Nanopore-based kinetics analysis of individual antibody-channel and antibody-antigen interactions.
BACKGROUND
The UNO/RIC Nanopore Detector provides a new way to study the binding and conformational changes of individual antibodies. Many critical questions regarding antibody function are still unresolved, questions that can be approached in a new way with the nanopore detector.


RESULTS
We present evidence that different forms of channel blockade can be associated with the same antibody, we associate these different blockades with different orientations of "capture" of an antibody in the detector's nanometer-scale channel. We directly detect the presence of antibodies via reductions in channel current. Changes to blockade patterns upon addition of antigen suggest indirect detection of antibody/antigen binding. Similarly, DNA-hairpin anchored antibodies have been studied, where the DNA linkage is to the carboxy-terminus at the base of the antibody's Fc region, with significantly fewer types of (lengthy) capture blockades than was observed for free (un-bound) IgG antibody. The introduction of chaotropic agents and its effects on protein-protein interactions have also been observed.


CONCLUSIONS
Nanopore-based approaches may eventually provide a direct analysis of the complex conformational "negotiations" that occur upon binding between proteins.
The Research Institute for Children, 200 Henry Clay Ave, New Orleans, LA 70118, USA. winters@cs.uno.edu
Stephen
S
Winters-Hilt
Eric
E
Morales
Iftekhar
I
Amin
Alexander
A
Stoyanov
D000465:Algorithms; D000936:Antigen-Antibody Complex / Q000032:analysis* / Q000276:immunology; D001185:Artificial Intelligence; D001427:Bacterial Toxins / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006460:Hemolysin Proteins / Q000737:chemistry*; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods; D007700:Kinetics; D036103:Nanotechnology / Q000295:instrumentation* / Q000379:methods; D010363:Pattern Recognition, Automated / Q000379:methods; D016062:Porosity; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014159:Transducers
D000936:Antigen-Antibody Complex; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; C021091:staphylococcal alpha-toxin