| AVS 64th International Symposium & Exhibition | |
| Advanced Ion Microscopy Focus Topic | Thursday Sessions |
| Session HI+NS+TR-ThA |
| Session: | Novel Beam Induced Surface Analysis and Nano-Patterning |
| Presenter: | Paul Pigram, La Trobe University, Australia |
| Authors: | N.G. Welch, CSIRO Manufacturing, Australia R.M.T. Madiona, La Trobe University, Australia J.A. Scoble, CSIRO Manufacturing, Australia B.W. Muir, CSIRO Manufacturing, Australia P.J. Pigram, La Trobe University, Australia |
| Correspondent: | Click to Email |
Antibody attachment, orientation and function at the solid interface are critical for the sensitive detection of biomolecules during immunoassays. Correctly oriented antibodies with solution-facing antigen binding regions have improved antigen capture in comparison with randomly oriented antibodies. Direct characterization of oriented proteins with surface analysis methods still remains a challenge. Surface sensitive techniques, however, such as Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) provide information-rich data that can be used to probe antibody attachment, orientation, denaturation and related characteristics.
Diethylene glycol dimethyl ether plasma polymers (DGpp) functionalized with chromium (DGpp+Cr) have improved immunoassay performance that is indicative of preferential antibody orientation. ToF-SIMS data from proteolytic fragments of anti-EGFR antibody bound to DGpp and DGpp+Cr have been used to construct artificial neural network (ANN) and principal component analysis (PCA) models indicative of correctly oriented systems. Whole antibody samples (IgG) tested against each of the models indicate preferential antibody orientation on DGpp+Cr. Cross-reference between ANN and PCA models yield 20 mass fragments associated with F(ab′)2 region representing correct orientation, and 23 mass fragments associated with the Fc region representing incorrect orientation. The mass fragments have been compared with amino acid fragments and amino acid composition in F(ab′)2 and Fc regions. A ratio of the sum of the ToF-SIMS ion intensities from the F(ab′)2 fragments to the Fc fragments demonstrated a 50% increase in intensity for IgG on DGpp+Cr as compared to DGpp.
This systematic data analysis methodology offers new opportunities for the investigation of antibody orientation on a range of substrates. It also yields good results for the characterization of antibody denaturation and for determining limits of detection.