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Christer Wingren

Publications and source records attributed to Christer Wingren.

14 recordsLinked to original sources
PubMed2006

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.

Adenocarcinoma↗
PubMed2006

Improved affinity coupling for antibody microarrays: engineering of double-(His)6-tagged single framework recombinant antibody fragments.

Antibody-based microarray is a novel technology with great promise in biomedicine that will provide unique means to perform global proteome analysis. In the process of designing the high-density antibody microarrays required, several critical key issues have been identified that remain to be resolved. In particular, there is a great need for specific and selective approaches enabling non-purified probes to be directly purified, orientated and coupled in a generic one-step procedure directly on the chip. In this study, we report on the successful design of affinity-tagged human recombinant single-chain fragment variable antibody fragments for improved affinity coupling in array applications. By replacing the standard single-histidine (His)(6)-tag with a consecutive double-(His)(6)-tag, the binding to Ni(2+)-nitrilotriacetic acid-coated substrates was significantly improved. Surface plasmon resonance analysis showed a significantly tighter binding with at least a threefold slower dissociation. The improved binding characteristics thus enabled non-purified probes even in the format of crude expression supernatants to be directly applied thereby eliminating the need for any time-consuming pre-purification step(s) prior to the immobilization. While the double-(His)(6)-tag probes were found to be expressed equally well as compared to the single-(His)(6)-tag probes, they displayed better long-term functional on-chip stability. Taken together, the results demonstrate the generic potential of double-(His)(6)-tag recombinant antibodies for the facile fabrication of high-density antibody microarrays.

Antibodies↗
PubMed2006

One-step fractionation of complex proteomes enables detection of low abundant analytes using antibody-based microarrays.

Antibody-based microarray is a novel technology with great promise within high-throughput proteomics. The tremendous complexity of all proteomes will, however, pose major technological challenges, especially when targeting low-abundant analytes that remains to be resolved. In this paper, we have shown that antibody microarrays readily could be used for screening of low-abundant low molecular weight analytes in complex proteomes by optimizing the sample format. Focused antibody microarrays, based on human recombinant single-chain Fv anti-cytokine antibodies on Ni2+-NTA functionalized glass slides or black polymer Maxisorp substrates, and crude cell supernatants from activated dendritic cells, containing low levels of secreted cytokines, was used for evaluation. The proteome was pre-fractionated based on size in a simple one-step procedure using centrifugal filter devices of various molecular weight cutoffs. The results showed that the generation of a nondiluted low molecular weight (LMW) fraction, corresponding to less than 2% of the original protein content, was critical for the successful screening of cytokines in the sub pg/mL range. The reduced complexity of the LMW fraction significantly improved the assay sensitivity, by improving the fluorescent tagging step and/or reducing the nonspecific binding to the substrates.

Chemical Fractionation↗
PubMed2006

Antibody microarrays: current status and key technological advances.

Antibody-based microarrays are among the novel classes of rapidly evolving proteomic technologies that holds great promise in biomedicine. Miniaturized microarrays (< 1 cm2) can be printed with thousands of individual antibodies carrying the desired specificities, and with biological sample (e.g., an entire proteome) added, virtually any specifically bound analytes can be detected. While consuming only minute amounts (< microL scale) of reagents, ultra- sensitive assays (zeptomol range) can readily be performed in a highly multiplexed manner. The microarray patterns generated can then be transformed into proteomic maps, or detailed molecular fingerprints, revealing the composition of the proteome. Thus, protein expression profiling and global proteome analysis using this tool will offer new opportunities for drug target and biomarker discovery, disease diagnostics, and insights into disease biology. Adopting the antibody microarray technology platform, several biomedical applications, ranging from focused assays to proteome-scale analysis will be rapidly emerging in the coming years. This review will discuss the current status of the antibody microarray technology focusing on recent technological advances and key issues in the process of evolving the methodology into a high-performing proteomic research tool.

Animals↗
PubMed2005

Gravimetric antigen detection utilizing antibody-modified lipid bilayers.

Lipid bilayers containing 5% nitrilotriacetic acid (NTA) lipids supported on SiO2 have been used as a template for immobilization of oligohistidine-tagged single-chained antibody fragments (scFvs) directed against cholera toxin. It was demonstrated that histidine-tagged scFvs could be equally efficiently coupled to an NTA-Ni2+-containing lipid bilayer from a purified sample as from an expression supernatant, thereby providing a coupling method that eliminates time-consuming protein prepurification steps. Irrespective of whether the coupling was made from the unpurified or purified antibody preparation, the template proved to be efficient for antigen (cholera toxin) detection, verified using quartz crystal microbalance with dissipation monitoring. In addition, via a secondary amplification step using lipid vesicles containing GM1 (the natural membrane receptor for cholera toxin), the detection limit of cholera toxin was less than 750 pM. To further strengthen the coupling of scFvs to the lipid bilayer, scFvs containing two histidine tags, instead of just one tag, were also evaluated. The increased coupling strength provided via the bivalent anchoring significantly reduced scFv displacement in complex solutions containing large amounts of histidine-containing proteins, verified via cholera toxin detection in serum.

Biological Assay↗
PubMed2005

Biocompatibility of surfaces for antibody microarrays: design of macroporous silicon substrates.

Major efforts to develop antibody microarray technology to enable global proteome analysis to be performed in a facile manner are under way. In this process, the design and the properties of the substrate will play crucial roles. In the present study, we have developed novel, highly biocompatible solid supports for microarrays, using adsorbed recombinant human single-framework antibody fragments as probes. Several silicon-based supports, including planar silicon, micro- and macroporous silicon, and nitrocellulose-coated variants thereof, were designed and evaluated in a stepwise procedure. The surfaces were scored based on biocompatibility and probe binding capacity as judged by spot morphology, signal intensities, signal to noise ratios, dynamic range, sensitivity, and reproducibility. A set of five commercially available substrates, selected to represent a set of supports providing different surface and coupling chemistries, was used as reference surfaces. The results showed that several well-performing silicon-based supports could be designed; in particular, a nitrocellulose-coated macroporous variant, MAP3-NC7, received the highest scores. In comparison, MAP3-NC7 displayed properties equal to or better than those of the reference substrates. Taken together, designed surfaces based on silicon can undoubtedly meet the requirements of the next generation of solid supports for antibody microarrays.

Antibodies↗
PubMed2005

Microarrays based on affinity-tagged single-chain Fv antibodies: sensitive detection of analyte in complex proteomes.

Protein-based microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform global proteome analysis. However, the process of designing adequate protein microarrays is a major inherent problem. In this study, we have evaluated a protein microarray platform based on nonpurified affinity-tagged single-chain (sc) Fv antibody fragments to generate proof-of-principle and to demonstrate the specificity and sensitivity of the array design. To this end, we used our human recombinant scFv antibody library genetically constructed around one framework, the n-CoDeR library containing 2 x 10(10) clones, as a source for our probes. The probes were immobilized via engineered C-terminal affinity tags, his- or myc-tags, to either Ni(2+)-coated slides or anti-tag antibody coated substrates. The results showed that highly functional microarrays were generated and that nonpurified scFvs readily could be applied as probes. Specific and sensitive microarrays were obtained, providing a limit of detection in the pM to fM range, using fluorescence as the mode of detection. Further, the results showed that spotting the analyte on top of the arrayed probes, instead of incubating the array with large sample volumes (333 pL vs. 40 microL), could reduce the amount of analyte required 4000 times, from 1200 attomole to 300 zeptomole. Finally, we showed that a highly complex proteome, such as human sera containing several thousand different proteins, could be directly fluorescently labeled and successfully analyzed without compromising the specificity and sensitivity of the antibody microarrays. This is a prerequisite for the design of high-density antibody arrays applied in high-throughput proteomics.

Humans↗
PubMed2004

Combined pseudo-merohedral twinning, non-crystallographic symmetry and pseudo-translation in a monoclinic crystal form of the gammadelta T-cell ligand T10.

T10 is a non-classical class Ib-like major histocompatibility complex (MHC) cell-surface antigen which binds directly to certain gammadelta T-cell receptors in the absence of any exogenous and endogenous ligands, such as peculiar lipids or glycolipids. The crystal structure at 2.5 A resolution of murine T10 was determined by molecular replacement using data from an almost perfectly twinned monoclinic crystal. The space group is P2(1), with unit-cell parameters a = 78.2, b = 70.0, c = 139.2 A, beta = 106.8 degrees. Self-rotation function analysis and various intensity statistics revealed the presence of pseudo-merohedral twinning, but these tests underestimated the true twin fraction of alpha approximately 0.46. Native Patterson analyses pointed to the presence of pseudo-translation among the four molecules present in the asymmetric unit. Data analysis, structure determination and model refinement are discussed.

Animals↗
PubMed2004

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.

Antibodies↗
PubMed2003

Designing proteins to crystallize through beta-strand pairing.

Inherent difficulties in growing protein crystals are major concerns within structural biology and particularly in structural proteomics. Here, we describe a novel approach of engineering target proteins by surface mutagenesis to increase the odds of crystallizing the molecules. To this end, we have exploited our recent triad-hypothesis using proteins with crystallographically defined beta-structures as the principal models. Crystal packing analyses of 182 protein structures belonging to 21 different superfamilies implied that the propensities to crystallize could be engineered into target proteins by replacing short segments, 5-6 residues, of their beta-strands with 'cassettes' of suitable packing motifs. These packing motifs will generate specific crystal packing interactions that promote crystallization. Key features of the primary and tertiary structures of such packing motifs have been identified for immunoglobulins. Further, packing motifs have been engineered successfully into six model antibodies without disturbing their capabilities to be produced, their immunoreactivity and their overall structure. Preliminary crystallization analyses have also been performed. Taken together, the procedures outline a rational protocol for crystallizing proteins by surface mutagenesis. The importance of these findings is discussed in relation to the crystallization of proteins in general.

Animals↗
PubMed2002

Single framework recombinant antibody fragments designed for protein chip applications.

High-throughput proteomics, based on the microarray platform, requires stable, highly functional components that will yield a highly sensitive read-out of low abundance proteins. Although antibodies are the best characterized binding molecules for this purpose, only a fraction of them appear to behave satisfactorily in the chip format. Therefore, high demands need to be placed on their molecular design. In the present study, we have focused on recombinant antibody design based on a single framework for protein chip applications, aiming at defining crucial molecular probe parameters. Our results show that engineered human recombinant scFv antibody fragments that displayed appropriate biophysical properties (molecular [functional] stability in particular) can be generated, making them prime candidates for high-density antibody arrays. In fact, a superior framework that displays both multifaceted adsorption properties and very high functional stability over several months on chips (stored in a dried-out state) was identified. Taken together, designed scFv fragments based on a single molecular scaffold, readily accessible in large phage display libraries, can undoubtedly meet the requirements of probe content in antibody microarrays, particularly for global proteome analysis.

Antibodies, Monoclonal↗