Improved anti-IgG and HSA affinity ligands: clinical application of VHH antibody technology.

Large scale, highly specific purification of valuable proteins from blood and removal of undesirable components promise to have wide therapeutic applications. Moreover, depletion of bulk proteins from blood is a prerequisite for clinical proteomics. Here we describe the development of specific, high affinity Camelid antibody fragments (VHH) derived from immune libraries for purification and depletion of the bulk protein HSA and IgG from human serum and plasma for therapeutic and research purposes. The anti-IgG VHH substantially improved depletion of IgGs from blood over the classical method based on protein A. To demonstrate the improved performance of VHH based IgG depletion, we analyzed the presence of auto-antibodies in human plasma before and after depletion from two groups of patients with auto-immune disease: Goodpasture syndrome (GP) and systemic lupus erythematosus (SLE). VHHs can be produced efficiently and cost effectively in Saccharomyces cerevisiae, a genetically regarded as safe (GRAS) microorganism. A good manufacturing process (GMP) for purification of these VHHs has also been developed. Moreover, as VHHs are single protein chains, they can be coupled relatively easily to solid matrices. These three factors are important for developing affinity purification medication.

Reliable and controllable antibody fragment selections from Camelid non-immune libraries for target validation.

With the completion of the sequence of the human genome, emphasis is now switching to the human proteome. However, the number of proteins is not only larger than mRNAs in the transcriptome, proteins need often to be in complex with other proteins to be functional. A favourable option to study proteins in their natural context is with a combination of biochemical and microscopic techniques using specific antibodies. Therefore, we designed a fast, reliable and controllable selection and screening of single-domain antibody fragments (VHH) from a Camelid non-immune library. We isolated VHH for four muscle disease related proteins; emerin, actin, tropomyosin-1, and nuclear poly(A)-binding protein. Important features of antibodies for target validation studies are recognition of the antigen in natural conformations and biologically relevant complexes. We show that selected antibody fragments are functional in various immunological techniques and prove useful in diagnostic applications. Our selection strategy is amenable to automation and to the establishment of proteomics platforms. It opens the way to quickly and cost-effectively obtain multiple antibody fragments for many antigens that can detect changes in their localization, level, and modification as well as subtle changes in supramolecular structures, which often associate with disease.