New approach to steroid separation based on a low affinity IgM antibody.
IgM antibodies are often of low affinity (dissociation constant (Kd) > 10(-5) M) and therefore they are usually neglected as tools in, e.g., immunoassays. Previous studies have shown that low affinity biological interactions can be studied and exploited in affinity chromatography, biosensor technology and capillary electrophoresis. In this study we have demonstrated that IgM can be a useful ligand for analytical separation of antigens in weak affinity chromatography (WAC). A low affinity human monoclonal IgM antibody, directed at digoxin, was produced in a hybridoma cell culture, purified to homogeneity and immobilized onto an HPLC support. The IgM HPLC column displayed specific weak affinity retention in the 0.01-0.1 mM range as evaluated with digoxin and ouabain. The specificity was not affected when samples of ouabain in a crude environment of diluted serum were separated on the IgM column. These findings suggest an approach in immunoadsorbent technology where biomolecules can be analyzed and separated with weak affinity chromatography using IgM as a general affinity ligand.
Department of Natural Sciences, University of Kalmar, Sweden. magnus.strandh@ng.hik.se
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Strandh
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Ohlin
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Borrebaeck
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Ohlson
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007075:Immunoglobulin M / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D012967:Sodium Dodecyl Sulfate; D013056:Spectrophotometry, Ultraviolet; D013256:Steroids / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M; D013256:Steroids; D012967:Sodium Dodecyl Sulfate