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Chapter 31 : Cool Tools 2: Development of a Cell Surface Protein Microarray

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Cool Tools 2: Development of a Cell Surface Protein Microarray, Page 1 of 2

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Abstract:

The cell surface of is necessary for colonization of the human host and is also the target of the immune system when enters the bloodstream as an opportunistic pathogen. In a study to develop cell surface protein microarray, cell surface antigens that are specific to different phases (i.e., acute phase and early and midconvalescence) of candidemia, were identified. The study identified a set of 13 cell surface antigens capable of distinguishing acute candidemia from healthy individuals and uninfected hospital patients with commensal colonization. Studies need to address whether the serodiagnostic antigens identified in this study could provide protection from hematogenously disseminated candidiasis and whether sero-logical differences exist between superficial (i.e., thrush and vaginal candidiasis) and systemic infections. The increasing number of candidal infections necessitates the assay development of protein microarrays that include the whole proteome of not just but also other pathogenic species, as these studies may elucidate additional sero-diagnostic antigens and/or vaccine candidates. A high-throughput in vivo transformation system using a pXT7 linear vector was employed for cloning PCR products of genes encoding cell surface proteins. For PCR amplification, all forward and reverse primers had common 33-nucleotide-long sequences at the 5' end, followed by a gene-specific sequence (20 to 26 nucleotides). The protein microarray was produced by printing the in vitro-expressed proteins in duplicate onto nitrocellulose-coated FAST glass slides at a density of 960 spots per slide using an automatic GMS417 robot and the OmniGrid 100.

Citation: Mochon A. 2012. Cool Tools 2: Development of a Cell Surface Protein Microarray, p 489-496. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch31

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Figures

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FIGURE 1

Verification of in vivo recombination. To confirm recombination of the pTX7 vector and PCR products, agarose gel electrophoresis was used. An empty vector (arrow) was used as a marker. Plasmids with cell surface coding sequence inserts migrate more slowly than the empty vector. doi:10.1128/9781555817176.ch31.f1

Citation: Mochon A. 2012. Cool Tools 2: Development of a Cell Surface Protein Microarray, p 489-496. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch31
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Image of FIGURE 2
FIGURE 2

In vitro transcription/translation of cell surface proteins. Template DNA of the gene of interest is transcribed into mRNA by T7 RNA polymerase, which is then translated into protein by the ribosomal machinery in the lysate after 6 h of incubation at 30°C. RBS, ribosomal binding site; T7P, T7 promoter; T7T, T7 terminator. doi:10.1128/9781555817176.ch31.f2

Citation: Mochon A. 2012. Cool Tools 2: Development of a Cell Surface Protein Microarray, p 489-496. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch31
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Image of FIGURE 3
FIGURE 3

cell surface protein microarray. Shown is a representative image of the cell surface protein microarray of hybridized with the sera of an acute candidemia patient. The array consisted of 16 subsets. Each of the cell surface peptides was printed in duplicate. Each white box indicates a duplicated print of buffer alone, and each gray box shows a duplicate print of reaction mixture with no DNA. Reprinted from reference with permission. doi:10.1128/9781555817176.ch31.f3

Citation: Mochon A. 2012. Cool Tools 2: Development of a Cell Surface Protein Microarray, p 489-496. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch31
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Tables

Generic image for table
TABLE 1

The 40 most serodominant antigens in acute candidemia patients

Citation: Mochon A. 2012. Cool Tools 2: Development of a Cell Surface Protein Microarray, p 489-496. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch31
Generic image for table
TABLE 2

Antigenic biomarkers of acute candidemia patients

Citation: Mochon A. 2012. Cool Tools 2: Development of a Cell Surface Protein Microarray, p 489-496. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch31
Generic image for table
TABLE 3

Antigenic biomarkers of early-convalescent-phase and mid-convalescent-phase candidemia patients

Citation: Mochon A. 2012. Cool Tools 2: Development of a Cell Surface Protein Microarray, p 489-496. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch31

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