Binding of IgG to MoFc gamma RII purified and reconstituted into supported planar membranes as measured by total internal reflection fluorescence microscopy.
Total internal reflection fluorescence microscopy (TIRFM) has been combined with functional reconstitution of the mouse IgG receptor moFc gamma RII in substrate-supported planar membranes to quantitatively probe IgG-moFc gamma RII interactions. MoFc gamma RII was purified from the macrophage-related cell line J774A.1 using affinity chromatography with Fab fragments of the anti-moFc gamma RII monoclonal antibody 2.4G2. Purified moFc gamma RII was reconstituted into liposomes by detergent dialysis, and the liposomes were fused on quartz substrates to form supported planar membranes containing moFc gamma RII. TIRFM measurements showed that fluorescently labeled 2.4G2 Fab specifically bound to the planar membranes, confirming the presence of moFc gamma RII. The receptor density in the planar membranes was sufficiently high to allow direct detection of bound, fluorescently labeled polyclonal and monoclonal mouse IgG with TIRFM, demonstrating that moFc gamma RII retained Fc-mediated IgG binding activity after planar membrane formation and permitting direct measurement of bound IgG as a function of the IgG solution concentration. Cross-inhibition measurements showed that polyclonal mouse IgG blocked the binding of labeled 2.4G2 Fab and that 2.4G2 Fab blocked the binding of labeled polyclonal IgG. This work provides a direct measure of the relatively weak IgG-moFc gamma RII association constant and demonstrates a new model system in which the chemical and physical properties of IgG-moFc gamma RII interactions can be quantitatively characterized as a function of membrane, antibody, and solution properties.
Poglitsch
C L
CL
Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290.
Sumner
M T
MT
Thompson
N L
NL
D000818:Animals; D000943:Antigens, Differentiation / Q000302:isolation & purification / Q000378:metabolism*; D001667:Binding, Competitive; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism*; D007700:Kinetics; D008567:Membranes, Artificial; D051379:Mice; D008856:Microscopy, Fluorescence; D011961:Receptors, Fc / Q000302:isolation & purification / Q000378:metabolism*; D017452:Receptors, IgG; D013379:Substrate Specificity; D013816:Thermodynamics
D000943:Antigens, Differentiation; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008567:Membranes, Artificial; D011961:Receptors, Fc; D017452:Receptors, IgG