Arnout Gerritsen, Associate Director, Genmab, The Netherlands

Marijn van der Neut Kolfschoten, Senior Scientist, Crucell, The Netherlands

The Octet system can be used for both quantitative and qualitative applications. Within research department of Crucell, BLI technology is broadly used to improve the selection of therapeutic mAbs against viral targets. Examples of these applications, such as prot A quantitation, binding characteristics (breadth of binding, kinetics etc), competition experiments and sometimes even mechanism of action experiments, will be discussed in this presentation.

Pim Hermans, Director of Ligand Discovery, BAC BV, The Netherlands

Since more and more antibody based therapeutics are lacking a regular protein A binding site it becomes a challenge to find a protein A equivalent for primary capture of antibody formats. For this reason BAC has developed a range of CaptureSelect affinity ligands directed against a unique panel of antibody sub-domains (e.g. present on Fab) to facilitate affinity purification and detection of virtually any antibody based format. Within the ligand development department of BAC BV, FortéBio's Octet platform has become an important and valuable tool for ligand candidate screening and lead selection by enabling verification of most optimal kinetic parameters. Examples of ligand screening processes will be presented in addition to antibody characterization assays that can be set-up in Octet by introducing CaptureSelect affinity ligands.

Philip Buckle, Application Manager, ForteBio Inc.

Kinetics analysis of protein-protein and protein-small molecule interactions is a key application for real time, label free systems such as the Octet family of instruments. In this talk, tips and tricks for obtaining the best possible data will be presented. This will include optimization of target immobilisation and analyte binding, along with biosensor regeneration strategies (if required) and sample plate and method design. The Octet analysis software options will also be discussed, giving details of the analysis models and their applicability to data interpretation.

Peter Coombs, Postdoctoral Fellow, MRC National Institute for Medical Research , UK

The influenza virus surface glycoprotein hemagglutinin (HA) mediates receptor binding and membrane fusion in influenza infections. The receptors to which HA binds are sialic acids, which are found in α2,3 or α2,6 linkages to galactose on cell surface glycoproteins and glycolipids. Avian influenza viruses preferentially bind α2,3-linked sialic acids, which predominate in the enteric tract of birds where the viruses replicate. Human influenza viruses preferentially bind to α2,6-linked sialic acids, which are the predominant form found in the human upper respiratory tract. We have developed a quantitative assay to measure the binding of influenza viruses to immobilized sialic acids. Using the Octet RED instrument system from ForteBio, we are able to measure virus binding in real time and determine the affinity, and therefore specificity, for different sugars. Analysis of different human, avian and swine influenza strains, as well as analysis of certain mutations in specific strains, has revealed which viruses are able to bind well to human receptors. Screening of emerging influenza virus strains for human receptor binding potential using this technique will provide essential information in preparation for future pandemics.

Alexander Mader, Renate Kunert, Department of Biotechnology, Institute for Applied Microbiology, BOKU – University of Natural Resources and Life Sciences

IgM antibodies are the first immunoglobulin class to be secreted by B cells upon primary stimulation by antigen. Therefore they play an important role in the body's defense mechanisms against bacterial infections and cancer cells. The IgM class naturally exists in pentameric and hexameric form. The molecular weight is approximately 950 kDa for the pentameric and 1150 kDa for the hexameric form. The high valency of IgMs gives them a high avidity for the target antigen. Further IgMs are highly active in cytotoxic and cytolytic reactions and have the ability for effective complement activation. Thus the IgM class of antibodies is attractive as therapeutic agents. However, recombinant expression of pentameric IgM in mammalian cells is considered to be difficult and has not been achieved routinely so far.
The group of professor Kunert previously established a modular methodology platform enabling the cultivation of the host cell line, the transfection procedure and selection of stable clones as well as cryopreservation and large scale cell propagation under serum- and protein free conditions. With this platform we established a number of different IgM producing mammalian cell lines in the last years. The method of choice for screening our producer cell lines is ELISA. To accelerate the screening process we established a one-step as well as a two-step quantification assay using our FortéBio Octet QK system equipped with the novel Protein L sensor tips. The obtained results are comparable with our well established ELISA assay, thus making quantification of IgMs using ForteBio's label-free technology a fast and reliable alternative.