1. Real-time measurement of spontaneous antigen-antibody dissociation
Simone Kulin, Rani Kishore, Joseph B Hubbard, Kristian Helmerson Biophys J. 2002 Oct;83(4):1965-73. doi: 10.1016/S0006-3495(02)73958-1.
We report observations in real time of thermally driven adhesion and dissociation between a monoclonal IgE antibody and its specific antigen N-epsilon-2,4-dinitrophenyl-L-lysine. Both molecules were attached to the surfaces of different polystyrene microspheres trapped by optical tweezers. Monitoring spontaneous successive attachment and detachment events allowed a direct determination of the reaction-limited detachment rate k(off) for a single bond and also for multiple bonds. We observed both positive and negative cooperativity between multiple bonds depending on whether the antigen was linked to the microsphere with or without a tether, respectively.
2. Targeted surface-enhanced Raman scattering nanosensors for whole-cell pH imagery
K L Nowak-Lovato, K D Rector Appl Spectrosc. 2009 Apr;63(4):387-95. doi: 10.1366/000370209787944406.
In this paper the ability to develop a gold-core, silver-shell surface-enhanced Raman scattering (SERS) nanosensor that has both a targeting component as well as a sensing component is demonstrated. The nanosensor is capable of targeting through the FcepsilonRI receptor-mediated endocytic pathway due to the adsorption of 2,4-epsilon-dinitrophenol-L-lysine (DNP) ligand on the nanoparticle surface. The nanosensor is also sensitive to pH changes in the endocytic vesicle within the pH range of 4.5-7.5 by 4-mercaptopyridine (4-MPy) adsorbed to the particle surface. The targeting and sensing moieties do not significantly interfere with each other's function. The sensing component of the nanosensor is calibrated with in vivo measurements of rat basophil leukemia (RBL-2H3) cells in standard buffer solutions using the ionophore nigericin, which serves to equilibrate the external [H(+)] concentration with that of the cell compartments. The targeting of the nanosensor is verified with a beta-hexosaminidase assay. It is also demonstrated that the targeted nanosensor is capable of making accurate cellular pH measurements in RBL-2H3 cells, out to ninety minutes after targeted nanosensor addition. Whole-cell, time-lapse, hyperspectral image cubes demonstrating endocytic vesicular pH changes during FcepsilonRI receptor mediated endocytosis are demonstrated.
