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Plasminogen-based capture combined with amplification technology for the detection of PrP(TSE) in the pre-clinical phase of infection.

Segarra C, Bougard D, Moudjou M, Laude H, Béringue V, Coste J - PLoS ONE (2013)

Bottom Line: We demonstrated the possibility of detecting PrP(TSE) in white blood cells, in buffy coat and in plasma isolated from the blood of scrapie-infected sheep collected at the pre-clinical stage of the disease.The test also allowed the detection of PrP(TSE) in human plasma spiked with a 10(-8) dilution of vCJD-infected brain homogenate corresponding to the level of sensitivity (femtogram) required for the detection of the PrP(TSE) in asymptomatic carriers.The 100% specificity of the test was revealed using a blinded panel comprising 96 human plasma samples.

View Article: PubMed Central - PubMed

Affiliation: EFS-PyMed (Etablissement Français du Sang de Pyrénées Méditerranée), R&D TransDiag, Sécurité Transfusionnelle et Innovation Diagnostique, Montpellier, France.

ABSTRACT

Background: Variant Creutzfeldt-Jakob disease (vCJD) is a neurodegenerative infectious disorder, characterized by a prominent accumulation of pathological isoforms of the prion protein (PrP(TSE)) in the brain and lymphoid tissues. Since the publication in the United Kingdom of four apparent vCJD cases following transfusion of red blood cells and one apparent case following treatment with factor VIII, the presence of vCJD infectivity in the blood seems highly probable. For effective blood testing of vCJD individuals in the preclinical or clinical phase of infection, it is considered necessary that assays detect PrP(TSE) concentrations in the femtomolar range.

Methodology/principal findings: We have developed a three-step assay that firstly captures PrP(TSE) from infected blood using a plasminogen-coated magnetic-nanobead method prior to its serial amplification via protein misfolding cyclic amplification (PMCA) and specific PrP(TSE) detection by western blot. We achieved a PrP(TSE) capture yield of 95% from scrapie-infected material. We demonstrated the possibility of detecting PrP(TSE) in white blood cells, in buffy coat and in plasma isolated from the blood of scrapie-infected sheep collected at the pre-clinical stage of the disease. The test also allowed the detection of PrP(TSE) in human plasma spiked with a 10(-8) dilution of vCJD-infected brain homogenate corresponding to the level of sensitivity (femtogram) required for the detection of the PrP(TSE) in asymptomatic carriers. The 100% specificity of the test was revealed using a blinded panel comprising 96 human plasma samples.

Conclusion/significance: We have developed a sensitive and specific amplification assay allowing the detection of PrP(TSE) in the plasma and buffy coat fractions of blood collected at the pre-clinical phase of the disease. This assay represents a good candidate as a confirmatory assay for the presence of PrP(TSE) in blood of patients displaying positivity in large scale screening tests.

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Detection of PrPTSE from plasma and buffy coat of scrapie-infected sheep at the preclinical stage 7A:500 µl of PG 127 infected and healthy plasma samples underwent the capture step in duplicate with 10 µl of coated beads at 10 µg of plasminogen/mg of beads, before PMCA amplification using two rounds of 80 cycles. Detection was performed after PK digestion of the amplified products using western blot analysis with 6D11 as the primary antibody. Pre-Cl-Plasma: plasma sample from PG127 infected sheep at the pre-clinical stage (120 days post oral challenge) Neg Plasma: plasma sample from healthy sheep 7B: 50 and 25 µl of PG 127 infected (120 days post oral challenge) and healthy BC (buffy coat) samples underwent the capture step in duplicate with 10 µl of coated beads at 10 µg of plasminogen/mg of beads, before PMCA amplification using one round (i.e. 80 cycles). Detection was performed after PK digestion of the amplified products, using western blot analysis with 6D11 as the primary antibody. Lane PC: 10−7 IBH dilution amplified by PMCA Lane NC: Buffy coat from healthy sheep.
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pone-0069632-g007: Detection of PrPTSE from plasma and buffy coat of scrapie-infected sheep at the preclinical stage 7A:500 µl of PG 127 infected and healthy plasma samples underwent the capture step in duplicate with 10 µl of coated beads at 10 µg of plasminogen/mg of beads, before PMCA amplification using two rounds of 80 cycles. Detection was performed after PK digestion of the amplified products using western blot analysis with 6D11 as the primary antibody. Pre-Cl-Plasma: plasma sample from PG127 infected sheep at the pre-clinical stage (120 days post oral challenge) Neg Plasma: plasma sample from healthy sheep 7B: 50 and 25 µl of PG 127 infected (120 days post oral challenge) and healthy BC (buffy coat) samples underwent the capture step in duplicate with 10 µl of coated beads at 10 µg of plasminogen/mg of beads, before PMCA amplification using one round (i.e. 80 cycles). Detection was performed after PK digestion of the amplified products, using western blot analysis with 6D11 as the primary antibody. Lane PC: 10−7 IBH dilution amplified by PMCA Lane NC: Buffy coat from healthy sheep.

Mentions: In four white blood cell concentrates of naturally scrapie-infected sheep (SWBC), 0/4 and 1/4 samples were detected positive respectively after one and two rounds of PMCA (results not shown). After a third round, a PrPTSE signal was obtained from all four infected SWBC amplified samples, in contrast to those from healthy sheep (0/4) (Fig. 6). These results were reproduced in three different assays. In one sheep at the preclinical phase of scrapie, a specific PrPTSE signal was detected in a 500 µl plasma sample after PrP capture and two rounds of PMCA (Fig. 7A) and in both 50 µl and 25 µl buffy coat samples (BC) after one PMCA round (Fig. 7B). These assays were reproduced twice and each sample was tested in duplicate.


Plasminogen-based capture combined with amplification technology for the detection of PrP(TSE) in the pre-clinical phase of infection.

Segarra C, Bougard D, Moudjou M, Laude H, Béringue V, Coste J - PLoS ONE (2013)

Detection of PrPTSE from plasma and buffy coat of scrapie-infected sheep at the preclinical stage 7A:500 µl of PG 127 infected and healthy plasma samples underwent the capture step in duplicate with 10 µl of coated beads at 10 µg of plasminogen/mg of beads, before PMCA amplification using two rounds of 80 cycles. Detection was performed after PK digestion of the amplified products using western blot analysis with 6D11 as the primary antibody. Pre-Cl-Plasma: plasma sample from PG127 infected sheep at the pre-clinical stage (120 days post oral challenge) Neg Plasma: plasma sample from healthy sheep 7B: 50 and 25 µl of PG 127 infected (120 days post oral challenge) and healthy BC (buffy coat) samples underwent the capture step in duplicate with 10 µl of coated beads at 10 µg of plasminogen/mg of beads, before PMCA amplification using one round (i.e. 80 cycles). Detection was performed after PK digestion of the amplified products, using western blot analysis with 6D11 as the primary antibody. Lane PC: 10−7 IBH dilution amplified by PMCA Lane NC: Buffy coat from healthy sheep.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3722129&req=5

pone-0069632-g007: Detection of PrPTSE from plasma and buffy coat of scrapie-infected sheep at the preclinical stage 7A:500 µl of PG 127 infected and healthy plasma samples underwent the capture step in duplicate with 10 µl of coated beads at 10 µg of plasminogen/mg of beads, before PMCA amplification using two rounds of 80 cycles. Detection was performed after PK digestion of the amplified products using western blot analysis with 6D11 as the primary antibody. Pre-Cl-Plasma: plasma sample from PG127 infected sheep at the pre-clinical stage (120 days post oral challenge) Neg Plasma: plasma sample from healthy sheep 7B: 50 and 25 µl of PG 127 infected (120 days post oral challenge) and healthy BC (buffy coat) samples underwent the capture step in duplicate with 10 µl of coated beads at 10 µg of plasminogen/mg of beads, before PMCA amplification using one round (i.e. 80 cycles). Detection was performed after PK digestion of the amplified products, using western blot analysis with 6D11 as the primary antibody. Lane PC: 10−7 IBH dilution amplified by PMCA Lane NC: Buffy coat from healthy sheep.
Mentions: In four white blood cell concentrates of naturally scrapie-infected sheep (SWBC), 0/4 and 1/4 samples were detected positive respectively after one and two rounds of PMCA (results not shown). After a third round, a PrPTSE signal was obtained from all four infected SWBC amplified samples, in contrast to those from healthy sheep (0/4) (Fig. 6). These results were reproduced in three different assays. In one sheep at the preclinical phase of scrapie, a specific PrPTSE signal was detected in a 500 µl plasma sample after PrP capture and two rounds of PMCA (Fig. 7A) and in both 50 µl and 25 µl buffy coat samples (BC) after one PMCA round (Fig. 7B). These assays were reproduced twice and each sample was tested in duplicate.

Bottom Line: We demonstrated the possibility of detecting PrP(TSE) in white blood cells, in buffy coat and in plasma isolated from the blood of scrapie-infected sheep collected at the pre-clinical stage of the disease.The test also allowed the detection of PrP(TSE) in human plasma spiked with a 10(-8) dilution of vCJD-infected brain homogenate corresponding to the level of sensitivity (femtogram) required for the detection of the PrP(TSE) in asymptomatic carriers.The 100% specificity of the test was revealed using a blinded panel comprising 96 human plasma samples.

View Article: PubMed Central - PubMed

Affiliation: EFS-PyMed (Etablissement Français du Sang de Pyrénées Méditerranée), R&D TransDiag, Sécurité Transfusionnelle et Innovation Diagnostique, Montpellier, France.

ABSTRACT

Background: Variant Creutzfeldt-Jakob disease (vCJD) is a neurodegenerative infectious disorder, characterized by a prominent accumulation of pathological isoforms of the prion protein (PrP(TSE)) in the brain and lymphoid tissues. Since the publication in the United Kingdom of four apparent vCJD cases following transfusion of red blood cells and one apparent case following treatment with factor VIII, the presence of vCJD infectivity in the blood seems highly probable. For effective blood testing of vCJD individuals in the preclinical or clinical phase of infection, it is considered necessary that assays detect PrP(TSE) concentrations in the femtomolar range.

Methodology/principal findings: We have developed a three-step assay that firstly captures PrP(TSE) from infected blood using a plasminogen-coated magnetic-nanobead method prior to its serial amplification via protein misfolding cyclic amplification (PMCA) and specific PrP(TSE) detection by western blot. We achieved a PrP(TSE) capture yield of 95% from scrapie-infected material. We demonstrated the possibility of detecting PrP(TSE) in white blood cells, in buffy coat and in plasma isolated from the blood of scrapie-infected sheep collected at the pre-clinical stage of the disease. The test also allowed the detection of PrP(TSE) in human plasma spiked with a 10(-8) dilution of vCJD-infected brain homogenate corresponding to the level of sensitivity (femtogram) required for the detection of the PrP(TSE) in asymptomatic carriers. The 100% specificity of the test was revealed using a blinded panel comprising 96 human plasma samples.

Conclusion/significance: We have developed a sensitive and specific amplification assay allowing the detection of PrP(TSE) in the plasma and buffy coat fractions of blood collected at the pre-clinical phase of the disease. This assay represents a good candidate as a confirmatory assay for the presence of PrP(TSE) in blood of patients displaying positivity in large scale screening tests.

Show MeSH
Related in: MedlinePlus