A highly efficient preparative-scale generic immunoaffinity chromatography method for the purification of antibodies to hydrophobic haptens: purification procedure and monitoring tests.
A generic affinity chromatography purification protocol for the isolation of preparative quantities of pure and stable polyclonal antibodies to hydrophobic haptenic analytes is described together with a panel of tests to monitor the purification process and assess the functional and structural purity of isolated antibodies. The purification method is based on the use of a mixture of acetonitrile and propionic acid to elute bound antibodies from Sepharose 4B-based immunoabsorbent gels. Highly specific and pure antibodies to steroid estrogens, pentachlorophenol and Irgarol 1051 were isolated in 50-150 mg quantities per preparation in a batch-wise method using appropriate ligands linked to the solid phase via a hydrophilic chemical arm, tetraethylene pentamine. The panel of ELISA tests together with SDS-PAGE enabled the monitoring of the absorption and elution steps and provided data relevant to the assessment of the degree of structural and functional purity of the isolated antibody preparations. The study demonstrates that the affinity purification procedure is practical, simple, generic for antibodies to hydrophobic haptens and suitable for scaling up. In addition, the study showed that the functional properties of the affinity-purified antibodies indicated improvements on the operational properties (specificity and assay detection limits) of the source antisera. The isolated IgG antibodies showed near 100% functional and structural purity and no deterioration of activity on storage for long periods. The method provides critical reagents for labelled-antibody immunoassays and immunosensors and antibody-dependent sample purification techniques.
Griffith
Hannah M T
HM
Analytical Research Group, Pharmaceutical Sciences Research Division, School of Health and Life Sciences, King's College London, University of London, 150 Stamford Street, London SE1 9NH, UK.
Abuknesha
Ramadan A
RA
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification*; D000918:Antibody Specificity; D002846:Chromatography, Affinity / Q000379:methods*; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004958:Estradiol / Q000031:analogs & derivatives / Q000276:immunology; D006241:Haptens / Q000276:immunology*; D057927:Hydrophobic and Hydrophilic Interactions; D010416:Pentachlorophenol / Q000276:immunology; D012756:Sheep / Q000276:immunology; D014227:Triazines / Q000276:immunology
D000906:Antibodies; D006241:Haptens; D014227:Triazines; C507460:estradiol-17-O-hemiglutarate; C103341:irgarol 1051; D004958:Estradiol; D010416:Pentachlorophenol