Biosensor-based Approach Research Articles

Engineering of the Lrp/AsnC-type transcriptional regulator DecR as a genetically encoded biosensor for multilevel optimization of L-cysteine biosynthesis pathway in Escherichia coli.

L-cysteine is an important sulfur-containing amino acid being difficult to produce by microbial fermentation. Due to the lack of high-throughput screening methods, existing genetically engineered bacteria have been developed by simply optimizing the expression of L-cysteine-related genes one by one. To overcome this limitation, in this study, a biosensor-based approach for multilevel biosynthetic pathway optimization of L-cysteine from the DecR regulator variant of Escherichia coli was applied. Through protein engineering, we obtained the DecRN29Y/C81E/M90Q/M99E variant-based biosensor with improved specificity and an 8.71-fold increase in dynamic range. Using the developed biosensor, we performed high-throughput screening of the constructed promoter and RBS combination library, and successfully obtained the optimized strain, which resulted in a 6.29-fold increase in L-cysteine production. Molecular dynamics (MD)simulations and electrophoretic mobility shift analysis (EMSA)showed that the N29Y/C81E/M90Q/M99E variant had enhanced induction activity. This enhancement may be due to the increased binding of the variant to DNA in the presence of L-cysteine, which enhances transcriptional activation. Overall, our biosensor-based strategy provides a promising approach for optimizing biosynthetic pathways at multiple levels. The successful implementation of this strategy demonstrates its potential for screening improved recombinant strains.

Silicon/Silver nanocomposite-based surface-enhanced Raman spectroscopy (SERS) biosensor for ultrasensitive direct detection of deafness mutations in real system

Surface-enhanced Raman scattering (SERS) is a promising emerging, highly sensitive technique for detecting biological samples. The current research aims to bring an effortless biosensor-based approach to detect and discriminate DNA mutations with their fingerprints for each deafness mutation within the GJB2 (gap junction beta 2) gene. Therefore, we developed silver (Ag) modified silicon-based SERS sensing platform to directly screen GJB2 gene mutations for UAE patients with hereditary hearing loss (HL). The prepared AgNPs@Si biosensor was utilized for establishing a silicon-based SERS biosensing protocol for further analyses toward label-free DNA deafness mutations screening. Furthermore, the comparative studies demonstrated the viability of the developed sensor to distinguish between the normal/mutant samples in the real-time system by investigating the DNA assignments and the peaks intensity. Noticeably, under 785 nm laser excitation distinguishable Raman peaks for cytosine bands (C) are enriched with normal DNA (DMF-34) at 780 cm−1, 1450 cm−1 and 1590 cm−1 whereas with the single nucleotide deletion of cytosine band for the mutant DNA correspondingly predicted with Quenched intensities at the parallel Raman peak positions. The sensitivity studies portrayed the limit of detection to be 1 fg/μL. Further their reproducibility studies evaluation conveyed with low values of % RSD as 10. By the end of this study, we expected to update the mutational profile of the GJB2 gene in the UAE population using the advanced simple SERS-based technique for further supplemental applications in terms of genetic counseling and prenatal diagnosis.

Biosensor-based approach to detect bovine mastitis

Biosensor-based approach to detect bovine mastitis

Multiplexed experimental strategies for fragment library screening against challenging drug targets using SPR biosensors

Surface plasmon resonance (SPR) biosensor methods are ideally suited for fragment-based lead discovery. However, generally applicable experimental procedures and detailed protocols are lacking, especially for structurally or physico-chemically challenging targets or when tool compounds are not available. Success depends on accounting for the features of both the target and the chemical library, purposely designing screening experiments for identification and validation of hits with desired specificity and mode-of-action, and availability of orthogonal methods capable of confirming fragment hits. The range of targets and libraries amenable to an SPR biosensor-based approach for identifying hits is considerably expanded by adopting multiplexed strategies, using multiple complementary surfaces or experimental conditions. Here we illustrate principles and multiplexed approaches for using flow-based SPR biosensor systems for screening fragment libraries of different sizes (90 and 1056 compounds) against a selection of challenging targets. It shows strategies for the identification of fragments interacting with 1) large and structurally dynamic targets, represented by acetyl choline binding protein (AChBP), a Cys-loop receptor ligand gated ion channel homologue, 2) targets in multi protein complexes, represented by lysine demethylase 1 and a corepressor (LSD1/CoREST), 3) structurally variable or unstable targets, represented by farnesyl pyrophosphate synthase (FPPS), 4) targets containing intrinsically disordered regions, represented by protein tyrosine phosphatase 1B (PTP1B), and 5) aggregation-prone proteins, represented by an engineered form of human tau (tau K18M). Practical considerations and procedures accounting for the characteristics of the proteins and libraries, and that increase robustness, sensitivity, throughput and versatility are highlighted. The study shows that the challenges for addressing these types of targets is not identification of potentially useful fragments per se, but establishing methods for their validation and evolution into leads.

Determination of Hydrogen Peroxide Concentration in Biological Samples: a Biosensor-Based Approach

Determination of Hydrogen Peroxide Concentration in Biological Samples: a Biosensor-Based Approach

SAT-034 Photometry Based Measurement of G Protein-Coupled Receptor-Mediated Signalling

Many hormones and neurotransmitters exert their effects by binding to G protein-coupled receptors (GPCRs). These membrane-spanning receptors couple primarily to heterotrimeric G proteins which can act on effector proteins to modulate cellular functions such as membrane excitability, second messenger production or gene expression. GPCRs represent the most druggable targets in the mammalian genome, however individual receptors can often activate multiple signaling pathways, only some of which may mediate therapeutic effects. Much of what is known about the signaling pathways linked to activation of specific GPCRs comes from studies using heterologous cell models, but evidence now suggests that the specific complement of effectors engaged by a given GPCR is dependent on the cellular and tissue context, such that the same receptor may induce different signaling patterns in different cell types. This phenomenon is particularly evident in the central nervous system which contains hundreds of specialized cell populations whose function are regulated by the combined action of hormones, neurotransmitters and neuromodulators, and disruption of select signaling pathways may contribute to disease. To study GPCR mediated signaling in cells of interest in their native context, we have developed a biosensor-based approach that would allow in vivo expression and recording of fluorescent reporters in behaving animals. Our approach uses adeno-associated viruses (AAV) to express genetically encoded sensors in animals using cell-type selective promoters and Cre-recombinase dependent expression. Biosensor responses are recorded in real time from live animals using a fiber-photometry-based FRET recording platform. Our approach allows simultaneous measurement of cell-specific signaling with behavioral or physiological measurements made from live animal subjects. This approach has wide applicability to the study of neuroendocrine cell populations, hormone action in the brain, and to evaluate drug action on target cells.

A biosensor-based approach reveals links between efflux pump expression and cell cycle regulation in pleiotropic drug resistance of yeast

Multidrug resistance is highly conserved in mammalian, fungal, and bacterial cells, is characterized by resistance to several unrelated xenobiotics, and poses significant challenges to managing infections and many cancers. Eukaryotes use a highly conserved set of drug efflux transporters that confer pleiotropic drug resistance (PDR). To interrogate the regulation of this critical process, here we developed a small molecule-responsive biosensor that couples transcriptional induction of PDR genes to growth rate in the yeast Saccharomyces cerevisiae Using diverse PDR inducers and the homozygous diploid deletion collection, we applied this biosensor system to genome-wide screens for potential PDR regulators. In addition to recapitulating the activity of previously known factors, these screens identified a series of genes involved in a variety of cellular processes with significant but previously uncharacterized roles in the modulation of yeast PDR. Genes identified as down-regulators of the PDR included those encoding the MAD family of proteins involved in the mitotic spindle assembly checkpoint (SAC) complex. Of note, we demonstrated that genetic disruptions of the mitotic spindle assembly checkpoint elevate expression of PDR-mediating efflux pumps in response to exposure to a variety of compounds that themselves have no known influence on the cell cycle. These results not only establish our biosensor system as a viable tool for investigating PDR in a high-throughput fashion, but also uncover critical control mechanisms governing the PDR response and a previously uncharacterized link between PDR and cell cycle regulation in yeast.

High-Throughput, Biosensor-Based Approach to Examine Bone Morphogenetic Protein (BMP)-Receptor Interactions.

Binding of a BMP to its cognate cell surface receptors is the initiating step in the BMP signaling cascade. Thus, knowing which BMP-receptor complexes form is vital for understanding the physiological activities of a particular BMP. Here, we describe a surface plasmon resonance (SPR)-based, high-throughput approach that allows fast identification and evaluation of BMP-receptor complexes. Briefly, the extracellular, BMP-binding domains of receptors are produced as human IgG1-Fc-fusion proteins. The Fc moiety enables simple capture of the Fc-receptor-fusion protein on the sensor chip, supports a highly reproducible, uniform approach of surface regeneration, and ensures full activity of the receptor moiety. BMPs are injected over the captured receptors at one concentration (approximately 60-100nM), permitting stratification of high-affinity, medium-affinity, and low-affinity binders. Using this concentration range, equilibrium dissociation constants for high-affinity and medium-affinity binders can be estimated with good accuracy and with great precision from the single injection binding curves.

An amperometric glutamate biosensor for monitoring glutamate release from brain nerve terminals and in blood plasma

An excess of the excitatory neurotransmitter, glutamate, in the synaptic cleft during hypoxia/ischemia provokes development of neurotoxicity and originates from the reversal of Na+-dependent glutamate transporters located in the plasma membrane of presynaptic brain nerve terminals. Here, we have optimized an electrochemical glutamate biosensor using glutamate oxidase and developed a biosensor-based methodological approach for analysis of rates of tonic, exocytotic and transporter-mediated glutamate release from isolated rat brain nerve terminals (synaptosomes). Changes in the extracellular glutamate concentrations from 11.5 ± 0.9 to 11.7 ± 0.9 μΜ for 6 min reflected a low tonic release of endogenous glutamate from nerve terminals. Depolarization-induced exocytotic release of endogenous glutamate was equal to 7.5 ± 1.0 μΜ and transporter reversal was 8.0 ± 1.0 μΜ for 6 min. The biosensor data correlated well with the results obtained using radiolabelled L-[14C]glutamate, spectrofluorimetric glutamate dehydrogenase and amino acid analyzer assays. The blood plasma glutamate concentration was also tested, and reliability of the biosensor measurements was confirmed by glutamate dehydrogenase assay. Therefore, the biosensor-based approach for accurate monitoring rates of tonic, exocytotic and transporter-mediated release of glutamate in nerve terminals was developed and its adequacy was confirmed by independent analytical methods. The biosensor measurements provided precise data on changes in the concentrations of endogenous glutamate in nerve terminals in response to stimulation. We consider that the glutamate biosensor-based approach can be applied in clinics for neuromonitoring glutamate-related parameters in brain samples, liquids and blood plasma in stroke, brain trauma, therapeutic hypothermia treatment, etc., and also in laboratory work to record glutamate release and uptake kinetics in nerve terminals.

Introduction of Intrinsic Kinetics of Protein-Ligand Interactions and Their Implications for Drug Design.

Structure-kinetic relationship analyses and identification of dominating interactions for optimization of lead compounds should ideally be based on intrinsic rate constants instead of the more easily accessible observed kinetic constants, which also account for binding-linked reactions. The intrinsic rate constants for sulfonamide inhibitors and pharmacologically relevant isoforms of carbonic anhydrase were determined by a novel surface plasmon resonance (SPR) biosensor-based approach, using chemodynamic analysis of binding-linked pH-dependent effects. The observed association rates ( kaobs) were pH-dependent and correlated with the fraction of deprotonated inhibitor and protonated zinc-bound water molecule. The intrinsic association rate constants ( kaintr) were pH independent and higher than kaobs. By contrast, the observed and intrinsic dissociation rate constants were identical and pH-independent, demonstrating that the observed association and dissociation mechanisms are inherently different. A model accounting for the differences between intrinsic and observed rate constants was developed, useful also for other interactions with binding-linked protonation reactions.

EULAR 2017: Annual European Congress of Rheumatology, June 14–17, 2017, Madrid, Spain

EULAR 2017: Annual European Congress of Rheumatology, June 14–17, 2017, Madrid, Spain

Characterization of binding and quantification of human autoantibodies to PDGFRα using a biosensor-based approach

Systemic sclerosis (SSc) is a chronic autoimmune disease of the connective tissue. The variety and clinical relevance of autoantibodies in SSc patients have been extensively studied, eventually identifying agonistic autoantibodies targeting the platelet-derived growth factor receptor alpha (PDGFRα), and representing potential biomarkers for SSc.We used a resonant mirror biosensor to characterize the binding between surface-blocked PDGFRα and PDGFRα-specific recombinant human monoclonal autoantibodies (mAbs) produced by SSc B cells, and detect/quantify serum autoimmune IgG with binding characteristics similar to the mAbs. Kinetic data showed a conformation-specific, high-affinity interaction between PDGFRα and mAbs, with equilibrium dissociation constants in the low-to-high nanomolar range. When applied to total serum IgG, the assay discriminated between SSc patients and healthy controls, and allowed the rapid quantification of autoimmune IgG in the sera of SSc patients, with anti-PDGFRα IgG falling in the range 3.20–4.67 neq/L of SSc autoantibodies. The test was validated by comparison to direct and competitive anti-PDGFRα antibody ELISA. This biosensor assay showed higher sensibility with respect to ELISA, and other major advantages such as the specificity, rapidity, and reusability of the capturing surface, thus representing a feasible approach for the detection and quantification of high affinity, likely agonistic, SSc-specific anti-PDGFRα autoantibodies.

Generation and Characterization of Microvesicles after Daratumumab Interaction with Myeloma Cells

Daratumumab (DARA) is an anti-CD38 human mAb in phase III clinical trials in myeloma patients. DARA binding induces killing of tumor cells via complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and apoptosis.This work reports on the results obtained while dissecting the events following in vivo CD38 ligation by DARA. Treatment of myeloma cells with DARA + anti-human IgG at 37 °C influenced myeloma cytoskeleton, with redistribution of the CD38 molecules and a formation of distinct polar aggregates visualized by confocal microscopy. DARA effects are different from those observed using 8 different murine anti-human CD38 mAbs, which tended to internalize. The findings observed after DARA ligation were confirmed by exposing myeloma cells to DARA immobilized on CHO cells modified to express 4 distinct human FcRs. CHO cells adhere to plastic and mimics the events that take place in the myeloma niche. First, the interaction between Dara and the different FcRs was determined according to a biosensor-based approach on an IAsys Plus equipment (Affinity Sensors). Results show that DARA possesses the highest avidity for CD64, due to the higher kinetic stability of the complex. Conversely, the differences in the FcR-DARA recognition phase (kass) had only minor effects on final stability of the complex. The results also indicated that NK cells and monocytes are the blood populations with higher kdiss for DARA. The effects observed at 37 °C on myeloma cells in the presence of immobilized DARA were amplified when compared to those with soluble mAb. Results indicate that DARA induces CD38 target to aggregate, polarize and to release microvesicles (MV) from extrusions of the myeloma membrane. MV in culture supernatants and in bone marrow plasma of myeloma patients were characterized for concentration (particles/ml) and size by means of Malvern NanoSight NS300 equipment. DARA treatment was followed by high amounts of MV of different sizes released from myeloma cells. Same experiments repeated using DARA + anti-human IgG labeled with Alexa 488 analyzed by Malvern equipment highlighted the presence of DARA on the surface of MV.The induction of MV may be relevant for in vivo therapy: MV are outward buds of the membrane, which host molecules clustered in microdomains. The presence of CD38 has been confirmed. MV phenotype was analyzed looking for the ectoenzymes that join CD38 in the regulation of adenosine in the myeloma niche. MV phenotype included not only the presence of the expected CD38, but also of CD203a/PC-1, CD39 and CD73, the ectoenzymatic pathway leading to ADO production.A first conclusion is that DARA treatment is followed in vivo by a marked release of MV at the tumor site. The fate of the MV bearing DARA on their surface is multiple: on one side, MV may interact locally with different cells and populations of the niche. Another possibility is that MV are released into the blood stream and interact with cell populations therein. The lipid bilayer of MV consents passive movements even through tissues. MV appear as minicellular signals delivering instructions at a distance from their place of origin. Further, the ectoenzymes analyzed are also involved in cell migration or in interaction with countereceptors (e.g., CD31) expressed by endothelial cells. This issue, was investigated by testing FITC-conjugated DARA on a Laboratory-established human myeloma line. MV-DARA-FITC were then exposed to PBMC preparations obtained from normal donors. The results from a cytofluorimetric analysis highlighted the tendency of the labeled MV to cluster around CD16+ (NK cells) and CD14+ subsets (monocytes). At the moment, it is only possible to conclude that MV are associated with cell membranes, a binding likely mediated by FcRs.Not known yet whether MV interact with other cell types (e.g., macrophages, dendritic cells or lymphocytes). DARA shows a high affinity to FcRs of immune cell types (NK cells, monocytes, B cells). Given clinical data that indicate a robust increase in T cell counts, activation and clonality following DARA treatment should be expected. MV containing DARA and portions of myeloma cell membranes could help drive antigen presentation and T cell response in some patients. This is being investigated further. DisclosuresMark:Bioinvent International: Consultancy, Research Funding. Giuliani:Janssen Pharmaceutica: Research Funding; Celgene Italy: Research Funding. Sasser:Janssen Pharmaceuticals: Employment. Malavasi:Janssen: Honoraria, Research Funding.

Kinetic and mechanistic differences in the interactions between caldendrin and calmodulin with AKAP79 suggest different roles in synaptic function

The kinetic and mechanistic details of the interaction between caldendrin, calmodulin and the B-domain of AKAP79 were determined using a biosensor-based approach. Caldendrin was found to compete with calmodulin for binding at AKAP79, indicating overlapping binding sites. Although the AKAP79 affinities were similar for caldendrin (K(D) = 20 nM) and calmodulin (K(D) = 30 nM), their interaction characteristics were different. The calmodulin interaction was well described by a reversible one-step model, but was only detected in the presence of Ca(2+). Caldendrin interacted with a higher level of complexity, deduced to be an induced fit mechanism with a slow relaxation back to the initial encounter complex. It interacted with AKAP79 also in the absence of Ca(2+), but with different kinetic rate constants. The data are consistent with a similar initial Ca(2+)-dependent binding step for the two proteins. For caldendrin, a second Ca(2+)-independent rearrangement step follows, resulting in a stable complex. The study shows the importance of establishing the mechanism and kinetics of protein-protein interactions and that minor differences in the interaction of two homologous proteins can have major implications in their functional characteristics. These results are important for the further elucidation of the roles of caldendrin and calmodulin in synaptic function.

Direct detection of genomic DNA by surface plasmon resonance imaging: An optimized approach

The direct detection of specific sequences in genomic DNA samples is very challenging in the biosensor-based approach. In this work we developed an optimized strategy for the direct detection of DNA sequences in human genomic samples by a surface plasmon resonance imaging technology. As model study, the target analyte was identified in a DNA sequence mapping the human ABCB1 gene. The computed-assisted approach was here applied for probe design. After a preliminary evaluation of the probe functioning by the complementary synthetic target, the system was applied to the direct detection of the target sequence in human genomic DNA extracted from lymphocytes. To achieve this result, several steps aimed to improve the analytical performances of the biosensor were studied and optimized. The immobilization chemistry, based on thiolated probes, was adapted here to non-amplified sequence detection. DNA sample pre-treatments, i.e. genomic fragmentation by ultrasounds and dsDNA denaturation by thermal treatment were also investigated. A sandwich-like strategy, by using a secondary probe, was also applied to understand and confirm the selectivity of the developed biosensor in detecting ABCB1 gene in genomic samples. Finally, a reliable calibration curve of ABCB1 was obtained with an experimental detection limit of 140aM. Furthermore, the biosensor was well regenerable, assuring up to thirty cycles of effective measurements.

Sanguisorba minor extract suppresses plasmin-mediated mechanisms of cancer cell migration

BackgroundSanguisorba minor, as well as several other edible herbs and vegetables, has been used extensively in traditional medicine. The observed beneficial effects can be attributed at least in part to the direct modulation of several enzymatic activities by its polyphenolic constituents. MethodsThe ethanol extract of Sanguisorba minor was characterized by reversed-phase liquid chromatography, and most relevant analytes were identified by multiple stage mass spectrometry. The whole extract and the most relevant isolated constituents were tested for their ability to modulate the activity of human plasmin both toward a synthetic substrate and in human breast cancer cell culture models. Kinetic and equilibrium parameters were obtained by a concerted spectrophotometric and biosensor-based approach. ResultsQuercetin-3-glucuronide was recognized as the compound mainly responsible for the in vitro plasmin inhibition by S. minor extract, with an inhibition constant in the high nanomolar range; in detail, our approach based on bioinformatic, enzymatic and binding analyses classified the inhibition as competitive. Most interestingly, cell-based assays showed that this flavonoid was effective in suppressing plasmin-induced loss of cancer cell adhesion. General significanceOur results show that the extract from Sanguisorba minor limits plasmin-mediated tumor cell motility in vitro, mostly due to quercetin-3-glucuronide. This glucuronated flavonoid is a promising template for rational designing of anticancer drugs to be used in the treatment of pathological states involving the unregulated activity of plasmin.

Biosensor-Based Approach to the Identification of Protein Kinase Ligands with Dual-Site Modes of Action

The authors have used a surface plasmon resonance (SPR)–based biosensor approach to identify and characterize compounds with a unique binding mode to protein kinases. Biacore was used to characterize hits from an enzymatic high-throughput screen of the Tec family tyrosine kinase, IL2-inducible T cell kinase (ITK). Complex binding kinetics was observed for some compounds, which led to identification of compounds that bound simultaneously at both the adenosine triphosphate (ATP) binding site and a second, allosteric site on ITK. The presence of the second binding site was confirmed by X-ray crystallography. The second site is located in the N-terminal lobe of the protein kinase catalytic domain, adjacent to but distinct from the ATP site. To enable rapid optimization of binding properties, a competition-based Biacore assay has been developed to successfully identify second site noncompetitive binders that have been confirmed by X-ray crystallographic studies. The authors have found that SPR technology is a key method for rapid identification of compounds with dual-site modes of action.

Computational Design of Auxotrophy-Dependent Microbial Biosensors for Combinatorial Metabolic Engineering Experiments

Combinatorial approaches in metabolic engineering work by generating genetic diversity in a microbial population followed by screening for strains with improved phenotypes. One of the most common goals in this field is the generation of a high rate chemical producing strain. A major hurdle with this approach is that many chemicals do not have easy to recognize attributes, making their screening expensive and time consuming. To address this problem, it was previously suggested to use microbial biosensors to facilitate the detection and quantification of chemicals of interest. Here, we present novel computational methods to: (i) rationally design microbial biosensors for chemicals of interest based on substrate auxotrophy that would enable their high-throughput screening; (ii) predict engineering strategies for coupling the synthesis of a chemical of interest with the production of a proxy metabolite for which high-throughput screening is possible via a designed bio-sensor. The biosensor design method is validated based on known genetic modifications in an array of E. coli strains auxotrophic to various amino-acids. Predicted chemical production rates achievable via the biosensor-based approach are shown to potentially improve upon those predicted by current rational strain design approaches. (A Matlab implementation of the biosensor design method is available via http://www.cs.technion.ac.il/~tomersh/tools).

A Biosensor-Based Characterization of the Affinity Maturation of the Immune Response Against Interferon-β and Correlations with Neutralizing Antibodies in Treated Multiple Sclerosis Patients

The study of antibodies against Interferon-beta (IFN-beta) in treated multiple sclerosis (MS) patients has focused primarily on detection and quantification, with no single method of detection providing a comprehensive characterization. We assessed serial samples of 18 MS patients, treated with IFN-beta for between 66 and 198 months, to characterize the affinity maturation of these antibodies using a biosensor-based approach (Biacore). Biacore utilizes the principles of surface plasmon resonance (SPR) with data from the dissociation phase of the antigen-antibody reaction being inversely proportional to relative antibody affinity. In patients with neutralizing antibodies (NAbs+), mean antibody dissociation rates decreased from 0.00118 +/- 0.00030 s(-1) at month 6 to 0.00021 +/- 0.00008 s(-1) at month 36, followed by a slight increase to 0.00027 +/- 0.00003 s(-1) at month 60. In NAb- patients, mean antibody dissociation rates decreased only very slightly from 0.00130 +/- 0.00025 s(-1) to 0.00105 +/- 0.00020 s(-1) at month 18, followed by a gradual increase to 0.00243 +/- 0.00099 s(-1) at month 60. Our study shows little improvement in antibody affinity in NAb- patients, in contrast to a marked increase in antibody affinity over time in NAb+ patients with a significant correlation between NAb titers and relative antibody dissociation rates (Spearman's correlation, R(2) = -0.374, p < 0.001). The evaluation of relative antibody affinities will contribute to a thorough understanding of the anti-IFN-beta antibody response.

Transgenes monitoring in an industrial soybean processing chain by DNA-based conventional approaches and biosensors

The development of analytical methods for genetically modified organisms (GMO) screening is of great interest. In particular, since even highly processed GMO-derived food products are covered by new European legislations, a great effort has been devoted to the application of the analytical tests to these products. This work describes a polymerase chain reaction-based qualitative screening assay and a biosensor-based approach to detect transgenes in a Roundup Ready ® soybean processing line. Roundup Ready ® soybean was specifically analyzed in eight types of processed materials – seeds, crushed seeds, expander, crude flour, proteic flour, crude oil, degummed oil and lecithin – all derived from the same initial source and produced during the manufacturing process. Specific combinations of primers were used to differentiate sequences from the whole insert. The amplification of “marker” fragments with a maximum length of 500 bp was successfully achieved both in raw material (seeds) and in partially (crushed seeds, crude and proteic flours) and highly (crude and degummed oils and fluid lecithin) processed materials. Moreover, the extraction procedure was optimised and the polymerase chain reaction-electrophoresis analysis has been implemented by a biosensor-based approach.