affinity superantigens by phage display.

Protein L (PpL) is a B-cell superantigen from Peptostreptococcus magnus known to bind to mammalian Vkappa light chains. PpL from P.magnus strain 312 comprises five homologous immunoglobulin (Ig) binding domains. We first analysed the binding of the individual domains (B1-B5) of PpL(312) to human Vkappa light chains (huVkappa) subtypes 1 (huVkappaI) and 3 (huVkappaIII). Using a combination of rational design and phage selection we isolated mutants of the N-terminal B1 domain with a 14-fold increased affinity for huVkappa1 (B1kappa1) and >tenfold increased affinity for huVkappaIII (B1kappa3). We investigated the potential of the selected domains, in particular the B1kappa1 domain, as reagents in immunochemistry and immunotherapy. B1kappa1 proved a superior reagent than the wild-type domain, allowing up to tenfold more sensitive detection of human Vkappa antibody fragments in ELISA. A fusion protein of B1kappa1 with a human Vlambda antibody scFv fragment promoted the efficient recruitment of antibody encoded effector functions including complement, mononuclear phagocyte respiratory burst and phagocytosis through retargeting of IgGkappa and IgMkappa. Our results suggest that superantigens with improved affinity and/or specificity are easily accessible through protein engineering. Such engineered superantigens should prove useful as reagents in immunochemistry and may have potential as agents in immunotherapy.

Amino Acid Sequence↗
PubMed2003

Use of phage display and high-density screening for the isolation of an antibody against the 51-kDa subunit of complex I.

Monoclonal antibodies play an increasingly important role in structural biology. In this report, we develop the use of phage display technology for the isolation of an antibody that binds to a specific subunit of a macromolecular assembly. Antibodies that bind to the intact complex are selected from a phage display library and screened with a high-density Western blot to identify a subunit-specific binder. Conventional Western blotting and competition ELISA are then used to confirm the identity of the target subunit and that the antibody binds to the native protein complex and not to an epitope that is only revealed when the antibody is immobilized for phage selection. Using this technique, monoclonal scFv and Fab fragments have been produced that bind to the 51-kDa subunit of bovine complex I, a large integral membrane protein complex from mitochondria.

Animals↗
PubMed2003

Domain antibodies: proteins for therapy.

Occurring naturally in "heavy chain" immunoglobulins from camels, and now produced in fully human form, domain antibodies (dAbs) are the smallest known antigen-binding fragments of antibodies, ranging from 11 kDa to 15 kDa. dAbs are the robust variable regions of the heavy and light chains of immunoglobulins (VH and VL respectively). They are highly expressed in microbial cell culture, show favourable biophysical properties including solubility and temperature stability, and are well suited to selection and affinity maturation by in vitro selection systems such as phage display. dAbs are bioactive as monomers and, owing to their small size and inherent stability, can be formatted into larger molecules to create drugs with prolonged serum half-lives or other pharmacological activities.

Animals↗
PubMed2002

Isolation of receptor-ligand pairs by capture of long-lived multivalent interaction complexes.

We have combined phage display and array screening for the rapid isolation of pairs of interacting polypeptides. Our strategy, named SAC (selection by avidity capture), is based on the avidity effect, the formation of highly stable complexes formed by multivalent interactions; in our case, between a receptor (multivalently displayed on phage) and a ligand (coexpressed as a multimeric fusion protein). Capture of the long-lived interaction complex allows the isolation of phage bearing cognate interaction pairs, as we demonstrate for a range of interactions, including Ab-antigen pairs and the rapamycin-dependent interaction of FKBP-12 and FRAP. Cognate phage are enriched by SAC up to 1000-fold and interacting pairs can be identified by array screening. Application of SAC to Ab-antigen interactions as a model system yielded over 140 specific Abs to a single antigen and 92 Abs to three different fetal human brain antigens in a single round of SAC each. Our results suggest that SAC should prove useful for the identification and study of receptor-ligand interactions in particular among extracellular proteins, as well as for the rapid generation of specific Abs to multiple antigens.

Antibodies, Monoclonal↗