Abstract
For accurate micro-scale quantification of a specific protein in biological fluids, immunoaffinity chromatography (IAC) and isoelectric focusing (IEF) were combined in a single fused-silica capillary. The inner wall of the capillary was coated with an anti-E-tag antibody at the inlet side to form an IAC column, and polydimethylacrylamide, a neutral polymer, at the outlet side to form the capillary for IEF. After loading a sample, the whole capillary was filled with a carrier ampholyte solution. An anode solution, an acid, was then introduced to fill only the IAC column segment. Focusing was started with a pressure that balances with the electroosmotic flow produced in the acidified IAC column. Fluorescence-labeled recombinant Fab with an E-tag spiked at 16 pM to 10 nM in 50% serum was separated and detected with high precision. The coupling principle allows rapid and high-resolution IEF analysis of a protein in a biological sample without any loss of the immunoaffinity captured protein.
Highlights
For accurate micro-scale quantification of a specific protein in biological fluids, immunoaffinity chromatography (IAC) and isoelectric focusing (IEF) were combined in a single fused-silica capillary
The need for a combination is because immunochemical detection alone could be affected by false signals, and electrophoretic separation alone is not enough to detect a protein that is present at very low concentration in complex biological samples
When rFab, which is E-tagged and labeled with tetramethylrhodamine, at 100 nM was applied on the streptavidin-immobilized column at a linear flow rate of 6.0 cm/min for 15 min, which corresponds to an imaginary sample plug-length of 90 cm and a volume of 1.8 μL, a plateau of the fluorescence signal was observed, and no fluorescence material was eluted by subsequent irrigation with 100 mM phosphoric acid (Fig. 2, blue broken line)
Summary
For accurate micro-scale quantification of a specific protein in biological fluids, immunoaffinity chromatography (IAC) and isoelectric focusing (IEF) were combined in a single fused-silica capillary. The inner wall of the capillary is coated with an iminodiacetate-derivative, poly(3-N,N-dicarboxymethylamino-2-hydroxypropyl methacrylate), at the inlet side and polydimethylacrylamide (PDMA) at the outlet side The former coating holds nickel ions that act as an affinity ligand for hexahistidine (6xHis)-tagged proteins, and the latter coating suppresses electroosmotic flow (EOF) in a fused-silica capillary, providing a suitable environment for IEF. The affinity column segment due to the counteracting EOF and pressure-driven flow, but this turmoil does not extend to the neutral polymer coated capillary (Fig. 1) The merits of this unified separation system are: (1) a sample volume larger than the capillary volume can be loaded; (2) salts and unrelated highly abundant proteins that may compromise IEF separation can be removed; (3) the whole proteins that were captured in the affinity column can be analyzed by CIEF without loss; and (4) CIEF is performed under optimal conditions to achieve high resolution separation without any compromise
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