Abstract Gene expression analysis in the oncology space is largely performed with the classic and widely accepted methods real-time RT-PCR, microarray and next generation sequencing. These technologies are, however, too time consuming and expensive to be used in large scale screens with high sample numbers. TRAC (Transcription analysis with the aid of Affinity Capture) technology provides a solution for focused gene expression screening with unique combination of multiplex analysis, high-throughput sample processing, high precision and flexibility. Lysates of cells or tissues are directly applicable as sample material in TRAC assay without RNA extraction or cDNA conversion. The assay enables detection of up to 30 targets per sample and 96 samples per assay with 3-4h run time. In TRAC assay, each target mRNA of interest is recognized by a specific labeled ssDNA probe. Probes with different lengths and labels can be used for high-level of multiplexing. Hybridization of probes and targets takes place in solution, after which the probe-transcript complexes are captured by streptavidin coated magnetic beads. Unbound material is washed off and the probes are eluted for detection. The probes are then detected and quantified by capillary electrophoresis, which resolves the probes according to both size and fluorescence. The aim of this study was to apply TRAC assay to analyze the expression fingerprints of 20 gene markers related to angiogenesis, cell adhesion, protease activity and plasminogen activation, in cultured cells treated with different drug candidates. The expression of the target genes was analyzed from four different colon cancer cell lines (COLO, HT-29, CaCo2, DLD) to study culture age dependent effects and responses to drug candidates. Out of the 20 genes analyzed, curcumin caused consistent variation to cyclooxygenase-1 (COX-1) gene expression as a function of time in all tested cell lines. By comparison of expression fingerprints between non-treated cell line cultures, altogether four genes were identified as being expressed at higher level in one or two of the cell lines compared to the others. These genes were trypsinogen (PRSS1-3) in COLO, Serine protease inhibitor (SPINK) in HT-29, Protease Cathepsin B (CTSB) in COLO and cyclin D1 (CCDN1) in DLD and CaCo2 cell lines. The data showed good correlation with real-time RT-PCR results and reproducibility (CV <12%) and functioned as proof-of-concept for suitability of TRAC for screening purposes. Multiplex detection with TRAC from lysates offered high efficiency and significant cost and time savings compared to single-plex qPCR assay in large scale evaluation of drug candidates. Citation Format: Laura Mattinen, Jani Salmivaara, Jussi Halleen, Hans Söderlund, Jari Rautio. TRAC, a novel time and cost saving gene expression analysis technology with improved efficacy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3610.

We present a high-throughput roll-to-roll (R2R) manufacturing process for foil-based polymethyl methacrylate (PMMA) chips of excellent optical quality. These disposable, R2R hot embossed microfluidic chips are used for the identification of the antibiotic resistance genemecA in Staphylococcus epidermidis. R2R hot embossing is an emerging manufacturing technology for polymer microfluidic devices. It is based on continuous feeding of a thermoplastic foil through a pressurized area between a heated embossing cylinder and a blank counter cylinder. Although mass fabrication of foil-based microfluidic chips and their use for biological applications were foreseen already some years ago, no such studies have been published previously.

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Allergic contact dermatitis is a complex syndrome representing immunological responses to cutaneous exposure to protein-reactive chemicals. Although many contact sensitizers directly can elicit this disorder, others (prohaptens) require activation. Knowledge regarding the activating mechanisms remains limited, but one possibility is metabolic activation by cytochrome P450 (CYP) enzymes in the skin. We have, after quantitative reverse transcriptase-PCR studies of the CYP content in 18 human skin samples, developed an enriched skin-like recombinant human (rh) CYP cocktail using CYP1A1, 1B1, 2B6, 2E1, and 3A5. To validate the rhCYP cocktail, a prohaptenic conjugated diene ((5R)-5-isopropenyl-2-methyl-1-methylene-2-cyclohexene) was investigated using: the skin-like rhCYP cocktail, a liver-like rhCYP cocktail, single rhCYP enzymes, liver microsomes, keratinocytes, and a dendritic cell (DC) assay. The diene was activated to sensitizing epoxides in all non-cell-based incubations including the skin-like rhCYP cocktail. An exocyclic epoxide metabolite ((7R)-7-isopropenyl-4-methyl-1-oxaspiro[2.5]oct-4-ene) was found to be mainly responsible for the allergenic activity of the diene. This epoxide also induced pronounced DC activation indicated by upregulation of IL-8. The skin-like rhCYP cocktail provides a simplified alternative to using skin tissue preparations in mechanistic studies of CYP-mediated skin metabolism of prohaptens and offers the future possibility of designing in vitro predictive assays for assessment of allergenic activity of prohaptens.

Background: Allergen-mediated cross-linking of IgE antibodies bound to the Fc epsilon RI receptors on the mast cell surface is the key feature of the type I allergy. If an allergen is a homodimer, its allergenicity is enhanced because it would only need one type of antibody, instead of two, for cross-linking.Methodology/Principal Findings: An analysis of 55 crystal structures of allergens showed that 80% of them exist in symmetric dimers or oligomers in crystals. The majority are transient dimers that are formed at high protein concentrations that are reached in cells by colocalization. Native mass spectrometric analysis showed that native allergens do indeed form transient dimers in solution, while hypoallergenic variants of them exist almost solely in the monomeric form. We created a monomeric Bos d 5 allergen and show that it has a reduced capability to induce histamine release.Conclusions/Significance: The results suggest that dimerization would be a very common and essential feature for allergens. Thus, the preparation of purely monomeric variants of allergens could open up novel possibilities for specific immunotherapy.

Information derived from “omics” data in life science research are frequently limited by specific spatial or temporal scales these data describe. As a case study of integrating physiological and molecular data in human, here we study associations between the heart magnetic resonance images and serum lipidomic profiles. In the best case, such associations could help infer the physiologic state of the heart from a blood serum sample without need to use expensive imaging techniques. Strong marginal and partial correlations are found between the lipid profiles and parameters derived from the heart images. Regression analyses are applied to study these dependencies in more detail. This study demonstrates the feasibility of mapping lipid profiles to heart images, and thus combining information from two very different scales, small molecules and macroscopic physiologic features. Such mappings could be generalized to other “omics” data as well to complete our picture of the holistic function of a living organism.

A testosterone binding scFv antibody was isolated from a naive human library with a modest size of 10(8) clones. The crystal structure of the Fab fragment form of the 5F2 antibody clone complexed with testosterone determined at 1.5 angstrom resolution shows that the hapten is bound deeply in the antibody binding pocket. In addition to the interactions with framework residues only CDR-L3 and CDR-H3 loops interact with testosterone and the heavy chain forms the majority of the contacts with the hapten. The testosterone binding site of the 5F2 antibody with a high abundance of aromatic amino acid residues shows similarity with an in vitro affinity matured antibody having around 300 times higher affinity. The moderate affinity of the 5F2 antibody originates from the different orientation of the hapten and few light chain contacts. This is the first three-dimensional structure of a human steroid hormone binding antibody that has been isolated from a naive human repertoire. Copyright (C) 2010 John Wiley & Sons, Ltd.

(-)-Delta(9)-Tetrahydrocannabinol (THC) is the main psychoactive compound found in cannabis. In this study, an anti-THC Fab fragment, designed T3, was isolated from a display library cloned from the spleen cells of a mouse immunized with a THC bovine serum albumin conjugate, and the crystal structures of the T3 Fab in its free form and in complex with THC were determined at 1.9 angstrom and 2.0 angstrom resolution, respectively. The THC binding site of the T3 Fab is a narrow cavity: the n-pentyl group of THC protrudes deep into the interface area between the variable domains and the C-10 monoterpene moiety of the hapten is partially exposed to solvent. The metabolites of THC, with modifications in the C-10 monoterpene moiety, 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol and 11-hydroxy-Delta(9)-tetrahydrocannabinol, are bound by the T3 Fab with a higher affinity than THC. The crystal structures suggest that Ser52H and Arg53H of the T3 Fab are able to make hydrogen bonds with the metabolites, which leads to an increased binding against these metabolites. By developing a T3 Fab-Delta(9)-THC immunocomplex binding antibody from a nave antibody phage display library, the specificity of the Delta(9)-THC binding is highly increased, which allows a one-step, homogeneous, fluorescence resonance energy transfer-based sensitive immunoassay, with a detection limit of 20 ng/ml from saliva samples. (C) 2010 Elsevier Ltd. All rights reserved.

Antibody phage display technology is well established and widely used for selecting specific antibodies against desired targets. Using conventional manual methods, it is laborious to perform multiple selections with different antigens simultaneously. Furthermore, manual screening of the positive clones requires much effort. The authors describe optimized and automated procedures of these processes using a magnetic bead processor for the selection and a robotic station for the screening step. Both steps are performed in a 96-well microplate format. In addition, adopting the antibody phage display technology to automated platform polyethylene glycol precipitation of the enriched phage pool was unnecessary. For screening, an enzyme-linked immunosorbent assay protocol suitable for a robotic station was developed. This system was set up using human gamma-globulin as a model antigen to select antibodies from a VTT naive human single-chain antibody (scFv) library. In total, 161 gamma-globulin-selected clones were screened, and according to fingerprinting analysis, 9 of the 13 analyzed clones were different. The system was further tested using testosterone bovine serum albumin (BSA) and beta-estradiol-BSA as antigens with the same library. In total, 1536 clones were screened from 4 rounds of selection with both antigens, and 29 different testosterone-BSA and 23 beta-estradiol-BSA binding clones were found and verified by sequencing. This automated antibody phage display procedure increases the throughput of generating wide panels of target-binding antibody candidates and allows the selection and screening of antibodies against several different targets in parallel with high efficiency.

Cow's milk allergy (CMA) is a common food allergy, especially among infants and young children. Approximately 85% of milk-allergic children outgrow their allergy by the age of three but the remaining 15% remain allergic. Bovine β-lactoglobulin (BLG) is one of the major allergens in cow's milk. There is a definite need for the specific and sensitive detection of allergenic substances. Validated methods are obligatory to demonstrate allergen contamination and even fatal hidden allergens and, thus, to prevent life-threatening conditions of allergic persons. In this study, we constructed human IgE scFv libraries from an adult milk-allergic patient and isolated the first recombinant IgE antibodies specific to a food allergen, BLG. The selection of the IgE antibody libraries with two distinct panning procedures resulted in the enrichment of four clones having different BLG-binding profiles; two of the clones recognize the native BLG whereas the other two recognize only the heat-denatured form of BLG. For further characterization, the scFv fragments were converted to Fab fragments with human IgG1 isotype. The D1 Fab fragment, binding native BLG with nanomolar affinity, also partially inhibited serum IgE binding to BLG. These BLG-specific IgE antibodies can be applied for the detection of both native and denatured BLG in cow's milk products and furthermore, for the optimization of manufacturing processes to develop safe hypoallergenic milk products.

More than a decade of intensive use of microarray technology has flooded the scientific community with genome-wide expression data of diverse biological states. As a result, connection of the expression signatures of a relatively small number of genes related to, for example, disease states, patient responses or toxicological responses has become possible. Development of tools that enable cost- and time-efficient analysis of such signatures from large sample numbers is currently of major interest for research, drug screening and diagnostic purposes. A method named transcript analysis with aid of affinity capture (TRAC) is a novel solution hybridization and bead-based assay enabling multiplex mRNA target detection simultaneously from large sample numbers. Functionality of TRAC has been shown in a number of applications, including microbial quantification, gene expression-based monitoring of biotechnical processes, cell-based cancer marker gene screening and siRNA validation, which are reviewed here.

Silicon–glass microchips were designed and fabricated for on-chip solid phase extraction (SPE) and zone electrophoresis studies. The solvent channels for extraction and the separation channels for analyses were fabricated sequentially on the silicon device. Electrical contacts were integrated in a fused silica glass lid. Amorphous silicon thin film electrodes were fabricated for high voltage and conductivity detection. A chip installation rack with electrical and fluidic contacts was constructed to facilitate the experiments. Simulation was used to elucidate both the liquid flow and the electric field distribution. The operational performance of the microchips was demonstrated by using a fluorescein isothiocyanate (FITC)-labelled testosterone derivative as the model analyte and fluorescein as both the negative control and the calibration compounds. In SPE an immunosorbent, based on recombinant anti-testosterone Fab-fragments, was immobilized to activated Sepharose gel. Simultaneous monitoring of the movement of FITC-testosterone from SPE cavity through the channel to the detection point was performed with a laser-induced fluorescence detector. The observed limit of detection for FITC-testosterone was 2 μM.

Improved ways to cleave peptide chains at engineered sites easily and specifically would form useful tools for biochemical research. Uses of such methods include the activation or inactivation of enzymes or the removal of tags for enhancement of recombinant protein expression or tags used for purification of recombinant proteins. In this work we show by gel electrophoresis and mass spectroscopy that salts of Co(II) and Cu(II) can be used to cleave fusion proteins specifically at sites where sequences of His residues have been introduced by protein engineering. The His residues could be either consecutive or spaced with other amino acids in between. The cleavage reaction required the presence of low concentrations of ascorbate and in the case of Cu(II) also hydrogen peroxide. The amount of metal ions required for cleavage was very low; in the case of Cu(II) only one to two molar equivalents of Cu(II) to protein was required. In the case of Co(II), 10 molar equivalents gave optimal cleavage. The reaction occurred within minutes, at a wide pH range, and efficiently at temperatures ranging from 0 degrees C to 70 degrees C. The work described here can also have implications for understanding protein stability in vitro and in vivo.