Identification and minimization of nonideal binding effects in BIAcore analysis: ferritin/anti-ferritin Fab' interaction as a model system.
The interaction of human spleen ferritin with a monoclonal antibody Fab' fragment has been studied as a model system for BIAcore analysis. In particular, the influence of nonideal binding effects has been examined both experimentally and by the theoretical simulation of sensorgram curves. Mass transfer effects were found to have a small but significant influence on the observed binding kinetics of the ferritin/antiferritin Fab' interaction; however, this nonideal behavior could be overcome by systematic manipulation of experimental conditions such as the flow rate and the surface density of the immobilized antigen. Because of the multivalent nature of ferritin with 12 antiferritin Fab' binding sites per molecule, immobilization of the antigen by amine coupling had little effect on the majority of free binding sites on the molecule. Consequently, the binding data for both ferritin and apoferritin correlated well with an ideal binding model which assumes binding homogeneity. On the other hand, when ferritin was dissociated to its subunit dimer form (containing one Fab' binding site) prior to surface immobilization significant deviation from this model was observed. This nonideal behavior was probably due to heterogeneity of the immobilized ferritin subunit dimer on the sensor surface, resulting from the nonspecific amine coupling procedure.
Oddie
G W
GW
CSIRO, Division of Biomolecular Engineering, Parkville, Victoria, Australia.
Gruen
L C
LC
Odgers
G A
GA
King
L G
LG
Kortt
A A
AA
D000478:Alkylation; D000911:Antibodies, Monoclonal; D001052:Apoferritins / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine; D005293:Ferritins / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008970:Molecular Weight; D011487:Protein Conformation; D013696:Temperature
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D005293:Ferritins; D001052:Apoferritins; D003545:Cysteine