Capillary Electrophoresis-Based Noncompetitive Immunoassay for the Prion Protein Using Fluorescein-Labeled Protein A as a Fluorescent Probe

2005 ◽  
Vol 77 (14) ◽  
pp. 4489-4494 ◽  
Author(s):  
Wen-Chu Yang ◽  
Mary Jo Schmerr ◽  
Roy Jackman ◽  
Walter Bodemer ◽  
Edward S. Yeung
Keyword(s):  
Capillary Electrophoresis ◽  
Fluorescent Probe ◽  
Prion Protein ◽  
Protein A

scholarly journals Evaluation of a Preclinical Blood Test for Scrapie in Sheep Using Immunocapillary Electrophoresis

2006 ◽  
Vol 89 (3) ◽  
pp. 720-727 ◽  
Author(s):  
Roy Jackman ◽  
David J Everest ◽  
Mary Jo Schmerr ◽  
Mohammed Khawaja ◽  
Pat Keep ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Prion Protein ◽  
Clinical Signs ◽  
Test System ◽  
Buffy Coat ◽  
Infected Sheep ◽  
Test Method ◽  
Peptide Sequence ◽  
Transmissible Spongiform Encephalopathies ◽  
Blood Samples

Abstract An analytical method is described for detection of endogenous disease-associated prion protein in the buffy coat fraction from the blood of sheep infected with scrapie. The method has been improved and evaluated for its performance in the preclinical diagnosis of ovine transmissible spongiform encephalopathies. The test system uses a protocol for sample preparation that includes extraction and concentration and a test method that uses a liquid-phase competitive immunoassay for prion protein. Antibodies directed to a peptide sequence at the C-terminus of the prion protein (PrP) and a fluorescein-labeled peptide conjugate are used in the assay. Free zone capillary electrophoresis with laser-induced fluorescence for detection is used to separate the antibody-bound fluorescently labeled peptide and free labeled peptide. In this assay, the PrP competes with the fluorescently labeled peptide for limited antibody binding sites, which results in a reduction of the peak representing the immunocomplex of the antibody bound to the fluorescently labeled peptide. When blood samples from scrapie-infected sheep aged 712 months and of the scrapie-susceptible PrP genotypes VRQ/VRQ and VRQ/ARQ were analyzed, the abnormal PrP was found in blood samples. These results correlated with the post-mortem diagnosis of scrapie. The sheep were preclinical and appeared normal at the time of testing but later died with clinical disease approximately 12 months after testing. In older animals, and those with clinical signs, a smaller percentage of animals tested positive. This study has demonstrated that this technology can be used as a sensitive, rapid preclinical test to detect the disease-associated PrP in the blood of scrapie-infected sheep. Improvements in the extraction protocol and capillary electrophoresis conditions will enhance the robustness of this test.

The complex-formation behaviour of His residues in the fifth Cu2+ binding site of human prion protein: a close look

2009 ◽  
Vol 33 (11) ◽  
pp. 2300 ◽  
Author(s):  
Maurizio Remelli ◽  
Daniela Valensin ◽  
Dimitri Bacco ◽  
Ewa Gralka ◽  
Remo Guerrini ◽  
...  
Keyword(s):  
Complex Formation ◽  
Binding Site ◽  
Prion Protein ◽  
Protein A ◽  
Human Prion Protein

Monitoring of proteinase activation in cell apoptosis by capillary electrophoresis with bioengineered fluorescent probe

2006 ◽  
Vol 569 (1-2) ◽  
pp. 176-181 ◽  
Author(s):  
Shixia Zhou ◽  
Juqiang Lin ◽  
Wei Du ◽  
Zhihong Zhang ◽  
Qingming Luo ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Fluorescent Probe ◽  
Cell Apoptosis ◽  
Proteinase Activation

scholarly journals Is indeed the prion protein a Harlequin servant of "many" masters?

Prion ◽  
2009 ◽  
Vol 3 (4) ◽  
pp. 202-205 ◽  
Author(s):  
M. Catia Sorgato ◽  
Caterina Peggion ◽  
Alessandro Bertoli
Keyword(s):  
Prion Protein ◽  
Protein A

Prion protein: a different concept of replication

1993 ◽  
Vol 16 (11) ◽  
pp. 425-426 ◽  
Author(s):  
Rosalind M. Ridley ◽  
Harry F. Baker
Keyword(s):  
Prion Protein ◽  
Protein A

3PT172 Dynamics of normal prion protein, a raft-associated GPI-anchored molecule, in the live neuronal plasma membrane(The 50th Annual Meeting of the Biophysical Society of Japan)

2012 ◽  
Vol 52 (supplement) ◽  
pp. S170-S171
Author(s):  
Yuri L. Nemoto ◽  
Chieko Nakada ◽  
Hiroko Hijikata ◽  
Takahiro K. Fujiwara ◽  
Rinshi S. Kasai ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Annual Meeting ◽  
Prion Protein ◽  
Protein A ◽  
Biophysical Society ◽  
Neuronal Plasma Membrane

scholarly journals The cellular prion protein interacts with and promotes the activity of Na,K-ATPases

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0258682
Author(s):  
Declan Williams ◽  
Mohadeseh Mehrabian ◽  
Hamza Arshad ◽  
Shehab Eid ◽  
Christopher Sackmann ◽  
...  
Keyword(s):  
Prion Protein ◽  
Prion Diseases ◽  
Protein A ◽  
Neuroblastoma Cells ◽  
Cellular Prion Protein ◽  
Affinity Capture ◽  
Endoproteolytic Cleavage ◽  
Relative Quantitation ◽  
Steady State Levels ◽  
Uptake Activity

The prion protein (PrP) is best known for its ability to cause fatal neurodegenerative diseases in humans and animals. Here, we revisited its molecular environment in the brain using a well-developed affinity-capture mass spectrometry workflow that offers robust relative quantitation. The analysis confirmed many previously reported interactions. It also pointed toward a profound enrichment of Na,K-ATPases (NKAs) in proximity to cellular PrP (PrPC). Follow-on work validated the interaction, demonstrated partial co-localization of the ATP1A1 and PrPC, and revealed that cells exposed to cardiac glycoside (CG) inhibitors of NKAs exhibit correlated changes to the steady-state levels of both proteins. Moreover, the presence of PrPC was observed to promote the ion uptake activity of NKAs in a human co-culture paradigm of differentiated neurons and glia cells, and in mouse neuroblastoma cells. Consistent with this finding, changes in the expression of 5’-nucleotidase that manifest in wild-type cells in response to CG exposure can also be observed in untreated PrPC-deficient cells. Finally, the endoproteolytic cleavage of the glial fibrillary acidic protein, a hallmark of late-stage prion disease, can also be induced by CGs, raising the prospect that a loss of NKA activity may contribute to the pathobiology of prion diseases.

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