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 p erformed in untreated fused-silica columns. Model proteins and complex protein mixtures with pI values ranging from 4.0 to 11.0 are separate d in such columns in less than 10 min in the presence of phosphate buf fer with a pH between 4.0 and 9.0. The application of CE separation pr ocedures for routine analysis of protein in serum, urine, and cerebros pinal fluid in berate-based buffer is also demonstrated. The detection of protein in CE is usually based on the intrinsic ultraviolet (UV) a bsorbance of the peptide bond at or near 200 nm, which provides a dete ction limit of about 10(-5) M. The same protein separation procedures can also be applied to immunochemical reaction systems in which one co mponent is labeled. Thus, an antigen analyte, or the antibody to the a nalyte, may be labeled with a fluor and detected by laser-induced fluo rescence (LIF). With a fluorescent-labeled reactant, the use of LIF de tection further extends the detection limit to 10(-11) M. The CE separ ation technique for proteins provides a means to separate the bound an d free species of the labeled antigen or antibody without the use of a solid support. The application of these separation techniques in conj unction with laser-induced fluorescence detection to make possible the homogeneous immunochemical measurement of species at concentrations i n the range of 10(-9) to 10(-10) M is shown.