| 1. |
- Zieba, Agata, et al.
(författare)
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Molecular tools for companion diagnostics
- 2012
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 29:6, s. 634-640
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Forskningsöversikt (refereegranskat)abstract
- The heterogeneous nature of cancer results in highly variable therapeutic responses even among patients with identical stages and grades of a malignancy. The move towards personalised medicine in cancer therapy has therefore been motivated by a need to customise therapy according to molecular features of individual tumours. Companion diagnostics serves to support early drug development, it can provide surrogate markers in clinical trials, and also guide selection of individual therapies and monitoring of responses in routine clinical care. The era of companion diagnostics can be said to have begun with the introduction of the HercepTest - a first-of-a-kind diagnostic tool developed by DakoCytomation in 1998 to select patients for therapy with the anticancer drug Herceptin (trastuzumab). Herceptin and the paired test proved that companion diagnostics can help guide patient-tailored therapies. We will discuss herein technologies to analyse companion diagnostics markers at the level of DNA, RNA or protein, focusing on a series of methods developed in our laboratory that can facilitate drug development and help stratify patients for therapy.
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| 2. |
- Padhan, Narendra, et al.
(författare)
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Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation
- 2017
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own.
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| 3. |
- Yan, Junhong, et al.
(författare)
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Highly sensitive and specific protein detection via proximity ligation in capillary westerns
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Detection and quantification of proteins and their post-translational modifications are crucial todecipher functions of complex networks in cell biology and medicine. Affinity-based protein analyseshave an important role in proteomic research, and western blotting is one of the most-widely usedtechniques. The NanoPro 1000 system developed by ProteinSimple is an instrument that can resolveand identify proteins and their isoforms through capillary isoelectric focusing with antibody baseddetection directly in the capillaries, in analogy to conventional western blotting. The in situ proximityligation assay (PLA) serves to detect the location of proteins using pairs of oligonucleotide-conjugatedantibodies that mediate formation of rolling circle amplification (RCA) products upon recognition ofthe target protein, or single or dual primary antibodies binding the target. PLA can offer improvedspecificity of detection, and increased sensitivity via, RCA initiated by oligonucleotides attached tothe antibody pairs. Here we have used in situ PLA to detect single or pairs of primary antibodiesbinding proteins separated in the NanoPro 1000 system for highly sensitive and specific detection ofproteins and their isoforms. We achieved at least 10-fold improved detection limits for Erk protein inHDMEC cell lysates, compared with the standard NanoPro 1000 assays, and we demonstrated greatlyenhanced specificity of detection by combining pairs of antibodies, each of which exhibited crossreactivesignals when used on their own.
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