Protein detection enhanced by 3DNA dendrimer signal amplification.

DNA dendrimers, conjugated with both anti-biotin antibodies and up to 350 labeling entities, were designed and adapted to protein microarray and enzyme-linked immunosorbent assay (ELISA) to improve the limits of protein detection with no additional steps or equipment. Application of conjugated dendrimers to standard ELISA cytokine detection resulted in up to threefold improvement of the limits of detection with no significant increase in the inter- and intra-assay coefficient of variation (CV) compared to streptavidin horseradish peroxidase (SA-HRP) detection. The adaptation of conjugated dendrimers to protein microarray cytokine detection resulted in up to 10-fold improvement of the limits of detection, but assay conditions would have to be optimized to decrease the intra- and inter-assay %CVs.

Native antigen fractionation protein microarrays for biomarker discovery.

In this protocol, we used the T24 human bladder cancer cell line as a source of native antigens to construct fractionated lysate microarrays. Subsequently, these microarrays were used to compare the autoantibody responses of individuals with interstitial cystitis/painful bladder syndrome (IC/PBS) to those of normal female controls. To accomplish this, T24 cells were lysed under nondenaturing conditions to obtain native antigens. These native antigens were then fractionated in 2D using a PF-2D liquid chromatography; the first dimension separated the proteins by their isoelectric points, and the second separated them according to hydrophobicity. The resulting protein fractions were printed onto nitrocellulose-coated glass slides (PATH slides) to create a set of fractionated lysate microarrays. To compare the autoantibody responses of IC/PBS patients with normal controls, the fractionated lysate arrays were competitively hybridized with fluorescently labeled IgG samples purified from both IC/PBS and control sera. This protocol presents a detailed description of the creation and use of native antigen fractionated lysate microarrays for autoantibody profiling.

Multiplexed antibody arrays for the discovery and validation of glycosylated protein biomarkers.

Protein glycosylation, the enzymatic linkage of mono- and poly-saccharides to proteins, is a critical determinant of protein function; however, there is a lack of tools for studying the glycosylation of specific proteins in complex samples. A new type of antibody-lectin sandwich assay enables the measurement of the glycosylation of specific proteins that have been captured from complex samples using antibody arrays combined with lectin-based detection probes. Antibody-lectin sandwich arrays have the potential to expand our understanding of the role of glycans and protein glycosylation in disease and to identify and investigate new biomarkers for early detection, disease prognosis and therapeutic response prediction. While antibody-lectin sandwich arrays yield less-detailed structural information regarding protein glycosylation than other available methods, they do provide a simple and reproducible method for investigating changes in protein abundance and glycosylation of multiple proteins and can be easily applied to large or small sample sets. By profiling protein and glycan variations, new disease-associated glycan alterations can be identified and validated for use as biomarkers.

Protein microarrays as an application for disease biomarkers.

Protein microarrays are an increasingly powerful technology in the hunt for new and novel diagnostic and prognostic biomarkers. Lending credit to the highly established DNA microarray, protein microarrays are versatile tools that utilize a variety of formats to facilitate the discovery of new biomarkers and our understanding of disease pathways. The aims of this review are: to detail a variety of protein microarray technologies currently used, including forward-phase technologies and reverse-phase technologies useful in both the discovery and validation of candidate biomarkers; to explore the strengths and weaknesses of various proteomic microarray platforms; to explain how bioinformatics helps compare data between microarray data sets; and to discuss the downstream applications of such technologies as they relate to the development of a highly personalized approach to medicine.

Label-free detection of 16S ribosomal RNA hybridization on reusable DNA arrays using surface plasmon resonance imaging.

In this paper, we describe the detection of bacterial cell-extracted 16S ribosomal RNA (rRNA) using an emerging technology, surface plasmon resonance (SPR) imaging of DNA arrays. Surface plasmon resonance enables detection of molecular interactions on surfaces in response to changes in the index of refraction, therefore eliminating the need for a fluorescent or radioactive label. A variation of the more common SPR techniques, SPR imaging enables detection from multiple probes in a reusable array format. The arrays developed here contain DNA probes (15-21 bases) designed to be complementary to 16S rRNA gene sequences of Escherichia coli and Bacillus subtilis as well as to a highly conserved sequence found in rRNAs from most members of the domain Bacteria. We report species-specific hybridization of cell-extracted total RNA and in vitro transcribed 16S rRNA to oligonucleotide probes on SPR arrays. We tested multiple probe sequences for each species, and found that success or failure of hybridization was dependent upon probe position in the 16S rRNA molecule. It was also determined that one of the probes intended to bind 16S rRNA also bound an unknown protein. The amount of binding to these probes was quantified with SPR imaging. A detection limit of 2 micro g ml-1 was determined for fragmented E. coli total cellular RNA under the experimental conditions used. These results indicate the feasibility of using SPR imaging for 16S rRNA identification and encourage further development of this method for direct detection of other RNA molecules.