Molecular design of recombinant scFv antibodies for site-specific photocoupling to ß-cyclodextrin in solution and onto solid support.

The ability to design and tailor-make antibodies to meet the biophysical demands required by the vast range of current and future antibody-based applications within biotechnology and biomedicine will be essential. In this proof-of-concept study, we have for the first time tailored human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the dock'n'flash technology. In more detail, we have successfully explored the possibility to expand the genetic code of E. coli and introduced the photoreactive UAA p-benzoyl-L-phenylalanine (pBpa), and showed that the mutated scFv antibody could be expressed in E. coli with retained structural and functional properties, as well as binding affinity. The pBpa group was then used for affinity capture of the mutated antibody by ß-cyclodextrin (ß-CD), which provided the hydrogen atoms to be abstracted in the subsequent photocoupling process upon irradiation at 365nm. The results showed that the pBpa mutated antibody could be site-specifically photocoupled to free and surface (array) immobilized ß-CD. Taken together, this paves the way for novel means of tailoring recombinant scFv antibodies for site-specific photochemical-based tagging, functionalization and immobilization in numerous applications.

Generation of miniaturized planar ecombinant antibody arrays using a microcantilever-based printer.

Miniaturized (Ø 10 µm), multiplexed (>5-plex), and high-density (>100 000 spots cm(-2)) antibody arrays will play a key role in generating protein expression profiles in health and disease. However, producing such antibody arrays is challenging, and it is the type and range of available spotters which set the stage. This pilot study explored the use of a novel microspotting tool, Bioplume(TM)-consisting of an array of micromachined silicon cantilevers with integrated microfluidic channels-to produce miniaturized, multiplexed, and high-density planar recombinant antibody arrays for protein expression profiling which targets crude, directly labelled serum. The results demonstrated that 16-plex recombinant antibody arrays could be produced-based on miniaturized spot features (78.5 um(2), Ø 10 µm) at a 7-125-times increased spot density (250 000 spots cm(-2)), interfaced with a fluorescent-based read-out. This prototype platform was found to display adequate reproducibility (spot-to-spot) and an assay sensitivity in the pM range. The feasibility of the array platform for serum protein profiling was outlined.

Combining crystallography and molecular dynamics: the case of Schistosoma mansoni phospholipid glutathione peroxidase.

Oxidative stress is a widespread challenge for living organisms, and especially so for parasitic ones, given the fact that their hosts can produce reactive oxygen species (ROS) as a mechanism of defense. Thus, long lived parasites, such as the flatworm Schistosomes, have evolved refined enzymatic systems capable of detoxifying ROS. Among these, glutathione peroxidases (Gpx) are a family of sulfur or selenium-dependent isozymes sharing the ability to reduce peroxides using the reducing equivalents provided by glutathione or possibly small proteins such as thioredoxin. As for other frontline antioxidant enzymatic systems, Gpxs are localized in the tegument of the Schistosomes, the outermost defense layer. In this article, we present the first crystal structure at 1.0 and 1.7 A resolution of two recombinant SmGpxs, carrying the active site mutations Sec43Cys and Sec43Ser, respectively. The structures confirm that this enzyme belongs to the monomeric class 4 (phospholipid hydroperoxide) Gpx. In the case of the Sec to Cys mutant, the catalytic Cys residue is oxidized to sulfonic acid. By combining static crystallography with molecular dynamics simulations, we obtained insight into the substrate binding sites and the conformational changes relevant to catalysis, proposing a role for the unusual reactivity of the catalytic residue.

Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays.

Immobilizing biomolecules with retained functionality and stability on solid supports is crucial for generation of sensitive immunoassays. However, upon use of conventional immobilization strategies, a major portion of the biomolecules (e.g. antibodies) frequently tends to lose their bioactivity. In this study, we describe a procedure to immobilize human single-chain variable fragment (scFv) via genetic fusion to partial spider silk, which have a high tendency to adhere to solid supports. Two scFvs, directed towards serum proteins, were genetically fused to partial spider silk proteins and expressed as silk fusion proteins in E. coli. Antigen binding ability of scFvs attached to a partial silk protein denoted RC was investigated using microarray analysis, whereas scFvs fused to the NC silk variant were examined using nanoarrays. Results from micro- and nanoarrays confirmed the functionality of scFvs attached to both RC and NC silk, and also for binding of targets in crude serum. Furthermore, the same amount of added scFv gives higher signal intensity when immobilized via partial spider silk compared to when immobilized alone. Together, the results suggest that usage of scFv-silk fusion proteins in immunoassays could improve target detection, in the long run enabling novel biomarkers to be detected in crude serum proteomes.

Photopatterning of self assembled monolayers on oxide surfaces for the selective attachment of biomolecules.

The immobilization of functional biomolecules to surfaces is a critical process for the development of biosensors for disease diagnostics. In this work we report the patterned attachment of single chain fragment variable (scFv) antibodies to the surface of metal oxides by the photodeprotection of self-assembled monolayers, using near-UV light. The photodeprotection step alters the functionality at the surface; revealing amino groups that are utilized to bind biomolecules in the exposed regions of the substrate only. The patterned antibodies are used for the detection of specific disease biomarker proteins in buffer and in complex samples such as human serum.

Multiplexing of miniaturized planar antibody arrays for serum protein profiling--a biomarker discovery in SLE nephritis.

In the quest to decipher disease-associated biomarkers, miniaturized and multiplexed antibody arrays may play a central role in generating protein expression profiles, or protein maps, of crude serum samples. In this conceptual study, we explored a novel, 4-times larger pen design, enabling us to, in a unique manner, simultaneously print 48 different reagents (antibodies) as individual 78.5 µm(2) (10 µm in diameter) sized spots at a density of 38,000 spots cm(-2) using dip-pen nanolithography technology. The antibody array set-up was interfaced with a high-resolution fluorescent-based scanner for sensitive sensing. The performance and applicability of this novel 48-plex recombinant antibody array platform design was demonstrated in a first clinical application targeting SLE nephritis, a severe chronic autoimmune connective tissue disorder, as the model disease. To this end, crude, directly biotinylated serum samples were targeted. The results showed that the miniaturized and multiplexed array platform displayed adequate performance, and that SLE-associated serum biomarker panels reflecting the disease process could be deciphered, outlining the use of miniaturized antibody arrays for disease proteomics and biomarker discovery.

Miniaturization of multiplexed planar recombinant antibody arrays for serum protein profiling.

BACKGROUND: Antibody-based microarrays are a developing tool for high-throughput proteomics in health and disease. However, in order to enable global proteome profiling, novel miniaturized high-density antibody array formats must be developed. RESULTS: In this proof-of-concept study, we have designed a miniaturized planar recombinant (single-chain Fragment variable). antibody array technology platform for multiplexed profiling of non-fractionated, directly labelled serum samples. The size of the individual spot features was reduced 225-times (78.5 µm(2)/spot) and the array density was increased 19-times (38,000 spots/cm(2)). These miniaturized, multiplexed arrays were produced, using a desktop nanofabrication system based on dip-pen nanolithography technology, and interfaced with a high-resolution fluorescent-based scanner. The reproducibility, sensitivity, specificity, and applicability of the set-up were demonstrated by profiling a set of well-characterized serum samples. CONCLUSION: The designed antibody array platform opens up new possibilities for large-scale, multiplex profiling of crude proteomes in a miniaturized fashion.

Hospital mortality due to pulmonary embolism and an evaluation of the usefulness of preventative interventions.

BACKGROUND: Mortality rates due to pulmonary embolism (PE) are difficult to estimate often due to the presence of comorbid disease. OBJECTIVES: To determine the accuracy of hospital records in identifying PE cases, PE-related mortality, and the number of PE-related deaths which are potentially preventable. METHODS: Retrospective chart review of PE cases hospitalized at The Ottawa Hospital over an 8 year period. Cases were reviewed to determine accuracy of coding, as well as the certainty with which PE was the cause of death. In PE-related deaths, a determination was made as to whether any interventions may have been life-saving. RESULTS: 498 cases of 612 (81%) cases coded as PE were correctly coded. 111 (22%) died during hospitalization, 63% of deaths were attributed to PE. The presence of a cardiorespiratory comorbidity or cancer was independently associated with an increased rate of death due to PE. 54% of PE-related deaths were determined to be potentially preventable, most commonly by appropriate DVT prophylaxis. A significantly higher number of cancer patients as compared to non-cancer patients may have potentially had their death due to PE prevented by an inferior vena cava filter (IVCF). Systemic thrombolysis was deemed to be potentially life-saving in 1/38 PE-related deaths. CONCLUSION: Hospital mortality due to clinically recognized PE can be determined by chart review of PE cases identified using the ICD coding system. Death due to PE is often potentially preventable; in the subgroup with cancer and DVT/PE, an IVCF may be a potentially useful intervention to prevent death due to PE. Prospective studies are needed to confirm these results.

Reductive openings of benzylidene acetals. Kinetic studies of borane and alane activation by Lewis acids.

The reaction kinetics for a number of reductive openings of methyl 2,3-di-O-benzyl-4,6-O-benzylidene-alpha-D-glucopyranoside have been investigated. Openings to give free HO-6 (using BH(3) x THF-AlCl(3)-THF or LiAlH(4)-AlCl(3)-Et(2)O) follow first order kinetics, while reactions yielding free HO-4 (using BH(3) x NMe(3)-AlCl(3)-THF or BH(3) x NMe(3)-BF(3) x OEt(2)-THF) follow higher order kinetics. The addition of water to the BH(3) x NMe(3)-AlCl(3)-THF results in faster reactions. The BH(3) x SMe(2)-AlCl(3)-THF system constitutes a borderline case, yielding both free HO-6 (by a first order reaction) and free HO-4 (by a higher order reaction). These results correlate well with the concept of regioselectivity by activation of borane complexes.