The Perfinity approach to separations is an immunoassay technique that replaces the second antibody of a sandwich pair with a chromatography column. The complete system includes trypsin digestion followed by chromatography and mass spectral analysis. There are significant benefits to this approach.
Differences among protein isoforms often occur in small hidden regions of the total structure. Immunological contact areas, or epitopes, are small, making it difficult to interrogate these domains with antibodies. Moreover, antibodies do not cleanly discriminate between single amino acid variants.
Chromatographic retention is generally determined by a relatively large portion of the surface of a protein or peptide. Variants that differ by a single amino acid can be resolved. However, serum cannot be analyzed directly. Proteins must be extracted or samples fractionated prior to analysis.
With immunochromatographic analyses, the structural selectivity of antibodies can be used to bind and purify antigens from biological extracts, then chromatography can be used to resolve proteins that differ by only small changes in structure. In a given run, hundreds of peaks can be resolved, which enables high degrees of multiplexing (Figure 2).
Researchers have found ways to leverage combinations of these features, using multiple apparatuses and transfer pipettes. Buffer exchanges and a solution-based digestion of proteins into more easily identifiable peptide fragments are performed off-line.
The Perfinity Workstation combines the selectivity of antibodies with the resolving power of chromatography. Coupling of affinity columns that select proteins of interest to immobilized trypsin generates requisite peptides, making it easy to distinguish between single amino acid variants via mass-spectral analysis. Buffer exchange and desalting are performed on-line. The reduction in sample-processing time enables users to screen a variety of conditions rapidly.
A major advantage of Perfinity’s sample-preparation approach, resulting from the work of Perfinity founder Fred Regnier, is that unlike other technologies users are not required to immobilize antibodies. Instead, antibodies are added to the sample and allowed to function as they would as part of the immune system—antibody-antigen complexes are formed in solution. This results in enhanced kinetics. Removal of the immobilization process makes for one less possible source of error.