Laura E DeForge | PubMed Health

PubMed2006

Synthetic anti-BR3 antibodies that mimic BAFF binding and target both human and murine B cells.

BR3, which is expressed on all mature B cells, is a specific receptor for the B-cell survival and maturation factor BAFF (B-cell-activating factor belonging to the tumor necrosis factor [TNF] family). In order to investigate the consequences of targeting BR3 in murine models and to assess the potential of BR3 antibodies as human therapeutics, synthetic antibody phage libraries were employed to identify BAFF-blocking antibodies cross-reactive to murine and human BR3, which share 52% identity in their extracellular domains. We found an antibody, CB1, which exhibits muM affinity for murine BR3 and very weak affinity for the human receptor. CB3s, an affinity-matured variant of CB1, has sub-nM affinity for BR3 from both species. Alanine scanning and crystallographic structural analysis of the CB3s/BR3 complex reveal that CB3s mimics BAFF by interacting with a similar region of the BR3 surface. Despite this similarity in binding epitopes, CB1 variants antagonize BAFF-dependent human B-cell proliferation in vitro and are effective at reducing murine B-cell populations in vivo, showing significant promise as therapeutics for human B-cell-mediated diseases.

Amino Acid Sequence↗

PubMed2004

Substrate capacity considerations in developing kinase assays.

In developing a screening assay for a serine/threonine kinase, we evaluated various formats of an in-plate enzyme-linked immunosorbent assay (ELISA), as well as solution-phase kinase assays using either ELISA or AlphaScreen detection. Substrate was available both as a biotinylated 15-residue peptide and as a 25-residue peptide containing the same sequence expressed as a glutathione S-transferase fusion protein. When increasing concentrations of either of these substrates were coated directly onto ELISA plates, the rates of the kinase reactions progressively increased. In contrast, when the biotin-peptide was captured onto NeutrAvidin-coated plates, the finite peptide binding capacity of the plates limited the amount of substrate that could be incorporated into the assay system and thereby limited the rate of the reaction at a given kinase concentration. Solution-phase kinase reactions can tolerate high substrate concentrations; however, analysis of kinase reaction samples containing biotin-peptide concentrations higher than the binding capacity of NeutrAvidin-coated plates resulted in an inability to detect differences between reactions run at different substrate concentrations. For AlphaScreen detection following solution-phase kinase reactions, limitations in the binding capacity of the donor and acceptor beads caused loss of signal for substrate concentrations above the maximum binding capacity. Overall, the solution-phase assays required significantly more kinase than the in-plate assays (1-4 microg/ml versus <100 ng/ml, respectively). These studies demonstrate that the amount of substrate that can be incorporated into an assay system substantially affects the rate of the kinase reaction and therefore the amount of kinase required for the assay.

Enzyme-Linked Immunosorbent Assay↗