Superantigen Recognition and Interactions: Functions, Mechanisms and Applications

Superantigens are unconventional antigens which recognise immune receptors outside their usual recognition sites e.g. complementary determining regions (CDRs), to elicit a response within the target cell. T-cell superantigens crosslink T-cell receptors and MHC Class II molecules on antigen-presenting cells, leading to lymphocyte recruitment, induction of cytokine storms and T-cell anergy or apoptosis among many other effects. B-cell superantigens, on the other hand, bind immunoglobulins on B-cells, affecting opsonisation, IgG-mediated phagocytosis, and driving apoptosis. Here, through a review of the structural basis for recognition of immune receptors by superantigens, we show that their binding interfaces share specific physicochemical characteristics when compared with other protein-protein interaction complexes. Given that antibody-binding superantigens have been exploited extensively in industrial antibody purification, these observations could facilitate further protein engineering to optimize the use of superantigens in this and other areas of biotechnology.

Epitope Binning of Novel Monoclonal Anti F1 and Anti LcrV Antibodies and Their Application in a Simple, Short, HTRF Test for Clinical Plague Detection

Mouse monoclonal antibodies were raised against plague disease biomarkers: the bacterial capsular protein fraction 1 (F1) and the low-calcium response—LcrV virulence factor (Vag). A novel tandem assay, employing BioLayer Interferometry (BLI), enabled the isolation of antibodies against four different epitopes on Vag. The tandem assay was carried out with hybridoma supernatants, circumventing the need for antibody purification. The BioLayer assay was further adopted for characterization of epitope-repetitive antigens, enabling the discovery of two unique epitopes on F1. The selected antibodies were purified and applied as “oligo-clonal” reagents for the immuno-detection of both biomarkers. The developed Homogenous Time Resolved Fluorescence (HTRF) tests were short (10 min) and simple (no washing steps), allowing for detection of 10 ng/mL F1 and 2.5 ng/mL Vag. The tests were successfully applied for detection of disease biomarkers produced by various Y. pestis strains during growth in blood culture vials.

Efficiency assessment of ion-exchange chromatography as the final stage of anti-VEGF antibody purification

The article presents the results of chromatographic purification of the anti-VEGF antibody using the ion-exchange sorbent CaptoТМ S and considers the prospects for medical application of the antibody as a therapeutic agent for cancer treatment.

Analysis and Optimal Design of Batch and Two-Column Continuous Chromatographic Frontal Processes for Monoclonal Antibody Purification

Frontal chromatography has seen increased interest for protein purification, in particular as a polishing step in downstream processes for therapeutic proteins production, as for example the purification of monoclonal antibodies (mAbs) from high molecular weight impurities, e.g., aggregates, using cation exchange resins. In this work we introduce a new two-column continuous process implementing frontal chromatography. The design procedure and its performance, compared to classical batch technology, are discussed. This represents an additional option in the realisation of optimised continuous downstream processing of therapeutic proteins.