Insight into the dynamics of pathogenic infectivity has been greatly enhanced by studies using whole proteome microarray technology [1]. Techniques allowing high throughput production of recombinant proteins representing comprehensive antigenic potential of pathogenic bacteria and viruses allow examination of the degrees to which specific antigenic proteins are involved in human serological response.The true value of this potential is unlocked by the multiplex power of microarray technology, in which very large numbers of candidate antigenic proteins are screened in parallel for relative reactivities to samples of patient sera of known or unknown disposition. In comparison to traditional ELISA screening, multiplexed microarray testing enables comprehensive data acquisition and comparison from single serum samples, greatly facilitating determination of important components and mechanisms of humoral immune response to pathogenic challenges.
Protein microarray technology has seen steady advance since its implementation in the mid 1980s. Factors contributing to increased facility include improved detection and scanning capabilities, refined data analysis techniques, and process development improvements directed specifically at improving quality and breadth of raw data obtained. Of particular note is the introduction of microarray substrates that promote increased protein density per unit area at the site of deposition, exemplified by porous nitrocellulose membranes, a readily manufactured commercial product that directly substitutes for standard glass microarray slides in production methodologies.
Porous nitrocellulose is cast as a thin film to the surface of standard glass microarray slides, and provides a 3-dimensional scaffold to which printed antigens bind through ionic and van der Waals interactions. Because there is no covalent chemistry the tertiary protein structure remains intact resulting in more consistent binding interaction with antibodies or other ligands. Porous nitrocellulose membrane slides bind up to 500 times more protein per unit area than 2-dimensional substrates, allowing wider dynamic range of response, lower detection limits, and lower variability of intra and inter assay responses [2].
Recent high density whole proteome screening examples that demonstrate effectiveness of porous nitrocellulose as a protein microarray substrate include whole proteome antigenic response screening of Herpes Simplex 1 and 2 [3], identification of native and induced responses to P. falciparum immune challenges [4], identification of safety related antigenic characteristics of DryVax® MVA vaccine [5], and a comprehensive analysis of antigenic responses to M.tuberculosis relating active versus latent infections [6].
References
- 1. Vigil, A., Davies, D. H., and Felgner, P. L. (2010), Future Microbiol 5, 241-251.
- 2. Grace BioLabs, (2011), “ONCYTE Guide to Protein Microarrays”, www.gracebio.com
- Kalantari-Dehaghi, M., et al, (2012), J Virol.4328-4339.
- 4. Doolan, D. L., et al, (2008), Proteomics 8, 4680-4694.
- Hermanson, G., et al, (2012, Vaccine, 30(3): 614-625.
- Kunnath-Velayudhan, S., et al, (2012), Journal of Infectious Diseases, 206: 697-705.
