Antibody landscape of C57BL/6 mice cured of B78 melanoma via a combined radiation and immunocytokine immunotherapy regimen.

Sera of immune mice that were previously cured of their melanoma through a combined radiation and immunocytokine immunotherapy regimen consisting of 12 Gy of external beam radiation and the intratumoral administration of an immunocytokine (anti-GD2 mAb coupled to IL-2) with long-term immunological memory showed strong antibody-binding against melanoma tumor cell lines via flow cytometric analysis. Using a high-density whole-proteome peptide array (of 6.090.593 unique peptides), we assessed potential protein-targets for antibodies found in immune sera. Sera from 6 of these cured mice were analyzed with this high-density, whole-proteome peptide array to determine specific antibody-binding sites and their linear peptide sequence. We identified thousands of peptides that were targeted by these 6 mice and exhibited strong antibody binding only by immune (after successful cure and rechallenge), not naïve (before tumor implantation) sera and developed a robust method to detect these differentially targeted peptides. Confirmatory studies were done to validate these results using 2 separate systems, a peptide ELISA and a smaller scale peptide array utilizing a slightly different technology. To the best of our knowledge, this is the first study of the full set of germline encoded linear peptide-based proteome epitopes that are recognized by immune sera from mice cured of cancer via radio-immunotherapy. We furthermore found that although the generation of B-cell repertoire in immune development is vastly variable, and numerous epitopes are identified uniquely by immune serum from each of these 6 immune mice evaluated, there are still several epitopes and proteins that are commonly recognized by at least half of the mice studied. This suggests that every mouse has a unique set of antibodies produced in response to the curative therapy, creating an individual "fingerprint." Additionally, certain epitopes and proteins stand out as more immunogenic, as they are recognized by multiple mice in the immune group.

Use of surface plasmon resonance imaging to study viral RNA: protein interactions.

Surface plasmon resonance imaging (SPRi) is an emerging microarray technology that is label-free, rapid and extremely flexible. Here the capabilities of SPRi are demonstrated in results of proof-of-concept experiments detailing a method for studying viral genomic RNA:protein interactions in array format. The principal RNA is the well-characterized origin of assembly (OAS) containing region of Tobacco mosaic virus (TMV) RNA, whereas the principal protein is the primary subunit for TMV virion assembly, the 20S capsid protein aggregate. DNA probes complementary to TMV and non-TMV RNA fragments were covalently attached to a thin gold layer deposited on glass. These DNA probes were used to discreetly capture in vitro transcribed TMV and Red clover necrotic mosaic virus (RCNMV) RNA2 (used as a negative control for the subsequent protein binding). The 4S TMV capsid protein monomers were isolated from TMV particles purified from infected plants of Nicotiana tabacum L. and were induced to form 20S stacked disc aggregates. These 20S stacked disc aggregates were then injected onto the array containing the RNA fragments captured by the DNA probes immobilized on the microarray surface. The discrete and preferential binding of the 20S stacked disc aggregates to the array locations containing the TMV OAS RNA sequence was observed. The results demonstrate that SPRi can be used to monitor binding of large RNA molecules to immobilized DNA capture probes which can then be used to monitor the subsequent binding of complex protein structures to the RNA molecules in a single real-time, label-free microarray experiment. The results further demonstrate that SPRi can distinguish between RNA species that have or do not have an origin of assembly sequence specific for a particular viral capsid protein or protein complex. The broader implications of these results in virology research are found in other systems where the research goals include characterizing the specificity and kinetics of viral or host protein or protein complex interactions with viral nucleic acids.

Antibody microarray analysis of inflammatory mediator release by human leukemia T-cells and human non small cell lung cancer cells.

Cytokines and chemokines are responsible for regulating inflammation and the immune response. Cytokine and chemokine release is typically measured by quantitative enzyme-linked immunosorbant assay (ELISA) or Western blot analysis. To expedite the analysis of samples for multiple cytokines/chemokines, we have developed slide-based Thermo Scientific ExcelArray Antibody Sandwich Microarrays. Each slide consists of 16 subarrays (wells), each printed with 12 specific antibodies in triplicate and positive and negative control elements. This 16-well format allows for the analysis of 10 test samples using a six-point standard curve. The array architecture is based on the "sandwich" ELISA, in which an analyte protein is sandwiched between an immobilized capture antibody and a biotinylated detection antibody, using streptavidin-linked Thermo Scientific DyLight 649 Dye for quantitation. The observed sensitivity of this assay was <10 pg/mL. In our experiments, the Jurkat cell line was used as a model for human T-cell leukemia, and the A549 cell line was used as a model for human non-small cell lung cancer. To evoke a cytokine/chemokine response, cells were stimulated with tumor necrosis factor alpha (TNFalpha), phorbol-12-myristate-13-acetate (PMA, TPA), and phytohemagglutinin (PHA). Cell supernatants derived from both untreated and stimulated cells were analyzed on four different arrays (Inflammation I, Inflammation II, Angiogenesis, and Chemotaxis), enabling the quantitation of 41 unique analytes. Stimulated cells showed an increase in the expression level of many of the test analytes, including IL-8, TNF-alpha, and MIP-1alpha, compared to the non-treated controls. Our experiments clearly demonstrate the utility of antibody microarray analysis of cell-culture supernatants for the profiling of cellular inflammatory mediator release.