Search

Thousands of antibodies for diagnostic and other analytical purposes are on the market. However, it is often difficult to identify duplicates, reagent changes, and to assign the correct original publications to an antibody. This slows down scientific progress and might even be a cause of irreproducible research and a waste of resources. Recently, activities were started to suggest the sole use of recombinant antibodies in combination with the open communication of their sequence. In this case, such uncertainties should be eliminated. Unfortunately, this approach seems to be rather a long-term vision since the development and manufacturing of recombinant antibodies remain quite expensive in the foreseeable future. Also, nearly all commercial antibody suppliers may be reluctant to publish the sequence of their antibodies, since they fear counterfeiting. De-novo sequencing of antibodies is also not feasible today for a reagent user without access to the hybridoma clone. Nevertheless, it seems to be crucial for any scientist to have the opportunity to identify an antibody undoubtedly to guarantee the traceability of any research activity using antibodies from a third party as a tool. For this purpose, we developed a method for the identification of antibodies based on a MALDI-TOF-MS fingerprint. To circumvent lengthy denaturation, reduction, alkylation, and enzymatic digestion steps, the fragmentation was performed with a simple formic acid hydrolysis step. Eighty-nine unknown monoclonal antibodies were used for this study to examine the feasibility of this approach. Although the molecular assignment of peaks was rarely possible, antibodies could be easily recognized in a blinded test, simply from their mass-spectral fingerprint. A general protocol is given, which could be used without any optimization to generate fingerprints for a database. We want to propose that in most scientific projects relying critically on antibody reagents, such a fingerprint should be established to prove and document the identity of the used antibodies and to assign a specific reagent to a datasheet of a commercial supplier, a public database record or an antibody ID.

Silicon photonic sensors are promising candidates for lab-on-a-chip solutions with versatile applications and scalable production prospects using complementary metal-oxide semiconductor (CMOS) fabrication methods. However, the widespread use has been hindered because the sensing area adjoins optical and electrical components making packaging and sensor handling challenging. In this work, a local back-side release of the photonic sensor is employed, enabling a separation of the sensing area from the rest of the chip. This approach allows preserving the compatibility of photonic integrated circuits in the front-end of line and metal interconnects in the back-end of line. The sensor is based on a micro-ring resonator and is fabricated on wafer-level using a CMOS technology. We revealed a ring resonator sensitivity for homogeneous sensing of 106 nm/RIU. �� 1989-2012 IEEE.

A 2-dimensional high-throughput screening method is presented to select peptide sequences from large peptide libraries for precision formulation additives, having a high capacity to specifically host a drug of interest and provide tailored drug release properties. The identified sequences are conjugated with poly(ethylene glycol) (PEG) to obtain peptide-PEG conjugates that proved to be valuable as solubilizers for small organic molecule drugs to overcome limitations of poor water-solubility and low bio-availability. The 2D-screening method selects peptide sequences on both (i) high loading capacities and (ii) preferred drug-release capabilities as demonstrated on an experimental Tau-protein aggregation inhibitor/Tau- deaggregator with potentials for an anti-Alzheimer disease drug (BB17). To enable 2D-screening, a one-bead one-compound (OBOC) peptide library was immobilized on a glass slide, allocating individual beads to permanent positions. While the first screening step involved incubation of the supported OBOC library with BB17 to identify beads with high drug binding capacities by fluorescence scanner readouts, the second step reveals release properties of the high capacity binders by incubation with blood plasma protein model solutions. Efficiently peptides with high BB17 capacities and either keeper or medium or fast releaser properties can be identified by direct sequence readouts from the glass slide supported resin beads via matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Four peptides are synthesized as peptide-PEG solubilizers representing strong, medium, weak releasers and non-binders. Loading capacities reached up to 1:3.4 (mol drug per mol carrier) and release kinetics (fast/medium/slow) are in agreement with the selection process as investigated by fluorescence anisotropy and fluorescence correlation spectroscopy. The ability of BB17/conjugate complexes to inhibit the aggregation of Tau4RDΔK (four repeat Tau ((M)Q244-E372 with deletion of K280), 129 residues) in N2a cells is studied by a Tau-pelleting assay showing the modulation of cellular Tau aggregation. Promising effects such as the reduction of 55% of total Tau load are observed for the strong releaser additive. Studies of in vitro Thioflavin S Tau-aggregation assays show half-maximal inhibitory activities (IC50 values) of BB17/conjugates in the low micro-molar range.

Objective The human copper-protein ceruloplasmin (Cp) is the major copper-containing protein in the human body. The accurate determination of Cp is mandatory for the reliable diagnosis of several diseases. However, the analysis of Cp has proven to be difficult. The aim of our work was a proof of concept for the determination of a metalloprotein-based on online immunocapture ICP-MS. The immuno-affinity step is responsible for the enrichment and isolation of the analyte from serum, whereas the compound-independent quantitation with ICP-MS delivers the sensitivity, precision, and large dynamic range. Off-line ELISA (enzyme-linked immunosorbent assay) was used in parallel to confirm the elution profile of the analyte with a structure-selective method. The total protein elution was observed with the 32S mass trace. The ICP-MS signals were normalized on a 59Co signal. Results The human copper-protein Cp could be selectively determined. This was shown with pure Cp and with a sample of human serum. The good correlation with off-line ELISA shows that Cp could be captured and eluted selectively from the anti-Cp affinity column and subsequently determined by the copper signal of ICP-MS. Electronic supplementary material The online version of this article (10.1186/s13104-018-3324-7) contains supplementary material, which is available to authorized users.

The allergenic potential of airborne proteins may be enhanced via post-translational modification induced by air pollutants like ozone (O3) and nitrogen dioxide (NO2). The molecular mechanisms and kinetics of the chemical modifications that enhance the allergenicity of proteins, however, are still not fully understood. Here, protein tyrosine nitration and oligomerization upon simultaneous exposure of O3 and NO2 were studied in coated-wall flow-tube and bulk solution experiments under varying atmospherically relevant conditions (5–200 ppb O3, 5–200 ppb NO2, 45–96% RH), using bovine serum albumin as a model protein. Generally, more tyrosine residues were found to react via the nitration pathway than via the oligomerization pathway. Depending on reaction conditions, oligomer mass fractions and nitration degrees were in the ranges of 2.5–25% and 0.5–7%, respectively. The experimental results were well reproduced by the kinetic multilayer model of aerosol surface and bulk chemistry (KM-SUB). The extent of nitration and oligomerization strongly depends on relative humidity (RH) due to moisture-induced phase transition of proteins, highlighting the importance of cloud processing conditions for accelerated protein chemistry. Dimeric and nitrated species were major products in the liquid phase, while protein oligomerization was observed to a greater extent for the solid and semi-solid phase states of proteins. Our results show that the rate of both processes was sensitive towards ambient ozone concentration, but rather insensitive towards different NO2 levels. An increase of tropospheric ozone concentrations in the Anthropocene may thus promote pro-allergic protein modifications and contribute to the observed increase of allergies over the past decades.

The primary screening of hybridoma cells is a time-critical and laborious step during the development of monoclonal antibodies. Often, critical errors occur in this phase, which supports the notion that the generation of monoclonal antibodies with hybridoma technology is difficult to control and hence, a risky venture. We think that it is crucial to improve the screening process to eliminate most of the critical deficits of the conventional approach. With this new microarray-based procedure, several advances could be achieved: Selectivity for excellent binders, high-throughput, reproducible signals, avoidance of misleading avidity (multivalency) effects, and performance of simultaneous competition experiments. The latter can also be used to select clones of desired cross-reactivity properties. In this paper, a model system with two excellent clones against carbamazepine, two weak clones, and blank supernatant containing fetal bovine serum was designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the immunoglobulin G (IgG) concentration, which is usually unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration are not feasible in most cases. Furthermore, in this approach, the enrichment, isolation, and purification of IgG for characterization is not necessary. Raw cell culture supernatant can be used directly, even when fetal calf serum (FCS) or other complex media is used. In addition, an improved method for the oriented antibody-immobilization on epoxy-silanized slides is presented. Based on the results of this model system with simulated hybridoma supernatants, we conclude that this approach should be preferable to most other protocols leading to many false positives, causing expensive and lengthy elimination steps to weed out the poor clones.

Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications.

Functional sequences of precision polymers based on thiolactone/Michael chemistry are identified from a large one-bead one-compound library. Single-bead readout by MALDI-TOF MS/MS identifies sequences that host m-THPC that is a second generation photo-sensitizer drug. The corresponding Tla/Michael-PEG conjugates make m-THPC available in solution and drug payload as well as drug release kinetics can be fine-tuned by the precision segment.

A novel stationary phase for affinity separations is presented. This material is based on sintered borosilicate glass readily available as semi-finished filter plates with defined porosity and surface area. The material shows fast binding kinetics and excellent long-term stability under real application conditions due to lacking macropores and high mechanical rigidity. The glass surface can be easily modified with standard organosilane chemistry to immobilize selective binders or other molecules used for biointeraction. In this paper, the manufacturing of the columns and their respective column holders by 3D printing is shown in detail. The model system protein A/IgG was chosen as an example to examine the properties of such monolithic columns under realistic application conditions. Several specifications, such as (dynamic) IgG capacity, pressure stability, long-term performance, productivity, non-specific binding, and peak shape, are presented. It could be shown that due to the very high separation speed, 250 mg antibody per hour and column can be collected, which surpasses the productivity of most standard columns of the same size. The total IgG capacity of the shown columns is around 4 mg (5.5 mg/mL), which is sufficient for most tasks in research laboratories. The cycle time of an IgG separation can be less than 1 min. Due to the glass material’s excellent pressure resistance, these columns are compatible with standard HPLC systems. This is usually not the case with standard affinity columns, limited to manual use or application in low-pressure systems. The use of a standard HPLC system also improves the ability for automation, which enables the purification of hundreds of cell supernatants in one day. The sharp peak shape of the elution leads to an enrichment effect, which might increase the concentration of IgG by a factor of 3. The final concentration of IgG can be around 7.5 mg/mL without the need for an additional nanofiltration step. The purity of the IgG was &gt, 95% in one step and nearly 99% with a second polishing run.

Environmental pollutants like fine particulate matter can cause adverse health effects through oxidative stress and inflammation. Reactive oxygen and nitrogen species (ROS/RNS) such as peroxynitrite can chemically modify proteins, but the effects of such modifications on the immune system and human health are not well understood. In the course of inflammatory processes, the Toll-like receptor 4 (TLR4) can sense damage-associated molecular patterns (DAMPs). Here, we investigate how the TLR4 response and pro-inflammatory potential of the proteinous DAMPs α-Synuclein (α-Syn), heat shock protein 60 (HSP60), and high-mobility-group box 1 protein (HMGB1), which are relevant in neurodegenerative and cardiovascular diseases, changes upon chemical modification with peroxynitrite. For the peroxynitrite-modified proteins, we found a strongly enhanced activation of TLR4 and the pro-inflammatory transcription factor NF-κB in stable reporter cell lines as well as increased mRNA expression and secretion of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-8 in human monocytes (THP-1). This enhanced activation of innate immunity via TLR4 is mediated by covalent chemical modifications of the studied DAMPs. Our results show that proteinous DAMPs modified by peroxynitrite more potently amplify inflammation via TLR4 activation than the native DAMPs, and provide first evidence that such modifications can directly enhance innate immune responses via a defined receptor. These findings suggest that environmental pollutants and related ROS/RNS may play a role in promoting acute and chronic inflammatory disorders by structurally modifying the body's own DAMPs. This may have important consequences for chronic neurodegenerative, cardiovascular or gastrointestinal diseases that are prevalent in modern societies, and calls for action, to improve air quality and climate in the Anthropocene., Graphical abstract Image 1, Highlights • Pollutants and oxidative stress can cause protein nitration and oligomerization. • Peroxynitrite amplifies inflammatory potential of disease-related proteins in vitro. • Chemical modification of damage-associated molecular patterns (DAMPs). • Positive feedback of modified DAMPs via pattern recognition receptor (TLR4). • Air pollution may promote inflammatory disorders in the Anthropocene.

The illegal use of explosives by terrorists and other criminals is an increasing issue in public spaces, such as airports, railway stations, highways, sports venues, theaters, and other large buildings. Security in these environments can be achieved by different means, including the installation of scanners and other analytical devices to detect ultra-small traces of explosives in a very short time-frame to be able to take action as early as possible to prevent the detonation of such devices. Unfortunately, an ideal explosive detection system still does not exist, which means that a compromise is needed in practice. Most detection devices lack the extreme analytical sensitivity, which is nevertheless necessary due to the low vapor pressure of nearly all explosives. In addition, the rate of false positives needs to be virtually zero, which is also very difficult to achieve. Here we present an immunosensor system based on kinetic competition, which is known to be very fast and may even overcome affinity limitation, which impairs the performance of many traditional competitive assays. This immunosensor consists of a monolithic glass column with a vast excess of immobilized hapten, which traps the fluorescently labeled antibody as long as no explosive is present. In the case of the explosive 2,4,6-trinitrotoluene (TNT), some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein, detectable by highly sensitive laser-induced fluorescence with the help of a Peltier-cooled complementary metal-oxide-semiconductor (CMOS) camera. Liquid handling is performed with high-precision syringe pumps and chip-based mixing-devices and flow-cells. The system achieved limits of detection of 1 pM (1 ppt) of the fluorescent label and around 100 pM (20 ppt) of TNT. The total assay time is less than 8 min. A cross-reactivity test with 5000 pM solutions showed no signal by pentaerythritol tetranitrate (PETN), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). This immunosensor belongs to the most sensitive and fastest detectors for TNT with no significant cross-reactivity by non-related compounds. The consumption of the labeled antibody is surprisingly low: 1 mg of the reagent would be sufficient for more than one year of continuous biosensor operation.

The primary screening of hybridoma cells is a time-critical and laborious step during the development of monoclonal antibodies. Often, critical errors occur in this phase, which supports the notion that the generation of monoclonal antibodies with hybridoma technology is difficult to control and hence, a risky venture. We think that it is crucial to improve the screening process to eliminate most of the critical deficits of the conventional approach. With this new microarray-based procedure, several advances could be achieved: Selectivity for excellent binders, high-throughput, reproducible signals, avoidance of misleading avidity (multivalency) effects, and performance of simultaneous competition experiments. The latter can also be used to select clones of desired cross-reactivity properties. In this paper, a model system with two excellent clones against carbamazepine, two weak clones, and blank supernatant containing fetal bovine serum was designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the immunoglobulin G (IgG) concentration, which is usually unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration are not feasible in most cases. Furthermore, in this approach, the enrichment, isolation, and purification of IgG for characterization is not necessary. Raw cell culture supernatant can be used directly, even when fetal calf serum (FCS) or other complex media is used. In addition, an improved method for the oriented antibody-immobilization on epoxy-silanized slides is presented. Based on the results of this model system with simulated hybridoma supernatants, we conclude that this approach should be preferable to most other protocols leading to many false positives, causing expensive and lengthy elimination steps to weed out the poor clones.

Background: Molecular-MRI is a promising imaging modality for the assessment of abdominal aortic aneurysms (AAAs). Interleukin-1β (IL-1β) represents a new therapeutic tool for AAA-treatment, since pro-inflammatory cytokines are key-mediators of inflammation. This study investigates the potential of molecular-MRI to evaluate therapeutic effects of an anti-IL-1β-therapy on AAA-formation in a mouse-model. Methods: Osmotic-minipumps were implanted in apolipoprotein-deficient-mice (N = 27). One group (Ang-II+01BSUR group, n = 9) was infused with angiotensin-II (Ang-II) for 4 weeks and received an anti-murine IL-1β-antibody (01BSUR) 3 times. One group (Ang-II-group, n = 9) was infused with Ang-II for 4 weeks but received no treatment. Control-group (n = 9) was infused with saline and received no treatment. MR-imaging was performed using an elastin-specific gadolinium-based-probe (0.2 mmol/kg). Results: Mice of the Ang-II+01BSUR-group showed a lower aortic-diameter compared to mice of the Ang-II-group and control mice (p < 0.05). Using the elastin-specific-probe, a significant decrease in elastin-destruction was observed in mice of the Ang-II+01BSUR-group. In vivo MR-measurements correlated well with histopathology (y = 0.34x-13.81, R2 = 0.84, p < 0.05), ICP-MS (y = 0.02x+2.39; R2 = 0.81, p < 0.05) and LA-ICP-MS. Immunofluorescence and western-blotting confirmed a reduced IL-1β-expression. Conclusions: Molecular-MRI enables the early visualization and quantification of the anti-inflammatory-effects of an IL-1β-inhibitor in a mouse-model of AAAs. Responders and non-responders could be identified early after the initiation of the therapy using molecular-MRI.

Hepcidin-25 was identified as themain iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II) binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandemmass spectrometry (MS/MS), high-resolutionmass spectrometry (HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D)model of hepcidin-25with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or referencematerial comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others., Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe, 701

Mass spectrometry-based methods play a crucial role in the quantification of the main iron metabolism regulator hepcidin by singling out the bioactive 25-residue peptide from the other naturally occurring N-truncated isoforms (hepcidin-20, -22, -24), which seem to be inactive in iron homeostasis. However, several difficulties arise in the MS analysis of hepcidin due to the "sticky" character of the peptide and the lack of suitable standards. Here, we propose the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces after testing several types of vials for the preparation of stock solutions and serum samples for isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions with the aim of developing a LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. A chromatographic separation based on usual acidic mobile phases was compared with a novel approach involving the separation of hepcidin-25 with solvents at high pH containing 0.1% of ammonia. Both methods were applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with good correlation of the results. Finally, we recommend a LC-MS/MS-based quantification method with a dynamic range of 0.5-40 μg/L for the assessment of hepcidin-25 in human serum that uses TFA-based mobile phases and silanized glass vials. Graphical abstract Structure of hepcidin-25 (Protein Data Bank, PDB ID 2KEF).

The quality of research antibodies is an issue for decades. Although several papers have been published to improve the situation, their impact seems to be limited. This publication makes the effort to simplify the description of validation criteria in a way that the occasional antibody user is able to assess the validation level of an immunochemical reagent. A simple, 1-page checklist is supplied for the practical application of these criteria.

An improved antibody against the explosive pentaerythritol tetranitrate (PETN) was developed. The immunogen was designed by the concept of bioisosteric replacement, which led to an excellent polyclonal antibody with extreme selectivity and immunoassays of very good sensitivity. Compounds such as nitroglycerine, 2,4,6-trinitrotoluene, 1,3,5-trinitrobenzene, hexogen (RDX), 2,4,6-trinitroaniline, 1,3-dinitrobenzene, octogen (HMX), triacetone triperoxide, ammonium nitrate, 2,4,6-trinitrophenol and nitrobenzene were tested for potential cross-reactivity. The detection limit of a competitive enzyme-linked immunosorbent assay was determined to be around 0.5 µg/l. The dynamic range of the assay was found to be between 1 and 1000 µg/l, covering a concentration range of three decades. This work shows the successful application of the bioisosteric concept in immunochemistry by exchange of a nitroester to a carbonate diester. The antiserum might be used for the development of quick tests, biosensors, microtitration plate immunoassays, microarrays and other analytical methods for the highly sensitive detection of PETN, an explosive frequently used by terrorists, exploiting the extreme difficulty of its detection.

N-Hydroxysuccinimide (NHS) esters are the most important activated esters used in many different bioconjugation techniques, such as protein labelling by fluorescent dyes and enzymes, surface activation of chromatographic supports, microbeads, nanoparticles, and microarray slides, and also in the chemical synthesis of peptides. Usually, reactions with NHS esters are very reliable and of high yield, however, the compounds are sensitive to air moisture and water traces in solvents. Therefore, the quantification of NHS would be a very helpful approach to identify reagent impurities or degradation of stored NHS esters. No robust and sensitive method for the detection of NHS (or the more hydrophilic sulfo-NHS) has been reported yet. Here, a chromatographic method based on HILIC conditions and UV detection is presented, reaching a detection limit of about 1 mg L−1, which should be sensitive enough for most of the applications mentioned above. Exemplarily, the hydrolytic degradation of a biotin-NHS ester and a purity check of a fluorescent dye NHS ester are shown. An important advantage of this approach is its universality, since not the structurally variable ester compound is monitored, but the constant degradation product NHS or sulfo-NHS, which avoids the necessity to optimize the separation conditions and facilitates calibration considerably.

The small heterobifunctional linker succinimidyl iodoacetate (SIA) was examined for the preparation of peptide–protein bioconjugates with predicable conjugation ratios. For many conjugation protocols, the protein is either treated with a reductant to cleave disulfide bonds or is reacted with thiolation chemicals, such as Traut’s reagent. Both approaches are difficult to control, need individual optimization and often lead to unsatisfactory results. In another popular approach, a heterobifunctional linker with a N-hydroxysuccinimide (NHS) and a maleimide functionality is applied to the protein. After the activation of some lysine ε-amino groups with the NHS ester functionality, a cysteine-containing peptide is attached to the activated carrier protein via maleimide. Particularly, the maleimide reaction leads to some unwanted byproducts or even cleavage of the linker. Many protocols end up with conjugates with unpredictable and irreproducible conjugation ratios. In addition, the maleimide-thiol addition product should be assumed immunogenic in vivo. To avoid these and other disadvantages of the maleimide approach, we examined the known linker succinimidyl iodoacetate (SIA) in more detail and developed two protocols, which lead to peptide–protein conjugates with predefined average conjugation ratios. This holds potential to eliminate tedious and expensive optimization steps for the synthesis of a bioconjugate of optimal composition.

Air pollution and climate change are potential drivers for the increasing burden of allergic diseases. The molecular mechanisms by which air pollutants and climate parameters may influence allergic diseases, however, are complex and elusive. This article provides an overview of physical, chemical and biological interactions between air pollution, climate change, allergens, adjuvants and the immune system, addressing how these interactions may promote the development of allergies. We reviewed and synthesized key findings from atmospheric, climate, and biomedical research. The current state of knowledge, open questions, and future research perspectives are outlined and discussed. The Anthropocene, as the present era of globally pervasive anthropogenic influence on planet Earth and, thus, on the human environment, is characterized by a strong increase of carbon dioxide, ozone, nitrogen oxides, and combustion- or traffic-related particulate matter in the atmosphere. These environmental factors can enhance the abundance and induce chemical modifications of allergens, increase oxidative stress in the human body, and skew the immune system toward allergic reactions. In particular, air pollutants can act as adjuvants and alter the immunogenicity of allergenic proteins, while climate change affects the atmospheric abundance and human exposure to bioaerosols and aeroallergens. To fully understand and effectively mitigate the adverse effects of air pollution and climate change on allergic diseases, several challenges remain to be resolved. Among these are the identification and quantification of immunochemical reaction pathways involving allergens and adjuvants under relevant environmental and physiological conditions.

Screening of one-bead-one-compound (OBOC) libraries is a proven procedure for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The traditional peptide screening involves tedious steps such as affinity selection, bead picking, sequencing, and characterization. Herein, we present a high-throughput &ldquo, all-on-one chip&rdquo, system to avoid slow and technically complex bead picking steps. On a traditional glass slide provided with an electrically conductive tape, beads of a combinatorial peptide library are aligned and immobilized by application of a precision sieve. Subsequently, the chip is incubated with a fluorophore-labeled target protein. In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry, high-affinity binders are directly and unambiguously sequenced with high accuracy without picking of the positive beads. The use of an optimized ladder sequencing approach improved the accuracy of the de-novo sequencing step to nearly 100%. The new technique was validated by employing a FLAG-based model system, identifying new peptide binders for the monoclonal M2 anti-FLAG antibody, and was finally utilized to search for IgG-binding peptides. In the present format, more than 30,000 beads can be screened on one slide.

Chemical modifications such as nitration and cross-linking may enhance the allergenic potential of proteins. The kinetics and mechanisms of the underlying chemical processes, however, are not yet well understood. Here, we present a size-exclusion chromatography/spectrophotometry method (SEC-HPLC-DAD) that allows a simultaneous detection of mono-, di-, tri-, and higher protein oligomers, as well as their individual nitration degrees (NDs). The ND results of proteins from this new method agree well with the results from an alternative well-established method, for the analysis of tetranitromethane (TNM)- and nitrogen dioxide and ozone (NO2/O3)-nitrated protein samples. Importantly, the NDs for individual oligomer fractions can be obtained from the new method, and also, we provide a proof of principle for the calculation of the concentrations for individual protein oligomer fractions by their determined NDs, which will facilitate the investigation of the kinetics and mechanism for protein tyrosine nitration and cross-linking.

Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports.

Caffeine is a useful indicator to quickly assess liver function. High-throughput tests are needed for single-point caffeine measurements, with low cross-reactivity toward its major metabolite, paraxanthine. A newly developed ELISA was compared with an LC-MS/MS reference method, using 60 saliva samples from 10 individuals, before and after caffeine intake. Bland-Altman plot, Student t-test and F-test were used to compare the two methods. Proteins were precipitated using organic solvent and the caffeine recoveries compared with those obtained using sample microfiltration. The antibody, with a low cross-reactivity toward paraxanthine (0.08%), allows quantification of caffeine in saliva samples from 2.5 µg/L to 125 µg/L with high precision and the ELISA shows comparable results to those obtained by LC-MS/MS. A one-step protein precipitation using an organic solvent provides comparable results to a more costly and time-consuming microfiltration pre-treatment of samples. The new ELISA is a fit-for-purpose metho...

A series of new epoxide-based monoliths is synthesized by self-polymerization of polyglycerol polyglycidyl ether as porous support for affinity chromatography. Porosity and mean pore sizes are investigated as a function of the porogen ratio and analyzed by mercury porosimetry. The morphologies are visualized by environmental scanning electron microscope. With a particularly rigid monolith prepared with 75 vol% porogen (66% porosity and 12 μm mean pore size), a capillary column (0.5 mm ID) is prepared and directly functionalized with recombinant protein A to extract immunoglobulin G from rabbit serum.

Antibodies are a promising tool for the fast and selective trace detection of explosives. Unfortunately, the production of high-quality antibodies is not trivial and often expensive. Therefore, excellent antibodies are a rare and limiting resource in fields such as biosensing, environmental analysis, diagnostics, cancer therapy, and proteomics. Here, we report the synthesis, bioconjugation, and application of the structurally optimized hapten 6-(2,4,6-trinitro)-phenylhexanoic acid to improve the selectivity and sensitivity of antibodies for the detection of one of the most important explosives, trinitrotoluene. With a conjugate of bovine serum albumin and a highly purified N-hydroxy-succinimide (NHS)–activated hapten, two rabbits were immunized to obtain polyclonal antibodies. The immunization process was monitored by enzyme-linked immunosorbent assay to gain information about the progress of antibody titer and affinity. Finally, the polyclonal antibodies reached an affinity constant of (5.1 ± 0.6) × 109 l/mol (rabbit R1) and (2.3 ± 0.2) × 109 l/mol (rabbit R2). The respective assays show a minimum test midpoint (IC50 value) of 0.1 ± 0.01 µg/l (R1) and 0.2 ± 0.02 µg/l (R2) and a working range of 0.005 to 150 µg/l (R1) and 0.007 to 200 µg/l (R2), which corresponds to more than four orders of magnitude for both. This is quite remarkable for a competitive immunoassay, which is often believed to have a narrow dynamic range. The limit of detection was calculated to 0.6 ng/l (R1) and 1.5 ng/l (R2), which is up to 100 times improvement in relation to the assay of Zeck et al. (1999) on the basis of a monoclonal antibody. The excellent selectivity of the polyclonal antibodies was comprehensively examined by determining the cross-reactivity to common explosives and other nitroaromatics including nitro musk components. The widely held belief that polyclonal antibodies generally display higher cross-reactivities than monoclonals could be disproved. Copyright © 2012 John Wiley & Sons, Ltd.

Triacetone triperoxide (TATP) is a primary explosive, which was used in various terrorist attacks in the past. For the development of biosensors, immunochemical µ-TAS, electronic noses, immunological test kits, or test strips, the availability of antibodies of high quality is crucial. Recently, we presented the successful immunization of mice, based on the design, synthesis, and conjugation of a novel TATP derivative. Here, the long-term immunization of rabbits is shown, which resulted in antibodies of extreme selectivity and more than 1,000 times better affinity in relation to the antibodies from mice. Detection limits below 10 ng L−1 (water) were achieved. The working range covers more than four decades, calculated from a precision profile. The cross-reactivity tests revealed an extraordinary selectivity of the antibodies—not a single compound could be identified as a relevant cross-reactant. The presented immunoreagent might be a major step for the development of highly sensitive and selective TATP detectors particularly for security applications.

The pharmaceutical compound carbamazepine (CBZ) is an emerging pollutant in the aquatic environment and may potentially be used as a wastewater marker. In this work, an enzyme-linked immunosorbent assay (ELISA) for the detection of carbamazepine in surface and sewage waters has been developed. The heterogeneous immunoassay is based on a commercially available monoclonal antibody and a novel enzyme conjugate (tracer) that links the hapten via a hydrophilic peptide (triglycine) spacer to horseradish peroxidase. The assay achieves a limit of detection of 24 ng/L and a quantitation range of 0.05-50 microg/L. The analytical performance and figure of merits were compared to liquid chromatography-tandem mass spectrometry after solid-phase extraction. For nine Berlin surface water samples and one wastewater sample, a close correlation of results was observed. A constant overestimation relative to the CBZ concentration of approximately 30% by ELISA is probably caused by the presence of 10,11-epoxy-CBZ and 2-hydroxy-CBZ in the samples. The ELISA displayed cross-reactivities for these compounds of 83% and 14%, respectively. In a first screening of 27 surface water samples, CBZ was detected in every sample with concentrations between 0.05 and 3.2 microg/L. Since no sample cleanup is required, the assay allowed for the determination of carbamazepine with high sensitivity at low costs and with much higher throughput than with conventional methods.

A new class of boron-dipyrromethene (BDP or BODIPY) dyes was obtained by phenanthrene fusion to the beta-pyrrole positions, absorbing in the wavelength range of important laser sources. Despite a 'propeller-like' distorted structure in the crystalline state, the chromophore absorbs (log epsilon > or = 5) and fluoresces (Phif > or = 0.8) strongly and can be easily turned into a fluorescence light-up probe. Incorporation into latex beads produces bright and photostable single-dye and Forster Resonance Energy Transfer (FRET) particles for microscopy applications.

A new type of biodetector was designed based on a bioluminescence test with the bacterium Vibrio fischeri performed in a liquid continuous flow-through system. Here we describe the modification of a commercial tube luminescence detector to work in the flow mode by building a new flow cell holder and a new case including "top cover" to connect the flow cell with the waste and the incubation capillary in a light-proof manner. As different samples were injected successively it was necessary to keep the individual peaks separated. This was done using an air-segmented flow in the reaction coil. To afford fast screening, the incubation time of the sample and the Vibrio fischeri, which equaled the dead time of the detection system, was set at 5.6 min. Rapid monitoring of toxic substances is achieved by using 20 microL of sample and flow-rates of 110-150 microL min(-1). As a proof-of-principle, we show results for the detection of five selected di-, tri- and tetrachlorophenols at different concentrations varying from 1 to 200 mg L(-1). Calculation of inhibition rates and EC50 values were performed and compared with corresponding values from the DIN EN ISO 11348-2 microplate format. Compared with the latter, the inhibition rates obtained with our flow-through biodetector for the compounds tested were generally about twofold lower, but importantly, a much faster detection is possible.

Aflatoxins, a group of structurally related mycotoxins, are well known for their toxic and carcinogenic effects in humans and animals. Aflatoxin derivatives and protein conjugates are needed for diverse analytical applications. This work describes a reliable and fast synthesis of novel aflatoxin derivatives, purification by preparative HPLC and characterisation by ESI-MS and one- and two-dimensional NMR. Novel aflatoxin bovine serum albumin conjugates were prepared and characterised by UV absorption and MALDI-MS. These aflatoxin protein conjugates are potentially interesting as immunogens for the generation of aflatoxin selective antibodies with novel specificities.

An improved antibody against the explosive pentaerythritol tetranitrate (PETN) was developed. The immunogen was designed by the concept of bioisosteric replacement, which led to an excellent polyclonal antibody with extreme selectivity and immunoassays of very good sensitivity. Compounds such as nitroglycerine, 2,4,6-trinitrotoluene, 1,3,5-trinitrobenzene, hexogen (RDX), 2,4,6-trinitroaniline, 1,3-dinitrobenzene, octogen (HMX), triacetone triperoxide, ammonium nitrate, 2,4,6-trinitrophenol and nitrobenzene were tested for potential cross-reactivity. The detection limit of a competitive enzyme-linked immunosorbent assay was determined to be around 0.5 µg/l. The dynamic range of the assay was found to be between 1 and 1000 µg/l, covering a concentration range of three decades. This work shows the successful application of the bioisosteric concept in immunochemistry by exchange of a nitroester to a carbonate diester. The antiserum might be used for the development of quick tests, biosensors, microtitration plate immunoassays, microarrays and other analytical methods for the highly sensitive detection of PETN, an explosive frequently used by terrorists, exploiting the extreme difficulty of its detection.

Zu den klassischen Aufgaben der analytischen Chemie wie Element- und Speziesanalytik kommen im Gefolge der Miniaturisierung immer mehr Anforderungen aus den Nachbardisziplinen. Angesichts der explosionsartigen Vermehrung von Nachweistechniken und Identifizierungsverfahren in Biologie und Molekularbiologie stellt sich die Frage nach einer Neuorientierung und Standortbestimmung der analytischen Chemie immer dringender.

Background: Recent investigations have shown that proteins, including Bet v 1a, are nitrated by exposure to polluted urban air. We have investigated immunogenic and allergenic properties of in vitro nitrated allergens in in vivo models. Methods: Untreated and nitrated samples of ovalbumin or Bet v 1a were compared for their ability to stimulate proliferation and cytokine secretion in splenocytes from DO11.10 or from sensitized BALB/c mice, and for their ability to induce specific immunoglobulin (Ig)G1, IgG2a and IgE in sensitized mice. Additionally, sera from birch pollen-allergic individuals were analysed for IgE and IgG specific for nitrated Bet v 1a. Results: Upon splenocyte stimulation with nitrated as compared with unmodified allergens, proliferation as well as interleukin 5 and interferon-γ production were enhanced. Sera of mice sensitized with nitrated allergens showed elevated levels of specific IgE, IgG1 and IgG2a, compared with sera from mice sensitized with unmodified allergens. Moreover, cross-reactivity of antibodies against unrelated, nitrated allergens was observed in mice. We also found higher amounts of functional, specific IgE against nitrated than against untreated Bet v 1a in sera from birch pollen-allergic patients. Conclusions: Our findings suggest that nitration enhances allergic responses, which may contribute to an increased prevalence of allergic diseases in polluted urban environments.

A reversed-phase high-performance liquid chromatography system with UV-detector was equipped with an on-line acetylcholinesterase inhibition assay to achieve effect-directed analysis of potentially toxic samples. The enzyme activity was detected colorimetrically using Ellman's reagent. The inhibition and substrate conversion took place in glass capillaries at a 100 microL/min flow rate. Extra-column band spreading in the reaction coils reduces the sensitivity and separation power of biochemical detectors severely. Knitted reactors exhibited no reduction of longitudinal dispersion in the tested flow range. The implementation of air-segmentation allowed an extended inhibition and substrate conversion time without a significant loss of chromatographic resolution. The limit of detection of two model compounds carbofuran (carbamate) and paraoxon-ethyl (organophosphate) was determined to be 13 ng (injected mass) and 7.4 ng, respectively, applying an isocratic chromatography method. A mixture of five insecticides was separated by a gradient elution and the inhibitory effect on the enzyme activity could be detected with high resolution. The band width at half height of the enzyme inhibition detector signal after a reaction time of about 8 min or 4.2 m of capillary, respectively, increased only by a factor of 1.4 compared to the UV-detector signal.

People suffering from food allergies are dependent on accurate food labeling, as an avoidance diet is the only effective countermeasure. Even a small amount of allergenic protein can trigger severe reactions in highly sensitized patients. Therefore, sensitive and reliable tests are needed to detect potential cross-contamination. In this paper two fast sandwich immunoassays are described for the determination of peanut (Arachis hypogaea) and hazelnut (Corylus avellana) traces in complex food matrices. Mouse monoclonal antibodies were used as capture antibodies, and labeled rabbit polyclonal antibodies were used as detection antibodies in both assays. The assay time was 30 min in total, and cross-reactivities against a variety of fruits and seeds were found to be in the low 10(-4)% (ppm) level or in some cases not detectable. The recoveries in all tested food matrices ranged from 86 to 127%, and the limits of detection were in the range of 0.2-1.2 mg/kg (ppm) in food for both peanut and hazelnut, respectively.

Der Trend zu hochmolekularen Analyten scheint Miniaturisierung zu stimulieren. Fragen der klinischen Chemie und der Lebensmittelchemie rucken in den Vordergrund. Apparativ dominiert die Massenspektrometrie, auch als Detektor fur die Flussigchromatographie und die Kapillarelektrophorese. Bei den Trennverfahren spielt die Mehrdimensionalitat eine immer grosere Rolle. Mehr und mehr Publikationen stammen aus dem asiatischen Raum.

A parallel affinity sensor array (PASA) for the rapid automated analysis of 10 antibiotics in milk is presented, using multianalyte immunoassays with an indirect competitive ELISA format. Microscope glass slides modified with (3-glycidyloxypropyl)trimethoxysilane were used for the preparation of hapten microarrays. Protein conjugates of the haptens were immobilized as spots on disposable chips, which were processed in a flow cell. Monoclonal antibodies against penicillin G, cloxacillin, cephapirin, sulfadiazine, sulfamethazine, streptomycin, gentamicin, neomycin, erythromycin, and tylosin allowed the simultaneous detection of the respective analytes. Antibody binding was detected by a second antibody labeled with horseradish peroxidase generating enhanced chemiluminescence, which was recorded with a sensitive CCD camera. All liquid handling and sample processing was fully automated, and one analysis was carried out in milk within less than 5 min. The detection limits ranged from 0.12 (cephapirin) to 32 microg/L (neomycin). Penicillin G could be detected at the maximum residue limit (MRL); the detection limits for all other analytes were far below the respective MRLs. The PASA system proved to be the first immunochemical biosensor platform having the potential to test for numerous antibiotics in parallel, such being of considerable interest for the control of milk in the dairy industry.

Post-translational nitration of proteins was analyzed by capillary reversed-phase high-performance liquid chromatography (RP-HPLC) on-line interfaced to electrospray ionization mass spectrometry (ESI--MS) or tandem mass spectrometry (ESI--MS/MS). Both methods were compared using a tryptic digest of bovine serum albumin (BSA) and yielded sequence coverages of 95% and 33% with RP-HPLC--ESI--MS and RP-HPLC--ESI--MS/MS, respectively. At least 95% of the tyrosines were covered by the former method, whereas the latter method only detected less than 50% of the tyrosine-containing peptides. Upon liquid-phase nitration of BSA in aqueous solution using an excess of tetranitromethane, at least 16 of the 20 tyrosine residues were found to be nitrated. After exposure of solid BSA samples to gaseous nitrogen dioxide and ozone at atmospherically relevant concentration levels, only 3 nitrated peptides were detected. By use of such a model system, RP-HPLC--ESI--MS proved to be a rapid and highly efficient method for the comprehensive and quantitative detection of protein nitration.

The described in vitro test system for allergy diagnosis is based on microscope glass slides activated with (3-glycidyloxypropyl)trimethoxysilane. Allergen solutions are immobilized as small droplets (approximately 10 nL) on the activated glass slides with a piezoelectric arrayer. In contrast to other tests for specific IgE, such as Pharmacia CAP FEIA, AlaSTAT, or FAST, only a 25-microL serum sample is needed for the screening of allergen-specific IgE against a multitude of allergens and the test can be performed in less than 1 h. Compared with multiallergen dipstick screening tests (e.g., IgEquick, CMG Immunodot) based on multiallergen-coated nitrocellulose strips, the measurement of the microarray-based system can be performed automatically. The chemiluminescence intensities are detected with a sensitive CCD camera. Allergen extracts and recombinant/purified allergens (24 preparations) have been used on the same modified surface for the screening of allergen-specific IgE. With these disposable microarray slides, it is possible to distinguish between patients with and without elevated levels of allergen-specific IgE. Repeated measurements of serum samples demonstrated a sufficient reproducibility. Detection limits (microg/L) of 0.35 (r Betvl), 0.16 (PLA2), and 1.9 (Der p1) were achieved.

Wenn sich giftige Algen stark vermehren, werden sie zu einer Gefahr fur Mensch und Tier. Die Komplexitat der Toxin-Analytik hat eine regelmasige Kontrolle von Algenbluten bisher verhindert. Inzwischen gibt es jedoch leistungsfahige immunologische Methoden.

Enzymatische und immunchemische Verfahren sowie Chemo- und Biosensoren prasentiert der abschliesende Teil des Trendberichts zur den wichtigsten Fortschritten in der Analytik der vergangenen zwei Jahre, Teil 1 beginnt auf Seite 455.

A monoclonal antibody (clone AD4G2) was generated against a common part of microcystins and nodularins, the unusual amino acid Adda [(2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4E,6E-dienoic acid]. A direct competitive ELISA based on this antibody was developed and the cross-reactivity pattern was measured. Different toxins showed a very similar response. The assay provides therefore a sum parameter of microcystins, nodularins and peptide fragments containing Adda. The IC50 for microcystin-LR was 0.33 μg L−1 which leads to a detection limit of 0.07 μg L−1. This is well below the concentration of 1 μg L−1 proposed by the World Health Organisation (WHO) as the limit for drinking water. Microcystin-LR spiked water samples in the concentration range between 0.1 and 1 μg L−1 were measured and a mean recovery of 113 ± 23% was found. The antibody is well suited for the determination of microcystins in drinking as well as surface water.

The production and characterization of a monoclonal antibody (clone (MC10E7)) with extraordinary sensitivity and high selectivity for [4-arginine]microcystins is described. The immunogen used for the production of the antibody was synthesized using a novel coupling chemistry to bind microcystin-LR(MC–LR) via dehydroalanine to the carrier protein. With a direct competitive enzyme-linked immunosorbent assay (ELISA) using MC10E7, IC50 values for MC–LR of 0.06 μg l−1 have been achieved. The detection limit for MC–LR was 6 ng l−1. The provisional guideline value proposed by the World Health Organization (WHO) is 1 μg l−1 for drinking water. All [4-arginine]microcystins show similar IC50 values and detection limits, whereas other MCs such as MC–LA, are not recognized. The affinity constant for MC10E7 was determined to be at least 7×1010 l mol−1. The antibody was tested for its robustness against interference of humic acids, pH, salt content, surfactants or organic solvents and was found to be very stable. MC–LR spiked water samples in the concentration range between 0.01 and 0.1 μg l−1 were measured and a mean recovery of 99.9±16.4% was found. The antibody is well suited for sensitive analysis for MCs in drinking as well as surface water.

Die Analytica in Munchen bietet alle zwei Jahre den richtigen Anlas, uber Veranderungen und Fortschritte in der Analytischen Chemie zu berichten. Dieser zweite Teil “Spektroskopie” vervollstandigt den Trendbericht “Analytische Chemie” [Nachr. Chem. 2000, 48, 348].

Die Analytica in Munchen bietet alle zwei Jahre den richtigen Anlas, uber die Veranderungen und Fortschritte in der Analytischen Chemie zu berichten. Die wichtigsten Stichworte im ersten Teil des Berichtes: Life Sciences, kombinierte Methoden, HTS, Chiptechniken…Ein Nachtrag “Spektroskopie” vervollstandigt im nachsten Monat den Trendbericht.

A test strip to quantify trinitrotoluene (TNT) in water has been developed using a homogenous apoenzyme reactivation immunoassay system (ARIS). In comparison with other test kits for the detection of TNT, the novel test strip is much simpler in application. The test strip has only to be dipped into the aqueous sample. A blue color develops on the reagent strips proportional to the TNT concentration. The concentration of TNT is determined either by visual comparison with a color card, or more precisely using a reflectometer. A measuring range of about 1–10,000 μg l−1 TNT in water was demonstrated. The reagent strips were prepared by sequential impregnation of a cellulose pad with two solutions, one based on acetone and the other based on aqueous buffer. All necessary components are included in the reagent strip in dry form.