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Serial analysis of 38 proteins during the progression of human breast tumor in mice using an antibody colocalization microarray.

Li H, Bergeron S, Annis MG, Siegel PM, Juncker D - Mol. Cell Proteomics (2015)

Bottom Line: The profiles of 38 proteins detected in sera from these animals were analyzed by clustering, and we identified 10 proteins with the greatest relative increase in serum concentration that correlated with growth of the primary mammary tumor.Next, the sensitivity and specificity of individual and optimal protein panels were calculated, showing high accuracy as early as week 2.These results provide a foundation for studies of tumor growth through measuring serial changes of protein concentration in animal models.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Biomedical Engineering Department, §McGill University and Genome Quebec Innovation Centre.

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Related in: MedlinePlus

Schematic outlining the process flow for preparing the slides and performing an antibody colocalization microarray in a snap chip format.a, dAbs are spotted onto an aminosilane-coated slide that is stored in a freezer. cAbs are spotted onto another aminosilane-coated slide with the same spotting layout and transferred to a nitrocellulose-coated assay slide followed by blocking and storage in a freezer. b, both slides are removed from the freezer prior to use. The assay slide is incubated with sample solutions, and then the dAbs are transferred to the assay slide by snapping followed by incubation with streptavidin-Cy 5. Next, the assay results are imaged with a fluorescence microarray scanner, and the data are analyzed.
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Figure 1: Schematic outlining the process flow for preparing the slides and performing an antibody colocalization microarray in a snap chip format.a, dAbs are spotted onto an aminosilane-coated slide that is stored in a freezer. cAbs are spotted onto another aminosilane-coated slide with the same spotting layout and transferred to a nitrocellulose-coated assay slide followed by blocking and storage in a freezer. b, both slides are removed from the freezer prior to use. The assay slide is incubated with sample solutions, and then the dAbs are transferred to the assay slide by snapping followed by incubation with streptavidin-Cy 5. Next, the assay results are imaged with a fluorescence microarray scanner, and the data are analyzed.

Mentions: The procedure for making antibody microarrays and performing the multiplex immunoassay in an ACM format with a snap chip (22) is illustrated in Fig. 1. In ACMs, each dAb is delivered only to the corresponding cAb spot, thus avoiding cross-reactivity and false positive signals. Whereas previously only one snap and transfer were performed (22), we implemented a novel “double transfer” protocol that we recently developed to improve the alignment between cAb and dAb microarrays.2 Briefly, whereas previously only the dAbs were transferred to the assay slide by snapping, here both cAb and dAb microarrays are transferred to minimize misalignment and thus increase the density of spots that may be transferred (Fig. 1). We established a snap chip targeting 50 human proteins (supplemental Table 1) comprising cancer biomarkers, cancer-related proteins, and cytokines. For all assays, standard curves were established for each target protein.


Serial analysis of 38 proteins during the progression of human breast tumor in mice using an antibody colocalization microarray.

Li H, Bergeron S, Annis MG, Siegel PM, Juncker D - Mol. Cell Proteomics (2015)

Schematic outlining the process flow for preparing the slides and performing an antibody colocalization microarray in a snap chip format.a, dAbs are spotted onto an aminosilane-coated slide that is stored in a freezer. cAbs are spotted onto another aminosilane-coated slide with the same spotting layout and transferred to a nitrocellulose-coated assay slide followed by blocking and storage in a freezer. b, both slides are removed from the freezer prior to use. The assay slide is incubated with sample solutions, and then the dAbs are transferred to the assay slide by snapping followed by incubation with streptavidin-Cy 5. Next, the assay results are imaged with a fluorescence microarray scanner, and the data are analyzed.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4390249&req=5

Figure 1: Schematic outlining the process flow for preparing the slides and performing an antibody colocalization microarray in a snap chip format.a, dAbs are spotted onto an aminosilane-coated slide that is stored in a freezer. cAbs are spotted onto another aminosilane-coated slide with the same spotting layout and transferred to a nitrocellulose-coated assay slide followed by blocking and storage in a freezer. b, both slides are removed from the freezer prior to use. The assay slide is incubated with sample solutions, and then the dAbs are transferred to the assay slide by snapping followed by incubation with streptavidin-Cy 5. Next, the assay results are imaged with a fluorescence microarray scanner, and the data are analyzed.
Mentions: The procedure for making antibody microarrays and performing the multiplex immunoassay in an ACM format with a snap chip (22) is illustrated in Fig. 1. In ACMs, each dAb is delivered only to the corresponding cAb spot, thus avoiding cross-reactivity and false positive signals. Whereas previously only one snap and transfer were performed (22), we implemented a novel “double transfer” protocol that we recently developed to improve the alignment between cAb and dAb microarrays.2 Briefly, whereas previously only the dAbs were transferred to the assay slide by snapping, here both cAb and dAb microarrays are transferred to minimize misalignment and thus increase the density of spots that may be transferred (Fig. 1). We established a snap chip targeting 50 human proteins (supplemental Table 1) comprising cancer biomarkers, cancer-related proteins, and cytokines. For all assays, standard curves were established for each target protein.

Bottom Line: The profiles of 38 proteins detected in sera from these animals were analyzed by clustering, and we identified 10 proteins with the greatest relative increase in serum concentration that correlated with growth of the primary mammary tumor.Next, the sensitivity and specificity of individual and optimal protein panels were calculated, showing high accuracy as early as week 2.These results provide a foundation for studies of tumor growth through measuring serial changes of protein concentration in animal models.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Biomedical Engineering Department, §McGill University and Genome Quebec Innovation Centre.

Show MeSH
Related in: MedlinePlus