Purification of prostate-specific antigen from human serum by indirect immunosorption and elution with a hapten.
When isolating proteins from complex biological material by immunosorption, the nonspecific binding and elution of other proteins present in much larger concentrations than the target protein are often a general problem, preventing the isolation of a pure protein. To improve this situation we have developed a new indirect immunosorption method, which makes use of a digoxigenylated anti-analyte antibody. This antibody is linked to streptavidin-coated magnetic beads via a biotinylated anti-digoxigenin antibody. After binding of the analyte to the affinity matrix, the complex composed of analyte and digoxigenylated anti-analyte antibody is specifically eluted with a solution of digoxigenin-lysine at pH 7.3. Coelution of nonspecifically bound proteins was highly reduced as revealed by SDS-PAGE when compared to the acidic eluates of the direct immunosorption. The efficiency of the indirect immunosorption method was demonstrated with the isolation of free prostate-specific antigen (PSA) and PSA/alpha(1)-antichymotrypsin complex from human serum and subsequent analysis of the intact proteins by SDS-PAGE and MALDI-TOF-mass spectrometry.
Peter
J
J
Technische Universität München, Lichtenbergstrasse 4, Garching, 85748, Germany.
Unverzagt
C
C
Lenz
H
H
Hoesel
W
W
D004591:Electrophoresis, Polyacrylamide Gel; D006241:Haptens; D006801:Humans; D007164:Immunosorbents; D008297:Male; D017430:Prostate-Specific Antigen / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006241:Haptens; D007164:Immunosorbents; D017430:Prostate-Specific Antigen