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The impact of protein biochips and microarrays on the drug development process
Abstract
With the genome sequences of several organisms now in public databases, the scientific community has realized that it is time to prepare for the next step: the understanding of biological systems or systems biology. Whereas genes contain the information for life, the encoded proteins and RNAs fulfill nearly all the functions, from replication to regulation. At present, there is a perceived demand for high-throughput and parallel analytical devices as research tools in systems biology, and, in addition, for new concepts to extract knowledge and value from these data. Protein biochips will play a decisive role in meeting this need in the future.
... Using this method, we screened the DNA sequences bound by zNanog in theznlk1 promoter, which was demonstrated by ChIP-Seq. Compared with the yeast one-hybrid [26], ChIP, protein binding microarrays, and biochip [27], we consider that this method is The second pair of primers amplified the znlk1 promoter at the position of À1822 to À1692 bp, and the third pair amplified it at the position of À1816 to À1692 bp; the positions of À1996 to À1502 bp and À2000 to À1815 bp were not amplified by the first and fourth pairs of primers. The input and IgH3 antibody were used as positive controls. ...
Nanog is a homeodomain transcription factor that is essential for maintenance of pluripotency and self-renewal of embryonic stem cells (ESCs). In the present study, we demonstrate that zebrafish Nanog (zNanog) directly binds to the promoter region of zebrafish nlk1 (znlk1) by ChIP-Seq analysis and that it up-regulates the expression of znlk1 in fibroblast-like embryonic cells of Danio rerio (ZEM-2S cells) and in zebrafish embryos at 30% epiboly both at the mRNA and protein levels. In addition, compared with control (MO-C) embryos at 30% epiboly, the mRNA and protein expression of vasa and the numbers of vasa-positive cells were increased in embryos injected with zNanog morpholino (MO-zNanog). Further, injection of znlk1 mRNA into zNanog-depleted embryos restored the expression of vasa and the number of vasa-positive cells. These data indicated that zNanog up-regulates the expression of znlk1 through directly binding to the znlk1 promoter, thereby suppressing the expression of vasa. Vasa is a marker gene for PGCs. Our results suggest that zNanog plays a role in restraint of PGC cell number through regulating the expression of znlk1 in the early embryonic development. The current results provide fundamental information to support further investigation regarding the regulatory mechanism of zNanog during the development of PGCs.
... Наиболее популярны методы протеомного анализа [97], трансгенных нокаутов [98], методы с использованием siRNA [52], антисенс-технологии [58,63], аптамеры [91,94], CALI и FALI [78][79][80]. В последнее время в фармацевтической индустрии происходит настоящая революция в высокопроизводительном скрининге и валидации мишеней с использованием новейших технологий ДНК микроматриц (microarrays), ДНК чипов и микрожидкостных устройств [99][100][101][102][103]. ...
Review is devoted to the description of the main existing and developing technologies for experimental validation of potential targets predicted in silico by comparative genomics and bioinformatics methods. Since this problem has not been solved yet, the description of a wide set of methods, suitable for the validation of potential targets, is given. The following questions have been considered: (1) applications of proteomic technologies (control of potential targets expression and their variability, analysis of protein-protein interactions); (2) use of genomics technologies in experimental validation of targets (inactivation of the target genes, suppression of transcription, inactivation of the target mRNA, suppression of translation); (3) methods of direct inactivation of target proteins (monoclonal antibodies, light-inactivation, one-chain antibodies, intrabodies, aptamers); (4) high-throughput technologies; (5) targets validations in vivo.
... It is clear that the set of all combinations of all possible gene interactions would be too large to explore. Our task is to devise a computer strategy that can explore a very large space of gene interactions, enriched for plausibility, and reduce them to a manageable set for human consideration (e.g. Huels et al. 2002). ...
We use Backward Chaining Rule Induction (BCRI), a novel data mining method for hypothesizing causative mechanisms, to mine lung cancer gene expression array data for mechanisms that could impact survival. Initially, a supervised learning system is used to generate a prediction model in the form of "IF THEN " style rules. Next, each antecedent (i.e. an IF condition) of a previously discovered rule becomes the outcome class for subsequent application of supervised rule induction. This step is repeated until a termination condition is satisfied. "Chains" of rules are created by working backward from an initial condition (e.g. survival status). Through this iterative process of "backward chaining," BCRI searches for rules that describe plausible gene interactions for subsequent validation. Thus, BCRI is a semi-supervised approach that constrains the search through the vast space of plausible causal mechanisms by using a top-level outcome to kick-start the process. We demonstrate the general BCRI task sequence, how to implement it, the validation process, and how BCRI-rules discovered from lung cancer microarray data can be combined with prior knowledge to generate hypotheses about functional genomics.
... During the last years it has evolved to be a powerful technique concerning biological questions involved in the transcriptional state, e.g. oncology [5], development [6] and drug discovery [7]. In a common microarray hybridization, two pools of RNA (e.g. from control and treated cells) are differentially labeled (usually by Cy3-and Cy5-labeled nucleotides) and co-hybridized to a glass slide having either PCR products ("cDNA array") or gene-specific oligonucleotides ("oligo array") covalently attached to its surface. ...
The amplification of RNA with the T7-System is a widely used technique for obtaining increased amounts of RNA starting from limited material. The amplified RNA (aRNA) can subsequently be used for microarray hybridizations, warranting sufficient signal for image analysis. We describe here an amplification-time dependent degradation of aRNA in prolonged standard T7 amplification protocols, that results in lower average size aRNA and decreased yields.
A time-dependent degradation of amplified RNA (aRNA) could be observed when using the classical "Eberwine" T7-Amplification method. When the amplification was conducted for more than 4 hours, the resulting aRNA showed a significantly smaller size distribution on gel electrophoresis and a concomitant reduction of aRNA yield. The degradation of aRNA could be correlated to the presence of the T7 RNA Polymerase in the amplification cocktail. The aRNA degradation resulted in a strong bias in microarray hybridizations with a high coefficient of variation and a significant reduction of signals of certain transcripts, that seem to be susceptible to this RNA degrading activity. The time-dependent degradation of these transcripts was verified by a real-time PCR approach.
It is important to perform amplifications not longer than 4 hours as there is a characteristic 'quality vs. yield' situation for longer amplification times. When conducting microarray hybridizations it is important not to compare results obtained with aRNA from different amplification times.
... The number of analytes that can be assayed in a commercial high through-put clinical diagnostic analyzer ranges from 60-130 [Aller et al., 2002]. Based on the biological function these antigens/analytes can be categorized into amino acids, proteins [Lee et al., 2001;Mouradian et al., 2002;Doherty et al, 2003], cytokines [Morse et al, 2004], enzymes, carbohydrates, lipids, vitamins, electrolytes, blood gases, trace elements, drugs [Huels et al, 2002], tumor markers and nucleic acids [Burtis et al, 1999]. Among the various methods available, immunoassays are widely used in molecular biology providing the molecular basis for many clinical diagnoses [Eteshola et al.., 2001;Bernard et al.., 2001]. ...
ABSTRACT: The emerging paradigm of lab-on-a-chip powered by microfluidics is expected to revolutionize miniaturization, automation and integration in the point-of-care centers which require quick, efficient and reproducible results. Furthermore, high throughput requirement from the life sciences laboratories have made the development of lab-on-a-chip a major area of research over the past few years. Immunoassays, which are routinely used to determine the concentrations of various analytes in life sciences laboratories, are one of the most important laboratory tests that require efficient automation and integration. These are different from other laboratory tests in the clinical laboratories such as colorimetric tests because they involve formation of antibody-antigen complexes to generate a signal that can be measured. The immunoassays also involve the application of magnetically responsive beads to increase the surface area for the reactions thereby enhance the signal. Although miniaturization was started in the early 1990's there are no commercial devices that perform immunoassays involving magnetic beads. None of the state-of-the art commercial microfluidic technologies, which are based on continuous flow in etched microchannel, have been able to fully deliver the promised benefits of microfluidics. This is primarily due to their incompatability with common sample matrices and architectural inflexibility. Furthermore, the transport of the magnetic beads in microchannels is a difficult task in continuous mode of operation as magnetic susceptibility of the beads is rather weak and because of demagnetization of the particles. In this thesis, a droplet-based microfluidic lab-on-a-chip based on electrowetting actuation is developed to perform immunoassays using magnetic beads on human physiological samples. Text (Electronic thesis) in PDF format. Mode of access: World Wide Web. Advisor: Srinivas Palanki, Florida State University, FAMU-FSU College of Engineering, Dept. of Chemical Engineering. Title and description from dissertation home page (viewed Sept. 19, 2007). Document formatted into pages; contains xiv, 113 pages. Thesis (Ph. D.)--Florida State University, 2007. Includes bibliographical references.
... In proteomics study, much attention has been paid to protein chip technology due to its benefits for simultaneous and high-throughput screening of proteins. 1,2 In parallel with this, efforts to develop label-free methods coupled with protein chip technology have been made. 3,4 Among them, a promising combination is surface plasmon resonance (SPR) protein chip technology, which is capable of directly detecting biomolecular interactions. ...
Here we report an effective method for protein immobilization on a surface plasmon resonance (SPR) gold chip, describing the combination of cysteine- and oligomerization domain-mediated immobilization of enhanced green fluorescent protein (EGFP) as a model protein for the purpose of orientation-controlled surface density packing. In order to facilitate the oligomerization of EGFP, the dimeric and trimeric constructs derived from GCN4- leucine zipper domain were chosen for multimeric EGFP assembly. For orientation-controlled immobilization of the protein, EGFP modified with cysteine residues showing excellent orientation on a gold chip was used as a starting protein, as previously reported in our earlier study (Anal. Chem., 2007, 79, 2680-2687). Constructs of EGFP with oligomerization domains were genetically engineered, and corresponding fusion proteins were purified, applied to a gold chip, and then analyzed under SPR. The immobilized EGFP density on a gold chip increased according to the states of protein oligomerization, as dimeric and trimeric EGFPs displayed better adsorption capability than monomeric and dimeric forms, respectively. Fluorescence measurement corroborated the SPR results. Taken together, our findings indicated that the combination of cysteine- and oligomerization domain-mediated immobilization of protein could be used in SPR biosensor applications, allowing for an excellent orientation and high surface density simultaneously.
... These tools can be useful for both understanding molecular pathways in which the target protein participates, as well as screening model systems for changes that occur in disease states or following drug treatment (i.e. enzyme activity testing) ( Huels et al., 2002). Tools for screening interactions with entire "proteome sets" of both known and unknown proteins are also available in microarray format. ...
Proteins are the principal targets of drug discovery. Most large pharmaceutical companies now have a proteomics-oriented biotech or academic partner or have started their own proteomics division. Common applications of proteomics in the drug industry include target identification and validation, identification of efficacy and toxicity biomarkers from readily accessible biological fluids, and investigations into mechanisms of drug action or toxicity. Target identification and validation involves identifying proteins whose expression levels or activities change in disease states. These proteins may serve as potential therapeutic targets or may be used to classify patients for clinical trials. Proteomics technologies may also help identify protein-protein interactions that influence either the disease state or the proposed therapy. Efficacy biomarkers are used to assess whether target modulation has occurred. They are used for the characterization of disease models and to assess the effects and mechanism of action of lead candidates in animal models. Toxicity (safety) biomarkers are used to screen compounds in pre-clinical studies for target organ toxicities as well as later on in development during clinical trials. Complementary approaches such as metabolomics and genomics can be used in conjunction with proteomics throughout the drug development process to create more of a unified, systems biology approach.
... With the increasing investment in gene expression microarray technology, there has been a move toward a " systems biology " approach to understanding the coupling of gene networks and signaling cascades that describe the phenotypes of living matter (e.g., [13],[35],[24]). This has led to a call for tools to (semi-)automatically explore the space of genomic interactions (e.g., [14]) in order to reduce the set of interactions to a manageable set for examination. The goal of this exploration is to focus analysts on plausible interactions, pathways, and markers, which can then be scrutinized further with hypothesis testing methods. ...
Exploring the vast number of possible feature interactions in domains such as gene expression microarray data is an onerous task. We describe Backward-Chaining Rule Induction (BCRI) as a semi-supervised mechanism for biasing the search for IF-THEN rules that express plausible feature interactions. BCRI adds to a relatively limited tool-chest of hypothesis generation software and is an alternative to purely unsupervised association-rule learning. We illustrate BCRI by using it to search for gene-to-gene causal mechanisms that underlie lung cancer. Mapping hypothesized gene interactions against prior knowledge offers support and explanations for hypothesized interactions, and suggests gaps in current knowledge that induction might help fill. BCRI is implemented as a wrapper around a base supervised-rule-learning algorithm. We summarize our prior work with an adaptation of C4.5 as the base algorithm (C45-BCRI), extending this in the current study to use Brute as the base algorithm (Brute-BCRI). In contrast to C4.5's greedy strategy, Brute extensively searches the rule space. Moreover, Brute returns many more rules (i.e., hypothesized feature interactions) than does C4.5. To remain an effective hypothesis-generation tool requires that Brute-BCRI more carefully rank and prune hypothesized interactions than does C45-BCRI. Prior knowledge serves to evaluate final Brute- BCRI rules just as it does with C45-BCRI, but prior knowledge also serves to evaluate and prune intermediate search states, thus maintaining a manageable number of rules for evaluation by a domain expert.
... With the increasing investment in gene expression microarray technology, there has been a move toward a "systems biology" approach to understanding the coupling of gene networks and signaling cascades that describe the phenotypes of living matter (e.g., [1], [2], [3]). This has led to a call for tools to (semi-)automatically explore the space of genomic interactions (e.g., [4]) in order to reduce the set of interactions to a manageable set for examination. The goal of this exploration is to focus analysts on plausible interactions, pathways, and markers, which can then be scrutinized further with hypothesis testing methods. ...
Exploring the vast number of possible feature interactions in domains such as gene expression microarray data is an onerous
task. We propose Backward-Chaining Rule Induction (BCRI) as a semi-supervised mechanism for biasing the search for plausible
feature interactions. BCRI adds to a relatively limited tool-chest of hypothesis generation software, and it can be viewed as an alternative to purely unsupervised association rule learning. We illustrate BCRI by
using it to search for gene-to-gene causal mechanisms. Mapping hypothesized gene interactions against a domain theory of prior
knowledge offers support and explanations for hypothesized interactions, and suggests gaps in the current domain theory, which
induction might help fill.
... Applications of protein array technology such as target identification and characterization, target validation, diagnostic marker identification and validation, preclinical study monitoring, and patient typing seem to be feasible. We have recently reviewed the value of recent combinations of efforts in genomics, proteomics, and biochip technology and their impact on the overall drug development process (Huels et al ., 2002). For the first time, tools are available to study disturbances within biological systems, such as disease or drug treatment, on the gene and protein expression levels. ...
Protein array technology is becoming an increasingly important tool in the drive toward proteome-scale analysis of protein activity and interactions. Presently, this technology compliments the more traditional methods for proteomic analysis, including two-dimensional gel electrophoresis/chromatography and mass spectrometry. While the task of producing a “whole-proteome” chip, containing active proteins, is a daunting one, current protein and antibody arrays represent the first steps toward that goal. In this review, we discuss current approaches for the generation of protein arrays, and their applications, including their use in the study of protein–protein, protein–nucleic acid, enzyme–substrate, and so on, interactions. Potential applications of protein arrays in interaction screening, such as compound–protein interactions are also discussed.
... DNA microarray is an innovative technology that can measure the expression level of thousands of genes in parallel (1,2,3). Due to the huge throughput microarray experiments provide, computational methods that extract the knowledge from the large sets of experimental results become important. ...
Biclustering algorithm on Gibbs sampling strategy is a recruit in the field of the analysis of gene expression data of microarray experiments. Its feasibility and validity still need to be researched not only for synthetic datasets but also for real datasets. Here we investigated a biclustering algorithm on a microarray dataset of Yeast genome through building a database for storing microarray datasets and MIPS data, and running the scripts on Matlab platform to discover gene patterns. In contrast with standard clusterings that reveal genes behaving similarly over all the conditions, biclustering groups genes over only a subset of conditions for which those genes have a sharp probability distribution. It has the key advantage of providing a transparent probabilistic interpretation of the biclusters. Its basic strategy of Gibbs sampling does not suffer from the problem of local minima that often characterizes expectation maximization, so that the patterns should be more global and accurate. Also we tested it with the known explanation of genes in MIPS, objectively to demonstrate the effectiveness and deficiencies of biclustering approach, and the functions of a few unknown ORFs in some bicluster can be deduced in the present research. In addition, the result of similarity searching in Blast-Search can be an assistant evidence for its effectivity.
... A listing of the different types of microarray fabrication techniques as well as advantages and disadvantages are shown in Table 1. Long-standing biomedical applications for the technology beyond medical research 10 include clinical diagnostics 178 , drug testing 176,177,179 , disease monitoring 180,181 , drug discovery [182][183][184] , medical, expression profiling 6,185,186 , protein function characterization 3,176,177 , and characterization of protein molecular interactions 3,176,177 . The greatest promise for the assay format is in direct sample-to-answer multiplexed and ultra-sensitive assay formats that reliably target several specific analytes in a biological complex milieu without target amplification or pre-purification tasks 187 . ...
Of the diverse analytical tools used in proteomics, protein microarrays possess the greatest potential for providing fundamental information on protein, ligand, analyte, receptor, and antibody affinity-based interactions, binding partners and high-throughput analysis. Microarrays have been used to develop tools for drug screening, disease diagnosis, biochemical pathway mapping, protein-protein interaction analysis, vaccine development, enzyme-substrate profiling, and immuno-profiling. While the promise of the technology is intriguing, it is yet to be realized. Many challenges remain to be addressed to allow these methods to meet technical and research expectations, provide reliable assay answers, and to reliably diversify their capabilities. Critical issues include: (1) inconsistent printed microspot morphologies and uniformities, (2) low signal-to-noise ratios due to factors such as complex surface capture protocols, contamination, and static or no-flow mass transport conditions, (3) inconsistent quantification of captured signal due to spot uniformity issues, (4) non-optimal protocol conditions such as pH, temperature, drying that promote variability in assay kinetics, and lastly (5) poor protein (e.g., antibody) printing, storage, or shelf-life compatibility with common microarray assay fabrication methods, directly related to microarray protocols. Conventional printing approaches, including contact (e.g., quill and solid pin), non-contact (e.g., piezo and inkjet), microfluidics-based, microstamping, lithography, and cell-free protein expression microarrays, have all been used with varying degrees of success with figures of merit often defined arbitrarily without comparisons to standards, or analytical or fiduciary controls. Many microarray performance reports use bench top analyte preparations lacking real-world relevance, akin to "fishing in a barrel", for proof of concept and determinations of figures of merit. This review critiques current protein-based microarray preparation techniques commonly used for analytical and function-based proteomics and their effects on array-based assay performance.
... Il fine della proteomica è di definire la funzione di ogni proteina nella cellula e come questa funzione cambi a seconda del microambiente, delle modifiche strutturali e conformazionali, della localizzazione cellulare e del tipo di interazioni a cui la proteina può andare incontro. I microarray proteici costituiscono solo una parte delle tecnologie che si stanno sviluppando per analizzare e comprendere il proteoma e le sue alterazioni (12,13); risultano, tuttavia, uno degli approcci attualmente più innovativi per potenzialità di applicazioni e quantità di informazioni che possono fornire. È logico quindi prevedere che lo sviluppo di questa tecnologia determinerà un sostanziale aumento della conoscenza delle malattie e dei loro meccanismi patogenetici e, conseguentemente, permetterà diagnosi precoci ed approcci terapeutici mirati. ...
La ricerca biomedica ha compiuto notevoli e rapidi progressi negli ultimi anni grazie al sequenziamento del genoma umano ed alla disponibilità di tecnolo-gie quali i DNA array: la rappresentazione di ogni gene umano in un singolo chip, infatti, permette, al-meno teoricamente, la quantificazione dell'espres-sione genica in ogni tessuto (1). In questo contesto, la proteomica è figlia naturale della genomica poiché, se da un lato i DNA array dipendono dalla retrotrascrizione dell'RNA messaggero, dall'altro gli array proteici permettono una misurazione diret-ta dei prodotti genici, con gli obiettivi dichiarati di definire la quantità, le modificazioni, l'attività, la localizzazione e le interazioni di tutte le proteine del campione in esame. Attualmente le tecnologie disponibili limitano le nostre analisi alla valutazione contemporanea di uno o due di questi parametri e soltanto in relazione ad una frazione proteica. Esistono, perciò, array che permettono una pro-teomica quantitativa ed altri che sviluppano invece una proteomica funzionale, volta a definire la fun-zione di ogni proteina di un dato organismo (2). Proprio grazie alle sue caratteristiche ed alla sua versatilità, la tecnologia dei microarray proteici si inserisce sia nella valutazione quantitativa che fun-zionale del proteoma. Un microarray proteico è cos-tituito da un supporto solido (slide) sul quale diversi reagenti (proteine purificate, peptidi, anticorpi, al-lergeni,…) sono depositati ("spottati", in gergo tec-nico) in maniera ordinata e ad una specifica e definita densità (fino a 500 molecole/spot di 150 mm). Ognuno di questi agenti cattura la propria pro-teina target, isolandola così da una miscela comp-lessa, quale può essere, per esempio, un lisato cellu-lare, e le proteine catturate vengono successiva-mente evidenziate e quantificate o valutate per quanto concerne la loro attività, le modificazioni, le interazioni proteina-proteina. Nonostante l'esistenza di diversi microarray proteici e di differenti approcci per la loro generazione, un comune passaggio critico nella loro produzione è legato alla distribuzione del set proteico che deve avvenire senza provocare la denaturazione delle proteine ed in modo che le quantità depositate siano sufficienti per la realizzazione del test. A questo scopo, recentemente sono comparsi array proteici costruiti su supporti di vetro, d'oro, di polistirene, al fine di minimizzare la perdita di materiale proteico (soprattutto anticorpi) durante le procedure di legame e di lavaggio. Inoltre, attraverso sofisticate tecniche di microingegneria, sono stati costruiti chips la cui superficie risulta modificata dalla pre-senza di micropozzetti, microcanali e mi-crochiusure, la cui funzione è quella di ridurre l'e-vaporazione e la denaturazione degli agenti "spot-tati" sul supporto, di aumentare la capacità di bind-ing-protein e di prevenire la cross-contaminazione tra i vari agenti depositati (proprio perché fisica-mente separati) (3-5). Cimentato il campione con il microarray e bloccata la proteina target, è necessario rivelare la sua pre-senza sulla slide e, possibilmente, quantificarla. Generalmente, la visualizzazione di un microarray proteico è affidata all'utilizzo di fluorocromi coniu-gati allo stesso target o ad un anticorpo. I fluorocro-mi attualmente più utilizzati sono Cy3, Cy5 ed Alexa. Cy3 e Cy5 sono gli stessi fluorocromi mag-giormente utilizzati anche nei DNA microarray. La fluorescenza emessa in seguito all'eccitazione viene raccolta da un apposito apparato scanner o da un microscopio a fluorescenza o da un microscopio confocale. L'immagine ottenuta da un array proteico è innanzi-tutto una conferma qualitativa dell'avvenuto legame tra l'agente sulla slide ed il suo target proteico. La quantificazione del target è un aspetto più comp-lesso sia per l'analisi statistica necessaria per at-tribuire una corretta significatività al segnale ac-Corrispondenza a: Dott.
... Protein microarrays offer means of conducting massive screening of drugs [93,94]. Although not applied directly on the verification of anticancer drug targets, protein microarray is engaged in the profiling of enzyme activity of the secreted and membrane proteome of cancer cell lines [95] and the analysis of inhibitory properties of lead compounds on enzymatic drug targets [96]. Compared to traditional assays, protein microarrays help to speed up lead identification as well as downstream processes such as lead optimization and validation. ...
Cancer biomarkers are of potential use in early cancer diagnosis, anti-cancer therapy development and monitoring the responses to treatments. Protein-based cancer biomarkers are major forms in use as they are much easier to be monitored in body fluids or tissues. For cancer biomarker discovery, high-throughput techniques such as protein microarrays hold great promises, because they are capable of global unbiased monitoring but with a miniaturized format. In doing so, novel and cancer type specific biomarkers can be systematically discovered at an affordable cost. In this review, we give a relatively complete picture on protein microarrays applied to clinical samples for cancer biomarker discovery, and conclude this review with the future perspectives. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
... Immunoassays are among the most active research topics in several paramount fields, such as medical diagnosis [1], drug discovery [2], food safety testing [3], and environmental monitoring [4]. By utilizing the specific recognition of antibody/antigen interactions, the immunoassay test provides an extraordinarily selective and sensitive way to quantitate target analytes of interest [5]. ...
A simple DNA-redox cation interaction enhancement strategy has been developed to improve the sensitivity of electrochemical immunosensors for protein detection. Instead of labeling with fluorophores or redox-active groups, the detection antibodies were tethered with DNA single strands. Based on the electrostatic interaction between redox cations ([Ru(NH3)6](3+)) and negatively charged DNA backbone, enhanced electrochemical signals were obtained. Human chorionic gonadotropin (hCG) detection has been performed as a trial analysis. A linear response range up to the concentration of 25 mIU/mL and a detection limit of 1.25 mIU/mL have been achieved, both are comparable with the ultrasensitive enzyme-linked immunosorbent assay (ELISA) tests. The method also shows great selectivity towards hCG over other hormones such as thyroid stimulating hormone (TSH) and follicle stimulating hormone (FSH). By and large, our approach bears the merits of cost effectiveness and simplicity of instrumentation in comparison with conventional optical detection methods.
... Protein arrays offer one means of conducting massive screening of drugs (Greenbaum et al., 2002; Wilson and Nock, 2003). The inhibitory properties of lead compounds on enzymatic drug targets can be analyzed in chip-based systems (Huels et al., 2002). Protein arrays have been employed to measure the enzyme activity profiles of the secreted and membrane ...
Protein microarrays, one emerging class of proteomic technologies, have broad applications for discovery and quantitative analysis. A rapidly expanding use of this technology is the acquisition of information about the posttranslational modifications of proteins reflecting the activity state of signal pathways and networks, and is now employed for the analysis of biopsy samples in clinical trial research.
We describe a novel multiplexing technology using a library of small fluorescent molecules, termed eTag molecules, to code
and quantify mRNA targets. eTag molecules, which have the same fluorometric property, but distinct charge-to-mass ratios possess
pre-defined electrophoretic characteristics and can be resolved using capillary electrophoresis. Coupled with primary Invader®
mRNA assay, eTag molecules were applied to simultaneously quantify up to 44 mRNA targets. This multiplexing approach was validated
by examining a panel of inflammation responsive genes in human umbilical vein endothelial cells stimulated with inflammatory
cytokine interleukin 1β. The laser-induced fluorescence detection and electrokinetic sample injection process in capillary
electrophoresis allows sensitive quantification of thousands of copies of mRNA molecules in a reaction. The assay is precise,
as evaluated by measuring qualified Z′ factor, a dimensionless and simple characteristic for applications in high-throughput screening using mRNA assays. Our data
demonstrate the synergy between the multiplexing capability of eTag molecules by sensitive capillary electrophoresis detection
and the isothermal linear amplification characteristics of the Invader® assay. eTag multiplex mRNA assay presents a unique
platform for sensitive, high sample throughput and multiplex gene expression analysis.
We have compiled 819 articles published in the year 2002 that involved commercial optical biosensor technology. The literature demonstrates that the technology's application continues to increase as biosensors are contributing to diverse scientific fields and are used to examine interactions ranging in size from small molecules to whole cells. Also, the variety of available commercial biosensor platforms is increasing and the expertise of users is improving. In this review, we use the literature to focus on the basic types of biosensor experiments, including kinetics, equilibrium analysis, solution competition, active concentration determination and screening. In addition, using examples of particularly well-performed analyses, we illustrate the high information content available in the primary response data and emphasize the impact of including figures in publications to support the results of biosensor analyses.
A review is presented, covering the recent progress in miniaturized total analysis systems, or lab-on-a-chip, for clinical analysis. Sections include: immunoassays; proteins, peptides and amino-acids; nucleic acids; cell analysis; enzyme chips; and small molecules. Future directions are also briefly discussed. (146 references).
Dilated cardiomyopathy (DCM) is a myocardial disease characterized by progressive depression of myocardial contractile function and ventricular dilatation. Thirty percent of DCM patients belong to the inherited genetic form; the rest may be idiopathic, viral, autoimmune, or immune-mediated associated with a viral infection. Disturbances in humoral and cellular immunity have been described in cases of myocarditis and DCM. A number of autoantibodies against cardiac cell proteins have been identified in DCM. In this study, we have profiled the autoantibody repertoire of plasma from DCM patients against a human protein array consisting of 37,200 redundant, recombinant human proteins and performed qualitative and quantitative validation of these putative autoantigens on protein microarrays to identify novel putative DCM specific autoantigens. In addition to analyzing the whole IgG autoantibody repertoire, we have also analyzed the IgG3 antibody repertoire in the plasma samples to study the characteristics of IgG3 subclass antibodies. By combining screening of a protein expression library with protein microarray technology, we have detected 26 proteins identified by the IgG antibody repertoire and 6 proteins bound by the IgG3 subclass. Several of these autoantibodies found in plasma of DCM patients, such as the autoantibody against the Kv channel-interacting protein, are associated with heart failure.
We synthesized a temperature-responsive polymer, N-(isopropylacrylamide)-methacrylic acid copolymer, to which poly-l-lysine was introduced. The synthesized polymer as well as the parent polymer showed reversible soluble-insoluble changes in response to temperature changes across the lower critical solution temperature at 32 degree C in an aqueous solution. We found that the polymer efficiently captured acidic bio-macromolecules such as RNA, glycosaminoglycans and mucin-type glycoproteins in biological samples, and the captured molecules were recovered using aqueous NaCl solutions at high concentration. The target acidic molecules thus obtained will be employed for further studies such as structural analysis after brief desalting procedure. The proposed method does not require any chromatographic separations, but only needs a small volume of an aqueous salt solution for releasing captured molecules. Overall procedures are quite easy and simple, and are completed at least within 1 h. We show a few examples for capturing RNA and glycosaminoglycans from cultured cells using the polymer.
Modern medicine faces the challenge of developing safer and more effective therapies to treat human diseases. Many drugs currently in use were discovered without knowledge of their underlying molecular mechanisms. Understanding their biological targets and modes of action will be essential to design improved second-generation compounds. Here, we describe the use of a genome-wide pool of tagged heterozygotes to assess the cellular effects of 78 compounds in Saccharomyces cerevisiae. Specifically, lanosterol synthase in the sterol biosynthetic pathway was identified as a target of the antianginal drug molsidomine, which may explain its cholesterol-lowering effects. Further, the rRNA processing exosome was identified as a potential target of the cell growth inhibitor 5-fluorouracil. This genome-wide screen validated previously characterized targets or helped identify potentially new modes of action for over half of the compounds tested, providing proof of this principle for analyzing the modes of action of clinically relevant compounds.
Proteomics technologies have proven extremely effective for discovering potential biomarkers and drug targets, but this success is creating a bottleneck. High-output technologies for discovery give way to low-output and low-throughput methods for preliminary validation. This bottleneck can be reduced though the use of several strategies. The first is to constrict the number of potential biomarkers and drug targets by dividing the proteome into smaller, more biologically significant segments. The second is to widen the bottleneck with higher-output and higher-throughput screening technologies. The third is to incorporate more preliminary validation into the discovery process. New and emerging technologies provide promise for each of these strategies.
The post-genomic era is revolutionizing the drug discovery process. The new challenges in the identification of therapeutic targets require efficient technological tools in order to be properly addressed. Label-free detection systems use proteins or ligands coupled to materials of which the physical properties are measurably modified upon specific interactions. Among the label-free systems currently available, the use of metal nanocolloids offers enhanced throughput and flexibility for real-time biomolecular recognition monitoring at a reasonable cost.
In the past 10 yr, the field of bioinformatics has been characterized by the mapping of many genomes. These efforts have stimulated explosive development of novel bioinformatics and experimental approaches to predict the functions and metabolic role of the new proteins. The main application of the work is to search, validate, and prioritize new targets for designing a new generation of drugs. Modern computer and experimental methods for discovery of new lead compounds have also expanded and integrated into the process referred to as rational drug design. They are directed to accelerate and optimize the drug discovery process using experimental and virtual (computer-aided drug discovery) methods. Recently, these methods and approaches have merged into a "from gene to lead" platform that includes the processes from new target discovery through obtaining highly effective lead compounds. This chapter describes the strategies as employed by the "From Gene to Lead" platform, including the major computer and experimental approaches and their interrelationship. The latter part of the chapter contains some examples of the steps required for implementing this platform.
Microarray technology is readily available to scientists interested in gene expression. Commensurate with this availability is the growing market in accessory products offering convenience but potentially variable performance. Here we evaluate seven commercial kits for probe labeling against a human apoptosis oligonucleotide array. All kits were found to label probes successfully using the manufacturers' instructions. The Stratagene Fairplay Microarray Labeling Kit was the most sensitive, with an overall call rate of 74% and the lowest rate of indeterminant calls for the HEK and HepG2 cell lines. The Invitrogen SuperScript Indirect cDNA Labeling System showed the most reproducible gene expression pattern and the least technical variation, both in terms of signal strength and between replicates on each array. The Promega Pronto! Plus System showed the least dye bias however, a higher level of variation between replicates was observed. Pairwise comparisons revealed that the Promega Pronto! Plus System and Invitrogen SuperScript Indirect cDNA Labeling System had the most similarity in their patterns of gene expression. Results obtained suggest variability in the performance of commercial kits between different manufacturers. This study supports the need to conduct comparative evaluations of commercial microarray probe labeling kits and the need for validation prior to use.
Total internal reflection fluorescence (TIRF) coupled with hydrogel-DNA droplet microarrays covalently bound on PMMA substrates presents a reusable, sensitive platform for evaluating DNA hybridization and for rapid biochip development. Hydrogel microarrays, which contain covalently bound DNA probes, are created via a simple printing and photocross-linking process. TIRF measurements of the arrays display robust reusability, show linear sensitivity down to 5 fmol of fluorescently labeled target DNA, and are sensitive to single basepair mismatches. Additionally, the ability to interrogate larger DNA is shown through studies with PCR amplification hybridization. We conclusively demonstrate an efficient, reproducible, low cost platform for DNA hybridization studies that could be used for fast high-throughput diagnostics as well as biochip development.
Genomic sequencing has provided a tremendous amount of information that can be useful in vaccine target identification. The sheer volume of information available necessitates the use of new research disciplines and techniques. Using bioinformatics, researchers sift through available data to identify appropriate candidates for biological analysis. This review provides an overview of available bioinformatic techniques for vaccine candidate identification and a few examples of how these techniques are being applied to specific bacterial pathogens.
We propose an original concept for a sandwich immunoassay that is completely performed on-chip using streptavidin-coated beads as substrate. The latter are electrostatically self-assembled on aminosilane micropatterns at the bottom of a microfluidic channel. We use mouse IgG diluted in phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA) solution as target antigen. The fluorescent sandwich immunocomplex is formed on the beads during the operation of the chip both in stop-flow and continuous-flow modes. Target mouse IgG antigen is detected down to a concentration of 15 ng/mL in stop-flow mode and 250 pg/mL in continuous-flow mode, using only 1300 nL of sample volume. We also demonstrate the possibility of simultaneous detection of two different antigens in a PBS-BSA solution using a dual microfluidic channel structure.
Generating molecular information in a clinically relevant time frame is the first hurdle to truly integrating precision medicine in health care. Reverse phase protein microarrays are being utilized in clinical trials for quantifying posttranslationally modified signal transduction proteins and cellular signaling pathways, allowing direct comparison of the activation state of proteins from multiple specimens, or individual patient specimens, within the same array. This technology provides diagnostic and therapeutic information critical to precision medicine. To enhance accessibility of this technology, two hurdles must be overcome: data normalization and data acquisition. Herein we describe an unamplified, dual-color signal detection methodology for reverse phase protein microarrays that allows multiplex, within spot data normalization, reduces data acquisition time, simplifies automated spot detection, and provides a stable signal output. This method utilizes Quantum Nanocrystal fluorophore labels (Qdot) substituted for organic fluorophores coupled with an imager (ArrayCAM) that captures images of the microarray rather than sequentially scanning the array. Streamlining and standardizing the data analysis steps with ArrayCAM high-resolution, dual mode chromogenic/fluorescent array imaging overcomes the data acquisition hurdle. The spot location and analysis algorithm provides certain parameter settings that can be tailored to the particular microarray type (fluorescent vs. colorimetric), resulting in greater than 99 % spot location sensitivity. The described method demonstrates equivalent sensitivity for a non-amplified Qdot immunoassay when using automated vs. manual immunostaining procedures.
In this paper we propose a specific sandwich immunoassay method for human-immunoglobulin G (HIgG). This immunoassay protocol takes advantage of sandwich binding of primary and secondary antibodies for increased specificity. Polystyrene microspheres (PS) serve as immobilizing support, site for sandwich immunoassay and then subsequently used for chemiluminescence (CL) detections. In this sandwich immunoassay, PS microspheres were modified with the primary anti-HIgG (Ab1) via electrostatic interaction, while CdTe nanoparticles (CdTeNPs) were modified with horseradish peroxidase labeled anti-HIgG (Ab2) via covalent binding. Antigen HIgG (Ag) was specifically captured by the first and secondary antibody and form sandwich immunoassay format. Combination of the remarkable sensitivity of CL method and the use of CdTe NPs as anti-HIgG-HRP carrier for the enzymatic signal amplification, provide a linear response range of HIgG from 0.01 to 300 ng mL(-1) with an extremely low detection limit of 0.3 pg mL(-1). This immunoassay system has many desirable merits including sensitivity, accuracy, and little required instrumentation. The assay results were compared with enzyme-linked immunosorbent assay (ELISA), and showed relatively good reliability. Significantly the new protocol may become quite promising technique for protein immune-detection as well as DNA analysis and other biological analyses.
The Reverse Phase Protein Microarray (RPMA) is an array platform used to quantitate proteins and their posttranslationally modified forms. RPMAs are applicable for profiling key cellular signaling pathways and protein networks, allowing direct comparison of the activation state of proteins from multiple samples within the same array. The RPMA format consists of proteins immobilized directly on a nitrocellulose substratum. The analyte is subsequently probed with a primary antibody and a series of reagents for signal amplification and detection. Due to the diversity, low concentration, and large dynamic range of protein analytes, RPMAs require stringent signal amplification methods, high quality image acquisition, and software capable of precisely analyzing spot intensities on an array. Microarray detection strategies can be either fluorescent or colorimetric. The choice of a detection system depends on (a) the expected analyte concentration, (b) type of microarray imaging system, and (c) type of sample. The focus of this chapter is to describe RPMA detection and imaging using fluorescent and colorimetric (diaminobenzidine (DAB)) methods.
Since 1989, the National Institute for Occupational Safety and Health (NIOSH) considers the Laboratory Animal Allergy - LAA a risk for workers and in 1998 the LAA has been recognized as occupational risk in the USA. Rat and mouse are the most source of allergens, not so much for the higher power of allergy respect to the other animals, but because represent the more utilized species in the research. Most of the allergens are members of the lipocalin superfamily, small extracellular proteins represented by at least 50 proteins that mainly bind or carry small hydrophobic molecules. The recent and innovative molecular techniques, as the microarray, have allow the characterization of numerous allergens. The protein microarray gives the possibility to study of IgE profile for each individual, simultaneos analysis of a wide number of parameters concerning the allergy, giving new diagnostic and therapeutic opportunities for the allergies. In the study of occupational allergy--as LAA--the protein microarray could improve: the identification and characterization of new allergens; the individuation of susceptible workers; the study of immunological responses in exposed workers; the strategies of prevention and protection; the environmental and housing conditions. The participation, formation and information of the workers could improve the behavioural and occupational practices, the use of personal and collective protective devices in order to reduce the exposure to LAA in occupational context.
A novel lab-on-paper device combining the simplicity and low-cost of microfluidic paper-based analytical devices (μPADs) and the sensitivity and selectivity of chemiluminescence ELISA (CL-ELISA) for the high-throughput, rapid, stable and reusable point-of-care testing is presented here. Chitosan was used to modify μPADs to covalently immobilize antibodies on μPADs. Thus, sandwich CL-ELISA on μPADs can be easily realized for further development of this technique in sensitive, specific and low-cost application. The paper device was fabricated by a low-cost, simple, and rapid wax-screen-printing method. Using tumor markers and paper microzone plate as model, the application test of this paper-based CL-ELISA was successfully performed with a linear range of 0.1-35.0 ng mL(-1) for α-fetoprotein, 0.5-80.0 U mL(-1) for cancer antigen 125 and 0.1-70.0 ng mL(-1) for carcinoembryonic antigen. Since the cutoff values of the three tumor markers in clinical diagnosis are 25 ng mL(-1), 35 U mL(-1) and 5 ng mL(-1), the sensitivity and linear ranges of the proposed method were enough for clinical application. In addition, this lab-on-paper immunodevice can provide reproducible results upon storage at 4 °C (sealed) for at least 5 weeks. Ultimately, this novel chitosan modification and wax-screen-printing methodology for μPADs can be readily translated to other signal reporting mechanism including electrochemiluminescence and photoelectrochemistry, and other receptors such as enzyme receptors and DNA receptors for determination of DNA, proteins and small molecules in point-of-care testing.
Ultimately, disease-related genetic defects are manifested on a protein level, involving derangements in protein function and the information flow within diseased cells and the surrounding tissue microenvironment. New classes of protein microarrays have been recently developed using highly sensitive, specific technology, and can be used to generate a “wiring diagram” of information flow mediated through posttranslational modification driven networks. Through the combined use of laser capture microdissection of pure populations of tumor cells obtained from patient biopsies, these arrays can provide the foundation for the development of individualized combinatorial therapies of molecular inhibitors to target tumor-specific deranged pathways regulating key biologic processes including proliferation, differentiation, apoptosis, immunity, and metastasis. In the future, therapies can be tailored to the specific deranged molecular circuitry of an individual patient's disease. The successful transition of these technologies from research tools to integrated clinical diagnostic platforms will require ongoing continued development, development of reference standards, and optimization with rigorous standardization development and quality-control procedures.
The emerging technology of protein microarrays has a far-reaching potential to meet the needs for measuring not just the abundance of protein constituents but also their functional states and their interactions with other biomolecules, with the requisite high throughput and high sensitivity. While protein microarrays still present some challenging problems, recent progress makes it likely that it will be possible soon to interrogate proteins at a proteome scale, using protein microarrays, analogous to DNA microarrays, thus making a substantial impact on biomedical research.
Keywords:
protein microarray;
protein profiling;
antibody microarray;
proteome;
disease proteomics;
biomarker;
protein–protein interaction
We propose a technique for fabricating a carbohydrate-terminated monolayer (CM) surface, which is extremely flat at the angstrom level. The CM was immobilized onto a Si surface by Si−C covalent bond. Specific adsorption of the protein molecule from a contacting solution was observed on the CM area of a patterned CM substrate surface, while its nonspecific adsorption was observed on the Si−O area.
In order to keep subscribers up-to-date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly-published material on comparative and functional genomics. Each bibliography is divided into 16 sections. 1 Reviews & symposia; 2 General; 3 Large-scale sequencing and mapping; 4 Genome evolution; 5 Comparative genomics; 6 Gene families and regulons; 7 Pharmacogenomics; 8 Large-scale mutagenesis programmes; 9 Functional complementation; 10 Transcriptomics; 11 Proteomics; 12 Protein structural genomics; 13 Metabolomics; 14 Genomic approaches to development; 15 Technological advances; 16 Bioinformatics. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted
The demand for polymer-based DNA microarrays will increase because of their cost-effectiveness, biocompatibility and easy processing. However not all polymers are ideal substrates because of different chemical interactions of polymeric substrates with the DNA molecules. Results from AFM analysis of DNA immobilised on polymeric surfaces are evaluated using fractality, Gaussian distribution and lateral force imaging. It has been found that the nanosize defects in the substrate, such as poly-l-lysine, plays an important role in the growth of DNA aggregates in a vertical direction, whereas the covalent binding of DNA molecules on NHS-functionalised cyclo-olefin copolymer leads to the lateral growth of DNA aggregates.© (2005) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.
Cited By :5, Export Date: 18 June 2015, Correspondence Address: Ivanov, A.S., Chemicals/CAS: Ligands
Cited By :5, Export Date: 18 June 2015, Correspondence Address: Ivanov, A.S., Chemicals/CAS: Ligands
During the past five years, investigations employing a variety of proteomic technologies have yielded a wealth of information on a number of autoimmune disorders. Animal models of autoimmune disease have been examined and have provided clues that can be useful in elucidating molecular pathways and mechanisms that play a role in autoimmune disorders. Human sera and body fluids have been analyzed and have resulted in the identification of autoantibodies that can be used as diagnostic markers in specific autoimmune diseases, and proteomic fingerprints of tissues and body fluids have resulted in the identification of individual proteins or patterns of protein expression that are deregulated in autoimmune diseases. The information provided by these proteomic studies are of diagnostic and therapeutic potential. This review provides an overview of the approaches used in the proteomic analyses of autoimmune disease.
We have developed a technique to establish catalogues of protein products of arrayed cDNA clones identified by DNA hybridisation
or sequencing. A human fetal brain cDNA library was directionally cloned in a bacterial vector that allows IPTG-inducible
expression of His6-tagged fusion proteins. Using robot technology, the library was arrayed in microtitre plates and gridded
onto high-density in situ filters. A monoclonal antibody recognising the N-terminal RGSH6 sequence of expressed proteins (RGS·His antibody, Qiagen) detected 20% of the library as putative expression clones. Two
example genes, GAPDH and HSP90α, were identified on high-density filters using DNA probes and antibodies against their proteins.
A new generation biochip is described as capable of supporting high-throughput (HT), multiplexed enzyme-linked immunosorbent assays (ELISAs). These biochips consist of an optically flat, glass plate containing 96 wells formed by an enclosing hydrophobic Teflon mask. The footprint dimensions of each well and the plate precisely match those of a standard microplate. Each well contains four identical 36-element arrays (144 elements per well) comprising 8 different antigens and a marker protein. Arrays are formed by a custom, continuous flow, capillary-based print head attached to a precise, high-speed, X-Y-Z robot. The array printing capacity of a single robot exceeds 20,000 arrays per day. Arrays are quantitatively imaged using a custom, high-resolution, scanning charge-coupled device (CCD) detector with an imaging throughout of 96 arrays every 30 s. Using this new process, arrayed antigens were individually and collectively detected using standard ELISA techniques. Experiments demonstrate that specific multiplex detection of protein antigens arrayed on a glass substrate is feasible. Because of the open microarray architecture, the 96-well microarray format is compatible with automated robotic systems and supports a low-cost, highly parallel assay format. Future applications of this new high-throughput screening (HTS) format include direct cellular protein expression profiling, multiplexed assays for detection of infectious agents and cancer diagnostics.
We have developed a system to identify highly specific antibody–antigen interactions by protein array screening. This removes
the need for selection using animal immunisation or in vitro techniques such as phage or ribosome display. We screened an array of 27 648 human foetal brain proteins with 12 well-expressed
antibody fragments that had not previously been exposed to any antigen. Four highly specific antibody–antigen pairs were identified,
including three antibodies that bind proteins of unknown function. The target proteins were expressed at a very low copy number
on the array, emphasising the unbiased nature of the screen. The specificity and sensitivity of binding demonstrates that
this ‘naive’ screening approach could be applied to the high throughput isolation of specific antibodies against many different
targets in the human proteome.
To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.
Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.
We constructed miniaturized autoantigen arrays to perform large-scale multiplex characterization of autoantibody responses directed against structurally diverse autoantigens, using submicroliter quantities of clinical samples. Autoantigen microarrays were produced by attaching hundreds of proteins, peptides and other biomolecules to the surface of derivatized glass slides using a robotic arrayer. Arrays were incubated with patient serum, and spectrally resolvable fluorescent labels were used to detect autoantibody binding to specific autoantigens on the array. We describe and characterize arrays containing the major autoantigens in eight distinct human autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. This represents the first report of application of such technology to multiple human disease sera, and will enable validated detection of antibodies recognizing autoantigens including proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA and post-translationally modified antigens. Autoantigen microarrays represent a powerful tool to study the specificity and pathogenesis of autoantibody responses, and to identify and define relevant autoantigens in human autoimmune diseases.
Starting with the discovery of penicillin, other antibiotics, and insulin, the quest for understanding and use of biological systems, i. e. , microorganisms and ani mal tissue, for the production of value products has lead to a dramatic increase in microbiological and bioengineering research in the last decades. Chemical and pharmaceutical companies quickly realized the huge commercial potential of these bioproducts and have spent millions of US dollars on R &D as well as on a build up of production facilities. Although there was limited knowledge about the cell's molecular mechanisms, which are the basis for the formation of the desired products, products from fermentation and extraction of biological matrices were a success right from the start. R&D projects within industry and academia on the continuous improvement of production processes, especially microbial productivity and down stream processing, allowed a fast return of investment and secured competitiveness in the market. Whereas the focus of such research projects was mainly on the discovery of strains with higher pro ductivity for the product of interest, e. g. , antibiotics, a lot of expertise and knowledge was generated allowing the use of biotechnological products and processes outside the pharmaceutical arena. The tremendous increase in knowl edge and the technological developments in microbial genetics where driven by these research projects and, accompanied with the advancements in nucleotide chemistry leading to a much better understanding of intracellular processes, served as a basis for modern molecular biology and recombinant biotech nology.
Background We describe a method for printing protein microarrays, and using these microarrays in a comparative fluorescence assay to measure the abundance of many specific proteins in complex solutions. A robotic device was used to print hundreds of specific antibody or antigen solutions in an array on the surface of derivatized microscope slides. Two complex protein samples, one serving as a standard for comparative quantitation, and the other representing an experimental sample in which the concentrations of specific proteins were to be measured, were labeled by covalent attachment of spectrally-resolvable fluorescent dyes. Specific antibody-antigen interactions localized specific components of the complex mixtures to defined cognate spots in the array, where the relative intensity of the fluorescent signals representing the experimental sample and the reference standard provided a measure of each protein's abundance in the experimental sample. To characterize the specificity, sensitivity and accuracy of this assay, we analyzed the performance of 115 antibody/antigen pairs. Results 50% of the arrayed antigens, and 20% of the arrayed antibodies, provided specific and accurate measurements of their cognate ligands at or below concentrations of 1.6 mu g/ml and 0.34 mu g/ml, respectively. Some of the antibody/antigen pairs allowed detection of the cognate ligands at absolute concentrations below 1 ng/ml, and partial concentrations of less than 1 part in 106, sensitivities sufficient for measurement of many clinically important proteins in patient blood samples. Conclusion Protein microarrays can provide a simple and practical means to characterize patterns of variation in hundreds or thousands of different proteins, in clinical or research applications.
The immobilization of antibodies on solid-phase materials has been used in many areas such as purification, diagnostic immunoassays and immunosensors. Problems associated with the loss of biological activity of the antibodies upon immobilization have been noticed in many cases. One of the main reasons for such loss is attributed to the random orientation of the asymmetric macromolecules on support surfaces. In this paper, the approaches for achieving oriented coupling of antibodies to increase the antigen binding capacity are reviewed. Some issues such as steric hindrance caused by neighbouring antibody molecules, the distance between an antibody and the support surface and the use of antibody fragments are dealt with. Some applications of the oriented immobilized antibodies in immunoassays and immunosensors are examined.
The isoxazole derivative Leflunomide (HWA 486) is a novel immunoregulatory and anti-inflammatory drug. Affinity chromatography was used to purify and identify Leflunomide binding proteins, which might play a role as potential cellular targets in the molecular mode of action. The Leflunomide derivative A 0273 was covalently coupled to a Fractogel® matrix. This column was used to separate a cytosolic protein extract of the macrophage cell line RAW 264.7 by several selected and specific gradient elution steps. Proteins that were specifically eluted through the active metabolite of Leflunomide, A 1726, were identified by subsequent protein sequence analysis. This allowed us to specify 10 cytosolic proteins, which bind with high affinity to this matrix. Three of them, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and phosphoglycerate mutase belong to the second part of the glycolytic pathway. The binding specificity of these protein/drug interactions was further evaluated using BIAcore® analysis. Kd values of glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and lactic dehydrogenase were similar to the Kd value of a known Leflunomide target protein, dihydroorotate dehydrogenase. In order to elucidate the features as well as the overall relevance of these results, cytosolic fractions of three additional cell lines MOLT-4, A20.2J, HeLa were compared using the same chromatographic protocol. The elution profiles as well as subsequent Western blot analyses confirmed the data obtained previously for the macrophage cell line RAW 264.7.
Protein–protein interactions have been widely used to study gene expression pathways and may be considered as a new approach
to drug discovery. Here I report the development of a universal protein array (UPA) system that provides a sensitive, quantitative,
multi-purpose, effective and easy technology to determine not only specific protein–protein interactions, but also specific
interactions of proteins with DNA, RNA, ligands and other small chemicals. (i) Since purified proteins are used, the results
can be easily interpreted. (ii) UPA can be used multiple times for different targets, making it economically affordable for
most laboratories, hospitals and biotechnology companies. (iii) Unlike DNA chips or DNA microarrays, no additional instrumentation
is required. (iv) Since the UPA uses active proteins (without denaturation and renaturation), it is more sensitive compared
with most existing methods. (v) Because the UPA can analyze hundreds (even thousands on a protein microarray) of proteins
in a single experiment, it is a very effective method to screen proteins as drug targets in cancer and other human diseases.
A convenient method for the construction of site-specifically modified poly(ethylene glycol)-protein conjugates is described. This method relies on the ability to generate a reactive carbonyl group in place of the terminal amino group. If the protein has N-terminal serine or threonine, this can be done by very mild periodate oxidation and generates a glyoxylyl group. A method less restricted by the nature of the N-terminal residue, but which requires somewhat harsher conditions, is metal-catalyzed transamination, which gives a keto group. The N-terminal-introduced reactive carbonyl group specifically reacts, under mild acidic conditions, with an aminooxy-functionalized poly(ethylene glycol) to form a stable oxime bond. Using polymers of different size and shape (linear or multibranched), various conjugates of IL-8, G-CSF, and IL-1ra were constructed and further characterized with respect to their biological activity and pharmacokinetic behavior in rats. Unlike most previous methods, this approach places a single PEG chain at a defined site on the protein. It should therefore be more likely to conserve biological activity when the latter depends on interaction with another macromolecule (unlike enzymic activity which often survives multiple PEGylation).
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).
The main developments in the "ligand assay" field in which I have been involved are traced. These include the original development of "first generation" competitive assays relying on radiolabeled analyte markers; the development of the first "second generation", noncompetitive (ultrasensitive) methods, which rely on the use of labeled (monoclonal) antibodies and high specific activity nonisotopic labels (leading to the transformation of the immunodiagnostic field in the 1980s); and the development of the first "third generation" miniaturized, chip-based, microarray methods, which permit the simultaneous ultrasensitive measurement of many analytes in the same small sample. The latter--applicable both to immunoassay and to DNA/RNA analysis--are likely to revolutionize the diagnostic and pharmaceutical fields in the next decade.
Cystic fibrosis (CF) has become a paradigm disorder for the clinical testing of gene therapies in the treatment of inherited disease. In recent years, efforts directed at gene therapy of CF have concentrated on improving gene delivery systems to the airway. Surrogate endpoints for complementation of CFTR dysfunction in the lung have been primarily dependent on correction of chloride transport abnormalities. However, it is now clear that the pathophysiology of CF airways disease is far more complex than can be solely attributed to altered chloride permeability. For example, in addition to functioning as a chloride channel, CFTR also has been implicated in the regulation of other apical membrane conductance pathways through interactions with the amiloride sensitive epithelial sodium channel (ENaC) and the outwardly rectifying chloride channel (ORCC). Superimposed on this functional diversity of CFTR is a highly regulated pattern of CFTR expression in the lung. This heterogeneity occurs at both the level of CFTR protein expression within different cell types in the airway and the anatomical location of these cells in the lung. Potential targets for gene therapy of CF include ciliated, non-ciliated, and goblet cells in the surface airway epithelium as well as submucosal glands within the interstitium of the airways. Each of these distinct cellular compartments may have functionally distinct roles in processes which affect the pathogenesis of CF airways disease, such as fluid and electrolyte balance. However, it is presently unclear which of these cellular targets are most pathophysiologic relevant with regard to gene therapy. Elucidation of the underlying mechanisms of CFTR function in the airway will allow for the rational design of gene therapy approaches for CF lung diseases. This review will provide a summary of the field's current knowledge regarding CFTR functional diversity in the airway and the implications of such diversity for gene therapies of CF lung disease.
A fluorescence-based immunosensor has been developed for simultaneous analysis of multiple samples. A patterned array of recognition elements immobilized on the surface of a planar waveguide is used to "capture" analyte present in samples; bound analyte is then quantified by means of fluorescent detector molecules. Upon excitation of the fluorescent label by a small diode laser, a CCD camera detects the pattern of fluorescent antigen:antibody complexes on the sensor surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. This immunosensor was used to detect physiologically relevant concentrations of staphylococcal enterotoxin B (SEB), F1 antigen from Yersinia pestis, and D-dimer, a marker of sepsis and thrombotic disorders, in spiked clinical samples.
Driven by chemistry but increasingly guided by pharmacology and the clinical sciences, drug research has contributed more
to the progress of medicine during the past century than any other scientific factor. The advent of molecular biology and,
in particular, of genomic sciences is having a deep impact on drug discovery. Recombinant proteins and monoclonal antibodies
have greatly enriched our therapeutic armamentarium. Genome sciences, combined with bioinformatic tools, allow us to dissect
the genetic basis of multifactorial diseases and to determine the most suitable points of attack for future medicines, thereby
increasing the number of treatment options. The dramatic increase in the complexity of drug research is enforcing changes
in the institutional basis of this interdisciplinary endeavor. The biotech industry is establishing itself as the discovery
arm of the pharmaceutical industry. In bridging the gap between academia and large pharmaceutical companies, the biotech firms
have been effective instruments of technology transfer.
In order to quantify autoantibodies in the sera of patients with autoimmune disease, we have created a microarray-based immunoassay that allows the simultaneous analysis of 18 known autoantigens. The microarrays contain serial dilutions of the various antigens, thereby allowing accurate determination of autoantibody titer using minimal amounts of serum. The assay is very sensitive and highly specific: as little as 40 fg of a known protein standard can be detected with little or no cross-reactivity to nonspecific proteins. The signal intensities observed from serial dilutions of immobilized antigen correlate well with serial dilutions of autoimmune sera. Miniaturized and highly parallelized immunoassays like these will reduce costs by decreasing reagent consumption and improve efficiency by greatly increasing the number of assays that can be performed with a single serum sample. This system will significantly facilitate and accelerate the diagnostics of autoimmune diseases and can be adapted easily to any other kind of immunoassay.
This article presents current trends and advances in protein biochip technologies that rely upon extraction and retention of target proteins from liquid media. Analytical strengths as well as technical challenges for these evolving platforms are presented with particular emphasis on selectivity, sensitivity, throughput and utility in the post-genome era. A general review of protein biochip technology is provided, which delineates approaches for protein biochip format and operation, as well as protein detection. A focused discussion of three protein biochip technologies, Biomolecular Interaction Analysis (Biacore, Uppsala, Sweden), Surface Enhanced Laser Desorption/Ionisation (SELDI) ProteinChip Arrays (Ciphergen Biosystems, Fremont, CA, USA) and Fluorescent Planar Wave Guide (Zeptosens, Witterswil, Switzerland), follows along with examples of relevant applications.
We developed versatile low-cost arrays of sol-gel-encapsulated enzymes (referred to as solzymes) suitable for repeated assays of bioactivity or enzyme inhibition. Sol-gel microstructures containing active enzymes were stabilized on glass at moderate pH and room temperature without harsh calcination. A multi-well bilayer of polydimethylsiloxane was used to support the solzyme array and contain the reaction medium. Each of the 147 microwells has a working volume of 5 muL and contains 50 mug of immobilized enzyme. The solzyme arrays maintained high activity through repeated applications and exhibited superior thermostability compared to soluble enzymes. Among the enzymes used were lipases, glucose oxidase, and horseradish peroxidase. Twenty different lipases and proteases were also used to prepare a hydrolase array, for which bromthymol blue served as a generic indicator of activity. The relative activities of the encapsulated hydrolases correlated closely with those of the soluble hydrolases, illustrating that sol-gel encapsulation preserved the hierarchy of enzyme activity. The development of solzyme arrays paves the way to higher throughput screening of diverse proteins and enzymes, including those that are available only in trace amounts.
This paper describes the fabrication of microarrays consisting of G protein-coupled receptors (GPCRs) on surfaces coated with gamma-aminopropylsilane (GAPS). Microspots of model membranes on GAPS-coated surfaces were observed to have several desired properties-high mechanical stability, long range lateral fluidity, and a thickness corresponding to a lipid bilayer in the bulk of the microspot. GPCR arrays were obtained by printing membrane preparations containing GPCRs using a quill-pin printer. To demonstrate specific binding of ligands, arrays presenting neurotensin (NTR1), adrenergic (beta1), and dopamine (D1) receptors were treated with fluorescently labeled neurotensin (BT-NT). Fluorescence images revealed binding only to microspots corresponding to the neurotensin receptor; this specificity was further demonstrated by the inhibition of binding in the presence of excess unlabeled neurotensin. The ability of GPCR arrays to enable selectivity studies between the different subtypes of a receptor was examined by printing arrays consisting of three subtypes of the adrenergic receptor: beta1, beta2, and alpha2A. When treated with fluorescently labeled CGP 12177, a cognate antagonist analogue specific to beta-adrenergic receptors, binding was only observed to microspots of the beta1 and beta2 receptors. Furthermore, binding of labeled CGP 12177 was inhibited when the arrays were incubated with solutions also containing ICI 118551, and in a manner consistent with the higher affinity of ICI 118551 for the beta2 receptor relative to that for the beta1 receptor. The ability to estimate binding affinities of compounds using GPCR arrays was examined using a competitive binding assay with BT-NT and unlabeled neurotensin on NTR1 arrays. The estimated IC(50) value (2 nM) for neurotensin is in agreement with the literature; this agreement suggests that the receptor -G protein complex is preserved in the microspot. This first ever demonstration of direct pin-printing of membrane proteins and ligand-binding assays thereof fills a significant void in protein microchip technology--the lack of practical microarray-based methods for membrane proteins.
One of the current trends in breast cancer research is to identify markers that can predict response to specific anticancer therapies; intense laboratory research and therapeutic trials are exploiting this strategy. The combination of cytotoxic drugs directed at the tumor population with the highest probability of being sensitive to them with molecules targeted at intracellular signaling and cell cycle control pathways, which may be deregulated as part of the malignant process, represents our best hope for improved survival in both early and advanced disease. The transmembrane tyrosine kinase receptor, HER2/neu, has been the subject of much investigation with respect to its prognostic value, predictive value, and as a target of antibody-mediated therapy. Retrospective evidence strongly suggests that HER2 overexpression is associated with decreased disease-free and overall survival in node-positive, and possibly also node-negative, breast cancer. Prospective trials have demonstrated that antibodies to HER2 can produce tumor responses in women with advanced disease that overexpresses this molecule. Moreover, the combination of such antibodies with cytotoxic drugs has been one of the few recent strategies to improve survival duration in metastatic breast cancer. The evidence supporting the role of HER2 as a factor predictive of response to hormone therapy and cytotoxic drugs is more ambiguous and requires prospective assessment. The available literature is reviewed herein, with a focus on the predictive value of HER2, potential mechanisms of resistance and sensitivity to various drugs, and future research directions involving this important molecule.
Proteomics is the protein equivalent of genomics and is the study of gene expression at a functional level. The proteome of an organism is the protein complement of its genome. However, unlike the genome, the proteome is dynamic: it varies according to the cell type and the functional state of the cell. In addition, the proteome shows characteristic perturbations in response to disease and external stimuli. Proteomics combines state of the art analytical methods with bioinformatics. Here, we review the concept and technology of proteomics with specific reference to applications in medical microbiology, cellular pathology, clinical chemistry, haematology/immunology, pharmacology and toxicology.
Holding all the chips
- K Calkins
Calkins, K. (2001) Holding all the chips. BioCentury The Bernstein Report on
Proteomics: quantitative and physical mapping of cellular proteins
- W P Blackstock
Blackstock, W.P. (1999) Proteomics: quantitative and physical mapping
of cellular proteins. Trends Biotechnol. 17, 121–127