Protein Microarray Analysis Research Articles

AsymptomaticYersinia pestisInfection, China

To the Editor: Plague is one of the oldest identifiable diseases. Modern public health measures and effective antimicrobial treatments have led to a decrease in plague cases worldwide. However, plague remains endemic in many natural foci. Since the early 1990s, the World Health Organization (WHO) has reported a steadily increasing trend in human plague cases, which has resulted in the recognition of plague as a reemerging disease (1). The emergence of antimicrobial drug–resistant strains of Yersinia pestis, along with an increasing number of plague cases, remind us that plague still poses a serious public health threat (2,3). In China, human cases of plague have been reported to WHO nearly every year from 1989 to 2003; these account for 9.5% of cases and 15.5% of deaths from this disease in Asia (1). Human cases of plague in China are usually caused by contact with plague-infected rodents. Here, we report the results of a serologic survey by using 3 methods (passive hemagglutination assay, Western blot, and protein microarray analysis) in marmot hunters in Qinghai Province, China. One hundred twenty serum samples were collected in 2 villages in Huangyuan County, Qinghai Province, from marmot hunters (63 samples) and their family members (57 samples); none had a history of fever in the past 2 years. One hundred nineteen serum samples were collected from persons with no history of marmot hunting in 2 nearby counties in Qinghai Province in which plague was not endemic. Thirty serum samples were collected from persons in Beijing and used as negative controls. All serum samples were initially screened with a passive hemagglutination assay to detect immunoglobulin (Ig) G antibody against F1 antigen of Y. pestis, by using a standard protocol (4). We then used an F1 antigen–based Western blot to analyze all serum samples. The protein microarray analysis was performed with 149 purified recombinant proteins of Y. pestis (5). The results of the serologic survey are summarized in the Table. The passive hemagglutination assay showed 17 positive samples in the marmot hunter population. None of the control serum samples were positive for F1 antigen in this assay. Western blot identified 9 additional positive samples in the marmot hunter population, resulting in a seropositivity rate of 21.7% (26/120). We also found positive samples in 4 (3.4%) of 119 serum samples by using Western blot in persons from areas in which plague was not endemic. Identical results were also obtained by using protein microarray analysis, which validated the results of Western blot. Table Analysis of sera for plague antibody by 3 methods Previous studies have shown that plague antibodies were more prevalent in males in the exposed population, and differences in the age, sex, or ethnic group of plague patients are the result of variations in exposure to the pathogen, not intrinsic factors (6,7). Our study showed that in the marmot hunter population, the plague seropositivity rate was significantly higher in males (36.8%, 25/68) than in females (2.0%, 1/52, p<0.01). Among the marmot hunter population, 63 (92.6%) of 68 males were hunters. Plague antibodies were also more prevalent in marmot hunters (39.7%, 25/63) than in their family members (1.8%, 1/57, p<0.01). This is the first serologic survey of plague in the marmot hunter population. The plague seropositivity rate of 21.7% (26/120) in hunters and their families is much higher than the 3.4% (4/119) in the population from regions in which plague was not endemic (p<0.01). Seroprevalence in marmot hunters was even higher (39.7%), which suggests that marmot hunting is a risk factor for plague infection. The marmot (Marmota himalayana) is the main host of Y. pestis in Qinghai Province. Plague-infected marmots are more easily captured by hunters. When persons hunt and butcher marmots without any effective protection, Y. pestis can be transmitted through tiny wounds in the skin, by bites of infected fleas, or by the respiratory route. Asymptomatic plague infection in marmot hunters might be explained by prophylactic use of antimicrobial drugs. Most hunters usually take sulfamethoxazole or tetracycline as a prophylactic measure. Even if the hunters were infected with Y. pestis, they would likely not develop symptomatic plague. However, if the antimicrobial drugs are not effective or hunters do not use prophylaxis, symptomatic infections will occur. Most reported human cases of plague in Qinghai Province were caused by hunting or butchering marmots, as shown by a recent outbreak of plague in October 2004 in Qinghai, in which 19 cases were reported and 8 persons died (M. Li et al., unpub. data).

Non-contact laser microdissection and pressure catapulting: Automation via object-oriented image processing

Laser Microdissection and Pressure Catapulting (LMPC) is a well-known non-contact method to isolate and collect specific cells from complex animal or plant tissues or living cultured cells. Thus, it is enabling pure and homogeneous sample preparation resulting in an eminent increase in the specificity of versatile downstream molecular analyses, like e.g., different PCR, nucleic acid and protein microarray analysis. An important innovation is the integration of computer-assisted recognition methods by object-oriented image processing. Here we show the automated recognition of sample material by applying object-oriented image processing on parameters like, e.g., density, area, neighborhood and shape. Coupled with this kind of software modules, the LMPC system is able to detect, isolate and finally capture the specimen of interest in a fully automated manner. Differential analysis of individual tissues and single cells will eliminate the averaging effect and allow the discovery of detailed differences between various cell types. Laser Microdissection und Pressure Catapulting (LMPC) ist eine etablierte und berührungsfreie Methode, Zellen aus komplexen tierischen oder pflanzlichen Geweben oder aus lebenden Kulturen spezifisch zu isolieren und zu sammeln. Dadurch wird eine saubere und homogene Probenpräparation gewährleistet, bei der die Aussagekraft einer Vielzahl nachgeschalteter molekularer Analysen steigt. Beispiele hierfür stellen verschiedene PCR-, Nukleinsäure- und Protein-Microarray-Untersuchungen dar. Die Integration computergestützter Erkennungsmethoden ist eine wichtige Weiterentwicklung dieser Technologie. In dieser Arbeit zeigen wir die automatische Erkennung von Probenmaterial durch die Anwendung so genannter objektorientierter Bildverarbeitung. Hierbei werden Parameter wie Objektdichte, Fläche, Nachbarschaftsbeziehungen oder die Form zur Erkennung herangezogen. Die Verbindung derartiger Software mit einem LMPC-System erlaubt die Erkennung, Isolierung und das automatische Sammeln der Probe in nur einem Arbeitsgang. Differenzierte Analysen einzelner Gewebe und Zellen führen zum Ausschluss von Mittelungseffekten. Sie erlauben die detaillierte Untersuchung verschiedener Zelltypen, wobei Unterschiede deutlich zu Tage treten.

Protein microarray analysis of disease activity in pediatric inflammatory bowel disease demonstrates elevated serum PLGF, IL-7, TGF-beta1, and IL-12p40 levels in Crohn's disease and ulcerative colitis patients in remission versus active disease.

Cytokines and growth factors play a major role in the dysregulated immune response in inflammatory bowel disease (IBD). We hypothesized that significant differences exist between the serum cytokine and growth factor profiles of pediatric IBD patients with active disease (AD) and those in remission, and that levels of some of these soluble mediators may be used to define regulators in IBD and determine disease activity. Eighty-eight consecutive patients with confirmed Crohn's disease (CD) and ulcerative colitis (UC) seen at the Duke Children's Hospital were prospectively enrolled and a serum sample was obtained. Data were recorded at the time of serum collection to calculate disease activity indices. The relative expression of 78 cytokines, growth factors, and soluble receptors was determined using proprietary antibody-based protein microarrays amplified by rolling circle amplification. SPSS 8 (SPSS Inc., Chicago, IL) was used to compare protein profiles for CD and UC patients in clinical remission (CR) versus AD. Sixty-five CD patients and 23 UC patients were enrolled. Forty-one CD patients had available samples and PCDAI results. Twenty-two patients were in remission PCDAI < or = 12.5 (median 5), 19 patients had disease activity >15 (median 30). Univariate analysis revealed that PLGF, IL-7, IL-12p40, and TGF-beta1 cytokine levels were significantly elevated for patients in CR versus AD (p < 0.01). Twelve UC serum samples had Seo/Truelove Witt AI for analysis. Five patients were in remission by TW AI and Seo AI < or =110 and 7 patients had active mild-to-severe disease by TW and Seo AI >110. Only one cytokine, IL12p40, showed significance between CR versus AD (p < 0.02). Surprisingly, we found no differences in circulating levels of proinflammatory cytokines but found that pediatric IBD patients in remission compared to those with AD had higher levels of specific circulating cytokines, including the regulatory cytokines IL-12p40 and TGF-beta1. It may be that these cytokines directly regulate intestinal inflammation in IBD or reflect the activity of T regulatory cells in negatively regulating the inflammatory response. Further studies will be needed to validate our results to define the molecular pathways involved in the intestinal immune response in man.

Protein Microarray Analysis of Disease Activity in Pediatric Inflammatory Bowel Disease Demonstrates Elevated Serum PLGF, IL-7, TGF-beta1, and IL-12p40 Levels in Crohn's Disease and Ulcerative Colitis Patients in Remission versus Active Disease

Protein Microarray Analysis of Disease Activity in Pediatric Inflammatory Bowel Disease Demonstrates Elevated Serum PLGF, IL-7, TGF-beta1, and IL-12p40 Levels in Crohn's Disease and Ulcerative Colitis Patients in Remission versus Active Disease

DDI-microFIA--A readily configurable microarray-fluorescence immunoassay based on DNA-directed immobilization of proteins.

We describe a chip-based immunoassay for multiplex antigen detection, based on the self-assembly of semi-synthetic DNA-protein conjugates to generate an easily configurable protein microarray. The general principle of this microarray-fluorescence immunoassay (microFIA) is similar to that of a two-sided (sandwich) immunoassay. However, covalent single-stranded DNA-streptavidin conjugates are employed for the efficient immobilization of biotinylated capture antibodies through hybridization to complementary surface-bound DNA oligomers. In a model system, we use the DNA-directed immobilization (DDI) of antibodies to generate an antibody microarray for the parallel detection of the tumor marker human carcinoembryonic antigen (CEA), recombinant mistletoe lectin rViscumin (rVis), ceruloplasmin (CEP), and complement-1-inactivator (C1A) in human blood serum samples. Detection limits down to 400 pg mL(-1) are reached. In addition, we describe a method for the internal standardization of protein microarray analyses, based on the simultaneous measurement of constant amounts of the blood proteins CEP and C1 A, intrinsically present in human serum, to compensate for interexperimental variations usually occurring in microarray analyses. The standardization leads to a significantly higher data reliability and reproducibility in intra- and interassay measurements. We further demonstrate that the DDI-microFIA can also be carried out in a single step by tagging of the analyte simultaneously with both capture and detection antibody and subsequent immobilization of the immunocomplex formed, on the DNA microarray capture matrix. This protocol significantly reduces handling time and costs of analysis.

Site-specific immobilization of biotinylated proteins for protein microarray analysis.

The postgenome era has led to a new frontier of proteomics that requires the development of protein microarray, which enables us to unravel the biological function of proteins in a massively parallel fashion. Several ways of immobilizing proteins onto surfaces have been reported, but many of these attachments are unspecific, resulting in the unfavorable orientation of the immobilized proteins. His6 tag has been used to site-specifically immobilize proteins onto nickel-coated slides, which presumably oriented proteins uniformly on the surface of the slide. However, the binding between Ni-NTA and His tag proteins is not strong, causing the immobilized proteins to dissociate from the slide even under simple wash conditions. The authors have developed a novel strategy of using an intein-mediated expression system to generate biotinylated proteins suitable for immobilization onto avidin-functionalized glass slides. Array-scan results not only show successful immobilization of proteins onto slides by antibody detection method but also full retention of biological activities of the immobilized proteins. The strong and specific interaction between biotin and avidin also permits the use of stringent incubation and washing conditions on the protein microchip, thus making it a highly robust method for array studies.

Effect of power-frequency magnetic fields on genome-scale gene expression in Saccharomyces cerevisiae.

To estimate the effect of 50 Hz magnetic-field exposure on genome-wide gene expression, the yeast Saccharomyces cerevisiae was used as a model for eukaryotes. 2D PAGE (about 1,000 spots) for protein and cDNA microarray (about 5,900 genes) analysis for mRNA were performed. The cells were exposed to 50 Hz vertical magnetic fields at 10, 150 or 300 mT r.m.s. for 24 h. As positive controls, the cells were exposed to aerobic conditions, heat (40 degrees C) or minimal medium. The 2D PAGE and microarray analyses for the positive controls showed high-confidence differential expression of many genes including those for known or unknown proteins and mRNAs. For magnetic-field exposure, no high-confidence changes in expression were observed for proteins or genes that were related to heat-shock response, DNA repair, respiration, protein synthesis and the cell cycle. Principal component analysis showed no statistically significant difference in principal components, with only insignificant differences between the magnetic-field intensities studied. In contrast, the principal components for the positive controls were significantly different. The results indicate that a 50 Hz magnetic field below 300 mT did not act as a general stress factor like heat shock or DNA damage, as had been reported previously by others. This study failed to find a plausible differential gene expression that would point to a possible mechanism of an effect of magnetic fields. The findings provide no evidence that the magnetic-field exposure alters the fundamental mechanism of translation and transcription in eukaryotic cells.

Microfabrication of biomaterials by the sub-ps laser-induced forward transfer process

The precise fabrication of micro-dimensioned patterns of biomaterials by the laser microprinting technique using a sub-ps UV laser is described. An ultrashort UV laser has been used to transfer the biomaterial, with low angular divergence, and deposit it onto the substrate with minimum spread and high spatial resolution. The laser-transferred features of 100 μm×100 μm size have been studied by means of scanning electron microscopy and scanning laser confocal fluorescence microscopy. The analysis of DNA and protein microarrays provides an excellent tool to expand our knowledge of genome functions.

RERG is a novel ras-related, estrogen-regulated and growth-inhibitory gene in breast cancer.

Using microarray analysis, we identified a unique ras superfamily gene, termed RERG (ras-related and estrogen-regulated growth inhibitor), whose expression was decreased or lost in a significant percentage of primary human breast tumors that show a poor clinical prognosis. Importantly, high RERG expression correlated with expression of a set of genes that define a breast tumor subtype that is estrogen receptor-positive and associated with a slow rate of tumor cell proliferation and a favorable prognosis for these cancer patients. RERG mRNA expression was induced rapidly in MCF-7 cells stimulated by beta-estradiol and repressed by tamoxifen treatment. Like Ras, RERG protein exhibited intrinsic GDP/GTP binding and GTP hydrolysis activity. Unlike Ras proteins, RERG lacks a known recognition signal for COOH-terminal prenylation and was localized primarily in the cytoplasm. Expression of RERG protein in MCF-7 breast carcinoma cells resulted in a significant inhibition of both anchorage-dependent and anchorage-independent growth in vitro and inhibited tumor formation in nude mice. These features of RERG are strikingly different from most Ras superfamily GTP-binding pro-teins and suggest that the loss of RERG expression may contribute to breast tumorigenesis.

Micro-Patterning of Polymers for High Resolution Microscopy Analysis

Micro-patterned surfaces are of interest in biology and chemistry due to the ability to confine functional sample materials to specific areas. If patterns are visible, either through optical microscopy, or topographically, with scanning probe microscopy, investigating samples in a manner that is cheap and accessible to most laboratories is possible. Examples include micropatterning cells in tissue engineering, and protein micro-array analysis. We have created a micro-patterned surface with tailored optical and topographic properties. These were investigated using the microscopy techniques, confocal microscopy (CM), atomic force microscopy (AFM) and near field scanning optical microscopy (NSOM). AFM and CM were used to investigate different aspects of the micro-pattern. to confirm the properties of the micro-pattern, we show the advantages of NSOM in investigating surfaces with both optical and topographic properties simultaneously. Micro-patterns were fabricated using a soft lithography technique, micro-contact printing, where reagents are ‘stamped’ upon substrates using an elastomeric moulding of a micro-template.

DNA and Protein Microarrays and their Contributions to Proteomics and Genomics

Knowledge in genomics and proteomics has exploded in the last two decades. This is in part due to key developments that have revolutionized the possibilities of bioanalytics such as the introduction of polymerase chain reaction (PCR) in the mid 80s that formed the base for the massively parallel sequencing of the genomes.A few years ago DNA and protein microarray analysis were added to the toolbox of life sciences analytics. These technologies already proved to be ideal tools for the identification of gene targets, the simultaneous measurement of the expression of a high number of genes or proteins, and the increase of the level of understanding of the biological functions of genes and proteins. A small number of experiments are now sufficient to obtain information on gene or protein expression which could not be obtained by using conventional bioanalytical technologies or which required an extremely high experimental effort. In the future applications, high sensitivity DNA and protein microarrays will allow low abundant genes and proteins to be monitored that so far have been inaccessible to current microarray technologies and thus will generate a new dimension of genomic and proteomic information.