To further characterize the proteins eluting from the Anti-HSA cartridge, peptide mass fingerprinting was performed on low intensity bands that did not correspond to the intact molecular weight of albumin. Figure 4 shows the mass spectra performed on peptide digests from the bands with corresponding protein identification using SwissProt. Most of these bands were identified as human serum albumin and are most likely proteolytic fragments of albumin in the sample since most migrated at a lower molecular weight than expected for intact HSA.
Removal of albumin and IgG were quantified using commercially available ELISA assays. Figure 5 shows results of measuring the removal of IgG and albumin from the flow. The removal of both IgG and HSA was shown to be greater than 99%. The cross reactivity of the Anti-HSA antibody was also examined to determine binding capacity for various species of albumin. Table 1 shows the capacity measurements made on the media using purified forms of albumin from various species. Although albumin from other species bind with some specificity, this antibody demonstrates the highest specificity for Human Serum Albumin.
POROS Protein G and Anti-HSA cartridges can quickly and efficiently remove both IgG and HSA, which are the two most abundant proteins in human serum. Using this immunoaffinity technique greater than 99% of both proteins can be removed from serum with little to zero non-specific binding of other proteins in the sample. Using a convenient cartridge format allows for samples to be processed manually or automated with an LC system to increase throughput.
The cartridges can be used as an effective sample prep device and are comp