Antibody binding to a tethered vesicle assembly using QCM-D.
The bilayer-tethered vesicle assembly has recently been proposed as a biomimetic model membrane platform for the analysis of integral membrane proteins. Here, we explore the binding of antibodies to membrane components of the vesicle assembly through the use of quartz crystal microbalance with dissipation monitoring (QCM-D). The technique provides a quantitative, label-free avenue to study binding processes at membrane surfaces. However, converting the signal generated upon binding to the actual amount of antibody bound has been a challenge for a viscoelastic system such as the tethered vesicle assembly. In this work, we first established an empirical relationship between the amount of bound antibody and the corresponding QCM-D response. Then, the results were examined in the context of an existing model describing the QCM-D response under a variety of theoretical loading conditions. As a model system, we investigated the binding of monoclonal antidinitrophenyl (DNP) IgG(1) to tethered vesicles displaying DNP hapten groups. The measured frequency and dissipation responses upon binding were compared to an independent measure of the amount of bound antibody obtained through the use of an in situ ELISA assay. At saturation, the surface mass density of bound antibody was approximately 900 ng/cm(2). Further, through the application of QCM-D models that describe the response of the quartz when loaded by either a single homogeneous viscoelastic film or by a two-layered viscoelastic film, we found that a homogeneous, one-layer model accurately predicts the amount of antibody bound to the tethered vesicles near antibody surface saturation, but a two-layer model must be invoked to accurately describe the kinetic response of the dissipation factor, which suggests that the binding of the antibody results in a stiffening of the top layer of the film.
Patel
Ankit R
AR
Department of Chemical Engineering, Stanford University, 381 North-South Mall, Stauffer III, Stanford, California 94305-5025, USA.
Kanazawa
Kay K
KK
Frank
Curtis W
CW
D019297:2,4-Dinitrophenol / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D001666:Binding Sites, Antibody; D015374:Biosensing Techniques; D003460:Crystallization; D004797:Enzyme-Linked Immunosorbent Assay; D006241:Haptens / Q000276:immunology / Q000378:metabolism; D007136:Immunoglobulins / Q000276:immunology* / Q000378:metabolism; D011791:Quartz / Q000737:chemistry*; D020349:Surface Plasmon Resonance; D013499:Surface Properties; D053835:Unilamellar Liposomes / Q000276:immunology / Q000378:metabolism*; D014867:Water / Q000737:chemistry
D000911:Antibodies, Monoclonal; D006241:Haptens; D007136:Immunoglobulins; D053835:Unilamellar Liposomes; D014867:Water; D011791:Quartz; D019297:2,4-Dinitrophenol