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Seeing Better through a MIST: Evaluation of Monoclonal Recombinant Antibody Fragments on Microarrays
Abstract
Automation is the key approach for genomewide and proteomewide screening of function and interaction. Especially for proteomics, antibody microarrays are a useful tool for massive parallel profiling of complex samples. To meet the requirements of antibody microarrays and to obtain a great variety of antibodies, new technologies such as phage display have partly replaced the classical hybridoma method. While the selection process for phage-displayed antibody fragments itself has been automated, the bottleneck was shifted further downstream to the identification of monoclonal binders obtained from the selections. Here, we present a new approach to reduce time, material, and waste to extend automation beyond the selection process by application of conventional microarray machinery. We were able to express recombinant antibody fragments in a single inoculation and expression step and subjected them without purification directly to an automated high-throughput screening procedure based on the multiple spotting technique (MIST). While obtaining comparable sensitivities to enzyme-linked immunosorbent assays, we minimized manual interaction steps and streamlined the technique to be accessible within the automated selection procedure.
... 32 However, while it is relatively straightforward to carry out automated ELISAs with a few microtiter plates, the assay is intrinsically complex and technically laborious. By the elimination of liquid handling, more recent array-based methods using filters 33 or microscope slides 34,35 have significantly increased the number of antibody clones that can be screened at one time. Although these formats are very effective for screening large numbers of antibodies against a single target, or single antibodies against many targets, with one exception, 35 they cannot be easily adapted to the screening of hundreds of antibodies in multiplex, that is, where individual antibody clones are analyzed against many targets in a single analytical step. ...
... By the elimination of liquid handling, more recent array-based methods using filters 33 or microscope slides 34,35 have significantly increased the number of antibody clones that can be screened at one time. Although these formats are very effective for screening large numbers of antibodies against a single target, or single antibodies against many targets, with one exception, 35 they cannot be easily adapted to the screening of hundreds of antibodies in multiplex, that is, where individual antibody clones are analyzed against many targets in a single analytical step. The inability to multiplex is a significant bottleneck for high-throughput screening, especially when contemplating generation and characterization of antibodies against all proteins encoded by a genome. ...
... Although microarrays are usually used to screen single antibodies against many antigens, or vice versa, a microarray method has been published, 34,35 in which antigens are arrayed first and antibodies subsequently arrayed in perfect register. Although this is not a true multiplex assay, in the sense that different binding events are analyzed pairwise, rather than simultaneously in a single test under identical conditions, it is capable of generating similar data. ...
We have developed a screening method that has the potential to streamline the high-throughput analysis of affinity reagents for proteomic projects. By using multiplexed flow cytometry, we can simultaneously determine the relative expression levels, the identification of nonspecific binding, and the discrimination of fine specificities to generate a complete functional profile for each clone. The quality and quantity of data, combined with significant reductions in analysis time and antigen consumption, provide notable advantages over standard ELISA methods and yield much information in the primary screen which is usually only obtained in later screens. By combining high-throughput screening capabilities with multiplex technology, we have redefined the parameters for the initial identification of affinity reagents recovered from combinatorial libraries and removed a significant bottleneck in the generation of affinity reagents on a proteomic scale.
... Finally, antibody phage display, because not dependent on animals, is not only ethically preferable in largescale projects, but also amenable to automation and miniaturisation, thus significantly shortening the time from antigen delivery to antibody production and lowering the cost per binder [17]. This also conveniently fits to the growing list of miniaturised and parallelised antibody validation assays, in particular using microarray-based methods [18][19][20]. SH2 (Src-homology 2) domains are internal regions of a wide variety of proteins, which can contain one or several of these modules of $100 amino acids that bind to specific phosphotyrosine-containing peptide motifs of other proteins in many different signal transduction cascades [21,22] (http://pawsonlab. mshri.on.ca). ...
... Microarray assays were generated and analysed as described [19]. The microarrays were spotted onto epoxy-coated slides (Corning Life Sciences) with 14 identical subarrays on each slide utilising a non-contact arrayer, Nanoplotter 2.0E (GeSim). ...
... Clearly, clones with broad crossreactivity within a homologous protein family can evade exclusion in the primary screening ELISA when simple negative controls, like BSA or lysozyme, are employed, as in most current primary assays. Here, protein arrays [18][19][20] will prove to be valuable in the future, because they allow one to assess binding to a large number of proteins using only minute amounts of scFvs. ...
In vitro antibody generation by panning a large universal gene library with phage display was employed to generate antibodies to more than 60 different antigens. Of particular interest was a comparison of pannings on 20 different SH2 domains provided by the Structural Genomics Consortium (SGC). Streamlined methods for high throughput antibody generation developed within the 'Antibody Factory' of the German National Genome Research Network (NGFN) were demonstrated to minimise effort and provide a reliable and robust source for antibodies. For the SH2 domains, in two successive series of selections, 2668 clones were analysed, resulting in 347 primary hits in ELISA. Half of these hits were further analysed, and more than 90 different scFv antibodies to all antigens were identified. The validation of selected antibodies by cross-reactivity ELISA, western blot and on protein microarrays demonstrated the versatility of the in vitro antibody selection pipeline to generate a renewable resource of highly specific monoclonal binders in proteome scale numbers with substantially reduced effort and time.
... 32 However, while it is relatively straightforward to carry out automated ELISAs with a few microtiter plates, the assay is intrinsically complex and technically laborious. By the elimination of liquid handling, more recent array-based methods using filters 33 or microscope slides 34,35 have significantly increased the number of antibody clones that can be screened at one time. Although these formats are very effective for screening large numbers of antibodies against a single target, or single antibodies against many targets, with one exception, 35 they cannot be easily adapted to the screening of hundreds of antibodies in multiplex, that is, where individual antibody clones are analyzed against many targets in a single analytical step. ...
... By the elimination of liquid handling, more recent array-based methods using filters 33 or microscope slides 34,35 have significantly increased the number of antibody clones that can be screened at one time. Although these formats are very effective for screening large numbers of antibodies against a single target, or single antibodies against many targets, with one exception, 35 they cannot be easily adapted to the screening of hundreds of antibodies in multiplex, that is, where individual antibody clones are analyzed against many targets in a single analytical step. The inability to multiplex is a significant bottleneck for high-throughput screening, especially when contemplating generation and characterization of antibodies against all proteins encoded by a genome. ...
... Although microarrays are usually used to screen single antibodies against many antigens, or vice versa, a microarray method has been published, 34,35 in which antigens are arrayed first and antibodies subsequently arrayed in perfect register. Although this is not a true multiplex assay, in the sense that different binding events are analyzed pairwise, rather than simultaneously in a single test under identical conditions, it is capable of generating similar data. ...
The development of high-throughput screening (HTS) technologies has become essential for initial characterization of recombinant antibodies and alternative affinity reagents, selected from large combinatorial libraries. Such binding ligands are routinely selected against a single antigen and screened for desired binding specificities. Recent progress with genome sequencing projects has led to widespread efforts to study corresponding proteomes; requiring selection of ligands against large numbers of gene products in a highly parallel manner. The capabilities of many routine HTS methods such as enzyme-linked immunosorbent assay (ELISA), or array-based methods, are limited to analysis of numerous different antibody clones against a single target or, individual antibody clones against many different targets.
We have developed a multiplexed flow cytometry screening method that allows analysis of individual binding ligands against numerous targets in the same analytical sample. The method produces a complex analytical profile for each antibody clone in the primary screen, by allowing simultaneous determination of relative expression levels, identification of non-specific binding, and discrimination of fine specificities. The quality and quantity of data, combined with significant reductions in analysis time and antigen consumption, provide notable advantages over other standard screening methods, such as ELISA. By combining HT screening capabilities with multiplex technology, we have redefined the parameters for the initial identification of affinity reagents recovered from combinatorial libraries and removed a significant bottleneck in the generation of affinity reagents on a proteomic scale.
... The first important advancement is the use of an antibody-immobilized format, which in this context has been proposed in [19], in contrast to antigen-immobilized formats, which are recommended in most textbooks and articles. The second improvement is the miniaturization of the assay, which is achieved by the use of a microarray format, which has been used favorably in many applications, e.g., [20][21][22][23][24]. This enables the fast and easy performance of screening, sometimes with only a single chip. ...
... In the context of hybridoma technology, microarray-based screening formats have been presented in several publications [21,[27][28][29][30][31][32][33][34][35][36]. Nearly all used antigen-immobilized formats have all of the limitations mentioned above. ...
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 immanent 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 simultaneous performance of competition experiments. The latter can directly 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 has been designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the IgG concentration, which is unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration is not possible 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 had been 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, we conclude that this approach should be preferable to most other protocols leading to many of false positives, causing expensive and lengthy confirmation steps to weed out the poor clones.
... The first important advancement is the use of an antibody-immobilized format, which in this context has been proposed in [19], in contrast to antigen-immobilized formats, which are recommended in most textbooks and articles. The second improvement is the miniaturization of the assay, which is achieved by the use of a microarray format, which has been used favorably in many applications, e.g., [20][21][22][23][24]. This enables the fast and easy performance of screening, sometimes with only a single chip. ...
... In the context of hybridoma technology, microarray-based screening formats have been presented in several publications [21,[27][28][29][30][31][32][33][34][35][36]. Nearly all used antigen-immobilized formats have all of the limitations mentioned above. ...
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.
... [6][7][8] Antibodies and antibody fragments originating from phage display are widely used in therapeutic development and diagnostics. [9][10][11] They are commonly used in methods such as ELISA, 12 immunoblotting, 13 immunofluorescence microscopy, 14 affinity chromatography, 15 flow cytometry, 16 microarrays assays, 17 hemagglutination assays, 18 bead based assays, 19 proximity ligation assays, 20,21 molecular imaging, 22 lateral flow strip assays, 23 immuno-PCR 24 and fluorescence resonance energy transfer (FRET). 25 Phage display is a powerful technique in medical and health biotechnology. ...
... 27 Antibody phage display provides direct access to the genetic information of the binder, allowing a fast adaptation of the antibody format to the desired diagnostic assay. 12,13,17,22 The sensitivity of the diagnostic test could be increased by displaying on phage surface many copies of antibody fragments. Also the usage of the secondary antibodies with specificity for phage coat proteins could improve assay quality. ...
Phage display is a powerful technique in medical and health biotechnology. This technology has led to formation of antibody libraries and has provided techniques for fast and efficient search of these libraries. The phage display technique has been used in studying the protein-protein or protein-ligand interactions, constructing of the antibody and antibody fragments and improving the affinity of proteins to receptors. Recently phage display has been widely used to study immunization process, develop novel vaccines and investigate allergen-antibody interactions. This technology can provide new tools for protection against viral, fungal and bacterial infections. It may become a valuable tool in cancer therapies, abuse and allergies treatment. This review presents the recent advancements in diagnostic and therapeutic applications of phage display. In particular the applicability of this technology to study the immunization process, construction of new vaccines and development of safer and more efficient delivery strategies has been described.
... Here, we describe the application of MIST for the simultaneous evaluation of phage display derived soluble monoclonal antibody fragments on protein microarrays. The multiple spotting technique allows simultaneous evaluation of phage display derived soluble monoclonal antibody fragments on protein microarrays (Angenendt et al. 2004). The technique is based on the concept of printing multiple solutions in a sequel of spotting processes onto the same single positions on a microarray slide (Fig. 34.1, Table 34.1). ...
The generation of recombinant antibodies by phage display in high-throughput demands fast downstream technologies and streamlined processes for the identification and initial characterisation of individual binders. Next to standard immunological methods such as enzyme-linked immunosorbent assays (ELISA) and Western-blot, protein microarrays offer a wide range of possibilities in the evaluation process of monoclonal binders. Here, we describe the application of a special protein microarray method – the multiple spotting technique (MIST) – for the simultaneous evaluation of hundreds of phage display derived soluble monoclonal antibody fragments on protein microarrays. The standard operating procedures provided include the expression of soluble antibody fragments in microtitre plates, the spotting protocols and data evaluation schemes. Additionally, we show the comparability of this protein microarray application to conventional ELISA on a recent target antigen in our semi-automated selection pipeline. Applying MIST allows to reduce time, material and waste, and extends automation beyond the selection process applying conventional microarray machinery.
... Here, the targets and the negative control proteins are spotted on a glass chip first. Then, after blocking, the antibody fragments are spotted on top of the first spots and detected with a secondary antibody (Angenendt et al., 2004). Similarly, selected recombinant antibodies can be screened for monospecificity on recombinant protein micro-and macroarrays (Lü king et al., 1999;Holt et al., 2000b). ...
After the completion and refinement of the human genome, the characterization of individual gene products in respect of their functions, their modifications, their cellular localization and regulation in both space and time has generated an increased demand for antibodies for their analysis. Taking into account that the human genome contains approximately 25,000 genes, and that their products are found in different splice variants and produce proteins with post-translational modifications, it can be estimated that at least 100,000 different protein products have to be investigated to gain a complete picture of what's going on in the proteome of a cell. Antibodies are preferred tools helping with the characterization and detection of proteins as well as with elucidating their individual functions. The generation of antibodies to all available human protein products by immunization and/or the hybridoma technology is not only logistically and financially enduring, but may prove to be a difficult task, as quite a number of interesting targets may evade the immune response of experimental animals, for example, allosteric variants dependent on fragile interactions to cofactors, highly conserved antigens etc. For this reason, alternative methods for the generation of antibodies have to supplement these approaches. In vitro methods for antibody generation are seen to offer this capability. In addition, they may provide a cost effective and large scale production alternative for detection reagents for the research community in their own right. Among in vitro techniques, phage display has been evolved as the most efficient option for tackling this problem and approaches optimised for automation are emerging. Maximum benefit for proteomic research could be generated by judicious and preferably international coordination of the ongoing efforts to combine the strengths of the well established animal based approaches and the novel opportunities offered by in vitro methods.
... These high-throughput screening platforms consist of a combination of robotic colony-pickers and workstations, incubators for high-throughput expression, fluid-handling robots for performing ELISAs, high-throughput cloning and high-throughput purification, detection devices, PCR-machines, sequencing apparatus and data handling systems and software to integrate the data from all steps 64 . Further efforts to streamline the screening procedures 65,66 , including miniaturization, in vitro expression 67 , multiplexing and signal detection and data processing, will increase the throughput of these screening systems. Finally, high-density gridding of bacteria followed by protein array screening 68 and filter-based colony screening 69,70 have been used to bypass the selection step and directly screen antibody libraries. ...
During the past decade several display methods and other library screening techniques have been developed for isolating monoclonal antibodies (mAbs) from large collections of recombinant antibody fragments. These technologies are now widely exploited to build human antibodies with high affinity and specificity. Clever antibody library designs and selection concepts are now able to identify mAb leads with virtually any specificity. Innovative strategies enable directed evolution of binding sites with ultra-high affinity, high stability and increased potency, sometimes to a level that cannot be achieved by immunization. Automation of the technology is making it possible to identify hundreds of different antibody leads to a single therapeutic target. With the first antibody of this new generation, adalimumab (Humira, a human IgG1 specific for human tumor necrosis factor (TNF)), already approved for therapy and with many more in clinical trials, these recombinant antibody technologies will provide a solid basis for the discovery of antibody-based biopharmaceuticals, diagnostics and research reagents for decades to come.
... To overcome this bottleneck, in vitro selection methods such as phage display were implemented . To advance the effectiveness of the in vitro selection, scFv's can be spotted directly onto microarrays (Wingren et al. 2005) or analyzed by doublespotting the scFv directly onto the prior insolublezed target proteins (Angenendt et al. 2004). ...
Within the last 5 years, protein microarrays have been developed and applied to multiple approaches: identification of protein-protein interactions or protein-small molecule interactions, cancer profiling, detection of microorganisms and toxins, and identification of antibodies due to allergens, autoantigens, and pathogens. Protein microarrays are small size (typically in the microscopy slide format) planar analytical devices with probes arranged in high density to provide the ability to screen several hundred to thousand known substrates (e.g., proteins, peptides, antibodies) simultaneously. Due to their small size, only minute amounts of spotted probes and analytes (e.g., serum) are needed; this is a particularly important feature, for these are limited or expensive. In this review, different types of protein microarrays are reviewed: protein microarrays (PMAs), with spotted proteins or peptides; antibody microarrays (AMAs), with spotted antibodies or antibody fragments (e.g., scFv); reverse phase protein microarrays (RPMAs), a special form of PMA where crude protein mixtures (e.g., cell lysates, fractions) are spotted; and nonprotein microarrays (NPMAs) where macromolecules other than proteins and nucleic acids (e.g., carbohydrates, monosaccharides, lipopolysaccharides) are spotted. In this study, exemplary experiments for all types of protein arrays are discussed wherever applicable with regard to investigations of microorganisms.
... Microarrays comprising a large number of spots, each with a different chemical or biochemical probe, allow for high-throughput biochemical assays to be performed using minute amounts of reagents [1][2][3][4] . However, the on-site delivery of reagents for microarray production requires expensive liquid handling robotics, such as inkjet or pin spotters, as well as skilled operators and maintenance. ...
The coordinated delivery of minute amounts of different reagents is important for microfluidics and microarrays, but is dependent on advanced equipment such as microarrayers. Previously, we developed the snap chip for the direct transfer of reagents, thus realizing fluidic operations by only manipulating microscope slides. However, owing to the misalignment between arrays spotted on different slides, millimeter spacing was needed between spots and the array density was limited. In this work, we have developed a novel double transfer method and have transferred 625 spots cm(-2), corresponding to >10000 spots for a standard microscope slide. A user-friendly snapping system was manufactured to make liquid handling straightforward. Misalignment, which for direct transfer ranged from 150-250 μm, was reduced to <40 μm for double transfer. The snap chip was used to quantify 50 proteins in 16 samples simultaneously, yielding limits of detection in the pg/mL range for 35 proteins. The versatility of the snap chip is illustrated with a 4-plex homogenous enzyme inhibition assay analyzing 128 conditions with precise timing. The versatility and high density of the snap chip with double transfer allows for the development of high throughput reagent transfer protocols compatible with a variety of applications.
... On CHO transformed cell surface selection scFv Abs against non-dominant epitopes [33] Human blood group antigens On red cell surface scFv Abs against ABO blood group system & C,c,D,E,e antigens Rh system [34] CD3 and CD20 subsets of blood leukocytes Blood leukocytes isolated by FACS scFv Description method [35] RhD-positive RhD isolated by MACS Fab Description method [36] immobilized cDNA templates, avoiding protein purification [47], or by using a phage-display array to facilitate identification of different antibodies fragments to known proteins have been described [48][49]. Recent developments of high-throughput sequencing technologies have made the use of next generation sequencing (NGS) widespread for the analysis of the sequences of the antibodies from a phage library display, in order to characterize the library in terms of diversity and quality, as well as during in vitro selection [50][51][52]. ...
The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described.
... Kibat et al. [25] demonstrated the utility of microarrays in the selection of scFv clones by making a fusion protein with a human IgG1 Fc part, using direct printing of the purified antibody and incubation with clinical samples. Angenendt et al. [26] screened scFv supernatants against printed target protein in spot-on-spot arrays demonstrating a higher-throughput assay. ...
We have developed a combinatory antibody–antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a “spot-on-spot” print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcα-serine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation.
... The open microarray architecture is one of the major advantages of microarray technology allowing several antibodies to be screened on several antigens at the same time which requires complicated liquid handling. This is highlighted by the multiple spotting techniques (MIST), which comprises immobilization of a binder onto a surface and subsequent spotting of the second compound on the same spot, on the top of the immobilized binder [32,33]. A major advantage of microarray technology is the production of functional proteins with methods such as the protein in situ microarray (PISA). ...
Phage-display technology constitutes a powerful tool for the generation of specific antibodies against a predefined antigen. The main advantages of phage-display technology in comparison to conventional hybridoma-based techniques are: (1) rapid generation time and (2) antibody selection against an unlimited number of molecules (biological or not). However, the main bottleneck with phage-display technology is the validation strategies employed to confirm the greatest number of antibody fragments. The development of new high-throughput (HT) techniques has helped overcome this great limitation. Here, we describe a new method based on an array technology that allows the deposition of hundreds to thousands of phages by micro-contact on a unique nitrocellulose surface. This setup comes in combination with bioinformatic approaches that enables simultaneous affinity screening in a HT format of antibody-displaying phages.
... Automated enzyme-linked immunosorbent assay (ELISA) 2 and microarray methods have been shown to be eVective primary screens for the initial identiWcation of phage hits [15][16][17][18][19]. Discovery of the most potent antibodies identiWed in the primary screen depends on the development and use of suitable secondary screening assays. ...
A method was developed to rapidly identify high-affinity human antibodies from phage display library selection outputs. It combines high-throughput Fab fragment expression and purification with surface plasmon resonance (SPR) microarrays to determine kinetic constants (kon and koff) for 96 different Fab fragments in a single experiment. Fabs against human tissue kallikrein 1 (hK1, KLK1 gene product) were discovered by phage display, expressed in Escherichia coli in batches of 96, and purified using protein A PhyTip columns. Kinetic constants were obtained for 191 unique anti-hK1 Fabs using the Flexchip SPR microarray device. The highest affinity Fabs discovered had dissociation constants of less than 1 nM. The described SPR method was also used to categorize Fabs according to their ability to recognize an apparent active site epitope. The ability to rapidly determine the affinities of hundreds of antibodies significantly accelerates the discovery of high-affinity antibody leads.
We established a label-free method of measuring proteins in crude cell lysate using antibody arrays and surface plasmon resonance (SPR) imaging. The refractivity of the running buffer was adjusted with that of the lysate to overcome the bulk effect. The chemistries of the fabricated arrays were investigated to reduce nonspecific adsorption on the array surface. We found that the hydrophilicity of the poly(ethylene glycol) moiety and lower electrostatic charge on the surface provided a specific measurement of antigen-antibody interaction. We validated the system by measuring the expression of eight proteins in the mouse brain and comparing the results to those by conventional Western blotting. The detection limit of the antibody array was approximately 30 ng/mL in crude cell lysate, on the same order as that of previous SPR research. The system enabled quick, label-free, and high-throughput analysis of abundant proteins with minimal sample volume ( approximately 200 muL). It is expected that our SPR antibody array will be applicable for direct protein expression profiling of cell lysate, as well as for cell phenotyping, food analysis, discovery of new biomarkers, and immunological disease diagnostics.
High-throughput methods to detect and quantify antibodies in sera and other patient specimens have use for many clinical and laboratory studies, including those associated with cancer detection, microbial exposures, and autoimmune diseases. We developed a new technique, termed layered peptide array (LPA), to serve as a screening tool to detect antibodies in a highly multiplexed format. We demonstrate here that a prototype LPA was capable of producing approximately 5000 measurements per experiment and appeared to be scalable to higher throughput levels. Sera and saliva from Sjögren's syndrome patients served as a test set to examine antibody titers in clinical samples. The LPA platform exhibited both a high sensitivity (100%) and high specificity (94%) for correctly identifying SSB antigen-positive samples. The multiplex capability of the platform was also confirmed when serum and saliva samples were analyzed for antibody reactivity to several peptides, including Sjögren's syndrome antigens A and B. The data indicate that LPA analysis will be a useful method for a number of screening applications.
The precise determination of affinity and specificity is a crucial step in the development of new protein reagents for therapy and diagnostics. Paradoxically, the selection of protein binders, e.g., antibody fragments, from large combinatorial repertoires is a rapid process compared to the subsequent characterization of selected clones. Here we demonstrate the use of suspension bead arrays (SBA) in combination with flow cytometry to facilitate the post-selection analysis of binder affinities. The array is designed to capture the proteins of interest (POIs) covalently on the surface of superparamagnetic color-coded microbeads directly from expression cell lysate, based on SpyTag-SpyCatcher coupling by isopeptide bond formation. This concept was validated by analyzing the affinities of a typical phage display output, i.e., clones consisting of single-chain variable fragment antibodies (scFvs), as SpyCatcher fusions in 12- and 24-plex SBA formats using a standard three-laser flow cytometer. We demonstrate that the equilibrium dissociation constants (Kd) obtained from multiplexed SBA assays correlate well with experiments performed on a larger scale, while the antigen consumption was reduced >100-fold compared to the conventional 96-well plate format. Protein screening and characterization by SBAs is a rapid and reagent-saving analytical format for combinatorial protein engineering to address specificity maturation and cross-reactivity profiling of antibodies.
The problems we face today in public health as a result of the -- fortunately -- increasing age of people and the requirements of developing countries create an urgent need for new and innovative approaches in medicine and in agronomics. Genomic and functional genomic approaches have a great potential to at least partially solve these problems in the future. Important progress has been made by procedures to decode genomic information of humans, but also of other key organisms. The basic comprehension of genomic information (and its transfer) should now give us the possibility to pursue the next important step in life science eventually leading to a basic understanding of biological information flow; the elucidation of the function of all genes and correlative products encoded in the genome, as well as the discovery of their interactions in a molecular context and the response to environmental factors. As a result of the sequencing projects, we are now able to ask important questions about sequence variation and can start to comprehensively study the function of expressed genes on different levels such as RNA, protein or the cell in a systematic context including underlying networks. In this article we review and comment on current trends in large-scale systematic biological research. A particular emphasis is put on technology developments that can provide means to accomplish the tasks of future lines of functional genomics.
Over the last years microarray technology has become one of the principal platform technologies for the high-throughput analysis of biological systems. Starting with the construction of first DNA microarrays in the 1990s, microarray technology has flourished in the last years and many different new formats have been developed. Peptide and protein microarrays are now applied for the elucidation of interaction partners, modification sites and enzyme substrates. Antibody microarrays are envisaged to be of high importance for the high-throughput determination of protein abundances in translational profiling approaches. First cell microarrays have been constructed to transform microarray technology from an in vitro technology to an in vivo functional analysis tool. All of these approaches share a common prerequisite: the solid support on which they are generated. The demands on this solid support are thereby as manifold as the applications themselves. This review is aimed to display the recent developments in surface chemistry and derivatization, and to summarize the latest developments in the different application areas of microarray technology.
Due to the success of DNA microarrays and the growing numbers of available protein expression clones, protein microarrays have become more and more popular for the high throughput screening of protein interactions. However, the widespread applicability of protein microarrays is currently hampered by the large effort associated with their production. Apart from the requirement for a protein expression library, expression and purification of the proteins themselves and the lacking stability of many proteins remain the bottleneck. Here we present an approach that allows the generation of high density protein microarrays from unbound DNA template molecules on the chip. It is based on the multiple spotting technique and comprises the deposition of a DNA template in a first spotting step and the transfer of a cell-free transcription and translation mixture on top of the same spot in a second spotting step. Using wild-type green fluorescent protein as a model protein, we demonstrated the time and template dependence of this coupled transcription and translation and showed that enough protein was produced to yield signals that were comparable to 300 microg/ml spotted protein. Plasmids as well as unpurified PCR products can be used as templates, and as little as 35 fg of PCR product ( approximately 22,500 molecules) were sufficient for the detectable expression of full-length wild-type green fluorescent protein in subnanoliter volumes. We showed that both aminopropyltrimethoxysilane and nickel chelate surfaces can be used for capture of the newly synthesized proteins. Surprisingly we observed that nickel chelate-coated slides were binding the newly synthesized proteins in an unspecific manner. Finally we adapted the system to the high throughput expression of libraries by designing a single primer pair for the introduction of the required T7 promoter and demonstrated the in situ expression using 384 randomly chosen clones.
Antibody-based microarrays are among the novel classes of rapidly evolving proteomic technologies that holds great promise in biomedicine. Miniaturized microarrays (< 1 cm2) can be printed with thousands of individual antibodies carrying the desired specificities, and with biological sample (e.g., an entire proteome) added, virtually any specifically bound analytes can be detected. While consuming only minute amounts (< microL scale) of reagents, ultra- sensitive assays (zeptomol range) can readily be performed in a highly multiplexed manner. The microarray patterns generated can then be transformed into proteomic maps, or detailed molecular fingerprints, revealing the composition of the proteome. Thus, protein expression profiling and global proteome analysis using this tool will offer new opportunities for drug target and biomarker discovery, disease diagnostics, and insights into disease biology. Adopting the antibody microarray technology platform, several biomedical applications, ranging from focused assays to proteome-scale analysis will be rapidly emerging in the coming years. This review will discuss the current status of the antibody microarray technology focusing on recent technological advances and key issues in the process of evolving the methodology into a high-performing proteomic research tool.
Antibody microarrays have shown great potential for measurement of either a spectrum of target proteins in proteomics or disease-associated antigens in molecular diagnostics. Despite its importance, the applications of antibody microarrays are still limited by a variety of fundamental problems. Among them, cross-reactivity significantly limits the multiplexing ability in parallel sandwich immunoassays. As a result, it is very important to design new capture probes in order to incorporate a universal label into the assay configuration. In this report, an antibody fragments (F(ab')2) microarray platform for serum tumor markers was developed. Each antigen was detected at different concentrations to assemble its calibration curve, and combinations of different markers were tested to examine the specificity of simultaneous detection based on the F(ab')2 microarrays. Diagnostics of serum samples with this cancer antibody microarray platform and immunoradiometric assays (IRMA) were also performed. Wide range calibration curves (0-1280 U mL(-1)) were obtained for each tumor marker. Comparative studies demonstrated that such F(ab')2 microarrays exhibited both moderately improved sensitivity and better specificity than full-sized monoclonal antibody microarrays. It is also demonstrated that this microarray platform is quantitative, highly specific and reasonably sensitive. More importantly, clinical applications of our F(ab')2 microarray platform for upwards of 100 patient serum samples clearly show its potential in cancer diagnostics.
Using a large phage antibody library, a protein microarray spotted directly with phage-displayed antibody clones was created to discriminate between recognition profiles of samples from healthy donors and leukemia patients. The protocol for preparing antibody-displaying phage chips was presented. Some conditions such as substrates and blocking buffers were compared and optimized. The major improvements of this microarray are higher throughput and lower cost compared to previous antibody chips. Due to its convenience and sensitivity, it can be extensively used for rapid and high throughput detection of protein profiles of experimental and clinical samples.
The layered peptide array (LPA) is a recently developed technique designed to measure antibody levels in a multiplex, high-throughput manner. LPAs can assess antibody presence either in fluid samples or from tissues while maintaining the two-dimensional orientation of the life science platform. In this manuscript, we evaluated and assessed the performance of the LPA platform, focusing on throughput capability, sensitivity, and specificity of the assay in several different systems.
Antibody-based microarrays are a rapidly emerging technology that has advanced from the first proof-of-concept studies to demanding serum protein profiling applications during recent years, displaying great promise within disease proteomics. Miniaturized micro- and nanoarrays can be fabricated with an almost infinite number of antibodies carrying the desired specificities. While consuming only minute amounts of reagents, multiplexed and ultrasensitive assays can be performed targeting high- as well as low-abundance analytes in complex nonfractionated proteomes. The microarray images generated can then be converted into protein expression profiles or protein atlases, revealing a detailed composition of the sample. The technology will provide unique opportunities for fields such as disease diagnostics, biomarker discovery, patient stratification, predicting disease recurrence and drug target discovery. This review describes an update of high-throughput proteomics, using antibody-based microarrays, focusing on key technological advances and novel applications that have emerged over the last 3 years.
On the basis of discussions with representatives from all sectors of the cancer research community, the National Cancer Institute (NCI) recognizes the immense opportunities to apply proteomics technologies to further cancer research. Validated and well characterized affinity capture reagents (e.g. antibodies, aptamers, and affibodies) will play a key role in proteomics research platforms for the prevention, early detection, treatment, and monitoring of cancer. To discuss ways to develop new resources and optimize current opportunities in this area, the NCI convened the "Proteomic Technologies Reagents Resource Workshop" in Chicago, IL on December 12-13, 2005. The workshop brought together leading scientists in proteomics research to discuss model systems for evaluating and delivering resources for reagents to support MS and affinity capture platforms. Speakers discussed issues and identified action items related to an overall vision for and proposed models for a shared proteomics reagents resource, applications of affinity capture methods in cancer research, quality control and validation of affinity capture reagents, considerations for target selection, and construction of a reagents database. The meeting also featured presentations and discussion from leading private sector investigators on state-of-the-art technologies and capabilities to meet the user community's needs. This workshop was developed as a component of the NCI's Clinical Proteomics Technologies Initiative for Cancer, a coordinated initiative that includes the establishment of reagent resources for the scientific community. This workshop report explores various approaches to develop a framework that will most effectively fulfill the needs of the NCI and the cancer research community.
Antibody-based microarrays are a new powerful proteomic technology that can be used to generate rapid and detailed expression profiles of defined sets of protein analytes in complex samples as well as high-resolution portraits of entire proteomes. Miniaturized micro- and nanoarrays can be printed with numerous antibodies carrying the desired specificities. Multiplexed and ultra-sensitive assays, specifically targeting several analytes in a single experiment, can be performed, while consuming only minute amounts of the sample. The array images generated can then be converted into protein expression profiles, or maps, revealing the detailed composition of the sample. This promising proteomic research tool will thus provide unique opportunities for e.g. disease proteomics, biomarker discovery, disease diagnostics, and patient stratification. This review describes the antibody-based microarray technology and applications thereof.
The success of genome sequencing projects has provided the basis for systematic analysis of protein function and has led to a shift from the description of single molecules to the characterization of complex samples. Such a task would not be possible without the provision of appropriate high-throughput technologies, such as protein microarray technology. In addition, the increasing number of samples necessitates the adaptation of such technologies to a multiplex format. This review will discuss protein microarray technology in the context of multiplex analysis and highlight its current prospects and limitations.
IntroductionGeneral Considerations for the Automation of Antibody GenerationDevelopment of an Antibody Generation PipelineConclusion
Die Entwicklung der DNA-Sequenzierungstechnologien wurde durch die Aufgabe, das ge-
samte menschliche Genom zu entschlüsseln, vorangetrieben. Fünf Jahre nach Ende des
Humangenomprojektes ist das Verständnis der Funktion der durch die rund 23.000 Gene
kodierten Proteine des menschlichen Genoms jedoch immer noch rudimentär. Einer der
limitierenden Faktoren dabei ist das Fehlen einer Hochdurchsatzmethode zur Herstellung
von Antikörpern für die zellbiologische und biochemische Charakterisierung der vielen
unbekannten Genprodukte und ihrer Interaktionspartner. In dem innerhalb des Nationalen
Genomforschungsnetzes (NGFN) geförderten Projektes „Antibody Factory“ werden deshalb
Methoden entwickelt, um Antikörper in vitro in großer Zahl herstellen zu können. Die in
vitro-Herstellung mittels Phagendisplay bietet die Vorteile einer einfachen Automatisierung
und der preiswerten Produktion in E. coli, kommt vollständig ohne Versuchstiere aus, ist
weitgehend miniaturisierbar und benötigt nur sehr geringe Mengen der meist begrenzt
verfügbaren Antigene. Begleitend werden verschiedene neuartige Methoden zur Antigen-
herstellung und für die Validierung der Antikörper in höherem Durchsatz untersucht.
Peptide and protein arrays have gained increasing attention due to their potential application
in many areas of research, clinical diagnosis, and pharmacy. Atypical array consists of a support containing
immobilized peptides or proteins positioned in an addressable format. The greatest advantage of the
arrays is the possibility for miniaturization, which relies on dividing the surface into miniature spots,
thus allowing for hundreds/thousands of analyses to be simultaneously performed using minimal
amounts of a precious biological material. The quality of assays with the use of peptide and protein
arrays depends on the surface properties, e.g., hydrophilicity, homogeneity, density of functional groups,
surface morphology, etc. In recent years, itwas shown that the quality of the assays might be improved by
introducing polymers acting as spacers between the peptide and the solid support. This approach causes
changes in the surface properties, e.g., it reduces the undesirable non-specific adsorption of biomolecules,
increases the density of functional groups, or can improve the biological activity of biomolecules attached
to the surface. In this review, various types of polymers that are used for peptide and protein arrays and
their impact on the assay quality are discussed.
The biomedical community has the imperative to develop reliable, clinically relevant and generalizable bioassays that can be used to accurately recognize those individuals with early-stage disease or those patients who will respond to therapy, with the ultimate aim of achieving individualized medicine. In recent years, increasingly sophisticated proteomic screening technologies have been introduced, providing the biomedical community with a valuable new approach for the systematic discovery and validation of novel diagnostic, prognostic and therapeutic tools. Nevertheless, the complexity of the cellular milieu wherein a variety of macromolecules interact in dynamic fashion, combined with the complex clinical manifestation of chronic pathologies and widespread diversity of patient populations, mean that universal biomarkers will not be easily developed. In this review, the five key challenges that must be surmounted in order to advance the clinical impact of this nascent field are described, and plausible solutions based on the authors' own ongoing proteomic profiling of cardiovascular disease is outlined.
From the time that Kohler and Milstein first published their technique of using a hybridoma to produce monoclonal antibodies (Mabs), a major and rapid progression occurred in the field of medicine, specifically immunology. Mabs began to be used as tools for diagnosing and treating disease and have proven to be valuable agents in the research laboratory. When tagged with fluorescent dyes, radioactive or heavy atoms (gold), enzymes or toxins, they help identify molecules of biological and medical importance. They can be used with microscopic techniques to identify structures or molecules within a cell. Mabs are being tested experimentally to inhibit transplant rejection, to alleviate and suppress autoimmune disease, and to detect and treat cancer. For example, Mabs can be used to remove undesirable lymphocytes from donor bone marrow cells prior to transplantation to avoid Graft versus Host disease. However, the use of Mabs produced by the mouse hybridoma can interfere with the effectiveness of the antibodies or cause allergic reactions in humans. Thus, new strategies are being explored for the construction of Mabs that can better penetrate the target tissue and are not recognized as foreign by the human immune system. Among these approaches is the cloning of human antibodies, or the placing of human antibody genes into the genome of mice that have been bred without an antibody-producing mechanism of their own. Mabs are being used in both the diagnosis and treatment of cancer because many of the same cancer types present in different patients were shown to have similar cell surface molecules. Mabs, tagged with chemotherapeutic agents or radioactive atoms, when targeted to these tumor antigens, have the potential to destroy tumor cells. Studies are being conducted to evaluate the effectiveness of Mabs to block growth factors or receptors on tumor cells; to target specific tissue components of the tumor or its blood vessels; to interfere with cell signals; or with apoptosis (programmed cell death). When labeled with radioactive isotopes, Mabs can also be used in the diagnosis of disease in nuclear medicine procedures. They are being used in the food industry to identify either contaminating molecules, or the presence of harmful bacteria. They are also being used to create strategic reserves of vaccines and antibodies for infectious agents that could be used in biowarfare.
Proteomic approaches, such as protein microarray technology, play an
important role in the study of complex biological systems. Their application in
plant science has been strongly supported by the completion of genome sequence
projects in the model plants Arabidopsis thaliana and rice. This chapter focuses
on the identification of substrate phosphorylation by protein kinases using protein
microarrays. Protein microarrays are widely used to profile antibodies and sera for
their specificity, and/or to screen entire proteomes for new protein interactions.
Here, we emphasise the feasibility of using protein microarrays in the discovery
of potential protein kinase substrates in plants. Our group has identified potential
substrates in two different plant species: first barley and later Arabidopsis. A signal
quantification and threshold-based selection method was introduced during optimisation
of protein microarray technology for substrate identification in order to
better evaluate the microarray data, and to provide a preliminary list of candidate
substrates for further investigation.
Over the past decade, the accumulation of detailed knowledge of antibody structure and function has enabled antibody phage display to emerge as a powerful in vitro alternative to hybridoma methods for creating antibodies. Many antibodies produced using phage display technology have unique properties that are not obtainable using traditional hybridoma technologies. In phage display, selections are performed under controlled, in vitro conditions that are tailored to suit demands of the antigen and the sequence encoding the antibody is immediately available. These features obviate many of the limitations of hybridoma methodology, and because the entire process relies on scalable molecular biology techniques, phage display is also suitable for high-throughput applications. Thus, antibody phage display technology is well suited for genome-scale biotechnology and therapeutic applications. This review describes the antibody phage display technology and highlights examples of antibodies with unique properties that cannot easily be obtained by other technologies.
Antibodies that bind their respective targets with high affinity and specificity have proven to be essential reagents for
biological research. Antibody phage display has become the leading tool for the rapid isolation of single-chain variable fragment
(scFv) antibodies in vitro for research applications, but there is usually a gap between scFv isolation and its application
in an array format suitable for high-throughput proteomics. In this chapter, we present our antibody phage display system
where antibody isolation and scFv immobilization are facilitated by the design of the phagemid vector used as platform. In
our system, the scFvs are fused at their C-termini to a cellulose-binding domain (CBD) and can be immobilized onto cellulose-based
filters. This made it possible to develop a unique filter lift screen that allowed the efficient screen for multiple binding
specificities, and to directly apply library-derived scFvs in an antibody spotted microarray.
Neisseria meningitidis is the most common cause of meningitis and causes epidemic outbreaks. One trait of N. meningitidis, which is associated with most of the currently recognized virulence determinants, is the presence of phase-variable genes that are suspected to enhance its ability to cause an invasive disease. To detect the immune responses to phase-variable expressed proteins, we applied protein microarray technology for the screening of meningitis patient sera. We amplified all 102 known phase-variable genes from N. meningitidis serogroup B strain MC58 by polymerase chain reaction and subcloned them for expression in Escherichia coli. With this approach, we were able to express and purify 67 recombinant proteins representing 66% of the annotated genes. These were spotted robotically onto coated glass slides to generate protein microarrays, which were screened using 20 sera of patients suffering from meningitis, as well as healthy controls. From these screening experiments, 47 proteins emerged as immunogenic, exhibiting a variable degree of seroreactivity with some of the patient sera. Nine proteins elicited an immune response in more than three patients, with one of them, the phase-variable opacity protein OpaV (NMB0442), showing responses in 11 patient sera. This is the first time that protein microarray technology has been applied for the investigation of genetic phase variation in pathogens. The identification of disease-specific proteins is a significant target in biomedical research, as such proteins may have medical, diagnostic, and commercial potential as disease markers.
Proteomic approaches play an important role in the study of complex biological systems. The application of
proteomic technologies in plant science has been strongly supported by the completion of genome sequence projects of
the model plants Arabidopsis thaliana and rice. This review focuses on the state of proteomic technologies with special
emphasis on their application in plant biology. An overview of recent developments in 2-dimensional gel electrophoresis
and liquid chromatography-based multidimensional protein identification technology, MudPIT is provided. These
techniques are commonly combined with mass spectrometric methods for identification of proteins. Furthermore, protein
expression profiling by antibody arrays and the selection of required recombinant antibodies by phage display are
described. Interaction studies, using functional protein microarrays or the yeast two-hybrid system are presented as
powerful techniques to gain insights into the function of proteins. Advantages and limitations of the described methods as
well as their current and potential future applications in plant research are discussed.
Affinity proteomics, mainly represented by antibody microarrays, has in recent years been established as a powerful tool for high-throughput (disease) proteomics. The technology can be used to generate detailed protein expression profiles, or protein maps, of focused set of proteins in crude proteomes and potentially even high-resolution portraits of entire proteomes. The technology provides unique opportunities, for example biomarker discovery, disease diagnostics, patient stratification and monitoring of disease, and taking the next steps toward personalized medicine. However, the process of designing high-performing, high-density antibody micro- and nanoarrays has proven to be challenging, requiring truly cross-disciplinary efforts to be adopted. In this mini-review, we address one of these key technological issues, namely, the choice of probe format, and focus on the use of recombinant antibodies vs. polyclonal and monoclonal antibodies for the generation of antibody arrays.
The analysis of self-assembled protein microarrays, using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, combines two high-throughput platforms for investigation of the proteome. In this article, we describe the fabrication in situ of protein arrays optimized for MALDI characterization. Using the green fluorescent protein (GFP) both as an epitope for immobilization and as a gauge for relative protein expression, we were able to generate amounts of protein on the array slides sufficient for MALDI identification. In addition, expression of N-terminal protein constructs fused to GFP demonstrated mass shifts consistent with that of the full-length protein. We envision this technology to be important for the functional screening of protein interactions.
In vitro antibody generation technologies have now been available for two decades. Research reagents prepared via phage display are becoming available and several recent studies have demonstrated that these technologies are now sufficiently advanced to facilitate generation of a comprehensive renewable resource of antibodies for any protein encoded by the approximately 22,500 human protein-coding genes. Antibody selection in vitro offers properties not available in animal-based antibody generation methods. By adjusting the biochemical milieu during selection, it is possible to control the antigen conformation recognized, the antibody affinity or unwanted cross-reactivity. For larger-scale antibody generation projects, the handling, transport and storage logistics and bacterial production offer cost benefits. Because the DNA sequence encoding the antibody is available, modifications, such as site-specific in vivo biotinylation and multimerization, are only a cloning step away. This opinion article summarizes opportunities for the generation of antibodies for proteome research using in vitro technologies.
Many areas of research today are based on enzymatic assays most of which are still performed as enzyme-linked immunosorbent assays in microtiter plates. The demand for highly parallel screening of thousands of samples eventually led to a miniaturization and automation of these assays. However, the final transfer of enzymatic assays from a microtiter-based technology to microarrays has proven to be difficult for various reasons, such as the inability to maintain unbound reaction products on the spot of reaction or the missing capability of multiplexing. Here, we have conducted multiplex enzymatic assays in subnanoliter volumes on a single microarray using the multiple spotting technology. We were able to measure enzymatic activity with a sensitivity down to 35 enzyme molecules, applying only conventional flat microarray surfaces and standard microarray hardware. We have performed assays of inhibition and applied this format for the detection of prognostic markers, such as cathepsin D. The new approach allows the rapid and multiplex screening of thousands of samples on a single microarray with applications in drug screening, metagenomics, and high-throughput enzyme assays.
Proteome-wide sets of antibodies would be an invaluable research resource for use in highly parallel assays such as microarrays. Such assays could provide deeper insights into biology and a wealth of information for clinical diagnostics. However, the rate of discovery of new proteins far exceeds the antibody supply currently produced from traditional animal-based systems. To address this problem, a variety of improvements in antibody production have been developed, including improved animal-based technologies, new antibody structures with superior performances, faster and more discriminating screening techniques, and rapid validation methods. Many of these technologies are amenable to automation, allowing antibody production throughput to significantly increase.
The success of genome sequencing projects has led to a shift from the description of single molecules to the characterisation of complex samples. At the same time, there is growing interest not only in studying organisms at the genomic level, but in the characterization of their proteome. Such a task would not be possible without the availability of appropriate technologies. Protein and antibody microarray technologies are, in addition to two-dimensional gel electrophoresis followed by mass spectrometry, two of the most propitious technologies for the screening of complex protein samples. Nevertheless, to succeed, protein and antibody microarrays have to overcome their current limitations. This review aims to introduce these new technologies and highlights their current prospects and limitations.
Antibody phage display, coupled with automated screening, facilitates and potentiates the mining of complex combinatorial libraries and the identification of potent drug leads. In managing phage screening data, the behavior of individual phage isolates in binding assays must be linked to their antibody identities as deduced from DNA sequencing. Reviewed here are recently reported approaches for high-throughput screening of clones isolated from phage antibody libraries after selection on a defined antigen. Specific information management challenges, and possible solutions, are described for organizing screening data to enable rapid lead discovery using these antibody libraries.
Thousands of genes are being discovered for the first time by sequencing the genomes of model organisms, an exhilarating reminder that much of the natural world remains to be explored at the molecular level. DNA microarrays provide a natural vehicle for this exploration. The model organisms are the first for which comprehensive genome-wide surveys of gene expression patterns or function are possible. The results can be viewed as maps that reflect the order and logic of the genetic program, rather than the physical order of genes on chromosomes. Exploration of the genome using DNA microarrays and other genome-scale technologies should narrow the gap in our knowledge of gene function and molecular biology between the currently-favoured model organisms and other species.
We report the design, construction, and use of the first very large non-immunized phage antibody library in Fab format, which allows the rapid isolation and affinity analysis of antigen-specific human antibody fragments. Individually cloned heavy and light chain variable region libraries were combined in an efficient two-step cloning procedure, permitting the cloning of a total of 3.7 x 10(10) independent Fab clones. The performance of the library was determined by the successful selection of on average 14 different Fabs against 6 antigens tested. These include tetanus toxoid, the hapten phenyl-oxazolone, the breast cancer-associated MUC1 antigen, and three highly related glycoprotein hormones: human chorionic gonadotropin, human luteinizing hormone, and human follicle-stimulating hormone. In the latter category, a panel of either homone-specific or cross-reactive antibodies were identified. The design of the library permits the monitoring of selections with polyclonal phage preparations and to carry out large scale screening of antibody off-rates with unpurified Fab fragments on BIAcore. Antibodies with off-rates in the order of 10(-2) to 10(-4) s-1 and affinities up to 2.7 nM were recovered. The kinetics of these phage antibodies are of the same order of magnitude as antibodies associated with a secondary immune response. This new phage antibody library is set to become a valuable source of antibodies to many different targets, and to play a vital role in target discovery and validation in the area of functional genomics.
With the human genome project approaching completion, there is a growing interest in functional analysis of gene products. The characterization of large numbers of proteins, their expression patterns and in vivo localisations, demands the use of automated technology that maintains a logistic link to the encoding genes. As a complementary approach, phage display is used for recombinant protein expression and the selection of interacting (binding) molecules. Cloning of libraries in filamentous bacteriophage or phage mid vectors provides a physical link between the expressed protein and its encoding DNA sequence. High-throughput technology for automated library handling and phage display selection has been developed using picking-spotting robots and a module for pin-based magnetic particle handling. This system enables simultaneous interaction screening of libraries and the selection of binders to different target molecules at high throughput. Target molecules are either displayed on high-density filter membranes (protein filters) or tag-bound to magnetic particles and can be handled as native ligands. Binding activity is confirmed by magnetic particle ELISA in the microtitre format. The whole procedure from immobilisation of target molecules to confirmed clones of binders is automatable. Using this technology, we have selected human scFv antibody fragments against expression products of human cDNA libraries.
Plants respond to pathogen attack by deploying several defense reactions. Some rely on the activation of preformed components, whereas others depend on changes in transcriptional activity. Using cDNA arrays comprising 13,000 unique expressed sequence tags, changes in the transcriptome of Arabidopsis thaliana were monitored after attempted infection with the bacterial plant pathogen Pseudomonas syringae pv. tomato carrying the avirulence gene avrRpt2. Sampling at four time points during the first 24 h after infiltration revealed significant changes in the steady state transcript levels of approximately 650 genes within 10 min and a massive shift in gene expression patterns by 7 h involving approximately 2,000 genes representing many cellular processes. This shift from housekeeping to defense metabolism results from changes in regulatory and signaling circuits and from an increased demand for energy and biosynthetic capacity in plants fighting off a pathogenic attack. Concentrating our detailed analysis on the genes encoding enzymes in glycolysis, the Krebs cycle, the pentose phosphate pathway, the biosynthesis of aromatic amino acids, phenylpropanoids, and ethylene, we observed interesting differential regulation patterns. Furthermore, our data showed potentially important changes in areas of metabolism, such as the glyoxylate metabolism, hitherto not suspected to be components of plant defense.
Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously on glass arrays with signal amplification by RCA with high specificity, femtomolar sensitivity, 3 log quantitative range, and economy of sample consumption. A 51-feature RCA cytokine glass array was used to measure secretion from human dendritic cells (DCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha). As expected, LPS induced rapid secretion of inflammatory cytokines such as macrophage inflammatory protein (MIP)-1beta, interleukin (IL)-8, and interferon-inducible protein (IP)-10. We found that eotaxin-2 and I-309 were induced by LPS; in addition, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), soluble interleukin 6 receptor (sIL-6R), and soluble tumor necrosis factor receptor I (sTNF-RI) were induced by TNF-alpha treatment. Because microarrays can accommodate approximately 1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys.
Antibody microarrays are becoming a major tool for the parallel analysis of complex samples. So far, many efforts have been made to increase the complexity and sensitivity of antibody microarrays. In contrast to enzyme-linked immunosorbent assay (ELISA) experiments, not all antibodies remain functional in the microarray format. Sensitivity is very much dependent on the type of coating and its application. The method described in this chapter is a quick and reliable method that has been very useful in determining the functionality of antibodies and the suitability of coatings for antibody microarrays. At the same time, a detailed description of how to prepare an inexpensive and highly efficient antibody microarray surface is given.
Microarray technology allows the simultaneous analysis of thousands of parameters within a single experiment. Microspots of capture molecules are immobilised in rows and columns onto a solid support and exposed to samples containing the corresponding binding molecules. Readout systems based on fluorescence, chemiluminescence, mass spectrometry, radioactivity or electrochemistry can be used to detect complex formation within each microspot. Such miniaturised and parallelised binding assays can be highly sensitive, and the extraordinary power of the method is exemplified by array-based gene expression analysis. In these systems, arrays containing immobilised DNA probes are exposed to complementary targets and the degree of hybridisation is measured. Recent developments in the field of protein microarrays show applications for enzyme-substrate, DNA-protein and different types of protein-protein interactions. This article discusses theoretical advantages and limitations of any miniaturised capture-molecule-ligand assay system and discusses how the use of protein microarrays will change diagnostic methods and genome and proteome research.
The high-molecular-weight melanoma-associated antigen, HMW-MAA, has been demonstrated to be of potential interest for diagnosis
and treatment of malignant melanoma. Murine monoclonal antibodies (mAb) generated in response to different epitopes of this
cell-surface molecule efficiently localise to metastatic lesions in patients with disseminated disease. In this work, phage-display-driven
selection for melanoma-reactive antibodies generated HMW-MAA specificities capable of targeting bacterial superantigens (SAg)
and cytotoxic T cells to melanoma cells. Cynomolgus monkeys were immunised with a crude suspension of metastatic melanoma.
A strong serological response towards HMW-MAA demonstrated its role as an immunodominant molecule in the primate. Several
clones producing monoclonal scFv antibody fragments that react with HMW-MAA were identified using melanoma cells and tissue
sections for phage selection of a recombinant antibody phage library generated from lymph node mRNA. One of these scFv fragments,
K305, was transferred and expressed as a Fab-SAg fusion protein and evaluated as the tumour-targeting moiety for superantigen-based
immunotherapy. It binds with high affinity to a unique human-specific epitope on the HMW-MAA, and demonstrates more restricted
crossreactivity with normal smooth-muscle cells than previously described murine mAb. The K305 Fab was fused to the superantigen
staphylococcal enterotoxin A (D227A) [SEA(D227A)], which had been mutated to reduce its intrinsic MHC class II binding affinity,
and the fusion protein was used to demonstrate redirection of T cell cytotoxicity to melanoma cells in vitro. In mice with
severe combined immunodeficiency, carrying human melanoma tumours, engraftment of human lymphoid cells followed by treatment
with the K305Fab-SEA(D227A) fusion protein, induced HMW-MAA-specific tumour growth reduction. The phage-selected K305 antibody
demonstrated high-affinity binding and selectivity, supporting its use for tumour therapy in conjunction with T-cell-activating
superantigens.
Antibodies are important tools for the detection of diagnostic markers and are the most well-known examples of specific molecular interactions. We have developed automated technology that enables the selection of antibodies and other interacting molecules from large recombinant libraries. The physical link between phenotype and genotype in phage display allows selective isolation and amplification of a particular phage encoding a desired antibody fragment. Successive rounds of phage selection, amplification and screening are performed at high throughput, using a pin-based magnetic particle processor. The integration of this with existing DNA and protein array technology enables industrial screening of complex libraries and opens up a new level of functional genomic analysis.
In order to investigate the role of the constant domains on the antigen-binding property of the variable domains, we have carried out a comparative thermodynamic study of the anti-dansyl Fv, Fab* and Fab fragments that possess the identical amino acid sequence of the variable domains. The thermodynamic analyses have shown that binding constants, enthalpy changes and entropy changes are similar for the three antigen-binding fragments, whereas the thermal stability of Fab is much higher than that of Fv and Fab*. We have concluded that (i) the variable domains of the three antigen-binding fragments possess identical intrinsic capability for antigen binding and (ii) the two constant domains serve to improve the stability of the variable domains.
In order to obtain an estimate of the overall level of correlation between mRNA and protein abundances for a well-characterized pharmaceutically relevant biological system, we have analyzed human liver by quantitative two-dimensional electrophoresis (for protein abundances) and by Transcript Image methodology (for mRNA abundances). Incyte's LifeSeq database was searched for expressed sequence tag (EST) sequences corresponding to a series of 23 proteins identified on 2-D maps in the Large Scale Biology (LSB) Molecular Anatomy database, resulting in estimated abundances for 19 messages (4 were undetected) among 7926 liver clones sequenced. A correlation coefficient of 0.48 was obtained between the mRNA and protein abundances determined by the two approaches, suggesting that post-transcriptional regulation of gene expression is a frequent phenomenon in higher organisms. A comparison with published data (Kawamoto, S., et al., Gene 1996, 174, 151-158) on the abundances of liver mRNAs for plasma proteins (secreted by the liver) suggests that higher abundance messages are strongly enriched in secreted sequences. Our data confirms this: of the 50 most abundant liver mRNAs, 29 coded for secreted proteins, while none of the 50 most abundant proteins appeared to be secreted products (although four plasma and red blood cell proteins were present in this group as contaminants from tissue blood).
By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven V(H) and seven V(L) (four Vkappa and three Vlambda) germline families cover more than 95 % of the human antibody diversity used. A consensus sequence was derived for each family and optimized for expression in Escherichia coli. In order to make all six complementarity determining regions (CDRs) accessible for diversification, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites flanking the CDRs. Molecular modeling verified that all canonical classes were present. We could show that all master genes are expressed as soluble proteins in the periplasm of E. coli. A first set of antibody phage display libraries totalling 2x10(9) members was created after cloning the genes in all 49 combinations into a phagemid vector, itself devoid of the restriction sites in question. Diversity was created by replacing the V(H) and V(L) CDR3 regions of the master genes by CDR3 library cassettes, generated from mixed trinucleotides and biased towards natural human antibody CDR3 sequences. The sequencing of 257 members of the unselected libraries indicated that the frequency of correct and thus potentially functional sequences was 61 %. Selection experiments against many antigens yielded a diverse set of binders with high affinities. Due to the modular design of all master genes, either single binders or even pools of binders can now be rapidly optimized without knowledge of the particular sequence, using pre-built CDR cassette libraries. The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes.
The first version of the Human Combinatorial Antibody Library (HuCAL) is a single-chain Fv-based phage display library (HuCAL-scFv) with 2x10(9) members optimised for high-throughput generation and targeted engineering of human antibodies. 61% of the library genes code for functional scFv as judged by sequencing. We show here that since HuCAL-scFv antibodies are expressed in high levels in Escherichia coli, automated panning and screening in miniaturised settings (96- and 384-well format) have now become feasible. Additionally, the unique modular design of HuCAL-genes and -vectors allows the distinctly facilitated conversion of scFv into Fab, miniantibody and immunoglobulin formats, and the fusion with a variety of effector functions and tags not only convenient for therapeutic applications but also for high-throughput purification and detection. Thus, the HuCAL principle enables the rapid and high-throughput development of human antibodies by optimisation strategies proven useful in classical low molecular weight drug development. We demonstrate in this report that HuCAL is a very convenient source of human antibodies for various applications.
A microarray comprising 21,024 different PCR products spotted on glass slides was constructed for gene expression studies on Trypanosoma brucei. The arrayed fragments were generated from a T. brucei shotgun clone library, which had been prepared from randomly sheared and size-fractionated genomic DNA. For the identification of stage-specific gene activity, total RNA from in vitro cultures of the human, long slender form and the insect, procyclic form of the parasite was labelled and hybridised to the microarray. Approximately 75% of the genomic fragments produced a signal and about 2% exhibited significant differences between the transcript levels in the bloodstream and procyclic forms. A few results were confirmed by Northern blot analysis or reverse-transcription and PCR. Three hundred differentially regulated clones have been selected for sequencing. So far, of 33 clones that showed about 2-fold or more over-expression in bloodstream forms, 15 contained sequences similar to those of VSG expression sites and at least six others appeared non-protein-coding. Of 29 procyclic-specific clones, at least eight appeared not to be protein-coding. A surprisingly large proportion of known regulated genes was already identified in this small sample, and some new ones were found, illustrating the utility of genomic arrays.
With the advent of protein and antibody microarray technology several different coatings and protocols have been published, which may be broadly divided into two types: gel-coated surfaces and plain non-gel-coated glass or plastic surfaces, some with chemical groups attached. We have screened 11 different array surfaces of both types and compared them with respect to their detection limit, inter- and intrachip variation, and storage characteristics. Five different antibodies were immobilized onto each type of microarray support, with total protein concentrations ranging from 40 fmol to 25 amol per spot. From these results, it was seen that some antibodies were more suited for use on antibody arrays. All measurements were performed in quadruplicate, and the results revealed high signal uniformity and reproducibility of most plain glass and plastic slides. Lower detection limits were obtained with polyacrylamide-coated slides, making them more suitable for the detection of very low concentrations of antigen. All microarray coatings could be stored for a period of 8 weeks; however, improved results were seen after 2 weeks of storage. In conclusion, the results indicate the need to test each antibody to be used on an antibody array and to select the microarray coating based on experimental requirements.
The system-wide study of proteins presents an exciting challenge in this information-rich age of whole-genome biology. Although traditional investigations have yielded abundant information about individual proteins, they have been less successful at providing us with an integrated understanding of biological systems. The promise of proteomics is that, by studying many components simultaneously, we will learn how proteins interact with each other, as well as with non-proteinaceous molecules, to control complex processes in cells, tissues and even whole organisms. Here, I discuss the role of microarray technology in this burgeoning area.
Antibody microarrays could have an enormous impact on the functional analysis of cellular activity and regulation, especially at the level of protein expression and protein-protein interaction, and might become an invaluable tool in disease diagnostics. The array surface is bound to have a tremendous influence on the findings from such studies. Apart from the basic issue of how to attach antibodies optimally without affecting their function, it is also important that the cognate antigens, applied within a complex protein mixture, all bind to the arrayed antibodies irrespective of their enormous variety in structure. In this study, various factors in the production of antibody microarrays on glass support were analysed: the modification of the glass surface; kind and length of cross-linkers; composition and pH of the spotting buffer; blocking reagents; antibody concentration and storage procedures, in order to evaluate their effect on array performance. Altogether, data from more than 700 individual array experiments were taken into account. In addition to home-made slides, commercially available systems were also included in the analysis.
We introduce a quantitative method that utilizes scanning electron microscopy for the analysis of protein chips (SEMPC). SEMPC is based upon counting target-coated gold particles interacting specifically with ligands or proteins arrayed on a derivative microscope glass slide by utilizing backscattering electron detection. As model systems, we quantified the interactions of biotin and streptavidin and of an antibody with its cognate hapten. Our method gives quantitative molecule-counting capabilities with an excellent signal-to-noise ratio and demonstrates a broad dynamic range while retaining easy sample preparation and realistic automation capability. Increased sensitivity and dynamic range are achieved in comparison to currently used array detection methods such as fluorescence, with no signal bleaching, affording high reproducibility and compatibility with miniaturization. Thus, our approach facilitates the determination of the absolute number of molecules bound to the chip rather than their relative amounts, as well as the use of smaller samples.
Stimulated by the achievements of the first phase in genomics and the resulting need of assigning functions to the acquired sequence information, novel formats of immunoassays are being developed for high-throughput multi-analyte studies. In principle, they are similar in nature to the microarray assays already established at the level of nucleic acids. However, the biochemical diversity and the sheer number of proteins are such that an equivalent analysis is much more complex and thus difficult to accomplish. The wide range of protein concentration complicates matters further. Performing microarray immunoassays already represents a challenge at the level of preparing a working chip surface. Arrays have been produced on filter supports, in microtiter plate wells and on glass slides, the last two usually coated with one-, two- or three-dimensionally structured surface modifications. The usefulness and suitability of all these support media for the construction and application of antibody microarrays are reviewed in this manuscript in terms of the different kinds of immunoassay and the various detection procedures. Additionally, the employment of microarrays containing alternative sensor molecules is discussed in this context. The sensitivity of microspot immunoassays predicted by the current analyte theory is not yet a reality, indicating the extent of both the technology's potential and the size of the task still ahead.
The performance of protein and antibody microarrays is dependent on various factors, one of which is the use of an appropriate microarray surface for the immobilisation of either protein or antibody samples. We have investigated the properties of seven new surfaces in the context of both protein and antibody microarray technology. We have demonstrated the functionality of all new slide coatings and investigated the mean signal to spotted concentration ratio, determined detection limits and calculated coefficients of variation. Moreover, new concepts for slide coatings such as dendrimer and poly(ethylene glycol)-epoxy slides were evaluated and improved qualities of novel slide surfaces were observed. Optimal slide coatings for antibody and protein chips were proposed and the requirements for both technologies were discussed.
Although approximately 10,000 antibodies are available from commercial sources, antibody reagents are still unavailable for most proteins. Furthermore, new applications such as antibody arrays and monoclonal antibody therapeutics have increased the demand for more specific antibodies to reduce cross-reactivity and side effects. An array containing every protein for the relevant organism represents the ideal format for an assay to test antibody specificity, because it allows the simultaneous screening of thousands of proteins for possible cross-reactivity. As an initial test of this approach, we screened 11 polyclonal and monoclonal antibodies to approximately 5,000 different yeast proteins deposited on a glass slide and found that, in addition to recognizing their cognate proteins, the antibodies cross-reacted with other yeast proteins to varying degrees. Some of the interactions of the antibodies with noncognate proteins could be deduced by alignment of the primary amino acid sequences of the antigens and cross-reactive proteins; however, these interactions could not be predicted a priori. Our findings show that proteome array technology has potential to improve antibody design and selection for applications in both medicine and research.
Following the age of genomics having sequenced the human genome, interest is shifted towards the function of genes. This new age of proteomics brings about a change of methods to study the properties of gene products on a large scale. Protein separation technologies are now applied to allow high-throughput purification and characterisation of proteins. Two-dimensional-gel electrophoresis (2DE) and mass spectrometry (MS) have become widely used tools in the field of proteomics. At the same time, protein and antibody microarrays have been developed as successor of DNA microarrays to soon allow the proteome-wide screening of protein function in parallel. This review is aimed to introduce this new technology and to highlight its current prospects and limitations.
The enzyme-linked immunosorbent assay (ELISA) is typically applied in the format of microtiter plates. To increase throughput and reduce consumption of precious samples, efforts have been made to transfer ELISA to the microchip format using conventional microarrays, microfluidic systems, and chips bearing microwells. However, all three formats lack the possibility to screen several analytes on several immobilized binders at a time or require complicated liquid handling, surface modifications, and additional equipment. Here, we describe an immunoassay performed on a standard microscope slide without the requirement for wells or tubes to separate the samples using standard surfaces and machinery already available for microarray technology. The new multiple spotting technique (MIST) comprises immobilization of a binder onto a surface and subsequent spotting of the second compound on the same spot, on top of the immobilized binder. We show that the analytes bind their ligands immediately within the confined space of separate droplets on the chip surface, thereby eliminating the need for extra incubation time. We illustrate the feasibility of the new technique by spotting dilution rows of proteins or monoclonal and polyclonal antibodies on top of their immobilized binders. Moreover, we demonstrate specificity by applying a mixture of antibodies in a multiplex format and demonstrate that the technique is compatible with conventional microarray protocols, such as total incubation. Finally, we indicate that the technique is capable of quantifying as little as 400 zmol (240,000 molecules) of analyte.
- A Lueking
- A Possling
- O Huber
- A Beveridge
- M Horn
- H Eickhoff
- J Schuchardt
- H Lehrach
- D Cahill
(19) Lueking, A.; Possling, A.; Huber, O.; Beveridge, A.; Horn, M.; Eickhoff, H.;
Schuchardt, J.; Lehrach, H.; Cahill, D. J. Mol. Cell. Proteomics 2003, 2,
1342-9. Epub 2003 Sep 29.
- B Krebs
- R Rauchenberger
- S Reiffert
- C Rothe
- M Tesar
- E Thomassen
- M Cao
- T Dreier
- D Fischer
17) Krebs, B.; Rauchenberger, R.; Reiffert, S.; Rothe, C.; Tesar, M.; Thomassen,
E.; Cao, M.; Dreier, T.; Fischer, D.; Hoss, A.; Inge, L.; Knappik, A.; Marget,
M.; Pack, P.; Meng, X. Q.; Schier, R.; Sohlemann, P.; Winter, J.; Wolle, J.;
Kretzschmar, T. J. Immunol. Methods 2001, 254, 67-84.
- P Angenendt
- J Glökler
Angenendt, P.; Glökler, J. Methods Mol. Biol. 2004, 278, 123-34.
- P Angenendt
- J Glökler
- Z Konthur
- H Lehrach
- D Cahill
Angenendt, P.; Glökler, J.; Konthur, Z.; Lehrach, H.; Cahill, D. J. Anal. Chem.
2003, 75, 4368-72.
- W Kusnezow
- A Jacob
- A Walijew
- F Diehl
- J D Hoheisel
Kusnezow, W.; Jacob, A.; Walijew, A.; Diehl, F.; Hoheisel, J. D. Proteomics
2003, 3, 254-64.
- N Levit-Binnun
- A B Lindner
- O Zik
Levit-Binnun, N.; Lindner, A. B.; Zik, O.; Eshhar, Z.; Moses, E. Anal. Chem.
2003, 75, 1436-41.
- S F Kingsmore
Kingsmore, S. F. Nat. Biotechnol. 2002, 20, 359-65.
- P Angenendt
- J Glökler
- J Sobek
- H Lehrach
- D J Cahill
Angenendt, P.; Glökler, J.; Sobek, J.; Lehrach, H.; Cahill, D. J. J. Chromatogr.,
A 2003, 1009, 97-104.