Perfusible and non-perfusible supports with monoclonal antibodies for bioaffinity chromatography of Escherichia coli penicillin amidase within its pH stability range.
Several monoclonal antibodies (mABs) have been prepared and immobilized for the biospecific isolation of penicillin amidase (PA) from Escherichia coli (EC 3.5.1.11), an enzyme without S-S bridges and a pH stability range of 4-9. During the immobilization the fluorescence emission maxima of the mABs were found to change from 336 nm to ca. 350 nm. Only one of these mABs was found to be suitable for preparative bioaffinity chromatography of PA within the pH stability range. This mAB was immobilized on different spherical supports (Eupergit C 250 L and Sepharose) and one perfusible support (Knauer Quick Disc) and used for analytical and preparative bioaffinity chromatography. Under isocratic conditions the plate height for the perfusible biospecific adsorbent was found to be an order of magnitude lower than for the other supports. The different forms of this proteolytically processed bacterial enzyme could not be separated, however, by the biospecific adsorbents. At the mAB density used in the adsorbents (10-30 microM), less than 30% of the theoretical binding capacity of the immobilized mABs could be used to adsorb the enzyme.
Kasche
V
V
AB Biotechnologie II, TU Hamburg-Harburg, Germany.
Gottschlich
N
N
Lindberg
A
A
Niebuhr-Redder
C
C
Schmieding
J
J
D000818:Animals; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D006825:Hybridomas; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D051379:Mice; D008807:Mice, Inbred BALB C; D010399:Penicillin Amidase / Q000302:isolation & purification*; D010477:Perfusion; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D010399:Penicillin Amidase