Application of an ELISA-elution assay to dissociate digoxin-antibody complexes in immunoaffinity chromatography.
In this study, we used a modified enzyme-linked immunosorbent assay (ELISA)-elution technique as a screening tool to select specific elution conditions. We examined 12 different elution conditions for the removal of antibodies from a complex on an ELISA plate; 0.2 mol/l glycine-HCl (pH 2.5), 1.0 mol/l acetic acid (pH 2.5), 25% methanol (pH 2.5) and 3 mol/l NaSCN showed a higher elution efficiency. We conducted affinity chromatography with these four conditions for the purification of anti-digoxin antibodies from hyperimmune sera with a digoxin-specific column using omega-aminoalkyl derivatives of Sepharose 4B, whose elution efficiency was similar to that of ELISA. We also monitored the relative specific activities during elution from the digoxin-specific column. The optimum, general-purpose dissociation reagent for this immunoaffinity system was identified as 25% methanol (pH 2.5) with an elution efficiency and relative specific activity of 88.40% and 62.25%, respectively. The high purity of the purified antibodies was demonstrated with sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
Xu
R
R
Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China.
Lin
G
G
Wang
W
W
Liu
M
M
Zhan
S
S
Wang
L
L
Zhang
K
K
Zhang
R
R
Li
J
J
D000906:Antibodies / Q000302:isolation & purification*; D000936:Antigen-Antibody Complex / Q000276:immunology*; D002846:Chromatography, Affinity / Q000379:methods*; D004077:Digoxin / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D004077:Digoxin