Imanda Jayawardena1, Simon Corrie2,3, Lisbeth Grondahl1,3*


1. School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD 4072, Australia.
2. ARC Centre for BioNano Science, Monash University, Clayton, VIC 3168, Australia.\
3. Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, QLD 4072, Australia.


An emerging challenge is the development of point of care (POC) diagnostic devices that are compliant with the ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid, Equipment-free, broadly Deliverable) put forward by the World Health Organization, that can serve the needs of the end user, at home or at a remote location. However, if a technique that satisfies most of the aforementioned criteria already exists, developing it into a POC device becomes relatively less challenging. Immunodiffusion is a simple assay that can be used for the determination of a target protein level in a biological sample without the need for “reading” equipment, instead using a simple length based measurement. Furthermore, the assay is conceptually compliant with the ASSURED criteria making it a promising candidate as a POC device. The traditional 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 which is termed the ‘precipitin ring’.1-2 While this is a promising technique, it is severely diffusion-limited and has hence been overlooked in terms of POC assay development.
Using the malarial antigen HRP2 and antisera from immunized rabbits as the model system, for the first time, we have investigated transforming the current immunodiffusion assays into a more rapid and sensitive version. Our initial work has demonstrated the effect of agarose gel porosity on precipitin ring size and assay time. We have also investigated the applicability of cryo-SEM and AFM imaging of gels3-4, for accurate pore size determination of agarose hydrogels. We found the results obtained to be highly comparable. We further investigated two other widely used approaches in hydrogel pore size determination; CLSM5 and SEM imaging of freeze dried agarose gels and found both these approaches to be highly inaccurate due to issues relating to image resolution and drying artefacts, respectively. 
Herein we present our work on gel based immunodiffusion assays and the investigations on accurate pore size determination of agarose hydrogels for optimising immunodiffusion assays.




1.             Mancini, G.; Vaerman, J.; Carbonara, A.; Heremans, J., A single-radial-diffusion method for the immunological quantitation of proteins. Proceedings of the Biological Fluids. Amsterdam, The Netherlands: Elsevier 1964, 370-379.

2.             Mancini, G.; Carbonara, A. t.; Heremans, J., Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 1965, 2, 235IN235-254IN236.

3.             Pernodet, N.; Maaloum, M.; Tinland, B., Pore size of agarose gels by atomic force microscopy. Electrophoresis 1997, 18, 55-58.

4.             Maaloum, M.; Pernodet, N.; Tinland, B., Agarose gel structure using atomic force microscopy: gel concentration and ionic strength effects. Electrophoresis 1998, 19, 1606-1610.

5.             Russ, N.; Zielbauer, B. I.; Koynov, K.; Vilgis, T. A., Influence of nongelling hydrocolloids on the gelation of agarose. Biomacromolecules 2013, 14, 4116-4124.