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Shifts of isoelectric points between cellular and secreted antibodies as revealed by isoelectric focusing and immobilized pH gradients
Author(s) -
Wenisch Elisabeth,
Reiter Susanne,
Hinger Susanne,
Steindl Franz,
Tauer Christa,
Jungbauer Alois,
Katinger Hermann,
Righetti Pier Giorgio
Publication year - 1990
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150111116
Subject(s) - isoelectric focusing , isoelectric point , deamidation , monoclonal antibody , chemistry , glycosylation , antibody , biochemistry , chromatography , biology , enzyme , immunology
Charge microheterogeneity of monoclonal antibodies, as revealed by isoelectric focusing in carrier ampholytes, has been known for a long time. Here we demonstrate, in the case of monoclonals against the gp‐41 of the HIV‐1 virus, that this heterogeneity is already present within the cell sap of hybridoma cells during antibody synthesis. When the monoclonals are secreted extracellularly, the same isoelectric point (p I ) spectrum is maintained, but there is a marked redistribution of the relative isoform abundance towards the lower p I components. This suggests in vivo processing of such forms, possibly via glycosylation or deamidation. The secreted antibodies are also analyzed by immobilized pH gradients (IPG), where they demonstrate an even more extensive heterogeneity, due to the marked increment in resolving power. Single bands are purified by preparative IPGs in a multicompartment electrolyzer and are shown to be stable with time. Thus, artefactual heterogeneity produced by the focusing technique is completely excluded and cellular processing is clearly established.