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Although most cancer drugs modulate the activities of cellular pathways by changing posttranslational modifications (PTMs), little is known regarding the extent and the time- and dose-response characteristics of drug-regulated PTMs. In this work, we introduce a proteomic assay called decryptM that quantifies drug-PTM modulation for thousands of PTMs in cells to shed light on target engagement and drug mechanism of action. Examples range from detecting DNA damage by chemotherapeutics, to identifying drug-specific PTM signatures of kinase inhibitors, to demonstrating that rituximab kills CD20-positive B cells by overactivating B cell receptor signaling. DecryptM profiling of 31 cancer drugs in 13 cell lines demonstrates the broad applicability of the approach. The resulting 1.8 million dose-response curves are provided as an interactive molecular resource in ProteomicsDB.

Our group has previously developed immunoassays for noncompetitive detection of small molecules based on the use of phage borne anti-immunocomplex peptides. Recently, we substituted the phage particles by biotinylated synthetic anti-immunocomplex peptides complexed with streptavidin, and named these constructs nanopeptamers. In this work, we report the results of combining AlphaLisa, a commercial luminescent oxygen channeling bead system, with nanopeptamers for the development of a noncompetitive homogeneous assay for the detection of small molecules. The signal generation of AlphaLisa assays relies on acceptor-donor bead proximity induced by the presence of the analyte (a macromolecule) simultaneously bound by antibodies immobilised on the surface of these beads. In the developed assay, termed as nanoAlphaLisa, bead proximity is sustained by the presence of a small model molecule (Atrazine, MW = 215) using an anti-atrazine antibody captured on the acceptor bead, and an atrazine nanopeptamer on the donor bead. Atrazine, is one of the most used pesticides worldwide and its monitoring in water has relevant human health implications. NanoAlphaLisa allowed the homogeneous detection of atrazine down to to 0.3 ng/mL in undiluted water samples in one hour, which is ten-fold below the accepted limit in drinking water. NanoAlphaLisa has the intrinsic advantages for automation, high-throughput, simple, and fast homogeneous detection of target analytes that AlphaLisa assay provides.

We describe ProteomeTools, a project building molecular and digital tools from the human proteome to facilitate biomedical research. Here we report the generation and multimodal liquid chromatography-tandem mass spectrometry analysis of >330,000 synthetic tryptic peptides representing essentially all canonical human gene products, and we exemplify the utility of these data in several applications. The resource (available at http://www.proteometools.org) will be extended to >1 million peptides, and all data will be shared with the community via ProteomicsDB and ProteomeXchange.

Colorectal cancer (CRC) is one of the top three most common cancers and among the top four causes of cancer-related deaths worldwide (Torre et al., 2015). CRC patients are well characterised on the transcriptome and proteome level, but proteomics data on representative cell lines as model systems for pre-clinical drug sensitivity studies lag behind. Here, label-free quantitative mass spectrometry was used to characterise the kinomes and full proteomes of 65 CRC cell lines, collectively termed the CRC65 cell line panel. This data was integrated with proteomics data on patient samples, as well as public transcriptome and drug sensitivity datasets, which were reanalysed from raw data in order to unify and streamline the data processing. Protein/mRNA ratios were constant across these datasets, enabling linear prediction of protein abundance from mRNA abundance after appropriate adjustment, which was used for mRNA-guided missing value imputation. An exploration of secondary imputation methods prompted the development of a complementary method for minimum-guided missing value imputation. Combining the proteomics datasets on cell lines and patients led to the discovery of integrated proteomic subtypes of CRC and enabled the identification of representative cell lines for each of them. Modelling publicly available dose-response data generated by four large-scale drug sensitivity studies as a function of kinome/full proteome profiles fuelled the prediction of drug sensitivity for cell lines and patients, allowed the identification of drugs differentially effective between the different integrated proteomic subtypes and revealed MERTK as a predictive biomarker for resistance towards MEK1/2 inhibitors. This predictive role of MERTK was subsequently confirmed using in vitro experiments, while immunohistochemistry of TMAs from 1,074 tumours generated as part of the QUASAR2 clinical trial unveiled that MERTK is also a prognostic biomarker in CRC. This dataset will be made available to the scientific community to facilitate the design of prospective clinical studies.

A method for identifying probe modification of proteins via tandem mass spectrometry was developed. Azide bearing molecules are immobilized on functionalised sepharose beads via copper catalysed Huisgen-type click chemistry and selectively released under acidic conditions by chemical cleavage of the triazene linkage. We applied this method to identify the modification site of targeted-diazotransfer on BirA.

An atlas for drug interactions Kinase inhibitors are an important class of drugs that block certain enzymes involved in diseases such as cancer and inflammatory disorders. There are hundreds of kinases within the human body, so knowing the kinase “target” of each drug is essential for developing successful treatment strategies. Sometimes clinical trials can fail because drugs bind more than one target. Yet sometimes off-target effects can be beneficial, and drugs can be repurposed for treatment of additional diseases. Klaeger et al. performed a comprehensive analysis of 243 kinase inhibitors that are either approved for use or in clinical trials. They provide an open-access resource of target summaries that could help researchers develop better drugs, understand how existing drugs work, and design more effective clinical trials. Science , this issue p. eaan4368

The receptor tyrosine kinase DDR1 has been implicated in multiple human cancers and fibrosis and is targeted by the leukemia drug Gleevec. This suggests that DDR1 might be a new therapeutic target. However, further insight into the DDR1 signaling pathway is required in order to support its further development. Here, we investigated DDR1 proximal signaling by the analysis of protein-protein interactions using proteomic approaches. All known interactors of DDR1 were identified and localized to specific phosphotyrosine residues on the receptor. In addition, we identified numerous signaling proteins as new putative phosphotyrosine mediated interactors including RasGAP, SHIP1, SHIP2, STATs, PI3K and the SRC family kinases. Most of the new proteins contain SH2 and PTB domains and for all interactors we could directly point the site of interaction to specific phosphotyrosine residues on the receptor. The identified proteins have roles in the early steps of the signaling cascade, propagating the signal from the DDR1 receptor into the cell. The map of phosphotyrosine mediated interactors of DDR1 created in this study will serve as a starting point for functional investigations which will enhance our knowledge on the role of the DDR1 receptor in health and disease. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.

The development of a monoclonal antibody enables a specific and sensitive detection of a potentially allergenic epitope from proglycinin in soy and constitutes an appropriate tool for analytical purposes in medical diagnostics and food labeling. Proglycinin was utilized as immunogen. The most favorable antibody G17E detected in a hybridoma cell culture screening was mass produced, purified, and used for immunoaffinity purification of proglycinin. In a heterogeneous, non-competitive ELISA format, a 50% ELISA inhibition point of 13 µg/L for purified proglycinin was determined. Cross-reactivity with homologous epitopes could be excluded. Specificity of G17E was determined by epitope mapping.

Solid supported probes have proven to be an efficient tool for chemical proteomics. The kinobeads technology features kinase inhibitors covalently attached to Sepharose for affinity enrichment of kinomes from cell or tissue lysates. This technology, combined with quantitative mass spectrometry, is of particular interest for the profiling of kinase inhibitors. It often leads to the identification of new targets for medicinal chemistry campaigns where it allows a two-in-one binding and selectivity assay. The assay can also uncover resistance mechanisms and molecular sources of toxicity. Here we report on the optimization of the kinobead assay resulting in the combination of five chemical probes and four cell lines to cover half the human kinome in a single assay (∼ 260 kinases). We show the utility and large-scale applicability of the new version of kinobeads by reprofiling the small molecule kinase inhibitors Alvocidib, Crizotinib, Dasatinib, Fasudil, Hydroxyfasudil, Nilotinib, Ibrutinib, Imatinib, and Sunitinib.

Selectivity and sensitivity are considered as pivotal criteria for the quality of immunochemical assay designs in environmental analysis. They are essentially determined by the variable domains of the implemented antibody. The variable domains of a triazine-selective single-chain Fv (scFv) were genetically engineered by stringent molecular evolution in order to optimize analytical characteristics of the corresponding atrazine immunoassay. Gene variation of the template antibody by sequential shuffling against the variable heavy and light chain repertoire of a triazine-selective immunoglobulin library was enhanced by introducing additional point mutatons. Improved scFv variants were selected by phage display employing an atrazine derivative. By this means the paramounting affinity of the initial scFv to sebuthylazine was shifted for the mutant antibodies toward a preferential recognition of the envisaged target analyte atrazine. In addition, the detection limit of the atrazine assaywas significantly improved by factor 25 from 5.1 microg/L for the initial template antibody to 0.2 microg/L for the mutant antibodies. The contribution of the engineered antibody variants to the assay improvement is also reflected by a shift of the equilibrium dissociation constant KD from 1.27 x 10(-8) M of the template antibody to 7.46 x 10(-10) M of the optimized variant. Sequence analysis revealed a bias of amino acid substitutions in the first two complementarity-determining regions (CDR) and the flanking framework regions of both variable chains for the shuffled clones as well as a deletion in the CDR3 of the light chain. Particularly the mutations of the VL domain turned out to have a decisive impact on the alterations in the analytical performance of the engineered scFv mutants. The application of the mutant antibodies for the atrazine determination of soil samples revealed consistency with HPLC data within the experimental error.

A system was constructed for the production of alkaline phosphatase (aP)-labeled antibody single-chain Fv (scFv) fragments in Escherichia coli. The expression vector pASK75 was modified by sequentially inserting the E. coli aP coding region and the scFv cloning cassette. Engineering the cloning sites SfiI and NotI located at the 5' and 3' end of the scFv gene provides an easy means to insert scFv fragments. These cloning sites are widely used in recombinant antibody technology and, thus, enable the one-step cloning of scFv fragments derived from corresponding antibody phage libraries into the expression vector. An expressed herbicide-specific scFv aP fusion protein retained both, analyte binding and enzymatic activity, as determined by ELISA. Therefore, this system permits the production of scFv-aP conjugates in E. coli, which can replace conventionally prepared aP-labeled antibodies in immunoassays. These fusion proteins are designed to accelerate the immunochemical detection of analytes, since the assay duration is essentially reduced by omitting the use of enzyme labeled secondary antibodies.

Summary Listeria monocytogenes phage endolysins Ply118 and Ply500 share a unique enzymatic activity and specifically hydrolyse Listeria cells at the completion of virus multiplication in order to release progeny phage. With the aim of determining the molecular basis for the lytic specificity of these enzymes, we have elucidated their domain structure and examined the function of their unrelated and unique C-terminal cell wall binding domains (CBDs). Analysis of deletion mutants showed that both domains are needed for lytic activity. Fusions of CBDs with green fluorescent protein (GFP) demonstrated that the C-terminal 140 amino acids of Ply500 and the C-terminal 182 residues of Ply118 are necessary and sufficient to direct the murein hydrolases to the bacterial cell wall. CBD500 was able to target GFP to the surface of Listeria cells belonging to serovar groups 4, 5 and 6, resulting in an even staining of the entire cell surface. In contrast, the CBD118 hybrid bound to a ligand predominantly present at septal regions and cell poles, but only on cells of serovars 1/2, 3 and 7. Non-covalent binding to surface carbohydrate ligands occurred in a rapid, saturation-dependent manner. We measured 4 × 104 and 8 × 104 binding sites for CBD118 and CBD500 respectively. Surface plasmon resonance analysis revealed unexpected high molecular affinity constants for the CBD–ligand interactions, corresponding to nanomolar affinities. In conclusion, we show that the CBDs are responsible for targeting the phage endolysins to their substrates and function to confer recognition specificity on the proteins. As the CBD sequences contain no repeats and lack all known sequence motifs for anchoring of proteins to the bacterial cell, we conclude that they use unique structural motifs for specific association with the surface of Gram-positive bacteria.

Fab antibody fragments were constructed by subcloning single chain Fv variable regions from the phagemid vector pCANTAB 5E into the expression vector pASK99. The vector was designed for bacterial secretion of Fab fragments and bears coding sequences for murine constant domains including the Strep-tag II at the carboxyl-terminal end of the constant heavy chain domain. The cloning procedure was carried out with the scFv antibodies IPR-7, IPR-53 and IPR-23. The second and third clone originated from the molecular evolution of the s-triazine selective antibody IPR-7. The Fab fragments were expressed under the transcriptional control of the tetA promoter system. Large-scale production benefits from the anhydrotetracycline-inducible system because of the lower costs for the inducer compared to IPTG and the tightly regulated expression of the recombinant antibody fragments. The Strep-tag purification technology facilitates the isolation of Fab fragments from the E. coli periplasm. The characteristics of functionally expressed Fab fragments were determined by employing a BIAcore 2000 system. The KD of the Fab variant IPR-23 (K(D)= 1.1 2 x 10(-9) M) optimized by molecular evolution was improved by a factor of 24 compared to the Fab IPR-7 (K(D) = 2.73 x 10(-8) M), which was derived from the template scFv antibody IPR-7. The affinity alteration was also reflected in the 22-fold reduction of the IC50 values of the variants Fab IPR-7 (IC50 = 60.5 microg/L) and Fab IPR-23 (IC50=2.7 microg/L) in the corresponding atrazine ELISA.

Immunochemical analysis relies on the selective binding of antibodies to defined targets such as environmental compounds. Commercial applications in the environmental field are still restricted to a limited number of immunoassays and a few immunochromatographic applications. The main barrier to a broader exploitation is seen in the generation of a sufficient number of antibodies within an acceptable time period. Recombinant technologies are expected to eventually replace the circumstantial approach to obtain new Abs by new immunizations. Examples for recombinant singlechain fragments (scFv) and antigen binding fragments (Fab) directed against herbicidal s-triazines are given. If antibodies are replaced by receptors and other functional ligands, biological effects monitoring becomes available. As an example an enzyme-linked receptor assay (ELRA) for endocrine disruptors providing estrogen equivalents is presented. Finally, the concept of bioresponse-linked instrumental analysis is introduced for t...

Recombinant antibody (Rab) technology is expected to play an essential role in immunological research and basically in technologies which are utilizing the benefits of antibodies as a central component. Although commercial kits are already available for the synthesis of Rab, the Rab approach is far away from being considered a standard method. This is mainly due to the fact that the complex procedure frequently needs technical refinement in order to be adapted to individual experimental conditions and goals. In contrast, the manuals, supplied by the manufacturers, are appropriately designed to guide a general application of the Rab strategy. Here, we focus exclusively on the Recombinant Phage Antibody System (RPAS, Pharmacia). The RPAS kit is designed to provide a tool for the synthesis of phage-displayed and soluble Rab. This work is adressed to applicants of the RPAS, in order to encourage them to modify the manufacturers manual, especially if problems arise at particular steps when following t...

The selectivity of immunoassay is limited by the cross-reactivity of antibodies to structurally related analytes. This becomes a drawback for applications that require discrimination of slightly different analytes. An approach to overcoming this problem is the application of antibody arrays that show differences in their affinity patterns. We have investigated this method using systematic modelling of multianalyte systems based on test-independent affinity parameters. A model system of anti-s-triazine antibodies and s-triazine derivatives has been investigated. The immunoassay is carried out in an indirect test format using an optical transducer for label-free monitoring of antibody binding at an immobilized hapten. The concentration of free antibody in equilibrium with the analyte is probed in a flow-through system. This format allows simple modelling of the response and assessment of the affinity constant from the calibration curve. The affinity patterns of five monoclonal antibodies and a recombinant single-chain fragment with respect to five s-triazine derivatives are determined by this method. An array of three antibodies is selected and the response pattern to mixtures of three analytes determined. Measured and calculated pattern correspond in principle, but systematic deviations are observed due to the perturbation of equilibrium during detection. The correlation of the true analyte concentration and the analyte concentrations predicted from the signal pattern using the affinity constants strongly depend on the selectivity and the affinity of the antibodies.

Recombinant antibody (RAb) technology is expected to play an essential role in future food and environmental immunoanalysis. The RAb approach is not yet, however, considered as a standard method, mainly due to technical reasons. In order to establish a feasible protocol for the production of hapten specific RAbs, the essential steps of the procedure are considered in detail, especially the polymerase chain reaction amplification of antibody coding genes and the utilization of immunomagnetic selection of phages displaying selective RAbs. The synthesis of functional recombinant single‐chain Fv against s‐triazine herbicides is used as an example. Expressed RAbs are characterized by immunoassay. Although RAb‐based calibration curves were shifted toward higher concentrations by one order of magnitude, the cross‐reactivity pattern of RAbs corresponded to the original monoclonal antibodies. Sequences of different RAb clones derived from the same hybridoma line only differed in a single silent mutation. This conf...

Immunochemical techniques supplement traditional analytical methods in an ideal way because they are extremely sensitive, simple, and inexpensive. The most important types presently used for environmental analysis are immunoassays, immunosensors, immunochromatography, and immunolabeling. Representative examples are given. Standardized immuno-chemical methods for environmental monitoring profit from monoclonal antibodies because of defined selectivities and affinities toward the analyte. A critical step in the production of monoclonal antibodies is the development of efficient screening procedures to identify suitable hybridoma lines. Immunomagnetic beads offer an alternative to conventional screening with immunoassays because the problems of being time-consuming, escaping detection or loosing valuable cells can be solved. This technique is based on tagging surface receptors of hybridoma cells with binding properties of the secreted antibodies by magnetic beads, which are coated with hapten conjugates. In spite of the considerable progress in hybridoma technology, antibody development has entered an entirely new domain because of the potential of recombinant antibodies. Recombinant technology will eventually allow faster production of antibodies with new binding properties (mutant antibodies) and also a stepwise abandonment of animal experiments required for antibody production. The production of phage antibodies for s -triazines is described.

Two years after the atrazine ban in Germany, 417 soil samples from corn fields were screened for atrazine. The analyses were carried out by an enzyme immunoassay (EIA) using polyclonal antibodies from sheep. Thirty five samples contained more than 100 μg atrazine per kg of soil and were considered positive, indicating this year application. The concentrations of all positive samples and several negative samples were validated by HPLC. A correlation of r = 0.96 was obtained. When two different extraction methods using water or acetone were compared, no difference in atrazine yields were found with the EIA. The EIA proves to be a useful screening method, as long as positive samples can be validated by other methods such as HPLC or GC. *Dedicated to Prof. Dr. Dr. h. c. H. Ziegler, TU Munchen, on the occasion of his 70th birthday.

Standardized immunoassays for environmental monitoring require monoclonal antibodies (MAbs) with defined selectivities and affinities. An important step in the production of MAbs is the development of efficient screening procedures to identify suitable cell lines. We demonstrate the usefulness of immunomagnetic cell separation for the screening of hybridomas. It is based on the expression of surface receptors resembling the secreted antibodies on the membrane surface of hybridoma cells. Producers are tagged by the target molecule covalently linked to magnetic beads and subsequently removed by permanent magnet. Several examples are given surpassing conventional techniques for the screening of s‐triazine antibodies.

The application of immunoassays in pesticide residue analysis is of increasing interest due to the sensitivity, simple handling and fast throughput of samples. For a wide application of these assays, a sufficient supply of standardized antibodies over a long period of time is necessary. The monoclonal antibody technology is solving this problem with an increasing number of cell lines which produce antibodies against different pesticides. Hybridomas were produced by cell fusion of spleen cells from mice immunized with dichloroatrazine conjugated to bovine serum albumin and mouse myeloma cells (PAI-B3 Ag8I). After screening with a competitive enzyme immunoassay, a monoclonal antibody that was specific for terbuthylazine and was produced by a permanent hybridoma cell line was selected for immunoassay development and optimization. For this purpose, an antigen- and antibody-immobilized ELISA technique was improved by varying the test parameters. Comparing both methods, the latter turned out to be superior (50% binding = 0.8 microgram/l, detection limit = 0.14 microgram/l).

Even though rabbit antibodies (Abs) are known to exceed murine Abs with respect to specificity, affinity, and stability, cloned leporid immune repertoires have been rarely considered in recombinant Ab preparation for environmental analysis. We have developed a set of four tetp/o-based phasmid vectors that allow the efficient cloning of both murine and leporid Ab repertoires. These vectors differ in the design of the cloning sites, choice of signal peptides, and antibiotic selection markers. A set of 39 primer oligodeoxynucleotides has been developed for the PCR amplification of rabbit Ab genes, representing the most exhaustive coverage of the leporid immune repertoire described so far. The atrazine-specific murine Fab fragment K411B and a cloned V-gene repertoire from sulfonamide-immunized rabbits were used to compare these phasmids with respect to expression of Fab fragments, phagemid titers, and number of Fab displaying phagemid particles. Our results show that the ratio of recombinant phagemids could be increased up to 65% of total phage titer by utilizing the appropriate phasmid. Based on this system, the selection of two sulfonamide-specific rabbit Abs, SA2 23 and SA2 90, was accomplished after a single phagemid panning round.

A mutually compatible vector system was developed for the bench-top fermenter production of mouse and rabbit Fab fragments comprising pASK85-pro-K411B and pASK85Rab-pro-WR13. These vectors provide a mouse- and rabbit-specific cloning site, respectively, the tetA promoter/operator and the proBA operon that complements the Pro biosynthetic deficiency of the Escherichia coli strain JM83 and can serve as an additional selection marker. Fermentation at elevated cell density (OD600 = 2040) of the atrazine-specific mouse Fab fragment K411B using JM83 harboring pASK85-pro-K411B in a 2 L bench-top vessel resulted in a yield of 240 g/L OD600 affinity-purified protein (13.8 mg). In contrast, expression of leporid Fab fragments using pASK85Rab-pro-WR13 was unsuccessful due to the aggregation of rabbit light chains, which probably relates to a general problem of this specific class of immunoglobulins with their additional Cys residues. Coexpression of rabbit Fab fragments together with four periplasmic folding-helper proteins encoded on pTUM4 led to a significantly improved folding efficiency, resulting in a yield of 50 µg/L OD600 affinity-purified rabbit Fab fragment (3.3 mg) from the 2 L bench-top fermenter.

Biological or biomimetic components are crucial determinants of biosensors and bioanalytical systems. Although receptors isolated from natural sources showed a high potential for analytical applications, the advance in modern methods in genetic engineering offers new routes for tailoring appropriate receptors for particular analytical tasks. Beside enzymes, hormone receptors, nucleic acids, membrane components, organelles or entire cells antibodies are frequently incorporated as biosensing elements. Due to their variable selectivity Abs are effective tools for the analysis of a wide variety of analytes ranging from low molecular weight agrochemicals (e.g., herbicides, insecticides) to complex proteins (e.g., structures of pathogenic microorganisms). The combination with up to nanomolar or even higher affinity enables the reliable trace analysis of complex sample mixtures without the need for extensive sample pretreatment or large sample volumes. In this chapter state of the art technologies are presented for Ab engineering. This includes principles for establishing Ab libraries. From these libraries selective binders can be isolated and further modified by applying the Darwinian principle of variation and selection in vitro. Experimental examples from our laboratory are given for antibody libraries, which provide a basic source for isolating suitable immunoreagents for pesticides, genetically modified organisms (GMO) and pathogens. In contrast to conventional immunoreagents, these Abs are amenable to subsequent optimization by rational design or directed molecular evolution. Furthermore, the molecular format can be modified; additional functions for the directed immobilization on sensing surfaces or integration of markers are installed by engineering fusion proteins. Finally, stability, one of the weaker points of Abs compared to biomimetics such as molecularly imprinted polymers (MIPs), can be engineered to warrant Ab functionality even under unfavorable conditions, for instance, under thermal pressure or in presence of denaturating sample components. Finally, limitations of the molecular Ab approach are indicated, which are already apparent and may be overcome by new kinds of biosensors employing straightforward production of alternatives to the Ab scaffold. Keywords: directed evolution; recombinant antibodies; environmental analysis; pesticides; pathogens

Due to their simplicity, speed, low cost, and specificity, immunoassays have become a useful tool for the analysis of environmental pollutants. Once the anti-hapten antibodies are produced, the same hapten or a related molecule is conjugated to a tracer enzyme or coating protein to set up the assay. Here we report the use of peptides that mimic the analyte as advantageous substitutes of competing haptens. These peptides, which open opportunities for innovation in the development of tracer reagents, can be selected from phage display libraries in a straightforward systematic manner. The concept was proven using assays for the herbicides molinate and atrazine as model systems. Several characteristics of the selection process that may affect the final assay were analyzed, such as the phage coat proteins fused to the peptide, the use of linear or constrained peptide libraries, the effect of the concentration of analyte used during the selection process, and the use of monoclonal or polyclonal antibodies as selector molecules. In all cases we found that the selected peptides performed with improved sensitivity as compared with the chemical hapten conventional assays, showing an analogous cross-reactivity pattern. Interestingly, the phage particles perform as robust and highly standardized assay reagents, and due to their filamentous repetitive structure, they function as sensitive multienzymatic reporters.

Listeria monocytogenes phage endolysins Ply118 and Ply500 share a unique enzymatic activity and specifically hydrolyse Listeria cells at the completion of virus multiplication in order to release progeny phage. With the aim of determining the molecular basis for the lytic specificity of these enzymes, we have elucidated their domain structure and examined the function of their unrelated and unique C-terminal cell wall binding domains (CBDs). Analysis of deletion mutants showed that both domains are needed for lytic activity. Fusions of CBDs with green fluorescent protein (GFP) demonstrated that the C-terminal 140 amino acids of Ply500 and the C-terminal 182 residues of Ply118 are necessary and sufficient to direct the murein hydrolases to the bacterial cell wall. CBD500 was able to target GFP to the surface of Listeria cells belonging to serovar groups 4, 5 and 6, resulting in an even staining of the entire cell surface. In contrast, the CBD118 hybrid bound to a ligand predominantly present at septal regions and cell poles, but only on cells of serovars 1/2, 3 and 7. Non-covalent binding to surface carbohydrate ligands occurred in a rapid, saturation-dependent manner. We measured 4 x 104 and 8 x 104 binding sites for CBD118 and CBD500 respectively. Surface plasmon resonance analysis revealed unexpected high molecular affinity constants for the CBD-ligand interactions, corresponding to nanomolar affinities. In conclusion, we show that the CBDs are responsible for targeting the phage endolysins to their substrates and function to confer recognition specificity on the proteins. As the CBD sequences contain no repeats and lack all known sequence motifs for anchoring of proteins to the bacterial cell, we conclude that they use unique structural motifs for specific association with the surface of Gram-positive bacteria.

Apart from the decisive sensitivity and specificity of immunosensors, the employed antibodies essentially contribute to additional key factors like fabrication costs for sensor chips and sensor stability. A production scheme for recombinant antibody fragments has been optimised with respect to these particular issues of biosensor development. The phagemid vector pCANTAB 5 E is widely used for the selection of antibody fragments from corresponding libraries. However, large-scale production of the selected single-chain F(v) (scFv) fragments is substantially restricted by the high cost for the inducer IPTG and the anti-E-tag antibody. The latter is needed in significant amounts for the purification of the recombinant protein. A generic strategy was established for subcloning scFv variable regions from pCANTAB 5 E into the plasmid pASK85 for the expression of F(ab) fragments. pASK85 bears coding sequences for murine constant domains including a His(6) tag at the carboxyl-terminal end of the constant heavy chain domain. The anti-s-triazine antibody K47H served as a model system in this study. Biosynthesis of the F(ab) fragment in a high cell density fermenter was induced by addition of anhydrotetracycline. The F(ab) fragment was subsequently purified from the periplasmic extract in a single step by immobilized metal affinity chromatography (IMAC). A yield of 100 microg/lxOD(550) purified F(ab) fragment was obtained employing a standard fermentation scheme. The sensitivity and cross-reactivity of the F(ab) was comparable to the parent scFv when assayed by enzyme immunoassay. However, the F(ab) fragment exhibited significantly improved long-term stability.

Biosensor sensitivity and selectivity depend essentially on the properties of the biorecognition elements to be used for analyte binding. Two principally different applications are considered, (1) effects monitoring with biological components as targets for bioeffective substances, among them endocrine disruptors; and (2) immunochemical analysis employing antibodies as binding proteins for a wide variety of analytes such as pesticides. Genetic engineering provides an elegant way not only for providing unlimited amounts of biorecognition molecules but also for the alteration of existing properties and the supplementation with additional functions. Instrumental applications were carried out with the optical sensor BIAcore. The first example deals with the characterization of receptors. For this purpose, the human estrogen receptor alpha was used. Binding studies were carried out with natural as well as xenoestrogens. An equilibrium dissociation constant K(d) of 2.3x10(-10) (M) was derived for 17beta-estradiol. A competition assay was performed with a bovine serum albumin (BSA)-17beta-estradiol conjugate, immobilized at the optical sensor surface, and the free estrogen. The signals obtained represent estradiol equivalents. This format was transferred to a microplate-based enzyme-linked receptor assay. It reached a detection limit of 0.02 microg l(-1) 17beta-estradiol and proved suitable for the detection of natural and synthetic estrogens as well as xenoestrogens in field studies. The second example is targeted at kinetic and affinity measurements of recombinant antibody fragments derived from antibody libraries with s-triazine selectivities. Different strategies for the synthesis of antibody fragment libraries, followed by the selection of specific antibody variants, were examined. An antibody library was derived from a set of B cells. Chain shuffling of the heavy and light chains provided the best binders. An enzyme linked immunosorbent assay (ELISA) was achieved for atrazine with an IC(50) of 0.9 microg l(-1) and a detection limit of 0.2 microg l(-1). The close relations between the optimization of recombinant antibodies by evolutionary strategies and genetic algorithms are considered.

An enzyme-linked immunoassay (ELISA) was used for screening atrazine residues in soil. Samples were annually collected in Southern Germany between 1993 and 1998. An average of 419.5 samples was analyzed per year amounting to 2517 samples. The fraction of positive samples defined by atrazine concentrations >100 μg/kg soil decreased successively from 8% (corresponding to 33 samples) in 1993 to 0.6% (corresponding to 2 samples) in 1998. All positive samples and a selection of negative samples were subsequently validated by HPLC. Comparison of ELISA and HPLC data yielded correlation coefficient values of r= 0.958–0.981 (n= 18–47), except for 1995 when only a correlation of r= 0.864 (n= 18) was obtained. Four samples were overestimated and another 4 were underestimated with respect to the atrazine threshold value of 100 μg/kg soil as revealed by HPLC validation. Thus, 99.68% of 2517 analyzed samples were correctly evaluated. The precision and reproducibility of the ELISA were adequate for a prescreening tool. The low cost per sample and the high sample throughput are not yet achievable by conventional analytical methods. The described combination of ELISA and HPLC has the potential to take advantage of both methods and to restrict determination errors to a minimum.

Immunochemical analysis for environmental monitoring is so far predominantly based on antibodies derived from sera and hybridoma cultures. This conventional technology will be supported or even replaced in the near future by the recombinant antibody (Rab) approach, since Rab are accessible to precise modification of antibody properties by virtue of genetic engineering. Furthermore, the in vitro generation of Rab libraries bearing an antibody diversity comparable to the vertebrate immune repertoire paves the way to substitute animals as the antibody source of choice. Utilising the s-triazine herbicides as target molecules, various strategies for the synthesis of Rab libraries are presented comprising natural, semisynthetic and synthetic antibody genes. The Rab genes are expressed as fusion proteins at the surface of phage particles. Special attention is given to the establishment of an efficient selection procedure. Phages presenting specific Rab are isolated via immuno affinity chromatography employing s-triazine coated beads. The described libraries are designed as a gene pool to provide an array of antibody variants for future immunosensor applications in environmental analysis.

Tumors of the head and neck represent a molecularly diverse set of human cancers, but relatively few proteins have actually been shown to drive the disease at the molecular level. In order to identify new targets for individualized diagnosis or therapeutic intervention, we performed a kinase centric chemical proteomics screen and quantified 146 kinases across 34 head and neck squamous cell carcinoma (HNSCC) cell lines using intensity-based label-free mass spectrometry. Statistical analysis of the profiles revealed significant inter-cell line differences for 42 kinases (p

Gold concentrations in epidermis, dermis, and whole skin were measured by neutron activation analysis after formation of suction bullae in 8 patients who had received protracted cyrysotherapy. Epidermis contained 3% (median) of the gold content of whole skin. A direct correlation between cumulative gold dose and skin gold level was noted. These findings suggest that apparent gold concentrations in skin are influenced by the depth of the biopsy, that keratinous tissues have little affinity for gold, and that the gold storage capability of skin is not saturated by large cumulative doses of gold. The beneficial effect of gold in pemphigus may not be mediated at the site of blister formation.

Atypical rough cell filaments of Listeria monocytogenes (designated FR variants), isolated from clinical and food samples, form long filaments up to 96 μm in length and demonstrated wild-type levels of adherence, invasion, and cytotoxicity to human epithelial HEp-2, Caco-2, and HeLa cells. Unlike previously described avirulent rough mutants of L. monocytogenes that secrete diminished levels of the major extracellular protein p60 and that form long chains that consist of multiple cells of similar size (designated MCR variants), FR variants secreted wild-type or greater levels of p60. This study shows that virulent filamentous forms of L. monocytogenes occur in clinical and food environments and have atypical morphological characteristics compared to those of the wild-type form.

A study of cirrhosis mortality among young adults (ages 25 to 44) in Baltimore between 1957 and 1966 was completed by reviewing death certificates, medical examiners' reports, and autopsy ...