Affinity chromatography of antibodies directed against soybean lipoxygenase-1 and -2 and an enzyme-linked immunosorbent assay (ELISA) for antibodies and lipoxygenases.
Crude immunoglobulin G (IgG) fractions of antisera directed against soybean lipoxygenase-1 and -2 were purified by being passed through an immunoadsorbent column containing lipoxygenase coupled to CNBr-activated Sepharose 4B. Bound immunoglobulin was desorbed with pulses of 2 M or 3 M ammonium thiocyanate or 0.1 M glycine-HCl buffer (pH 2.5). The total column recoveries of anti-lipoxygenase-1 IgG and anti-lipoxygenase-2 IgG were 45% and 58%, respectively. The affinity for lipoxygenase of immunospecific antibodies was determined in an enzyme-linked immunosorbent assay (ELISA). In a reaction with lipoxygenase-1, anti-lipoxygenase-1 IgG, which was eluted with glycine-HCl buffer (pH 2.5) with recovery of 24%, had a 6.5-times higher affinity than the whole IgG fraction of antiserum. The affinity of anti-lipoxygenase-2 IgG for lipoxygenase-2 increased 2.2-times after chromatography of IgG over an immunoadsorbent column using 2 M ammonium thiocyanate as eluent (recovery 21%).
D000906:Antibodies / Q000302:isolation & purification*; D000936:Antigen-Antibody Complex; D002846:Chromatography, Affinity / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D007074:Immunoglobulin G / Q000302:isolation & purification*; D008084:Lipoxygenase / Q000032:analysis*; D010944:Plants / Q000201:enzymology*; D013025:Glycine max
D000906:Antibodies; D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D008084:Lipoxygenase