One of the major difficulties in analyzing the proteome of human serum is the dynamic range of the concentrations of the proteins present in the sample. Human serum albumin (HSA) constitutes 57-71% of total serum protein and gammaimmunoglobulin (IgG) ranges from 8-26%. Removal of these two proteins alone clears about 75% of the total protein present in serum, thereby allowing the enhanced detection of the remaining proteins that are present in far lower concentration. To address this problem, a new POROS Anti-HSA support has been developed using an antibody ligand that has been optimized to specifically bind human serum albumin. Using the 0.2mL cartridge device, we have shown that the media will completely bind the albumin in a 10-70uL sample of human serum that has been diluted 1:10 with an albumin concentration of 3.5mgs/mL. In combination with the Protein G cartridge, both albumin and IgG can be effectively removed in one step. Immobilization of this immunoaffinity ligand was developed on our POROS Perfusion chromatography media, which is proven robust and rigid support capable of rapid protein separations. The cartridges can be cleaned and re-used up to one hundred cycles.
Here we describe the characterization of Anti-HSA antibody and Protein G affinity chromatography cartridges, which effectively remove HSA and IgG from human serum, enabling detection and analysis of the less abundant proteins. These cartridges have been characterized with respect to their percentage of removal of these abundant serum proteins as well as non-specific binding and cross-reactivity.
Highly specific, immunoaffinity ligand, exhibiting little to zero non-specific binding
Proven POROS Anti-HSA support for high throughput processing and cleanability
Long lifetime and reusability