title: Alteration of antibody specificity during isolation and storage.
abstract: Isolation buffer characteristics and storage conditions could partially transform natural antibodies. 50 IgG factions were isolated from seven healthy donors' sera using various protein G columns and buffers. PAGE revealed no major antibody cleavages; purity of IgGs eluted at pH 2.4-3.5 was close to homogeneity, independent of buffer composition. Although eluting at pH 2.4 resulted in 3.5- or 17-fold higher antibody yield compared to pH 3.0 or 3.5, respectively, it distorted the antibody molecule. IgGs eluted at pH 2.4 acquired reactivity against diagnostically important autoantigens, confirmed by standardized ELISAs. Preserved antibodies' natural activity is important in further experiments with oxidatively-induced autoantibodies.
authors.affiliation: Department of Rheumatology, Division of Internal Medicine University Medical Centre, Ljubljana, Slovenia.
authors.forename: Jasna
authors.initials: J
authors.lastname: Omersel
authors.forename: Urska
authors.initials: U
authors.lastname: Zager
authors.forename: Tanja
authors.initials: T
authors.lastname: Kveder
authors.forename: Borut
authors.initials: B
authors.lastname: Bozic
mesh_terms: D000918:Antibody Specificity; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology*
chemical_list: D007074:Immunoglobulin G