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In this work, a cost-effective optofluidic system is proposed and preliminary experimental results are presented. A microfluidic channel monolithically integrated into a photonic integrated circuit technology is used in conjunction with a cyclic olefin copolymer (COC) substrate to provide fluidic in- and output ports. We report on initial experimental results as well as on the simple and cost-effective fabrication of this optofluidic system by means of micro-milling.
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.
A unifying review of bioassay-guided fractionation, effect-directed analysis and related techniques
(2012)
The success of modern methods in analytical chemistry sometimes obscures the problem that the ever increasing amount of analytical data does not necessarily give more insight of practical relevance. As alternative approaches, toxicity- and bioactivity-based assays can deliver valuable information about biological effects of complex materials in humans, other species or even ecosystems. However, the observed effects often cannot be clearly assigned to specific chemical compounds. In these cases, the establishment of an unambiguous cause-effect relationship is not possible. Effect-directed analysis tries to interconnect instrumental analytical techniques with a biological/biochemical entity, which identifies or isolates substances of biological relevance. Successful application has been demonstrated in many fields, either as proof-of-principle studies or even for complex samples. This review discusses the different approaches, advantages and limitations and finally shows some practical examples. The broad emergence of effect-directed analytical concepts might lead to a true paradigm shift in analytical chemistry, away from ever growing lists of chemical compounds. The connection of biological effects with the identification and quantification of molecular entities leads to relevant answers to many real life questions.
ADAMTS4-specific MR-probe to assess aortic aneurysms in vivo using synthetic peptide libraries
(2022)
The incidence of abdominal aortic aneurysms (AAAs) has substantially increased during the last 20 years and their rupture remains the third most common cause of sudden death in the cardiovascular field after myocardial infarction and stroke. The only established clinical parameter to assess AAAs is based on the aneurysm size. Novel biomarkers are needed to improve the assessment of the risk of rupture. ADAMTS4 (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 4) is a strongly upregulated proteoglycan cleaving enzyme in the unstable course of AAAs. In the screening of a one-bead-one-compound library against ADAMTS4, a low-molecular-weight cyclic peptide is discovered with favorable properties for in vivo molecular magnetic resonance imaging applications. After identification and characterization, it’s potential is evaluated in an AAA mouse model. The ADAMTS4-specific probe enables the in vivo imaging-based prediction of aneurysm expansion and rupture.
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.
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.
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.
High performance liquid chromatography (HPLC) was hyphenated with a previously reported carbamazepine-specific enzyme-linked immunosorbent assay (ELISA) as a screening approach to water analysis in order to identify possible interferences from transformation products. Treated wastewater was analysed and three substances were recognized by the antibody besides carbamazepine: the metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 2-hydroxycarbamazepine plus the structurally not obviously related antihistamine cetirizine. The molar cross-reactivity against cetirizine was found to be pH-dependent and assessed to be 400% at pH 4.5 and 22% at pH 10.5. Performing the ELISA at pH 10.5 greatly improved the accuracy when carbamazepine was determined in surface and wastewater samples.
Chemical modification by peroxynitrite enhances TLR4 activation of the grass pollen allergen Phl p 5
(2023)
The chemical modification of aeroallergens by reactive oxygen and nitrogen species (ROS/RNS) may contribute to the growing prevalence of respiratory allergies in industrialized countries. Post-translational modifications can alter the immunological properties of proteins, but the underlying mechanisms and effects are not well understood. In this study, we investigate the Toll-like receptor 4 (TLR4) activation of the major birch and grass pollen allergens Bet v 1 and Phl p 5, and how the physiological oxidant peroxynitrite (ONOO–) changes the TLR4 activation through protein nitration and the formation of protein dimers and higher oligomers. Of the two allergens, Bet v 1 exhibited no TLR4 activation, but we found TLR4 activation of Phl p 5, which increased after modification with ONOO– and may play a role in the sensitization against this grass pollen allergen. We attribute the TLR4 activation mainly to the two-domain structure of Phl p 5 which may promote TLR4 dimerization and activation. The enhanced TLR4 signaling of the modified allergen indicates that the ONOO–-induced modifications affect relevant protein-receptor interactions. This may lead to increased sensitization to the grass pollen allergen and thus contribute to the increasing prevalence of allergies in the Anthropocene, the present era of globally pervasive anthropogenic influence on the environment.
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.
Cardiac troponin I (cTnI) is a crucial biomarker for diagnosing cardiac vascular diseases, including acute myocardial infarction (AMI). This study presents a proof-of-concept chemiluminescence-based immunosensor for rapid and accurate measurement of cTnI, with the potential for online monitoring. The immunosensor incorporates a flow cell design and a sensitive complementary metal-oxide-semiconductor (CMOS) camera for optical readout. A microfluidic setup was established to enable selective and quasi-online determination of cTnI within ten minutes. The sensor was tested with recombinant cTnI in phosphate buffer, demonstrating measurements in the concentration range of 2–25 µg/L, with a limit of detection (LoD) of 0.6 µg/L (23 pmol/L) achieved using the optimized system. The immunosensor exhibited high selectivity, as no cross-reactivity was observed with other recombinant proteins such as cTnT and cTnC at a concentration of 16 µg/L. Measurements with diluted blood plasma and serum yielded an LoD of 60 µg/L (2.4 nmol/L) and 70 µg/L (2.9 nmol/L), respectively. This biosensor offers a promising approach for the rapid and sensitive detection of cTnI, contributing to the diagnosis and management of acute myocardial infarction and other cardiac vascular diseases.
CMOS-Compatible Silicon Photonic Sensor for Refractive Index Sensing Using Local Back-Side Release
(2020)
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.
The trafficking of illegal drugs by criminal networks at borders, harbors, or airports is an increasing issue for public health as these routes ensure the main supply of illegal drugs. The prevention of drug smuggling, including the installation of scanners and other analytical devices to detect small traces of drugs within a reasonable time frame, remains a challenge. The presented immunosensor is based on a monolithic affinity column with a large excess of immobilized hapten, which traps fluorescently labeled antibodies as long as the analyte cocaine is absent. In the presence of the drug, 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 microfluidic chip-based mixing devices and flow cells. The biosensor achieved limits of detection of 7 ppt (23 pM) of cocaine with a response time of 90 s and a total assay time below 3 min. With surface wipe sampling, the biosensor was able to detect 300 pg of cocaine. This immunosensor belongs to the most sensitive and fastest detectors for cocaine and offers near-continuous analyte measurement.
Phage display biopanning with Illumina next-generation sequencing (NGS) is applied to reveal insights into peptide-based adhesion domains for polypropylene (PP). One biopanning round followed by NGS selects robust PP-binding peptides that are not evident by Sanger sequencing. NGS provides a significant statistical base that enables motif analysis, statistics on positional residue depletion/enrichment, and data analysis to suppress false-positive sequences from amplification bias. The selected sequences are employed as water-based primers for PP-metal adhesion to condition PP surfaces and increase adhesive strength by 100% relative to nonprimed PP.
An immunoassay was developed for ochratoxin A (OTA) detection in wine and two completely different detection methods were compared: ICP-MS and photometry. For labelling, we applied secondary antibodies conjugated with gold nanoparticles for ICP-MS and with horseradish peroxidase for photometric detection, respectively. Detection limits of the assay were determined as 0.003 µg L-1 for both detection methods. OTA in wine was determined below the EU limit value of 2 µg kg-1. The measurement range was between 0.01 and 1 µg L-1 for ICP-MS and photometric detection. Assay precision detected by ICP-MS and photometry were similar showing that precision of the detection method has only a minor effect on total assay precision. By using BSA as a buffer additive to minimize nonspecific binding, we could also confirm that OTA strongly binds to BSA. In a comparison study of several additives, casein was found to be preferable.
Comprehensive evaluation of peptide de novo sequencing tools for monoclonal antibody assembly
(2023)
Monoclonal antibodies are biotechnologically produced proteins with various applications in research, therapeutics and diagnostics. Their ability to recognize and bind to specific molecule structures makes them essential research tools and therapeutic agents. Sequence information of antibodies is helpful for understanding antibody–antigen interactions and ensuring their affinity and specificity. De novo protein sequencing based on mass spectrometry is a valuable method to obtain the amino acid sequence of peptides and proteins without a priori knowledge. In this study, we evaluated six recently developed de novo peptide sequencing algorithms (Novor, pNovo 3, DeepNovo, SMSNet, PointNovo and Casanovo), which were not specifically designed for antibody data. We validated their ability to identify and assemble antibody sequences on three multi-enzymatic data sets. The deep learning-based tools Casanovo and PointNovo showed an increased peptide recall across different enzymes and data sets compared with spectrum-graph-based approaches. We evaluated different error types of de novo peptide sequencing tools and their performance for different numbers of missing cleavage sites, noisy spectra and peptides of various lengths. We achieved a sequence coverage of 97.69–99.53% on the light chains of three different antibody data sets using the de Bruijn assembler ALPS and the predictions from Casanovo. However, low sequence coverage and accuracy on the heavy chains demonstrate that complete de novo protein sequencing remains a challenging issue in proteomics that requires improved de novo error correction, alternative digestion strategies and hybrid approaches such as homology search to achieve high accuracy on long protein sequences.
Fast and accurate determination of the protein content of a sample is an important and non-trivial task of many biochemical, biomedical, food chemical, pharmaceutical, and environmental research activities. Different methods of total protein determination are used for a wide range of proteins with highly variable properties in complex matrices. These methods usually work reasonably well for proteins under controlled conditions, but the results for non-standard and complex samples are often questionable. Here, we compare new and well-established methods, including traditional amino acid analysis (AAA), aromatic amino acid analysis (AAAA) based on the amino acids phenylalanine and tyrosine, reversed-phase liquid chromatography of intact proteins with UV absorbance measurements at 220 and 280 nm (LC-220, LC-280), and colorimetric assays like Coomassie Blue G-250 dye-binding assay (Bradford) and bicinchoninic acid (BCA) assay. We investigated different samples, including proteins with challenging properties, chemical modifications, mixtures, and complex matrices like air particulate matter and pollen extracts. All methods yielded accurate and precise results for the protein and matrix used for calibration. AAA, AAAA with fluorescence detection, and the LC-220 method yielded robust results even under more challenging conditions (variable analytes and matrices). These methods turned out to be well-suited for reliable determination of the protein content in a wide range of samples, such as air particulate matter and pollen.
The employment of type-I pyrethroids for airplane disinfection in recent years underlines the necessity to develop sensing schemes for the rapid detection of these pesticides directly at the point-of-use. Antibody-gated indicator-releasing materials were thus developed and implemented with test strips for lateral-flow assay-based analysis employing a smartphone for readout. Besides a proper matching of pore sizes and gating macromolecules, the functionalization of both the material's outer surface as well as the strips with PEG chains enhanced system performance. This simple assay allowed for the detection of permethrin as a target molecule at concentrations down to the lower ppb level in less than 5 minutes.
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 (TATP), ammonium nitrate, 2,4,6-trinitrophenol and nitrobenzene were tested for potential cross-reactivity. The detection limit of a competitive enzyme-linked immunosorbent assay (ELISA) was determined to be around 0.5 µg/L. The dynamic range of the assay was found to be between 1 µg/L 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.
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.
Current trends in materials and life sciences are flanked by the need to push detection limits to single molecules or single cells, enable the characterization of increasingly complex matrices or sophisticated nanostructures, speed up the time of analysis, reduce instrument complexity and costs, and improve the reliability of data. This requires suitable analytical tools such as spectroscopic, separation and imaging techniques, mass spectrometry, and hyphenated techniques as well as sensors and their adaptation to application-specific challenges in the environmental, food, consumer product, health sector, nanotechnology, and bioanalysis. Increasing concerns about health threatening known or emerging pollutants in drinking water, consumer products, and food and about the safety of nanomaterials led to a new awareness of the importance of analytical sciences. Another important driver in this direction is the increasing demand by legislation, particularly in view of the 17 sustainable development goals by the United Nations addressing clean energy, industry, and innovation, sustainable cities, clean water, and responsible consumption and production. In this respect, also the development of analytical methods that enable the characterization of material flows in production processes and support recycling concepts of precious raw materials becomes more and more relevant. In the future, this will provide the basis for greener production in the chemical industry utilizing recycled or sustainable starting materials.
This makes analytical chemistry an essential player in terms of the circular economy helping to increase the sustainability of production processes. In the life sciences sector, products based on proteins, such as therapeutic and diagnostic antibodies, increase in importance. These increasingly biotechnologically produced functional biomolecules pose a high level of complexity of matrix and structural features that can be met only by highly advanced methods for separation, characterization, and detection. In addition, metrological traceability and target definition are still significant challenges for the future, particularly in the life sciences.
However, innovative reference materials as required for the health and food sector and the characterization of advanced materials can only be developed when suitable analytical protocols are available. The so-called reproducibility crisis in sciences underlines the importance of improved measures of quality control for all kinds of measurements and material characterization. This calls for thorough method validation concepts, suitable reference materials, and regular interlaboratory comparisons of measurements as well as better training of scientists in analytical sciences.
The important contribution of analytical sciences to these developments is highlighted by a broad collection of research papers, trend articles, and critical reviews from these different application fields. Special emphasis is dedicated to often-overlooked quality assurance and reference materials.
The cowpea chlorotic mottle virus (CCMV) is a plant virus explored as a nanotechnological platform. The robust self-assembly mechanism of its capsid protein allows for drug encapsulation and targeted delivery. Additionally, the capsid nanoparticle can be used as a programmable platform to display different molecular moieties. In view of future applications, efficient production and purification of plant viruses are key steps. In established protocols, the need for ultracentrifugation is a significant limitation due to cost, difficult scalability, and safety issues. In addition, the purity of the final virus isolate often remains unclear. Here, an advanced protocol for the purification of the CCMV from infected plant tissue was developed, focusing on efficiency, economy, and final purity. The protocol involves precipitation with PEG 8000, followed by affinity extraction using a novel peptide aptamer. The efficiency of the protocol was validated using size exclusion chromatography, MALDI-TOF mass spectrometry, reversed-phase HPLC, and sandwich immunoassay. Furthermore, it was demonstrated that the final eluate of the affinity column is of exceptional purity (98.4%) determined by HPLC and detection at 220 nm. The scale-up of our proposed method seems to be straightforward, which opens the way to the large-scale production of such nanomaterials. This highly improved protocol may facilitate the use and implementation of plant viruses as nanotechnological platforms for in vitro and in vivo applications.
Immobilized metal affinity chromatography (IMAC) is a widely used technique for purifying polyhistidine-tagged recombinant proteins. However, it often has practical limitations that require complex optimizations and additional steps for purification. In this study, we introduce functionalized corundum particles as a novel, efficient, and economical method for purifying recombinant proteins in a column-free format. The corundum surface is modified with amino silane APTES, followed by EDTA dianhydride, and then loaded with nickel ions. We used the Kaiser test to monitor the modification process and ICP-MS to quantify the metal-binding capacity. To evaluate the system, we used His-tagged protein A/G (PAG) mixed with bovine serum albumin (BSA). The corundum particles exhibited a binding capacity of approximately 3 mg of protein per gram of corundum or 2.4 mg per 1 mL of corundum suspension. We also examined cytoplasm obtained from different E. coli strains as an example of a complex matrix. Varying the imidazole concentration in the loading and washing buffers showed that higher concentrations during loading improved purity. Even with sample volumes as large as one liter, we successfully isolated recombinant proteins down to a concentration of 1 µg/mL. We found higher purity levels with corundum when comparing the corundum material to standard Ni–NTA agarose beads. We successfully purified His6-MBP-mSA2, a fusion protein comprising monomeric streptavidin and maltose-binding protein, from E. coli cytoplasm, demonstrating the method's applicability. We also purified SARS-CoV-2-S-RBD-His8 expressed in human Expi293F cells, confirming its suitability for mammalian cell culture supernatants. The material cost of the nickel-loaded corundum material (without regeneration) is estimated to be less than 30 cents per gram of functionalized support or 10 cents per milligram of isolated protein. Another advantage of this system is the exceptional physical and chemical stability of corundum particles. Overall, we have demonstrated that this novel material offers an efficient, robust, and cost-effective purification platform for His-tagged proteins, even in challenging, complex matrices and large sample volumes with low product concentrations. This method has potential applications in both small laboratories and large-scale industrial settings.
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 “all-on-one chip” 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.
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) × 109l/mol (rabbit R1) and (2.3 ± 0.2) × 109l/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.
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. Nearly all commercial antibody suppliers also 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, as well as to assign a specific reagent to a datasheet of a commercial supplier, public database record, or antibody ID.
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.
Nonporous corundum powder, known as an abrasive material in the industry, was functionalized covalently with protein binders to isolate and enrich specific proteins from complex matrices. The materials based on corundum were characterized by TEM, ESEM, BET, DLS, EDS, and zeta potential measurements. The strong Al-O-P bonds between the corundum surface and amino phosphonic acids were used to introduce functional groups for further conjugations. The common crosslinker glutaraldehyde was compared with a hyperbranched polyglycerol (PG) of around 10 kDa. The latter was oxidized with periodate to generate aldehyde groups that can covalently react with the amines of the surface and the amino groups from the protein via a reductive amination process. The amount of bound protein was quantified via aromatic amino acid analysis (AAAA). This work shows that oxidized polyglycerol can be used as an alternative to glutaraldehyde. With polyglycerol, more of the model protein bovine serum albumin (BSA) could be attached to the surface under the same conditions, and lower non-specific binding (NSB) was observed. As a proof of concept, IgG was extracted with protein A from crude human plasma. The purity of the product was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A binding capacity of 1.8 mg IgG per gram of corundum powder was achieved. The advantages of corundum include the very low price, extremely high physical and chemical stability, pressure resistance, favorable binding kinetics, convenient handling, and flexible application.
Algal blooms are a frequent phenomenon in nearly all kinds of fresh water. Global warming and eutrophication by waste water, air pollution and fertilizers seem to lead to an increased frequency of occurrence. Many cyanobacteria produce hazardous and quite persistent toxins, which can contaminate the respective water bodies. This may limit the use of the raw water for many purposes. The purification of the contaminated water might be quite costly, which makes a continuous and large scale treatment economically unfeasible in many cases. Due to the obvious risks of algal toxins, an online or mobile detection method would be highly desirable. Several biosensor systems have been presented in the literature for this purpose. In this review, their mode of operation, performance and general suitability for the intended purpose will be described and critically discussed. Finally, an outlook on current developments and future prospects will be given.
Carbamazepine (CBZ), caffeine and cetirizine were monitored by enzyme-linked immunosorbent assays (ELISAs) in surface and wastewaters from Berlin, Germany. This fast and cost-efficient method enabled to assess the spatial and temporal variation of these anthropogenic markers in a high-throughput screening. CBZ and cetirizine were detected by the same antibody, which selectively discriminates between both compounds depending on the pH value used in the incubation step. To our best knowledge, this is the first dual-analyte immunoassay working with a single antibody.
The frequent sampling with 487 samples being processed allowed for the repeated detection of unusually high concentrations of CBZ and caffeine. ELISA results correlate well with the ones obtained by liquid chromatography tandem mass spectrometry (LC-MS/MS). Caffeine concentrations found in surface waters were elevated by combined sewer overflows after stormwater events. During the hay fever season, the concentrations of the antihistamine drug cetirizine increased in both surface and wastewaters.
Caffeine was almost completely removed during wastewater treatment, while CBZ and cetirizine were found to be more persistent. The maximum concentrations of caffeine, CBZ and cetirizine found in influent wastewater by LCMS/MS were 470, 5.0 and 0.49 µg L-1, while in effluent wastewater the concentrations were 0.22, 4.5 and 0.51 µg L-1, respectively. For surface waters, concentrations up to 3.3, 4.5 and 0.72 µg L-1 were found, respectively.
In this work, the optimization of the immunochemical response of antibody-gated indicator delivery (gAID) systems prepared with mesoporous silica nanoparticles has been studied along various lines of system tailoring, targeting the peroxide-type explosive TATP as an exemplary analyte. The mechanism of detection of these gAID systems relies on a displacement of an antibody “cap” bound to hapten derivatives anchored to the surface of a porous hybrid material, allowing the indicator cargo stored in the mesopores to escape and massively amplify the analyte-related signal. Since our aim was to obtain gAID systems with the best possible response in terms of sensitivity, selectivity, and assay time, sera obtained from different immunization boosts were screened, the influence of auxiliary reagents was assessed, structural hapten modification (hapten heterology) was investigated, and various indicator dyes and host materials were tested. Considering that highly selective and sensitive immunological responses are best obtained with high-affinity antibodies which, however, could possess rather slow dissociation constants, leading to slow responses, the main challenge was to optimize the immunochemical recognition system for a rapid response while maintaining a high sensitivity and selectivity. The best performance was observed by grafting a slightly mismatching (heterologous) hapten to the surface of the nanoparticles in combination with high-affinity antibodies as “caps”, yielding for the first time gAID nanomaterials for which the response time could be improved from hours to <5 min. The materials showed favorable detection limits in the lower ppb range and discriminated TATP well against H2O2 and other explosives. Further optimization led to straightforward integration of the materials into a lateral flow assay without further treatment or conditioning of the test strips while still guaranteeing remarkably fast overall assay times.
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 an 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 an 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.
Hepcidin-25 was identified as the main 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 (tandem mass spectrometry (MS/MS), high-resolution mass 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 reference material 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.
An airborne high repetition rate laser-induced plasma was applied as a versatile ambient ionization source for mass-spectrometric determinations of polar and nonpolar analytes in solution. The laser plasma was sustained between a home-built pneumatic nebulizer and the inlet capillary of an Orbitrap mass spectrometer. To maintain stable conditions in the droplet-rich spray environment, the plasma was directly fed by the fundamental output (λ = 1064 nm) of a current state-of-the-art diode-pumped solid-state laser. Ionization by the laser-driven plasma resulted in signals of intact analyte ions of several chemical categories. The analyte ions were found to be fully desolvated since no further increase in ion signal was observed upon heating of the inlet capillary. Due to the electroneutrality of the plasma, both positive and negative analyte ions could be formed simultaneously without altering the operational parameters of the ion source. While, typically, polar analytes with pronounced gas phase basicities worked best, nonpolar and amphoteric compounds were also detected. The latter were detected with lower ion signals and were prone to a certain degree of fragmentation induced during the ionization process. All the described attests the laser-induced microplasma by a good performance in terms of stability, robustness, sensitivity, and general applicability as a self-contained ion source for the liquid sample introduction.
MALDI-TOF-MS-Based Identification of Monoclonal Murine Anti-SARS-CoV-2 Antibodies within One Hour
(2022)
During the SARS-CoV-2 pandemic, many virus-binding monoclonal antibodies have been developed for clinical and diagnostic purposes. This underlines the importance of antibodies as universal bioanalytical reagents. However, little attention is given to the reproducibility crisis that scientific studies are still facing to date. In a recent study, not even half of all research antibodies mentioned in publications could be identified at all. This should spark more efforts in the search for practical solutions for the traceability of antibodies. For this purpose, we used 35 monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied to the protein. First, we examined the intact and light chain masses of the antibodies relative to the reference material NIST-mAb 8671. Already half of the antibodies could be identified based solely on these two parameters. In addition, we developed two complementary peptide mass fingerprinting methods with MALDI-TOF-MS that can be performed in 60 min and had a combined sequence coverage of over 80%. One method is based on the partial acidic hydrolysis of the protein by 5 mM of sulfuric acid at 99 degrees C. Furthermore, we established a fast way for a tryptic digest without an alkylation step. We were able to show that the distinction of clones is possible simply by a brief visual comparison of the mass spectra. In this work, two clones originating from the same immunization gave the same fingerprints. Later, a hybridoma sequencing confirmed the sequence identity of these sister clones. In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0. This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context.
Besides protein identification via mass spectrometric methods, protein and peptide quantification has become more and more important in order to tackle biological questions. Methods like differential gel electrophoresis or enzyme-linked immunosorbent assays have been used to assess protein concentrations, while stable isotope labeling methods are also well established in quantitative proteomics. Recently, we developed metalcoded affinity tagging (MeCAT) as an alternative for accurate and sensitive quantification of peptides and
proteins. In addition to absolute quantification via inductively coupled plasma mass spectrometry, MeCAT also enables sequence analysis via electrospray ionization tandem mass spectrometry. In the current study, we developed a new labeling approach utilizing an iodoacetamide MeCAT reagent (MeCAT-IA). The MeCAT-IA approach shows distinct advantages over the previously used MeCAT with maleinimide reactivity such as higher labelling efficiency and the lack of diastereomer formation during labeling. Here, we present a careful characterization of this new method focusing on the labeling process, which yields complete tagging with an excess of reagent of 1.6 to 1, less complex chromatographic behavior, and fragmentation characteristics of the tagged peptides using the iodoacetamide MeCAT reagent.
Nicht zuletzt bedingt durch den Pferdefleischskandal im Jahr 2013 wurden in Deutschland die Aktivitäten bei der Methodenstandardisierung im Bereich der Tierarten-Differenzierung in den vergangenen Jahren intensiviert. Wenn auch positive Befunde bei dem Nachweis von Bestandteilen aus Pferd in Lebensmitteln wie Lasagne derzeit so gut wie nicht mehr anzutreffen sind, so hat die Tierartendifferenzierung insgesamt beim Nachweis von Verfälschungen in Lebensmitteln einen hohen Stellenwert.
Diese Arbeit fasst daher den aktuellen Stand der Analytik in Deutschland mit Schwerpunkt bei der Standardisierung zusammen. Sie wurde erstellt durch die Arbeitsgruppe „Biochemische und molekularbiologische Analytik“ der Lebensmittelchemischen Gesellschaft mit Unterstützung von Experten der Arbeitsgruppe „Molekularbiologische Methoden zur Pflanzen- und Tierartendifferenzierung“ (§ 64 LFGB) sowie der ALTS-Arbeitsgruppe „Immunologie und Molekularbiologie“ (jeweils D).
In the field of animal species differentiation, work on standardizing methods has been intensified in Germany in recent years, not least due to the horsemeat scandal in 2013. Even though there are now hardly ever any positive findings anymore in examinations to detect horse adulterations in foods such as lasagne, animal species differentiation altogether ranks high in detecting adulteration of foods. This article, therefore, summarises the current status of analytical techniques used in Germany with standardization at German level. It has been established by the working group "Biochemical and Molecular Biological Analytics" of the Lebensmittelchemische Gesellschaft (Food Chemistry Society within the German Chemical Society) with support of experts in the working group “Molecular biology techniques for differentiating plant and animal species" (§64 of the German Food and Feed Code - LFGB) and the "Immunology and molecular biology" task force of the food hygiene and food of animal origin working group (ALTS), both from Germany.
Work on standardising methods in the field of animal species differentiation has been intensified in Germany in recent years, not least due to the horsemeat scandal in 2013. Even though there are now hardly ever any positive findings any more in examinations to detect horse adulterations in foods such as lasagne, animal species differentiation altogether ranks high in detecting adulteration of foods. This article therefore summarises the current status of analytical techniques used in Germany with standardisation at German level. It has been established by the working group “Biochemical and Molecular Biological Analytics” of the Lebensmittelchemische Gesellschaft (Food Chemistry Society within the German Chemical Society) with support of experts in the working group “Molecular biology techniques for differentiating plant and animal species” (§ 64 of the German Food and Feed Code – LFGB) and the “Immunology and molecular biology” task force of the food hygiene and Food of animal origin working group (ALTS), both from Germany.
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.
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 method to accurately and precisely determine caffeine in saliva samples.
Monitoring carbamazepine in surface and wastewaters by an immunoassay based on a monoclonal antibody
(2009)
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 µg/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 µg/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.
Rapid testing methods for the use directly at apointof need are expected to unfold their true potential especiallywhen offering adequate capabilities for the simultaneousmeasurement of multiple analytes of interest. Considering theunique modularity,high sensitivity,and selectivity of antibody-gated indicator delivery (gAID) systems,amultiplexed assayfor three small-molecule explosives (TATP, TNT,PETN) wasthus developed, allowing to detect the analytes simultaneouslywith asingle test strip at lower ppb concentrations in the liquidphase in < 5min using afluorescence reader or asmartphonefor readout. While the TNT and PETN systems were newlydeveloped here,all the three systems also tolerated harshermatrices than buffered aqueous model solutions.Besidesasingle-track strip,the outstanding modularity of the hybridbiosensor materials in combination with strip-patterningtechnologies allowed us to obtain amultichannel strip inastraightforwardmanner,offering comparable analyticalperformance while allowing to be tailored even more to theusersneed.
A non-invasive method to monitor the humoral immune response in mice after immunization is described. From fecal pellets of an individual mouse, a sufficient amount of active immunoglobulins or their fragments can be extracted to perform a regular examination of the status of the immune response by immunoassay. Hapten-specific antibodies from the feces of mice from three immunization trials showed very similar characteristics to those obtained from serum at a given date. Therefore, it can be suspected that some serum IgG enters the intestinal lumen and ends up in the feces, where they appear to be considerably stable. Hapten-specific IgAs were not found in the feces. Being able to analyze antibody titers in feces could be an interesting animal welfare refinement to standard practice that does not entail repeated blood sampling.
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.
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.
The allergenic and inflammatory potential of proteins can be enhanced by chemical modification upon exposure to atmospheric or physiological oxidants. The molecular mechanisms and kinetics of such modifications, however, have not yet been fully resolved. We investigated the oligomerization and nitration of the grass pollen allergen Phl p 5 by ozone (O3), nitrogen dioxide (NO2), and peroxynitrite (ONOO–). Within several hours of exposure to atmospherically relevant concentration levels of O3 and NO2, up to 50% of Phl p 5 were converted into protein oligomers, likely by formation of dityrosine cross-links. Assuming that tyrosine residues are the preferential site of nitration, up to 10% of the 12 tyrosine residues per protein monomer were nitrated. For the reaction with peroxynitrite, the largest oligomer mass fractions (up to 50%) were found for equimolar concentrations of peroxynitrite over tyrosine residues. With excess peroxynitrite, the nitration degrees increased up to 40% whereas the oligomer mass fractions decreased to 20%. Our results suggest that protein oligomerization and nitration are competing processes, which is consistent with a two-step mechanism involving a reactive oxygen intermediate (ROI), as observed for other proteins. The modified proteins can promote pro-inflammatory cellular signaling that may contribute to chronic inflammation and allergies in response to air pollution.
Protein modifications such as oligomerization and tyrosine nitration alter the immune response to allergens and may contribute to the increasing prevalence of allergic diseases. In this mini-review, we summarize and discuss relevant findings for the major birch and grass pollen allergens Bet v 1 and Phl p 5 modified with tetranitromethane (laboratory studies), peroxynitrite (physiological processes), and ozone and nitrogen dioxide environmental conditions). We focus on tyrosine nitration and the formation of protein dimers and higher oligomers via dityrosine cross-linking and the immunological effects studied.