Monitoring plasma processing steps with a sensitive Western blot assay for the detection of the prion protein

Lee, Douglas C.; Stenland, Christopher J.; Hartwell, Randal C.; Ford, Elizabeth K.; Cai, Kang; Miller, Jeanette L.C.; Gilligan, Kevin; Rubenstein, Richard; Fournel, Michael A.; Petteway, Stephen R.

doi:10.1016/s0166-0934(99)00135-4

Cited by 121 publications

(91 citation statements)

References 34 publications

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“…Although the main objective was not a complete analytical study, we nonetheless compared this protocol with different reported processes using concentration steps as UC commonly used in our lab for CJD diagnosis, 27 a centrifugation protocol (LC) 29 and NaPTa step precipitation (NaPTa) 28 as previously described by others. In first series, these concentration protocols were tested as reported without modification prior to a unique WB analysis.…”

Section: Discussionmentioning

confidence: 99%

“…28 Nonetheless, NaPTa also succeeded in recovering PrP res in the different PrP sc strains tested here from 5 mg of brain tissue. Using the centrifugation method (LC), 29 centrifugation at 20 000 g reduced the preparation time (3 h 05 min) whatever the amount of tissue. However, with the recommended initial amount of 8 mg of tissue, PrP res was clearly recovered, but when decreased to only 5 mg, the sensitivity dropped.…”

Section: Discussionmentioning

confidence: 99%

“…The protocol corresponding to the 'Sample preparation II' described by Lee et al 29 was performed as previously described with 8 or 5 mg of prefrontal cortex.…”

Section: Centrifugation Step Concentration Protocol (Lc)mentioning

confidence: 99%

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Rapid diagnosis of human prion disease using streptomycin with tonsil and brain tissues

Quadrio

Ugnon-Café

Dupin

et al. 2009

Laboratory Investigation

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The use of streptomycin in the pathological prion protein (PrP sc ) detection procedures represents a new and attractive way for diagnostic purpose. With this agent, western blot readily detected PrP sc in 263K scrapie hamster and C57Bl/6 wild-type mice challenged with C506M3 scrapie strain. Our aim was to evaluate this new diagnosis procedure in the field of human transmissible spongiform encephalopathies (TSEs). First, we had confirmed the ability of streptomycin to precipitate PrP res from human brain of Creutzfeldt-Jakob disease (CJD) patient. Second, we compared the detection of PrP res with streptomycin against three other protocols using other precipitations. Then we assessed PrP res detection with streptomycin in 98 brain tissue samples from various aetiologies of human TSEs and 52 brain samples from other dementia. Finally, we applied this protocol for tonsils examination of five patients suspected of variant CJD (v-CJD). Sensitivity and specificity obtained with the streptomycin protocol were both 100% on brain tissue. For tonsil tissues, PrP res was clearly identified in the two post-mortem confirmed v-CJD cases, whereas no characteristic three-band pattern was seen in the three confirmed non-v-CJD samples. In this study, streptomycin demonstrated its efficiency to detect PrP res both in the central nervous system and in the lymphoid tissue without practical difficulty and with rapid preparation. Because of its ability to act as a good agent for PrP sc examination in different tissues, recovery of PrP sc in biological fluids using streptomycin should open further perspectives of applications in CJD diagnostics. Streptomycin effects in vivo might thus also be questioned.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Rapid diagnosis of human prion disease using streptomycin with tonsil and brain tissues

Quadrio

Ugnon-Café

Dupin

et al. 2009

Laboratory Investigation

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“…Brain tissue (frontal cortex) was analysed without further treatment. The PrP res in dental tissues was first concentrated by centrifugation at 21,000 G for 1 hour at 4°C using the method of Lee et al 24 Pellets were dissolved in SDS-PAGE sample buffer and electrophoresed through 12% T SDS-PAGE minigels (Bio-Rad). Separated proteins were transferred using a semi-dry blotter (Bio-Rad) to Hybond-P membranes (Amersham Pharmacia Biotechnology).…”

Section: Western Blotmentioning

confidence: 99%

Investigation of PrPres in dental tissues in variant CJD

et al. 2003

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“…The qualitative and quantitative measurement of protein abundance is one of the common tasks in biomedical diagnostics in the search for therapeutic targets and diagnostic or prognostic biomarkers [1] of such diseases as cancer [2][3][4][5], human immunodeficiency virus [6][7][8], various autoimmune disorders [9,10], Lyme [11], Creutzfeldt-Jakob diseases [12,13], syphilis [14], and many others.…”

Section: Introductionmentioning

confidence: 99%

Multistrip Western blotting to increase quantitative data output

et al. 2007

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The qualitative and quantitative measurement of protein abundance and protein modification states are essential in understanding their role in diverse cellular processes. Traditional Western blotting technique, though sensitive, is prone to produce substantial errors and is not readily adapted to highthroughput technologies. We propose a modified immunoblotting procedure, which is based on simultaneous transfer of proteins from multiple gel-strips onto the same membrane, and is compatible with any conventional gel electrophoresis system. As a result, the data output per single blotting cycle can readily be increased up to ten-fold. In contrast to the traditional "one protein detection per electrophoresis cycle", this procedure allows simultaneous monitoring of up to nine different proteins. In addition to maintaining the ability to detect picogram quantities of protein, the modified system substantially improves data accuracy by reducing signal errors by two-fold. Multi-strip Western blotting procedure allows making statistically reliable side-by-side comparisons of different or repeated sets of data. Compared to the traditional methods, this approach provides a more economical, reproducible and effective procedure, facilitating the generation of large amounts of high-quality quantifiable data.

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Monitoring plasma processing steps with a sensitive Western blot assay for the detection of the prion protein

Cited by 121 publications

References 34 publications

Rapid diagnosis of human prion disease using streptomycin with tonsil and brain tissues

Rapid diagnosis of human prion disease using streptomycin with tonsil and brain tissues

Investigation of PrPres in dental tissues in variant CJD

Multistrip Western blotting to increase quantitative data output

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