Application of a Time‐Resolved Fluoroimmunoassay for the Analysis of Normal Prion Protein in Human Blood and Its Components

MacGregor, Ian; Hope, James; Barnard, Geoff; Kirby, Louise; Drummond, Olive; Pepper, Duncan S.; Hornsey, V.; Barclay, Robin; Bessos, Hagop; Turner, Marc; Prowse, Christopher V.

doi:10.1046/j.1423-0410.1999.7720088.x

Cited by 112 publications

(111 citation statements)

References 34 publications

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“…Interestingly, this processed soluble form of PrP that starts at Gly 90 showed an electrophoretic mobility on SDS-PAGE similar to PrP [27][28][29][30] , the protease-resistant core of PrP Sc (15). Using a time-resolved fluoroimmunoassay, MacGregor et al (22) showed that soluble forms of PrP are present in various human blood fractions. It is still unclear whether soluble PrP can be converted into PrP Sc .…”

Section: Resultsmentioning

confidence: 99%

“…In addition, there is evidence for a soluble form of PrP C in the medium of cultured cells (9 -13), in human cerebrospinal fluid (14), in human blood (22), and released from activated human platelets (15).…”

Section: Discussionmentioning

confidence: 99%

“…C in the culture medium of splenocytes suggested that shedding of PrP C is part of a processing pathway in the lymphoid system leading to the accumulation of significant levels of soluble PrP in the blood (22). To verify the presence of soluble PrP C in serum, we examined mouse and human blood.…”

Section: Soluble Prp In Blood Of Mice and Humans-detection Of Solublementioning

confidence: 99%

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Similar Turnover and Shedding of the Cellular Prion Protein in Primary Lymphoid and Neuronal Cells

Parizek¹,

Roeckl²,

Weber³

et al. 2001

Journal of Biological Chemistry

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The cellular prion protein (PrP C ) is essential for pathogenesis and transmission of prion diseases. Although prion replication in the brain is accompanied by neurodegeneration, prions multiply efficiently in the lymphoreticular system without any detectable pathology. We have used pulse-chase metabolic radiolabeling experiments to investigate the turnover and processing of PrP C in primary cell cultures derived from lymphoid and nervous tissues. Similar kinetics of PrP C degradation were observed in these tissues. This indicates that the differences between these two organs with respect to their capacity to replicate prions is not due to differences in the turnover of PrP C . Substantial amounts of a soluble form of PrP that lacks the glycolipid anchor appeared in the medium of splenocytes and cerebellar granule cells. Soluble PrP was detected in murine and human serum, suggesting that it might be of physiological relevance.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Soluble Prp In Blood Of Mice and Humans-detection Of Solublementioning

confidence: 99%

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Similar Turnover and Shedding of the Cellular Prion Protein in Primary Lymphoid and Neuronal Cells

Parizek¹,

Roeckl²,

Weber³

et al. 2001

Journal of Biological Chemistry

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“…Furthermore, the direct detection of conformational changes of the prion protein can be excluded: firstly, the amide I and amide II regions were intentionally excluded from the analysis of the differentiation between BSE-positive and BSE-negative such that the observed discrimination potential is unlikely to be based on specific protein signatures. Secondly, the concentration of prion proteins in serum [22][23][24] is less than 50 ng ml 21 and, hence, well below the mid-infrared detection limit. In summary, we have shown that the automation of the DPR process was successfully implemented into a DPR research system resulting in added convenience, simplified sample and data logistics and an improvement in reproducibility.…”

Section: Discussionmentioning

confidence: 99%

Classification of signatures of Bovine Spongiform Encephalopathy in serum using infrared spectroscopy

Martin¹,

Moecks

Belooussov

et al. 2004

Analyst

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Signatures of Bovine Spongiform Encephalopathy (BSE) have been identified in serum by means of ''Diagnostic Pattern Recognition (DPR)''. For DPR-analysis, mid-infrared spectroscopy of dried films of 641 serum samples was performed using disposable silicon sample carriers and a semi-automated DPR research system operating at room temperature. The combination of four mathematical classification approaches (principal component analysis plus linear discriminant analysis, robust linear discriminant analysis, artificial neural network, support vector machine) allowed for a reliable assignment of spectra to the class ''BSEpositive'' or ''BSE-negative''. An independent, blinded validation study was carried out on a second DPR research system at the Veterinary Laboratory Agency, Weybridge, UK. Out of 84 serum samples originating from terminally-ill, BSE-positive cattle, 78 were classified correctly. Similarly, 73 out of 76 BSE-negative samples were correctly identified by DPR such that, numerically, an accuracy of 94.4 % can be calculated. At a confidence level of 0.95 (a~0.05) these results correspond to a sensitivity w 85% and a specificity w 90%. Identical class assignment by all four classifiers occurred in 75% of the cases while ambiguous results were obtained in only 8 of the 160 cases. With an area under the ROC (receiver operating charateristics) curve of 0.991, DPR may potentially supply a valuable surrogate marker for BSE even in cases in which a deliberate bias towards improved sensitivity or specificity is desired. To the best of our knowledge, DPR is the first andup to now-only method which has demonstrated its capability of detecting BSE-related signatures in serum.

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“…Therefore, the pathological diagnosis of TSE is currently made principally on the basis of postmortem preparations of CNS tissues, highlighting the need for the development of a more rapid diagnostic method using body fluids, especially blood (6,27). For this purpose, several methods have been proposed and examined for prophylactic use (23,30,32). However, none of these methods has proved to be sufficient for the purposes (5,7,20,29).…”

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confidence: 99%

A Potential Blood Test for Transmissible Spongiform Encephalopathies by Detecting Carbohydrate‐Dependent Aggregates of PrPres‐Like Proteins in Scrapie‐Infected Hamster Plasma

Tsukui

Takata

Tadokoro

2007

Microbiology and Immunology

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PrPres has rarely been detected in blood (except in leukocytes) even in diseased animal models that are known to contain a large amount of PrPres in infected tissues. It seems likely that PrPres detection in blood is difficult because of the low titer of infectious material within the blood. Here, we demonstrate the detection of proteinase K‐resistant 3F4‐reactive protein in the plasma of scrapie‐infected hamsters but not in the plasma of mock‐infected hamsters by partial purification using a novel method termed “acidic SDS precipitation,” in conjunction with a highly sensitive chemiluminescence detection system used to show the presence of PrP at a concentration equivalent to 1.4 ×10–9 g of brain homogenate or 1.5 × 10–12 g (6.5 ×10–17 mol) of rPrP by conventional Western blotting. The 3F4‐reactive proteins in scrapie‐infected hamster plasma often resulted in multiple Mw protein bands occurring at higher Mw positions than the position of the di‐glycosyl PrP molecule. Mixing scrapie‐infected hamster brain homogenate with mock‐infected hamster plasma resulted in the formation of similar Mw positions for multiple 3F4‐reactive proteins. Predigestion of carbohydrate side chains from the proteins in the plasma or brain homogenate before mixing resulted in failure to obtain these multiple 3F4‐reactive proteins. These observations indicate that PrPres aggregated with other proteins in the plasma through carbohydrate side chains and was successfully detected in the plasma of scrapie‐infected hamsters. Counterparts in these aggregates with PrPres‐like proteins in scHaPl are not known but any that exist should resist the PK digestion.

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Application of a Time‐Resolved Fluoroimmunoassay for the Analysis of Normal Prion Protein in Human Blood and Its Components

Abstract: The majority of blood PrP(c) is found in the platelet and plasma compartments.

Cited by 112 publications

References 34 publications

Similar Turnover and Shedding of the Cellular Prion Protein in Primary Lymphoid and Neuronal Cells

Similar Turnover and Shedding of the Cellular Prion Protein in Primary Lymphoid and Neuronal Cells

Classification of signatures of Bovine Spongiform Encephalopathy in serum using infrared spectroscopy

A Potential Blood Test for Transmissible Spongiform Encephalopathies by Detecting Carbohydrate‐Dependent Aggregates of PrPres‐Like Proteins in Scrapie‐Infected Hamster Plasma

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