ABSTRACT
The single largest proteomics market is in the fi eld of differential proteomics, where samples of serum from diseased and nondiseased populations are compared and contrasted to search for differences in protein levels. These proteins become diagnostic markers of disease, targets for clinical therapeutic intervention, or therapeutics themselves. More than half the human plasma proteome comprises housekeeping proteins such as human serum albumin (HSA) and immune gamma globulins (IgGs). These proteins have no relevance to disease state and mask the presence of important proteins. As the analytical tools for proteomic analysis offer increasingly improved sensitivity, the ability to differentiate important proteins from housekeeping proteins is become increasingly important and diffi cult. Ian Humphrey Smith of the Human Proteome Organization said in 2001 “. . . solving this problem will require designing affi nity reagents, or molecules that capture certain classes of proteins, to screen out the high abundance proteins found in cells before analysis . . .” 1
Scientists have struggled with technologies such as size-exclusion chromatography and isoelectric focusing to deplete samples of HSA and IgGs, yet these techniques are not specifi c to the unwanted molecules, and important proteins are removed along with the depleted fractions. Some attempts have been made at standard products based on dye-based ligands and conventional bioprocessing media such as Protein-A, but the poor specifi cities, low capture capacities, and high costs have stalled these products in the market. We have developed a technology based on affi nity chromatography that uses naturally occurring camelid single chain antibody (VHH) fragments as ligands. The application areas for this technology are numerous (e.g., the purifi cation of proteins from different sources).2,3 The camelid single domain antibodies can solve the problems in proteomics by providing high-affi nity, high-specifi city binders that can remove over 95% of these proteins. Unlike antibody reagents, VHH fragments can be easily manufactured and are very stable. Unlike conventional affi nity reagents such as blue dye and Protein-A, these molecules offer much better specifi city and higher capacities. Unlike antibody fragments such as Fabs and scFvs, VHHs are designed by nature and do not suffer the same stability and hydrophobic agglomeration problems. Finally, unlike peptides, VHHs offer multiple binding sites for multiple epitope recognition and better capacity, and they are far less expensive.
