Gregor Kijanka

Protein arrays as tools for serum autoantibody marker discovery in cancer

Protein array technology has begun to play a significant role in the study of protein-protein interactions and in the identification of antigenic targets of serum autoantibodies in a variety of autoimmune disorders. More recently, this technology has been applied to the identification of autoantibody signatures in cancer. The identification of tumour-associated antigens (TAAs) recognised by the patients immune response represents an exciting approach to identify novel diagnostic cancer biomarkers and may contribute towards a better understanding of the molecular mechanisms involved. Circulating autoantibodies have not only been used to identify TAAs as diagnostic/prognostic markers and potential therapeutic targets, they also represent excellent biomarkers for the early detection of tumours and potential markers for monitoring the efficacy of treatment. Protein array technology offers the ability to screen the humoral immune response in cancer against thousands of proteins in a high throughput technique, thus readily identifying new panels of TAAs. Such an approach should not only aid in improved diagnostics, but has already contributed to the identification of complex autoantibody signatures that may represent disease subgroups, early diagnostics and facilitated the analysis of vaccine trials.

Centrifugal microfluidics for cell analysis.

Over the past two decades, centrifugal microfluidic systems have successfully demonstrated their capability for robust, high-performance liquid handling to enable modular, multi-purpose lab-on-a-chip platforms for a wide range of life-science applications. Beyond the handling of homogeneous liquids, the unique, rotationally controlled centrifugal actuation has proven to be specifically advantageous for performing cell and particle handling and assays. In this review we discuss technologies to implement two important steps for cell handling, namely separation and capturing/counting.

Array-based capture, distribution, counting and multiplexed assaying of beads on a centrifugal microfluidic platform

We present a novel centrifugal microfluidic platform for the highly efficient manipulation and analysis of particles for applications in bead-based assays. The platform uses an array of geometrical V-cup barriers to trap particles using stopped-flow sedimentation under highly reproducible hydrodynamic conditions. The impact parameters governing the occupancy distribution and capture efficiency of the arrayed traps are investigated. The unique, nearly 100% capture efficiency paired with the capability to establish sharply peaked, single occupancy distributions enables a novel, digital readout mode for color-multiplexed, particle-based assays with low-complexity instrumentation. The presented technology marks an essential step towards a versatile platform for the integration of bead- and cell-based biological assays.

Human IgG antibody profiles differentiate between symptomatic patients with and without colorectal cancer

Objective: Patients with cancer have antibodies against tumour antigens. Characterising the antibody repertoire may provide insights into aberrant cellular mechanisms in cancer development, ultimately leading to novel diagnostic or therapeutic targets. The aim of this study was to characterise the antibody profiles in patients whose symptoms warranted colonoscopy, to see if there was a difference in patients with and without colorectal cancer. Methods: Patients were recruited from a colonoscopy clinic. Individual serum samples from 43 patients with colorectal cancer and 40 patients with no cancer on colonoscopy were profiled on a 37 830 clone recombinant human protein array. Antigen expression was evaluated by quantitative reverse transcription-PCR and by immunohistochemistry on tissue microarrays. Results: Using a sex- and age-matched training set, 18 antigens associated with cancer and 4 associated with the absence of cancer (p<0.05) were identified and confirmed. To investigate the mechanisms triggering antibody responses to these antigens, antigen expression was examined in normal colorectal mucosa and colorectal carcinoma of the same patients. The identified antigens showed cellular accumulation (p53), aberrant cellular expression (high mobility group B1 (HMGB1)) and overexpression (tripartite motif-containing 28 (TRIM28), p53, HMGB1, transcription factor 3 (TCF3), longevity assurance gene homologue 5 (LASS5) and zinc finger protein 346 (ZNF346)) in colorectal cancer tissue compared with normal colorectal mucosa. Conclusions: It is demonstrated for the first time that screening high-density protein arrays identifies unique antibody profiles that discriminate between symptomatic patients with and without colorectal cancer. The differential expression of identified antigens suggests their involvement in aberrant cellular mechanisms in cancer.

Integrated microfluidic array plate (iMAP) for cellular and molecular analysis

Just as the Petri dish has been invaluable to the evolution of biomedical science in the last 100 years, microfluidic cell assay platforms have the potential to change significantly the way modern biology and clinical science are performed. However, an evolutionary process of creating an efficient microfluidic array for many different bioassays is necessary. Specifically for a complete view of a cell response it is essential to incorporate cytotoxic, protein and gene analysis on a single system. Here we present a novel cellular and molecular analysis platform, which allows access to gene expression, protein immunoassay, and cytotoxicity information in parallel. It is realized by an integrated microfluidic array plate (iMAP). The iMAP enables sample processing of cells, perfusion based cell culture, effective perturbation of biologic molecules or drugs, and simultaneous, real-time optical analysis for different bioassays. The key features of the iMAP design are the interface of on-board gravity driven flow, the open access input fluid exchange and the highly efficient sedimentation based cell capture mechanism (∼100% capture rates). The operation of the device is straightforward (tube and pump free) and capable of handling dilute samples (5-cells per experiment), low reagent volumes (50 nL per reaction), and performing single cell protein and gene expression measurements. We believe that the unique low cell number and triple analysis capabilities of the iMAP platform can enable novel dynamic studies of scarce cells.

Rapid characterization of binding specificity and cross-reactivity of antibodies using recombinant human protein arrays.

Antibodies are routinely used as research tools, in diagnostic assays and increasingly as therapeutics. Ideally, these applications require antibodies with high sensitivity and specificity; however, many commercially available antibodies are limited in their use as they cross-react with non-related proteins. Here we describe a novel method to characterize antibody specificity. Six commercially available monoclonal and polyclonal antibodies were screened on high-density protein arrays comprising of ~10,000 recombinant human proteins (Imagenes). Two of the six antibodies examined; anti-pICln and anti-GAPDH, bound exclusively to their target antigen and showed no cross-reactivity with non-related proteins. However, four of the antibodies, anti-HSP90, anti-HSA, anti-bFGF and anti-Ro52, showed strong cross-reactivity with other proteins on the array. Antibody-antigen interactions were readily confirmed using Western immunoblotting. In addition, the redundant nature of the protein array used, enabled us to define the epitopic region within HSP90 of the anti-HSP90 antibody, and identify possible shared epitopes in cross-reacting proteins. In conclusion, high-density protein array technology is a fast and effective means for determining the specificity of antibodies and can be used to further improve the accuracy of antibody applications.

Centrifugo-Magnetophoretic Purification of CD4+ Cells from Whole Blood Toward Future HIV/AIDS Point-of-Care Applications

In medical diagnostics, detection of cells exhibiting specific phenotypes constitutes a paramount challenge. Detection technology must ensure efficient isolation of (often rare) targets while eliminating nontarget background cells. Technologies exist for such investigations, but many require high levels of expertise, expense, and multistep protocols. Increasing automation, miniaturization, and availability of such technologies is an aim of microfluidic lab-on-a-chip strategies. To this end, we present an integrated, dual-force cellular separation strategy using centrifugo-magnetophoresis. Whole blood spiked with target cells is incubated with (super-)paramagnetic microparticles that specifically bind phenotypic markers on target cells. Under rotation, all cells sediment into a chamber located opposite a co-rotating magnet. Unbound cells follow the radial vector, but under the additional attraction of the lateral magnetic field, bead-bound target cells are deflected to a designated reservoir. This multiforce separation is continuous and low loss. We demonstrate separation efficiently up to 92% for cells expressing the HIV/AIDS relevant epitope (CD4) from whole blood. Such highly selective separation systems may be deployed for accurate diagnostic cell isolations from biological samples such as blood. Furthermore, this high efficiency is delivered in a cheap and simple device, thus making it an attractive option for future deployment in resource-limited settings.

Rapid and cost‐efficient enumeration of rare cancer cells from whole blood by low‐loss centrifugo‐magnetophoretic purification under stopped‐flow conditions

We present a substantially improved design and functionality of a centrifugo‐magnetophoretic platform which integrates direct immunoseparation and cost‐efficient, bright‐field detection of cancer cells in whole blood. All liquid handling takes place in a disposable cartridge with geometry akin to a conventional compact disc (CD). The instrumentation required to process such a “lab‐on‐a‐disc” cartridge can be as simple and cost‐efficient as the rotor on a common optical disc drive. In a first step, target cells in a blood sample are specifically bound to paramagnetic microbeads. The sample is then placed into the disc cartridge and spun. In the second step, magnetically tagged target cells are separated by a co‐rotating, essentially lateral magnetic field from the background population of abundant blood cells, and also from unbound magnetic beads. A stream of target cells centrifugally sediments through a stagnant liquid phase into a designated detection chamber. The continuous, multiforce immunoseparation proceeds very gently, i.e. the mechanical and hydrodynamic stress to the target cells is minimized to mitigate the risk of cell loss by collective entrapment in the background cells or vigorous snapping against a wall. We successfully demonstrate the extraction of MCF7 cancer cells at concentrations as low as 1 target cell per μl from a background of whole blood, with capture efficiencies of up to 88%. Its short time‐to‐answer is a notable characteristic of this system, with 10% of target cells collected in the first minute after their loading to the system and the remainder captured within the following 10 min. All the above‐mentioned factors synergetically combine to leverage the development of a prospective point‐of‐care device for CTC detection.

Diagnostic Potential of Zinc Finger Protein-Specific Autoantibodies and Associated Linear B-Cell Epitopes in Colorectal Cancer

Colorectal cancer is one of the most common cancers worldwide with almost 700,000 deaths every year. Detection of colorectal cancer at an early stage significantly improves patient survival. Cancer-specific autoantibodies found in sera of cancer patients can be used for pre-symptomatic detection of the disease. In this study we assess the zinc finger proteins ZNF346, ZNF638, ZNF700 and ZNF768 as capture antigens for the detection of autoantibodies in colorectal cancer. Sera from 96 patients with colorectal cancer and 35 control patients with no evidence of cancer on colonoscopy were analysed for the presence of ZNF-specific autoantibodies using an indirect ELISA. Autoantibodies to individual ZNF proteins were detected in 10–20% of colorectal cancer patients and in 0–5.7% of controls. A panel of all four ZNF proteins resulted in an assay specificity of 91.4% and sensitivity of 41.7% for the detection of cancer patients in a cohort of non-cancer controls and colorectal cancer patients. Clinicopathological and survival analysis revealed that ZNF autoantibodies were independent of disease stage and did not correlate with disease outcome. Since ZNF autoantibodies were shared between patients and corresponding ZNF proteins showed similarities in their zinc finger motifs, we performed an in silico epitope sequence analysis. Zinc finger proteins ZNF700 and ZNF768 showed the highest sequence similarity with a bl2seq score of 262 (E-value 1E-81) and their classical C2H2 ZNF motifs were identified as potential epitopes contributing to their elevated immunogenic potential. Our findings show an enhanced and specific immunogenicity to zinc finger proteins, thereby providing a multiplexed autoantibody assay for minimally invasive detection of colorectal cancer.

Relationship between epithelial and stromal TRIM28 expression predicts survival in colorectal cancer patients

TRIM28 is a multi‐domain nuclear protein with pleotropic effects in both normal and tumor cells. In this study, TRIM28 expression in epithelial and stromal tumor microenvironment and its prognostic role in colorectal cancer were investigated.