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Corrigendum: ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome
... In fact, although presumably not as huge as in the case of small molecule pharmaceuticals – with their many enzymes and liganddependent receptors as targets – the spectrum of excreted and cell surface proteins that constitute viable targets of non-Ig binding proteins should actually be much larger than the close to 100 targets that are addressed by approved biopharmaceuticals to date[2]. Many more 'druggable' targets are likely to be identified by means of modern proteome analysis[64]. In addition, the application of antibody (and probably soon also scaffold) selection technology itself will increasingly aid in the discovery of novel targets, in particular those overexpressed on malignant tissues[65].response. ...
... In fact, although presumably not as huge as in the case of small molecule pharmaceuticals – with their many enzymes and liganddependent receptors as targets – the spectrum of excreted and cell surface proteins that constitute viable targets of non-Ig binding proteins should actually be much larger than the close to 100 targets that are addressed by approved biopharmaceuticals to date [2]. Many more 'druggable' targets are likely to be identified by means of modern proteome analysis [64] . In addition, the application of antibody (and probably soon also scaffold) selection technology itself will increasingly aid in the discovery of novel targets, in particular those overexpressed on malignant tissues [65]. ...
Antibodies have been the paradigm of binding proteins with desired specificities for more than one century and during the past decade their recombinant or humanized versions have entered clinical application with remarkable success. Meanwhile, a new generation of receptor proteins was born, which is derived from small and robust non-immunoglobulin "scaffolds" that can be equipped with prescribed binding functions using the methods of combinatorial protein design. Their ongoing development does not only provide valuable insights into the principles of molecular recognition and protein structure-function relationships but also yields novel reagents for medical use. This technology goes hand in hand with our expanding knowledge about the molecular pathologies of cancer, immunological, and infectious diseases. Currently, questions regarding the choice of suitable medically relevant targets with regard to a certain protein scaffold, the methodology for engineering high affinity, arming with effector functions, routes of administration, plasma half-life, and immunogenicity are in the focus. While many protein scaffolds have been proposed during the past years, the technology shows a trend toward consolidation with a smaller set of systems that are being applied against multiple targets and in different settings, with emphasis on the development of drug candidates for therapy or in vivo diagnostics: Adnectins, Affibodies, Anticalins, DARPins, and engineered Kunitz-type inhibitors, among others. Only few data from early clinical studies are available yet, but many more are likely to come in the near future, thus providing a growing basis for assessing the therapeutic potential--but possibly also some limitations--of this exciting new class of protein drugs.
... Scale bars represent 10 µm. antibodies by recombinant systems has been started in proteome binder projects (Konthur et al., 2005; Hust et al., 2007; Schofield et al., 2007; Taussig et al., 2007), so increasing amounts of recombinant antibody genes, in particular to uncharacterized ORFs from the human genome, are becoming available. In this study we aimed to investigate multiple parameters which may effect the functional knockdown of surface VCAM-1 by intracellular expression of ER retained antibodies. ...
... In summary, knockdown with ER retained antibodies seems to be a promising technology for biomedical application as well as for studies of protein function in vitro and in vivo. The high throughput generation of recombinant antibodies by phage display (Hust and Dübel, 2004; Hust et al., 2007; Taussig et al., 2007) opens the way for its widespread use. ...
Vascular cell adhesion molecule 1 (VCAM-1) is involved in the recruitment of leukocytes to inflammatory sites. In this study we present the first functional knockdown of VCAM-1 using an ER retained antibody construct. We generated a knockdown construct encoding the VCAM-1 specific single chain variable fragment scFv6C7.1 fused to the C-terminal ER retention sequence KDEL. HEK-293:VCAM-YFP cells stably expressing a VCAM-YFP fusion protein were transiently transfected with the knockdown construct and showed down-regulation of surface VCAM-1. Knockdown efficiency of the system is time-dependent due to used transient transfection of the intrabody construct. Furthermore, intrabody mediated knockdown of HEK-293:VCAM-YFP cells also impaired cell-cell interaction with Jurkat cells that are endogenously expressing VLA-4, the physiological partner of VCAM-1. Posttranslational knockdown with ER retained antibodies seems to be a promising technique, as shown here for VCAM-1.
... Anticalins which bind fluorescein with high affinity and exert an almost quantitative fluorescence quenching effect provide interesting reagents for biophysical studies [42]. Another field of applications is likely to open in conjunction with chip technologies, for example for the diagnostics of plasma proteins or in proteome research [43]. Affibodies, in various multimerized and detection formats, have already yielded promising results with protein capture microarrays [44]. ...
... So far, the initially high expenses for development were primarily invested with the aim of biomedical use. Furthermore, the 'unique selling proposition' for alternative binding proteins will have to be better defined in order to open novel applications, for example in upcoming areas such as modern proteome research, where many thousands of gene product specific binding reagents will be needed during the next years [43]. At the moment, one of the most pertinent questions in this field is as follows: what are the criteria of success for a particular protein scaffold? ...
Originally proposed one decade ago, the idea of engineering proteins outside the immunoglobulin family for novel binding functions has evolved as a powerful technology. Several classes of protein scaffolds proved to yield reagents with specificities and affinities in a range that was previously considered unique to antibodies. Such engineered protein scaffolds are usually obtained by designing a random library with mutagenesis focused at a loop region or at an otherwise permissible surface area and by selection of variants against a given target via phage display or related techniques. Whereas a plethora of protein scaffolds has meanwhile been proposed, only few of them were actually demonstrated to yield specificities towards different kinds of targets and to offer practical benefits such as robustness, smaller size, and ease of expression that justify their use as a true alternative to conventional antibodies or their recombinant fragments. Currently, the most promising scaffolds with broader applicability are protein A, the lipocalins, a fibronectin domain, an ankyrin consensus repeat domain, and thioredoxin. Corresponding binding proteins are not only of interest as research reagents or for separation in biotechnology but also as potential biopharmaceuticals, especially in the areas of cancer, autoimmune and infectious diseases as well as for in vivo diagnostics. The medical prospects have boosted high commercial expectations, and many of the promising scaffolds are under development by biotech start-up companies. Although some issues still have to be addressed, for example immunogenicity, effector functions, and plasma half-life in the context of therapeutic use or low-cost high-throughput selection for applications in proteomics research, it has become clear that scaffold-derived binding proteins will play an increasing role in biotechnology and medicine.
... One of the major challenges in biomarker validation and diagnostic assay development is the availability of high-quality capture molecules, e.g., antibodies. Several efforts have been seek for the assortment of well-characterized affinity-based molecules, not limited to antibodies, in one database [28,29]. But also different other aspects of the entire validation process need to be improved. ...
Due to insufficient biomarker validation and poor performances in diagnostic assays, the candidate biomarker verification process has to be improved. Multi-analyte immunoassays are the tool of choice for the identification and detailed validation of protein biomarkers in serum. The process of identification and validation of serum biomarkers, as well as their implementation in diagnostic routine requires an application of independent immunoassay platforms with the possibility of high-throughput. This review will focus on three main multi-analyte immunoassay platforms: planar microarrays, multiplex bead systems and, array-based surface plasmon resonance (SPR) chips. Recent developments of each platform will be discussed for application in clinical proteomics, principles, detection methods, and performance strength. The requirements for specific surface functionalization of assay platforms are continuously increasing. The reasons for this increase is the demand for highly sensitive assays, as well as the reduction of non-specific adsorption from complex samples, and with it high signal-to-noise-ratios. To achieve this, different support materials were adapted to the immobilized biomarker/ligand, allowing a high binding capacity and immobilization efficiency. In the case of immunoassays, the immobilized ligands are proteins, antibodies or peptides, which exhibit a diversity of chemical properties (acidic/alkaline; hydrophobic/hydrophilic; secondary or tertiary structure/linear). Consequently it is more challenging to develop immobilization strategies necessary to ensure a homogenous covered surface and reliable assay in comparison to DNA immobilization. New developments concerning material support for each platform are discussed especially with regard to increase the immobilization efficiency and reducing the non-specific adsorption from complex samples like serum and cell lysates.
... The methods with the highest sensitivity have the potential to detect, in principle, low abundant proteins, with zeptomole detection limits already demonstrated (Pawlak et al., 2002). However, the development of sets of reagents of suitable specificity and affinity to support the conclusive detection and quantification of target protein (s) remains challenging, expensive and arduous, and coordinated efforts to develop validated affinity reagents are just getting underway (Taussig et al., 2007; Uhlen and Hober, 2008). The methods based on affinity reagents are therefore limited by slow assay development and, usually, also by the inability to significantly multiplex detection of proteins in the same sample. ...
The rise of systems biology implied a growing demand for highly sensitive techniques for the fast and consistent detection and quantification of target sets of proteins across multiple samples. This is only partly achieved by classical mass spectrometry or affinity-based methods. We applied a targeted proteomics approach based on selected reaction monitoring (SRM) to detect and quantify proteins expressed to a concentration below 50 copies/cell in total S. cerevisiae digests. The detection range can be extended to single-digit copies/cell and to proteins undetected by classical methods. We illustrate the power of the technique by the consistent and fast measurement of a network of proteins spanning the entire abundance range over a growth time course of S. cerevisiae transiting through a series of metabolic phases. We therefore demonstrate the potential of SRM-based proteomics to provide assays for the measurement of any set of proteins of interest in yeast at high-throughput and quantitative accuracy.
... Additional techniques, like surface-enhanced Raman spectroscopy are also available for PTM discovery in biological samples (Sundararajan et al., 2006). However, there is a gap between the discovery of PTMs and the ability to detect them in biological samples due to the lack of detection reagents, such as antibodies, with suitable affinity and specificity for these protein-PTMs (Blow, 2007; Kazanecki et al., 2007; Taussig et al., 2007; Uhlen, 2007). Aptamers demonstrate specificity and affinity that can parallel good monoclonal antibodies and, as detection reagents , are an attractive alternative to antibodies. ...
Posttranslational modifications on proteins can serve as useful biomarkers for disease. However, their discovery and detection in biological fluids is challenging. Aptamers are oligonucleotide ligands that demonstrate high affinity toward their target proteins and can discriminate closely related proteins with superb specificity. Previously, we generated a cyclophilin B aptamer (M9-5) that could discriminate sera from pancreatic cancer patients and healthy volunteers with high specificity and sensitivity. In our present work we further characterize the aptamer and the target protein, cyclophilin B, and demonstrate that the aptamer could discriminate between cyclophilin B expressed in human cells versus bacteria. Using mass-spectrometric analysis, we discovered post-translational modifications on cyclophilin B that might be responsible for the M9-5 selectivity. The ability to distinguish between forms of the same protein with differing post-translational modifications is an important advantage of aptamers as tools for identification and detection of biomarkers.
... Another effort to congregate information about different affinity reagents developed in projects throughout the world is an infrastructure funded by the European Union. This coordination effort, named ProteomeBinder, started in 2006 and is a network between more than 20 research groups worldwide, focusing on differently generated affinity reagents [6] . Also, a similar effort focused on monoclonal antibodies, Protein Capture Tools, was selected during spring 2007 to be launched by the National Institute of Health [7]. ...
Currently one of the most challenging tasks in biological and medical research is to explore and understand the function of all proteins encoded by the genome of an organism. A systematic approach based on the genome sequences is feasible because the full genome of many organisms presently is available and many more are underway. For the production of expression atlases different strategies are used. Early attempts to acquire information about protein expression levels have focused on the analysis of mRNA levels within different tissues and cell types. Recently, novel strategies to focus directly on protein levels have been developed. To assess global protein expression in a systematic and high-throughput manner, methods based on design of specific affinity ligands to recognize the proteins have been presented. By subsequently using these affinity molecules for detection of the corresponding proteins in a wide range of platforms, important information can be gained. This article focuses on strategies to profile protein levels and in particular the human protein atlas initiative and the use of microarray technologies.
The demand on antigen binding reagents in research, diagnostics and therapy raises questions for novel antibody formats as well as appropriate production systems. Recently, the novel single chain Fab (scFab) antibody format combining properties of single chain Fv (scFv) and Fab fragments was produced in the Gram-negative bacterium Escherichia coli. In this study we evaluated the Gram-positive bacterium Bacillus megaterium for the recombinant production of scFab and scFvs in comparison to E. coli.
The lysozyme specific D1.3 scFab was produced in B. megaterium and E. coli. The total yield of the scFab after purification obtained from the periplasmic fraction and culture supernatant of E. coli was slightly higher than that obtained from culture supernatant of B. megaterium. However, the yield of functional scFab determined by analyzing the antigen binding activity was equally in both production systems. Furthermore, a scFv fragment with specificity for the human C reactive protein was produced in B. megaterium. The total yield of the anti-CRP scFv produced in B. megaterium was slightly lower compared to E. coli, whereas the specific activity of the purified scFvs produced in B. megaterium was higher compared to E. coli.
B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli.
We have created a high quality phage display library containing over 1010 human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online. Sensitive detection was demonstrated in a bead based flow cytometry assay. Furthermore, positive staining by immunohistochemistry on tissue microarrays was found for 37% (143/381) of antibodies. Thus, we have demonstrated the potential of and illuminated the issues associated with genome-wide monoclonal antibody generation.
The aim of this study was to construct a ribosome display library of single chain variable fragments (scFvs) associated with hepatocarcinoma and screen such a library for hepatocarcinoma-binding scFvs. mRNA was isolated from the spleens of mice immunized with hepatocellular carcinoma cell line HepG2. Heavy and k chain genes (VH and k) were amplified separately by RT-PCR, and an anti-HepG2 VH/k chain ribosome display library was constructed by assembling VH and k into the VH/k chain with a specially constructed linker by SOE-PCR. The VH/k chain library was transcribed and translated in vitro using a rabbit reticulocyte lysate system. In order to isolate specific scFvs, recognizing HepG2 negative selection on a normal hepatocyte line WRL-68 was carried out before three rounds of positive selection on HepG2. After three rounds of panning, cell enzyme-linked immunosorbent assay (ELISA) showed that one of the scFvs had high affinity for the HepG2 cell and lower affinity for the WRL-68 cell. In this study, we successfully constructed a native ribosome display library. Such a library would prove useful for direct intact cell panning using ribosome display technology. The selected scFv had a potential value for hepatocarcinoma treatment.
Recombinant antibody technology has revolutionized the development of antibody therapeutics. This minireview offers an overview of enabling technologies and future prospects of this rapidly progressing field.
Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for antibody synthesis. Due to the outer membrane of gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the gram-positive bacterium Bacillus megaterium was shown to efficiently secrete recombinant proteins into the growth medium. Here we evaluated B. megaterium for the recombinant production of antibody fragments.
The lysozyme specific single chain Fv (scFv) fragment D1.3 was successfully produced using B. megaterium. The impact of culture medium composition, gene expression time and culture temperatures on the production of functional scFv protein was systematically analyzed. A production and secretion at 41 degrees C for 24 h using TB medium was optimal for this individual scFv. Interestingly, these parameters were very different to the optimal conditions for the expression of other proteins in B. megaterium. Per L culture supernatant, more than 400 microg of recombinant His6-tagged antibody fragment were purified by one step affinity chromatography. The material produced by B. megaterium showed an increased specific activity compared to material produced in E. coli.
High yields of functional scFv antibody fragments can be produced and secreted into the culture medium by B. megaterium, making this production system a reasonable alternative to E. coli.
Modern tools in proteomics require access to large arrays of specific binders for use in multiplex array formats, such as microarrays, to decipher complex biological processes. Combinatorial protein libraries offer a solution to the generation of collections of specific binders, but unit operations in the process to isolate binders from such libraries must be automatable to ensure an efficient procedure. In the present study, we show how a microfluidic concept that utilizes particle separation in an acoustic force field can be used to efficiently separate antigen-bound from unbound members of such libraries in a continuous flow format. Such a technology has the hallmarks for incorporation in a fully automated selection system for the isolation of specific binders.
The use of affinity-based tools has become invaluable as a platform for basic research and in the development of drugs and diagnostics. Applications include affinity chromatography and affinity tag fusions for efficient purification of proteins as well as methods to probe the protein network interactions on a whole-proteome level. A variety of selection systems has been described for in vitro evolution of affinity reagents using combinatorial libraries, which make it possible to create high-affinity reagents to virtually all biomolecules, as exemplified by generation of therapeutic antibodies and new protein scaffold binders. The strategies for high-throughput generation of affinity reagents have also opened up the possibility of generating specific protein probes on a whole-proteome level. Recently, such affinity proteomics have allowed the detailed analysis of human protein expression in a comprehensive manner both in normal and disease tissue using tissue microarrays and confocal microscopy.
Genomic, transcriptomic and proteomic projects often suffer from a lack of functional validation creating a strong demand for specific and versatile antibodies. Antibody phage display represents an attractive approach to select rapidly in vitro the equivalent of monoclonal antibodies, like single chain Fv antibodies, in an inexpensive and animal free way. However, so far, recombinant antibodies have not managed to impose themselves as efficient alternatives to natural antibodies.
We developed a series of vectors that allow one to easily fuse single chain Fv antibodies to Fc domains of immunoglobulins, improving their sensitivity and facilitating their use. This series enables the fusion of single chain Fv antibodies with human, mouse or rabbit Fc so that a given antibody is no longer restricted to a particular species. This opens up unlimited multiplexing possibilities and gives additional value to recombinant antibodies. We also show that this multi-Fc species production system can be applied to natural monoclonal antibodies cloned as single chain Fv antibodies and we converted the widely used 9E10 mouse anti-Myc-tag antibody into a human and a rabbit antibody.
Altogether, this new expression system, that brings constant quality, sensitivity and unique versatility, will be important to broaden the use of recombinant and natural monoclonal antibodies both for laboratory and diagnosis use.
The use of combinatorial protein engineering to design proteins with novel binding specificities and desired properties has evolved into a powerful technology, resulting in the recent advances in protein library selection strategies and the emerge of a variety of new engineered affinity proteins. The need for different protein library selection methods is due to that each target protein pose different challenges in terms of its availability and inherent properties. At present, alternative engineered affinity proteins are starting to complement and even challenge the classical immunoglobulins in different applications in biotechnology and potentially also for in vivo use as imaging agents or as biotherapeutics. This review article covers the generation and use of affinity proteins generated through combinatorial protein engineering. The most commonly used selection techniques for isolation of desired variants from large protein libraries are described. Different antibody derivatives, as well as a variety of the most validated engineered protein scaffolds, are discussed. In addition, we provide an overview of some of the major present and future applications for these engineered affinity proteins in biotechnology and medicine.
Utilisation of 'omics' technologies, in particular gene expression profiling, has increased dramatically in recent years. In basic research, high-throughput profiling applications are increasingly used and may now even be considered standard research tools. In the clinic, there is a need for better and more accurate diagnosis, prognosis and treatment response indicators. As such, clinicians have looked to omics technologies for potential biomarkers. These prediction profiling studies have in turn attracted the attention of basic researchers eager to uncover biological mechanisms underlying clinically useful signatures. Here we highlight some of the seminal work establishing the arrival of the omics, in particular transcriptomics, in breast cancer research and discuss a sample of the most current applications. We also discuss the challenges of data analysis and integrated data analysis with emphasis on utilising the current publicly available gene expression datasets. (Part of a Multi-author Review).
Camelid’ s heavy-chain antibody (HCAb) consists of only two heavy chains and lacks the two light chains together with the CH1 domain usually found in conventional immunoglobulins. A recombinant single antigen-binding entity, named VHH (or Nanobody®) was generated by reengineering the variable domains from HCAb. This study focuses on the detection of camelid´s immunoglobulins as well as their derivative nanobodies using a universal anti-camel antibody produced in rabbit (rIgG). Starting from a crude rabbit serum, a standard stock of rIgG (1 mg/ml) was prepared after purification by affinity chromatography using protein-A column. As expected, rIgG was able to detect camel antibodies in ELISA and immunoblotting, and its reactivity was equal against all different camel IgG subclasses, which were purified from serum by differential affinity chromatography on protein-G and -A. Interestingly, rIgG also recognized nanobodies since they were originally part of camel HCAbs, providing an alternative method to detect the corpus of these recombinant proteins rather than targeting their artificial tags. These data suggest that the anti-camel rIgG described here could be efficiently applied at different stages of nanobody technology, including the quantitation of the issued nanobodies and their detection when bound to target antigens.
The incidence of cancer and its associated mortality are increasing globally, indicating an urgent need to develop even more effective and sensitive sets of biomarkers that could help in early diagnosis and consequent intervention. Given that many cellular processes are carried out by proteins, cancer research has recently shifted toward an exploration of the full proteome for such discovery. Among the advanced methodologies that are being developed for analyzing the proteome, antibody microarrays have become a prominent tool for gathering the information required for a better understanding of disease biology, early detection, discrimination of tumors and monitoring of disease progression. Here, we review the technical aspects and challenges in the development and use of antibody microarray assays and examine recently reported applications in oncoproteomics.
A mass spectrometric (MS)-based strategy for antigen (Ag) identification and characterization of globally produced monoclonal antibodies (mAbs) is described. Mice were immunized with a mixture of native glycoproteins, isolated from the pooled plasma of patients with nonsmall cell lung cancer (NSCLC), to generate a library of IgG-secreting hybridomas. Prior to immunization, the pooled NSCLC plasma was subjected to 3-sequential steps of affinity fractionation, including high abundant plasma protein depletion, glycoprotein enrichment, and polyclonal antibody affinity chromatography normalization. In this paper, to demonstrate the high quality of the globally produced mAbs, we selected 3 mAbs of high differentiating power against a matched, pooled normal plasma sample. After production of large quantities of the mAbs from ascites fluids, Ag identification was achieved by immunoaffinity purification, SDS-PAGE, Western blotting, and MS analysis of in-gel digest products. One antigen was found to be complement factor H, and the other two were mapped to different subunits of haptoglobin (Hpt). The 2 Hpt mAbs were characterized in detail to assess the quality of the mAbs produced by the global strategy. The affinity of one of the mAbs to the Hpt native tetramer form was found to have a K(D) of roughly 10(-9) M and to be 2 orders of magnitude lower than the reduced form, demonstrating the power of the mAb proteomics technology in generating mAbs to the natural form of the proteins in blood. The binding of this mAb to the beta-chain of haptoglobin was also dependent on glycosylation on this chain. The characterization of mAbs in this work reveals that the global mAb proteomics process can generate high-quality lung cancer specific mAbs capable of recognizing proteins in their native state.
Monoclonal antibodies with high affinity and selectivity that work on wholemount fixed tissues are valuable reagents to the cell and developmental biologist, and yet isolating them remains a long and unpredictable process. Here we report a rapid and scalable method to select and express recombinant mouse monoclonal antibodies that are essentially equivalent to those secreted by parental IgG-isotype hybridomas.
Increased throughput was achieved by immunizing mice with pools of antigens and cloning - from small numbers of hybridoma cells - the functionally rearranged light and heavy chains into a single expression plasmid. By immunizing with the ectodomains of zebrafish cell surface receptor proteins expressed in mammalian cells and screening for formalin-resistant epitopes, we selected antibodies that gave expected staining patterns on wholemount fixed zebrafish embryos.
This method can be used to quickly select several high quality monoclonal antibodies from a single immunized mouse and facilitates their distribution using plasmids.
Protein microarrays represent an emerging technology that promises to facilitate high-throughput proteomics. The major goal of this technology is to employ peptides, full-length proteins, antibodies, and small molecules to simultaneously screen thousands of targets for potential protein-protein interactions or modifications of the proteome. This article describes the performance of a set of peptide aptamers specific for the human papillomavirus (HPV) type 16 oncoproteins E6 and E7 in a microarray format. E6 and E7 peptide aptamer microarrays were probed with fluorescence-labeled lysates generated from HPV-infected cervical keratinocytes expressing both E6 and E7 oncoproteins. Peptide aptamer microarrays are shown to detect low levels of E6 and E7 proteins. Peptide aptamers specific for cellular proteins included on these microarrays suggested that expression of CDK2, CDK4, and BCL-6 may be affected by HPV infection and genome integration. We conclude that peptide aptamer microarrays represent a promising tool for proteomics and may be of value in biological and clinical investigations of cervical carcinogenesis.
Recent advances in antibody microarray technology have made it possible to measure the expression of hundreds of proteins simultaneously in a competitive dual-colour approach similar to dual-colour gene expression microarrays. Thus, the established normalisation methods for gene expression microarrays, e.g. loess regression, can in principle be applied to protein microarrays. However, the typical assumptions of such normalisation methods might be violated due to a bias in the selection of the proteins to be measured. Due to high costs and limited availability of high quality antibodies, the current arrays usually focus on a high proportion of regulated targets. Housekeeping features could be used to circumvent this problem, but they are typically underrepresented on protein arrays. Therefore, it might be beneficial to select invariant features among the features already represented on available arrays for normalisation by a dedicated selection algorithm.
We compare the performance of several normalisation methods that have been established for dual-colour gene expression microarrays. The focus is on an invariant selection algorithm, for which effective improvements are proposed. In a simulation study the performances of the different normalisation methods are compared with respect to their impact on the ability to correctly detect differentially expressed features. Furthermore, we apply the different normalisation methods to a pancreatic cancer data set to assess the impact on the classification power.
The simulation study and the data application demonstrate the superior performance of the improved invariant selection algorithms in comparison to other normalisation methods, especially in situations where the assumptions of the usual global loess normalisation are violated.
Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids (aptamers), polypeptides (engineered binding proteins) and inorganic matrices (molecular imprinted polymers) have received considerable attention. A major advantage of these alternatives concerns the efficient (microbial) production and in vitro selection procedures. The latter approach allows for the high-throughput optimization of aptamers and engineered binding proteins, e.g. aiming at enhanced chemical and physical stability. This has resulted in a rapid development of the fields of nucleic acid- and protein-based affinity tools and, although they are certainly not as widely used as antibodies, the number of their applications has steadily increased in recent years. In the present review, we compare the properties of the more conventional antibodies with these innovative affinity tools. Recent advances of affinity tool developments are described, both in a medical setting (e.g. diagnostics, therapeutics and drug delivery) and in several niche areas for which antibodies appear to be less attractive. Furthermore, an outlook is provided on anticipated future developments.
The identification of immunogenic polypeptides of pathogens is helpful for the development of diagnostic assays and therapeutic applications like vaccines. Routinely, these proteins are identified by two-dimensional polyacrylamide gel electrophoresis and Western blot using convalescent serum, followed by mass spectrometry. This technology, however, is limited, because low or differentially expressed proteins, e.g. dependent on pathogen-host interaction, cannot be identified. In this work, we developed and improved a M13 genomic phage display-based method for the selection of immunogenic polypeptides of Mycoplasma hyopneumoniae, a pathogen causing porcine enzootic pneumonia. The fragmented genome of M. hyopneumoniae was cloned into a phage display vector, and the genomic library was packaged using the helperphage Hyperphage to enrich open reading frames (ORFs). Afterwards, the phage display library was screened by panning using convalescent serum. The analysis of individual phage clones resulted in the identification of five genes encoding immunogenic proteins, only two of which had been previously identified and described as immunogenic. This M13 genomic phage display, directly combining ORF enrichment and the presentation of the corresponding polypeptide on the phage surface, complements proteome-based methods for the identification of immunogenic polypeptides and is particularly well suited for the use in mycoplasma species.
A new anticalin against estradiol (E(2)), a kind of endocrine disruptor, was obtained in the present study to detect E(2) levels. A member of the lipocalin family from Pieris brassicae called bilin-binding protein (BBP) was employed for the preparation of a random library to specifically complex E(2). Sixteen amino acid residues at the center of the binding site, which were formed by four loops on top of an eight-stranded β-barrel, were subjected to targeted random mutagenesis. Estradiol-binding BBP variants so-called 'anticalins', which exhibit binding activity for compounds, such as E(2), were selected from the resulting library by combining both ribosome display and screening techniques. Four variants of complex E(2) with high affinity were identified. These variants exhibited dissociation constants (KDs) as low as 54.265 nM. ELISA showed that ribosome displayed anticalin (E(2)-A) specifically bound E(2). The 50% inhibition concentration (IC(50)) for E(2) was 50 ng mL(-1) and the limit of detection (LOD:IC(10)) was 0.071 ng mL(-1). The experimental results suggest that E(2)-A can be used as a potential anticalin to detect E(2) in animals.
Human antibodies are valuable tools for proteome research and diagnostics. Furthermore, antibodies are a rapidly growing class of therapeutic agents, mainly for inflammation and cancer therapy. The first therapeutic antibodies are of murine origin and were chimerized or humanized. The later-developed antibodies are fully human antibodies. Here, two technologies are competing the hybridoma technology using transgenic mice with human antibody gene loci and antibody phage display. The starting point for the selection of human antibodies against any target is the construction of an antibody phage display gene library.
In this review we describe the construction of human naive and immune antibody gene libraries for antibody phage display.
The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility.
In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10--20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average.
Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline.
Immunoassays are indispensable for research and clinical analysis, and following the emergence of the omics paradigm, multiplexing of immunoassays is more needed than ever. Cross-reactivity (CR) in multiplexed immunoassays has been unexpectedly difficult to mitigate, preventing scaling up of multiplexing, limiting assay performance, and resulting in inaccurate and even false results, and wrong conclusions. Here, we review CR and its consequences in single and dual antibody single-plex and multiplex assays. We establish a distinction between sample-driven and reagent-driven CR, and describe how it affects the performance of antibody microarrays. Next, we review and evaluate various platforms aimed at mitigating CR, including SOMAmers and protein fractionation-bead assays, as well as dual Ab methods including (i) conventional multiplex assays, (ii) proximity ligation assays, (iii) immuno-mass spectrometry, (iv) sequential multiplex analyte capture, (v) antibody colocalization microarrays and (vi) force discrimination assays.
Challenges such as the rapid development of detection reagents for emerging or engineered pathogens, the goal of identifying probes for every protein in the human proteome, and the development of therapeutic molecules require systems for development of epitope binding molecules that are faster and cheaper than conventional antibody development. To be practical and effective, antibody mimics must be small, stable molecules that contain exposed loops or surfaces that can be randomized and screened using selective combinatorial assays. The tenth human fibronectin type III domain (10Fn3) fits these requirements and has recently been developed as an antibody mimic for use in detection and therapeutic platforms. Previously described systems for working with 10Fn3 used PCR-based approaches to anneal multiple oligonucleotides to generate randomized 10Fn3 libraries. Here we describe a simplified approach for creating randomized 10Fn3 libraries and report the first use of a T7-based phage display system for screening these libraries.
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