Direct measurement of the weak interactions between a mouse Fc receptor (Fc gamma RII) and IgG1 in the absence and presence of hapten: a total internal reflection fluorescence microscopy study.
Total internal reflection fluorescence microscopy (TIRFM) has been used to directly measure the weak dissociation constants of IgG with a mouse IgG receptor (moFc gamma RII) that has been purified and reconstituted into substrate-supported planar membranes. Dissociation constants were measured for three different mouse monoclonal anti-dinitrophenyl (DNP) IgG1 antibodies and for polyclonal mouse IgG, in the absence and presence of saturating amounts of hapten (DNP-glycine). The dissociation constant for polyclonal mouse IgG was 3 microM, which agrees well with previous results. The dissociation constants for the three monoclonal antibodies with moFc gamma RII ranged from 2 microM to 3 microM and were not statistically different, suggesting that changes in moFc gamma RII dissociation constants which may exist within the IgG1 subclass are less than the error of the TIRFM measurements (approximately 20%). The measured IgG1-moFc gamma RII dissociation constants were not different for individual monoclonal antibodies in the absence or presence of saturating concentrations of DNP-glycine, directly showing that possible allosteric changes which might occur upon hapten binding and affect the equilibrium characteristics of Fc receptor binding are small. This work demonstrates a new approach for quantitatively examining the effects of solution components on weak receptor-ligand interactions.
Hsieh
H V
HV
Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290.
Poglitsch
C L
CL
Thompson
N L
NL
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D006241:Haptens; D007074:Immunoglobulin G / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D011961:Receptors, Fc / Q000378:metabolism*; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D006241:Haptens; D007074:Immunoglobulin G; D011961:Receptors, Fc