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Anal Biochem. 1999 Aug 15;273(1):98-104.

Purification of prostate-specific antigen from human serum by indirect immunosorption and elution with a hapten.

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  • 1Technische Universit√§t M√ľnchen, Lichtenbergstrasse 4, Garching, 85748, Germany.

Abstract

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.

Copyright 1999 Academic Press.

PMID:
10452804
[PubMed - indexed for MEDLINE]
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