Characterization of proteins by capillary electrophoresis in fused-silica columns: review on serum protein analysis and application to immunoassays.
Protein mixtures can be characterized in terms of their separations by capillary electrophoresis (CE). The separation of proteins by CE is performed in untreated fused-silica columns. Model proteins and complex protein mixtures with pI values ranging from 4.0 to 11.0 are separated in such columns in less than 10 min in the presence of phosphate buffer with a pH between 4.0 and 9.0. The application of CE separation procedures for routine analysis of protein in serum, urine, and cerebrospinal fluid in borate-based buffer is also demonstrated. The detection of protein in CE is usually based on the intrinsic ultraviolet (UV) absorbance of the peptide bond at or near 200 nm, which provides a detection limit of about 10(-5) M. The same protein separation procedures can also be applied to immunochemical reaction systems in which one component is labeled. Thus, an antigen analyte, or the antibody to the analyte, may be labeled with a fluor and detected by laser-induced fluorescence (LIF). With a fluorescent-labeled reactant, the use of LIF detection further extends the detection limit to 10(-11) M. The CE separation technique for proteins provides a means to separate the bound and free species of the labeled antigen or antibody without the use of a solid support. The application of these separation techniques in conjunction with laser-induced fluorescence detection to make possible the homogeneous immunochemical measurement of species at concentrations in the range of 10(-9) to 10(-10) M is shown.