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- de Oliveira, Felipe Marques Souza
(författare)
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Development and Application of Proximity Assays for Proteome Analysis in Medicine
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Along with proteins, a myriad of different molecular biomarkers, such as post-translational modifications and autoantibodies, could be used in an attempt to improve disease detection and progression. In this thesis, I build on several iterations of the proximity ligation assay to develop and apply new adaptable methods to facilitate detection of proteins, autoantibodies and post-translational modifications.In paper I, we present an adaptation of the solid-phase proximity ligation assay (SP-PLA) for the detection of post-translational modification of proteins (PTMs). The assay was adapted for the detection of two of the most commons PTMs present in proteins, glycosylation and phosphorylation, offering the encouraging prospect of using detection of PTMs in a diagnostic or prognostic capacity. In paper II, we developed a variant of the proximity ligation assay using micro titer plate for detection and quantification of protein using optical density as readout in the fluorometer, termed PLARCA. With a detection limit considerably lower than ELISA, PLARCA detected femtomolar levels of these proteins in patient samples.In paper III, we aim to compare detection values of samples collected from earlobe capillary, venous plasma, as well as capillary plasma stored in dried plasma spots (DPS) assessed with a 92-plex inflammation panel using multiplex proximity extension assay (PEA). Despite the high variability in protein measurements between the three sample sources, we were able to conclude that earlobe capillary sampling is a suitable less invasive alternative, to venipuncture.In paper IV, we describe the application of PLARCA and proximity extension assay (PEA) for the detection of GAD65 autoantibodies (GADA). Thus, offering highly sensitive and specific autoimmunity detection.
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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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| 4. |
- 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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