| AVS 65th International Symposium & Exhibition | |
| Nanometer-scale Science and Technology Division | Wednesday Sessions |
| Session NS+MN+PC+SS-WeA |
| Session: | IoT Session: Bio at the Nanoscale |
| Presenter: | Imanda Jayawardena, University of Queensland, Australia |
| Authors: | I. Jayawardena, University of Queensland, Australia S. Corrie, Monash University, Australia L. Grondahl, University of Queensland, Australia |
| Correspondent: | Click to Email |
Immunodiffusion is a simple assay used for the determination of a target protein concentration in a biological sample using a distance-based measurement. The assay allows the sample containing the antigen of interest to combine with an antiserum in a gel-based substrate leading to the formation of a ring-shaped precipitate (‘precipitin ring’), the size of which is proportional to target protein concentration.1 Using the malarial antigen HRP2 and antisera from immunized rabbits as the model system, for the first time, we are investigating transforming the current immunodiffusion assays into a more rapid and sensitive format.
The traditional assay substrate, agarose, is a severely diffusion limited system. The porosity of the gel is a key determinant of diffusion properties and is an essential parameter required for the study and modification of the assay. However, the scientific data on agarose pore size determination is based on artefact laden microscopy images of agarose hydrogels. Thus, we have performed an in-depth investigation on best imaging techniques for accurate pore size determination. The precipitin ring structure is a band of antigen-antibody precipitate, in significant contrast with the surrounding gel substrate. A brief preliminary investigation has been reported by Fedorov et al. on precipitin ring structure2, and we have extended this work by applying microscopic imaging techniques.
For imaging hydrogels, high pressure frozen gels were subjected to cryo-SEM3, and was established as the most accurate technique to study the native structure of the gel. Atomic force microscopy was found to complement cryo-SEM data while CLSM due to its limited resolution was found to be inadequate for the imaging of hydrogels. For imaging the protein-rich precipitin ring structure, high pressure frozen ring sections were subjected to cryo-SEM, however, more meaningful insight on the ring structure was obtained in this instance through CLSM studies.
Herein, we aim to present our work described above on imaging agarose hydrogels for accurate pore size determination and imaging precipitin ring structures associated with immunodiffusion assays.
1. Mancini, G.; Carbonara, A. t.; Heremans, J. Immunochemistry 1965
2. Fedorov, A. A.; Kurochkin, V. E.; Martynov, A. I.; Petrov, R. V. Journal of Theoretical Biology 2010
3. Aston, R.; Sewell, K.; Klein, T.; Lawrie, G.; Grøndahl, L. European Polymer Journal 2016