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Protein microarrays and biomarkers of infectious disease.

Natesan M, Ulrich RG - Int J Mol Sci (2010)

Bottom Line: Protein microarrays are powerful tools that are widely used in systems biology research.Distinct formats of protein microarrays have been developed for different applications, including abundance-based and function-based methods.Depending on the application, design issues should be considered, such as the need for multiplexing and label or label free detection methods.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, USA; E-Mail: rulrich@bioanalysis.org.

ABSTRACT
Protein microarrays are powerful tools that are widely used in systems biology research. For infectious diseases, proteome microarrays assembled from proteins of pathogens will play an increasingly important role in discovery of diagnostic markers, vaccines, and therapeutics. Distinct formats of protein microarrays have been developed for different applications, including abundance-based and function-based methods. Depending on the application, design issues should be considered, such as the need for multiplexing and label or label free detection methods. New developments, challenges, and future demands in infectious disease research will impact the application of protein microarrays for discovery and validation of biomarkers.

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Related in: MedlinePlus

Pathogen proteome microarrays. Confocal laser scanner image of proteins spotted in duplicate onto microarray slides, visualized using a rabbit anti-GST antibody bound to Cy5-labeled anti-rabbit antibody. (a) Vaccinia virus; (b) Yersinia pestis.
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f2-ijms-11-05165: Pathogen proteome microarrays. Confocal laser scanner image of proteins spotted in duplicate onto microarray slides, visualized using a rabbit anti-GST antibody bound to Cy5-labeled anti-rabbit antibody. (a) Vaccinia virus; (b) Yersinia pestis.

Mentions: The host antibody response provides a signature of pathogen proteins displayed by infectious diseases that can be captured and detected by microarrays. Our studies with vaccinia virus [12] and the bacterium Yersinia pestis [11] exemplify the application of antigen microarrays for the analysis of immune responses (Figure 2). Vaccinia is a large DNA virus (Orthopoxviridae) that replicates in the cytoplasm of host cells from genomes encoding 150–300 proteins. The virion is comprised of approximately 100 proteins. Most phenotypic variability occurs in proteins encoded in the terminal regions of the genome that are associated with host virulence or immune evasion. Some of these terminal-region proteins are secreted during cell infection and interfere with host immunity by binding complement factors, cytokines, and chemokines, while others interfere with signaling pathways regulating host gene expression and apoptosis. To construct a proteome microarray, genomic DNA of the Copenhagen (NC_001559.1) vaccine strain (Dryvax) was used as the template for PCR amplification of the 273 open reading frames (ORF). Gateway recombination cloning (Invitrogen, Carlsbad, CA, USA) was employed to facilitate high-throughput production of proteins from all ORF clones. All DNA clones were sequence-verified through the entire length of their inserts. Baculovirus-based expression was used to produce the recombinant viral proteins as GST-tagged fusions to ensure high yield of properly folded proteins with posttranslational modifications that are similar to those encountered in the human host. The GST-tagged proteins from cell lysates were affinity purified to 90% homogeneity in a single step by using glutathione-agarose in 96-well plates and analyzed for correct size and abundance by Western blots. Virus and control proteins were printed onto glass slides coated with a thin layer of nitrocellulose. Ultimately, 95% of the proteome was successfully expressed, purified, and arrayed.


Protein microarrays and biomarkers of infectious disease.

Natesan M, Ulrich RG - Int J Mol Sci (2010)

Pathogen proteome microarrays. Confocal laser scanner image of proteins spotted in duplicate onto microarray slides, visualized using a rabbit anti-GST antibody bound to Cy5-labeled anti-rabbit antibody. (a) Vaccinia virus; (b) Yersinia pestis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3100839&req=5

f2-ijms-11-05165: Pathogen proteome microarrays. Confocal laser scanner image of proteins spotted in duplicate onto microarray slides, visualized using a rabbit anti-GST antibody bound to Cy5-labeled anti-rabbit antibody. (a) Vaccinia virus; (b) Yersinia pestis.
Mentions: The host antibody response provides a signature of pathogen proteins displayed by infectious diseases that can be captured and detected by microarrays. Our studies with vaccinia virus [12] and the bacterium Yersinia pestis [11] exemplify the application of antigen microarrays for the analysis of immune responses (Figure 2). Vaccinia is a large DNA virus (Orthopoxviridae) that replicates in the cytoplasm of host cells from genomes encoding 150–300 proteins. The virion is comprised of approximately 100 proteins. Most phenotypic variability occurs in proteins encoded in the terminal regions of the genome that are associated with host virulence or immune evasion. Some of these terminal-region proteins are secreted during cell infection and interfere with host immunity by binding complement factors, cytokines, and chemokines, while others interfere with signaling pathways regulating host gene expression and apoptosis. To construct a proteome microarray, genomic DNA of the Copenhagen (NC_001559.1) vaccine strain (Dryvax) was used as the template for PCR amplification of the 273 open reading frames (ORF). Gateway recombination cloning (Invitrogen, Carlsbad, CA, USA) was employed to facilitate high-throughput production of proteins from all ORF clones. All DNA clones were sequence-verified through the entire length of their inserts. Baculovirus-based expression was used to produce the recombinant viral proteins as GST-tagged fusions to ensure high yield of properly folded proteins with posttranslational modifications that are similar to those encountered in the human host. The GST-tagged proteins from cell lysates were affinity purified to 90% homogeneity in a single step by using glutathione-agarose in 96-well plates and analyzed for correct size and abundance by Western blots. Virus and control proteins were printed onto glass slides coated with a thin layer of nitrocellulose. Ultimately, 95% of the proteome was successfully expressed, purified, and arrayed.

Bottom Line: Protein microarrays are powerful tools that are widely used in systems biology research.Distinct formats of protein microarrays have been developed for different applications, including abundance-based and function-based methods.Depending on the application, design issues should be considered, such as the need for multiplexing and label or label free detection methods.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, USA; E-Mail: rulrich@bioanalysis.org.

ABSTRACT
Protein microarrays are powerful tools that are widely used in systems biology research. For infectious diseases, proteome microarrays assembled from proteins of pathogens will play an increasingly important role in discovery of diagnostic markers, vaccines, and therapeutics. Distinct formats of protein microarrays have been developed for different applications, including abundance-based and function-based methods. Depending on the application, design issues should be considered, such as the need for multiplexing and label or label free detection methods. New developments, challenges, and future demands in infectious disease research will impact the application of protein microarrays for discovery and validation of biomarkers.

Show MeSH
Related in: MedlinePlus