Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis

detection. by flow cytometry and whole-cell ELISA. strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA. F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of scFv antibodies

Analysis of putative thiamine biosynthetic genes from the phytopathogen Mycosphaerella graminicola for fungicide target validation

Available from British Library Document Supply Centre- DSC:DXN062715 / BLDSC - British Library Document Supply CentreSIGLEGBUnited Kingdo

Enhancement of Immune Effector Functions by Modulating IgG's Intrinsic Affinity for Target Antigen.

Antibody-mediated immune effector functions play an essential role in the anti-tumor efficacy of many therapeutic mAbs. While much of the effort to improve effector potency has focused on augmenting the interaction between the antibody-Fc and activating Fc-receptors expressed on immune cells, the role of antibody binding interactions with the target antigen remains poorly understood. We show that antibody intrinsic affinity to the target antigen clearly influences the extent and efficiency of Fc-mediated effector mechanisms, and report the pivotal role of antibody binding valence on the ability to regulate effector functions. More particularly, we used an array of affinity modulated variants of three different mAbs, anti-CD4, anti-EGFR and anti-HER2 against a panel of target cell lines expressing disparate levels of the target antigen. We found that at saturating antibody concentrations, IgG variants with moderate intrinsic affinities, similar to those generated by the natural humoral immune response, promoted superior effector functions compared to higher affinity antibodies. We hypothesize that at saturating concentrations, effector function correlates most directly with the amount of Fc bound to the cell surface. Thus, high affinity antibodies exhibiting slow off-rates are more likely to interact bivalently with the target cell, occupying two antigen sites with a single Fc. In contrast, antibodies with faster off-rates are likely to dissociate each binding arm more rapidly, resulting in a higher likelihood of monovalent binding. Monovalent binding may in turn increase target cell opsonization and lead to improved recruitment of effector cells. This unpredicted relationship between target affinity and effector function potency suggests a careful examination of antibody design and engineering for the development of next-generation immunotherapeutics

Equilibrium binding of anti-EGFR IgGs to FcγRIIIa isoforms.

<p>Equilibrium binding of anti-EGFR IgGs to FcγRIIIa isoforms.</p

Effect of bivalent <i>vs</i>. monovalent antigen binding on ADCC activity.

<p>(<b>A</b>) ADCC activity of anti-CD4 ibalizumab formatted as either bivalent IgG or monovalent DuetMab against CD4⁺ T cells. (<b>B</b>) ADCC activity of anti-EGFR GA201formatted as either bivalent IgG or monovalent DuetMab against MDA-MB-231 cells. (<b>C</b>) ADCC activity of anti-HER2 B1D2 formatted as either bivalent IgG or monovalent DuetMab against BT474 cells. Each point represents the mean values of triplicate wells and the standard deviation is represented by error bars.</p

Binding affinity of IgG to target antigens.

<p>Binding affinity of IgG to target antigens.</p

EGFR and HER2 receptor density on human tumor cell lines.

<p>EGFR and HER2 receptor density on human tumor cell lines.</p

Reporter-based cytotoxicity of anti-HER2 IgG variants.

<p>Reporter-based cytotoxicity of anti-HER2 IgG variants.</p

Reporter-based cytotoxicity of anti-EGFR IgG variants.

<p>Reporter-based cytotoxicity of anti-EGFR IgG variants.</p