Limits...
In situ Protein Detection for Companion Diagnostics.

Gremel G, Grannas K, Sutton LA, Pontén F, Zieba A - Front Oncol (2013)

Bottom Line: The emergence of targeted therapies for cancer has created a need for the development of companion diagnostic tests.We also explore the possibility of using IHC to detect specific protein mutations in addition to DNA-based tests.Finally, we review alternative protein binders and proximity ligation assays and discuss their potential to facilitate the development of novel, targeted therapies against cancer.

View Article: PubMed Central - PubMed

Affiliation: Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University , Uppsala , Sweden.

ABSTRACT
The emergence of targeted therapies for cancer has created a need for the development of companion diagnostic tests. Assays developed in recent years are aimed at determining both the effectiveness and safety of specific drugs for a defined group of patients, thus, enabling the more efficient design of clinical trials and also supporting physicians when making treatment-related decisions. Immunohistochemistry (IHC) is a widely accepted method for protein expression analyses in human tissues. Immunohistochemical assays, used to localize and quantitate relative protein expression levels within a morphological context, are frequently used as companion diagnostics during clinical trials and also following drug approval. Herein, we describe established immunochemistry-based methods and their application in routine diagnostics. We also explore the possibility of using IHC to detect specific protein mutations in addition to DNA-based tests. Finally, we review alternative protein binders and proximity ligation assays and discuss their potential to facilitate the development of novel, targeted therapies against cancer.

No MeSH data available.


Related in: MedlinePlus

Proximity ligation assay. (A) Two probes stay in close proximity by binding to a protein or two proteins present in one complex. (B) They are joined and circularized by DNA ligation upon introduction of linear connector oligonucleotides. After ligation, rolling-circle amplification (RCA) is initiated. One of the proximity probes is used as a primer. (C) The single-stranded RCA products are hybridized with labeled detection oligonucleotide complementary to a multiplied motif in the sequence of the RCA product. The detection oligonucleotide can be labeled with fluorophore (D) or a horse radish peroxidase (E).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3814083&req=5

Figure 3: Proximity ligation assay. (A) Two probes stay in close proximity by binding to a protein or two proteins present in one complex. (B) They are joined and circularized by DNA ligation upon introduction of linear connector oligonucleotides. After ligation, rolling-circle amplification (RCA) is initiated. One of the proximity probes is used as a primer. (C) The single-stranded RCA products are hybridized with labeled detection oligonucleotide complementary to a multiplied motif in the sequence of the RCA product. The detection oligonucleotide can be labeled with fluorophore (D) or a horse radish peroxidase (E).

Mentions: Using traditional IHC techniques, the level of protein expression can easily be determined. However, the functional status of a cell cannot be evaluated by the level of expressed protein alone. The activity of signaling pathways, as assessed by the analysis of post-translation modifications (PTMs) and protein interactions, needs to be determined and taken into consideration (122). Cancer does not consist of a homologous mass of cells but of complex, heterogeneous cell populations that are affected by interactions with each other and the surrounding environment. Therefore, the analysis of cancer tissue at single cell resolution provides a much better understanding of the differences in signaling status and activity (123). In situ PLA enables a localized and specific detection by utilizing oligonucleotide-conjugated antibodies to determine the proximity between one or more targeted epitopes. This makes it a suitable method for detecting molecular events in cells and tissue, for example the status of a signaling pathway. The use of two independent binders and the additional requirement of proximity for reporting enable the specific detection of proteins, protein–protein interactions, and PTMs (124). Proximity ligation converts the recognition of a protein, protein complex, or PTM by two or more antibodies into an amplifiable, circular DNA molecule (Figure 3). Upon proximal binding of a pair of oligonucleotide-conjugated antibodies (PLA probes), the oligonucleotides guide the formation of a circle after applying two additional, single-stranded DNA molecules. This circular DNA molecule is then ligated and amplified by phi29 polymerase within a rolling-circle amplification (RCA) reaction, resulting in a localized, concatameric product. The latter is visualized by hybridization of detection oligonucleotides labeled with fluorophores or horse radish peroxidase (Figure 3) (125). Due to the environment of fixed cells and tissue the amplification product will collapse into a bundle with a diameter of approximately 1 μM (126) that can then be visualized as a bright dot that is quantifiable and easily distinguished from the background (127).


In situ Protein Detection for Companion Diagnostics.

Gremel G, Grannas K, Sutton LA, Pontén F, Zieba A - Front Oncol (2013)

Proximity ligation assay. (A) Two probes stay in close proximity by binding to a protein or two proteins present in one complex. (B) They are joined and circularized by DNA ligation upon introduction of linear connector oligonucleotides. After ligation, rolling-circle amplification (RCA) is initiated. One of the proximity probes is used as a primer. (C) The single-stranded RCA products are hybridized with labeled detection oligonucleotide complementary to a multiplied motif in the sequence of the RCA product. The detection oligonucleotide can be labeled with fluorophore (D) or a horse radish peroxidase (E).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814083&req=5

Figure 3: Proximity ligation assay. (A) Two probes stay in close proximity by binding to a protein or two proteins present in one complex. (B) They are joined and circularized by DNA ligation upon introduction of linear connector oligonucleotides. After ligation, rolling-circle amplification (RCA) is initiated. One of the proximity probes is used as a primer. (C) The single-stranded RCA products are hybridized with labeled detection oligonucleotide complementary to a multiplied motif in the sequence of the RCA product. The detection oligonucleotide can be labeled with fluorophore (D) or a horse radish peroxidase (E).
Mentions: Using traditional IHC techniques, the level of protein expression can easily be determined. However, the functional status of a cell cannot be evaluated by the level of expressed protein alone. The activity of signaling pathways, as assessed by the analysis of post-translation modifications (PTMs) and protein interactions, needs to be determined and taken into consideration (122). Cancer does not consist of a homologous mass of cells but of complex, heterogeneous cell populations that are affected by interactions with each other and the surrounding environment. Therefore, the analysis of cancer tissue at single cell resolution provides a much better understanding of the differences in signaling status and activity (123). In situ PLA enables a localized and specific detection by utilizing oligonucleotide-conjugated antibodies to determine the proximity between one or more targeted epitopes. This makes it a suitable method for detecting molecular events in cells and tissue, for example the status of a signaling pathway. The use of two independent binders and the additional requirement of proximity for reporting enable the specific detection of proteins, protein–protein interactions, and PTMs (124). Proximity ligation converts the recognition of a protein, protein complex, or PTM by two or more antibodies into an amplifiable, circular DNA molecule (Figure 3). Upon proximal binding of a pair of oligonucleotide-conjugated antibodies (PLA probes), the oligonucleotides guide the formation of a circle after applying two additional, single-stranded DNA molecules. This circular DNA molecule is then ligated and amplified by phi29 polymerase within a rolling-circle amplification (RCA) reaction, resulting in a localized, concatameric product. The latter is visualized by hybridization of detection oligonucleotides labeled with fluorophores or horse radish peroxidase (Figure 3) (125). Due to the environment of fixed cells and tissue the amplification product will collapse into a bundle with a diameter of approximately 1 μM (126) that can then be visualized as a bright dot that is quantifiable and easily distinguished from the background (127).

Bottom Line: The emergence of targeted therapies for cancer has created a need for the development of companion diagnostic tests.We also explore the possibility of using IHC to detect specific protein mutations in addition to DNA-based tests.Finally, we review alternative protein binders and proximity ligation assays and discuss their potential to facilitate the development of novel, targeted therapies against cancer.

View Article: PubMed Central - PubMed

Affiliation: Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University , Uppsala , Sweden.

ABSTRACT
The emergence of targeted therapies for cancer has created a need for the development of companion diagnostic tests. Assays developed in recent years are aimed at determining both the effectiveness and safety of specific drugs for a defined group of patients, thus, enabling the more efficient design of clinical trials and also supporting physicians when making treatment-related decisions. Immunohistochemistry (IHC) is a widely accepted method for protein expression analyses in human tissues. Immunohistochemical assays, used to localize and quantitate relative protein expression levels within a morphological context, are frequently used as companion diagnostics during clinical trials and also following drug approval. Herein, we describe established immunochemistry-based methods and their application in routine diagnostics. We also explore the possibility of using IHC to detect specific protein mutations in addition to DNA-based tests. Finally, we review alternative protein binders and proximity ligation assays and discuss their potential to facilitate the development of novel, targeted therapies against cancer.

No MeSH data available.


Related in: MedlinePlus