Christer Wingren

Recombining germline-derived CDR sequences for creating diverse single- framework antibody libraries

We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 × 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and functional variation in antibody molecules.

Design of high-density antibody microarrays for disease proteomics: Key technological issues.

Antibody-based microarray is a novel proteomic technology setting a new standard for molecular profiling of non-fractionated complex proteomes. The first generation of antibody microarrays has already demonstrated its potential for generating detailed protein expression profiles, or protein atlases, of human body fluids in health and disease, paving the way for new discoveries within the field of disease proteomics. The process of designing highly miniaturized, high-density and high-performing antibody microarray set-ups have, however, proven to be challenging. In this mini-review we discuss key technological issues that must be addressed in a cross-disciplinary manner before true global proteome analysis can be performed using antibody microarrays.

Multiplexed Lipid Dip-Pen Nanolithography on Subcellular Scales for the Templating of Functional Proteins and Cell Culture

Molecular patterning processes taking place in biological systems are challenging to study in vivo because of their dynamic behavior, subcellular size, and high degree of complexity. In vitro patterning of biomolecules using nanolithography allows simplification of the processes and detailed study of the dynamic interactions. Parallel dip-pen nanolithography (DPN) is uniquely capable of integrating functional biomolecules on subcellular length scales due to its constructive nature, high resolution, and high throughput. Phospholipids are particularly well suited as inks for DPN since a variety of different functional lipids can be readily patterned in parallel. Here DPN is used to spatially pattern multicomponent micro- and nanostructured supported lipid membranes and multilayers that are fluid and contain various amounts of biotin and/or nitrilotriacetic acid functional groups. The patterns are characterized by fluorescence microscopy and photoemission electron microscopy. Selective adsorption of functionalized or recombinant proteins based on streptavidin or histidine-tag coupling enables the semisynthetic fabrication of model peripheral membrane bound proteins. The biomimetic membrane patterns formed in this way are then used as substrates for cell culture, as demonstrated by the selective adhesion and activation of T-cells.

Detection of pancreatic cancer using antibody microarray-based serum protein profiling

The driving force behind oncoproteomics is to identify protein signatures that are associated with a particular malignancy. Here, we have used a recombinant scFv antibody microarray in an attempt to classify sera derived from pancreatic adenocarcinoma patients versus healthy subjects. Based on analysis of nonfractionated, directly labeled, whole human serum proteomes we have identified a protein signature based on 19 nonredundant analytes, that discriminates between cancer patients and healthy subjects. Furthermore, a potential protein signature, consisting of 21 protein analytes, could be defined that was shown to be associated with cancer patients having a life expectancy of <12 months. Taken together, the data suggest that antibody microarray analysis of complex proteomes will be a useful tool to define disease associated protein signatures.

High-throughput proteomics using antibody microarrays: an update.

Antibody-based microarrays are a rapidly emerging technology that has advanced from the first proof-of-concept studies to demanding serum protein profiling applications during recent years, displaying great promise within disease proteomics. Miniaturized micro- and nanoarrays can be fabricated with an almost infinite number of antibodies carrying the desired specificities. While consuming only minute amounts of reagents, multiplexed and ultrasensitive assays can be performed targeting high- as well as low-abundance analytes in complex nonfractionated proteomes. The microarray images generated can then be converted into protein expression profiles or protein atlases, revealing a detailed composition of the sample. The technology will provide unique opportunities for fields such as disease diagnostics, biomarker discovery, patient stratification, predicting disease recurrence and drug target discovery. This review describes an update of high-throughput proteomics, using antibody-based microarrays, focusing on key technological advances and novel applications that have emerged over the last 3 years.

Serum proteome profiling of metastatic breast cancer using recombinant antibody microarrays

The driving force behind oncoproteomics is to identify biomarker signatures associated with a particular malignancy. Here, we have for the first time used large-scale recombinant scFv antibody microarrays in an attempt to classify metastatic breast cancer versus healthy controls, based on differential protein expression profiling of whole serum samples. Using this multiplexed and miniaturised assay set-up providing pM range sensitivities, breast cancer could be classified with a specificity and sensitivity of 85% based on 129 serum analytes. However, by adopting a condensed 11 analyte biomarker signature, composed of nine non-redundant serum proteins, we were able to distinguish cancer versus healthy serum proteomes with a 95% sensitivity and specificity, respectively. When a subgroup of patients, not receiving anti-inflammatory drugs, was analysed, a novel eight analyte biomarker signature with a further improved predictive power was indicated. In a longer perspective, antibody microarray analysis could provide a tool for the development of improved diagnostics and intensified biomarker discovery for breast cancer patients.

Patterns of DNA-Labeled and scFv-Antibody-Carrying Lipid Vesicles Directed by Material-Specific Immobilization of DNA and Supported Lipid Bilayer Formation on an Au/SiO2 Template

Much effort is currently concentrated on research devoted to biofunctional patterned surfaces, which constitute the fundament for the development of microarrays for high-throughput gene and protein analyses. DNA microarrays have proved very successful, and the concept is in the process of being applied to protein arrays. However, in contrast to DNA fragments, proteins are easily denatured in contact with solid supports, and robotic printing of proteins onto chemically reactive glass slides will not necessarily be applicable as a generic protocol for the preparation of protein arrays. Supported phosphatidylcholine lipid bilayers have emerged as interesting candidate substrates for protein chips, since they efficiently reduce nonspecific protein adsorption 5] and, at the same time, allow different strategies for protein immobilization with biospecific water, desalted with a NAP5 column (Amersham Pharmacia, USA) according to manufacturers protocols, and stored as working stock solutions at 20 C until use. Epoxy-derivatized slides were prepared from plain glass slides (Sigma, USA) as previously described. Nhydroxysuccinimide slides were also used to spot the proteins but consistently gave inferior results. Proteins were prepared in NaHCO3 buffer (0.1M, pH 9) and arrayed on epoxy slides with a spacing of 180 m between the spots by using an statistical microarray analysis arrayer (Engineering Services Inc. , Ontario, Canada). After a 2-hour incubation period the slides were either used immediately, or stored for future use at 4 C. The slides, if stored, were typically used within 48 h of printing.

Identification of protein expression signatures associated with Helicobacter pylori infection and gastric adenocarcinoma using recombinant antibody microarrays.

Antibody microarray based technology is a powerful emerging tool in proteomics, target discovery, and differential analysis. Here, we report the first study where recombinant antibody fragments have been used to construct large scale antibody microarrays, composed of 127 different antibodies against mostly immunoregulatory antigens. The arrays were based on single framework recombinant antibody fragments (SinFabs) designed for high on-chip stability and functionality and were used for the analysis of malignant and normal stomach tissue samples from Helicobacter pylori-positive and -negative patients. Our results demonstrate that distinct tumor- as well as infection-associated protein expression signatures could be identified from these complex tissue proteomes, as well as biomarkers such as IL-9, IL-11, and MCP-4, previously not found in these diseases. In a longer perspective, this study may improve the understanding of H. pylori-induced stomach cancer and lead to development of improved diagnostics.

High-throughput proteomics using antibody microarrays

Antibody-based microarrays are a novel technology that hold great promise in proteomics. Microarrays can be printed with thousands of recombinant antibodies carrying the desired specificities, the biologic sample (e.g., an entire proteome) and any specifically bound analytes detected. The microarray patterns that are generated can then be converted into proteomic maps, or molecular fingerprints, revealing the composition of the proteome. Using this tool, global proteome analysis and protein expression profiling will thus provide new opportunities for biomarker discovery, drug target identification and disease diagnostics, as well as providing insights into disease biology. Intense work is currently underway to develop this novel technology platform into the high-throughput proteomic tool required by the research community.

Identification of serum biomarker signatures associated with pancreatic cancer.

Pancreatic cancer is an aggressive disease with poor prognosis, due, in part, to the lack of disease-specific biomarkers that could afford early and accurate diagnosis. With a recombinant antibody microarray platform, targeting mainly immunoregulatory proteins, we screened sera from 148 patients with pancreatic cancer, chronic pancreatitis, autoimmune pancreatitis (AIP), and healthy controls (N). Serum biomarker signatures were derived from training cohorts and the predictive power was evaluated using independent test cohorts. The results identified serum portraits distinguishing pancreatic cancer from N [receiver operating characteristics area under the curve (AUC) of 0.95], chronic pancreatitis (0.86), and AIP (0.99). Importantly, a 25-serum biomarker signature discriminating pancreatic cancer from the combined group of N, chronic pancreatitis, and AIP was determined. This signature exhibited a high diagnostic potential (AUC of 0.88). In summary, we present the first prevalidated, multiplexed serum biomarker signature for diagnosis of pancreatic cancer that may improve diagnosis and prevention in premalignant diseases and in screening of high-risk individuals.