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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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Capture optimization of ovine brain PrPTSE.3A: Batches of nanobeads (1% suspension) coated with 10, 20, and 30 µg of plasminogen/mg of beads were used to test prion capture efficacy using 30, 60 and 90 µl of plasminogen-coupled beads per 500 µl of human plasma spiked with a 10−3 dilution of 127S IBH. After one round of PMCA, detection was performed on PK-digested and amplified products using western blot analysis with 6D11 as the primary antibody. Lane 1: NBH: normal brain homogenate without PK digestion Lane 2: F10−2 IBH dilution without PMCA (Frozen) Lane 6: negative control: plasma only 3B: Tg338 127S IBH dilution (10−4) in plasma was tested using different volumes of coated beads at 10 µg of plasminogen/mg of beads (1% suspension) for the prion capture. After one round of PMCA, detection was performed on PK-digested and amplified product using western blot analysis with 6D11 as the primary antibody. Lane 1: NBH: normal brain homogenate without PK digestion Lane 5: negative control: plasma only.
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pone-0069632-g003: Capture optimization of ovine brain PrPTSE.3A: Batches of nanobeads (1% suspension) coated with 10, 20, and 30 µg of plasminogen/mg of beads were used to test prion capture efficacy using 30, 60 and 90 µl of plasminogen-coupled beads per 500 µl of human plasma spiked with a 10−3 dilution of 127S IBH. After one round of PMCA, detection was performed on PK-digested and amplified products using western blot analysis with 6D11 as the primary antibody. Lane 1: NBH: normal brain homogenate without PK digestion Lane 2: F10−2 IBH dilution without PMCA (Frozen) Lane 6: negative control: plasma only 3B: Tg338 127S IBH dilution (10−4) in plasma was tested using different volumes of coated beads at 10 µg of plasminogen/mg of beads (1% suspension) for the prion capture. After one round of PMCA, detection was performed on PK-digested and amplified product using western blot analysis with 6D11 as the primary antibody. Lane 1: NBH: normal brain homogenate without PK digestion Lane 5: negative control: plasma only.

Mentions: Prior to PMCA, PrPTSE needs to be captured from blood samples and concentrated. The optimum plasminogen concentration and bead volume for PrPTSE capture were firstly calculated using a series of plasminogen-coated nanobeads. Scrapie IBH at 10−3 and 10−4 dilutions was diluted in human plasma (500 µl) and mixed with various volumes of beads with different plasminogen concentrations before PMCA amplification. Representative results are shown in Fig. 3A and B. It is worth noting that lower signals were observed with increasing bead volume (Fig. 3 B lanes 6–8) and plasminogen concentration (Fig. 3A lanes 3, 4, 5, 7, 8, 9, 10, 11, 12). The strongest PrPTSE signal was observed using a plasminogen (Plg) concentration of 10 µg Plg/mg of beads (Fig. 3A lanes 3) and a volume of 10 µl of the 1% bead suspension per 500 µl of spiked plasma (Fig. 3B lanes 4, 6). Under these conditions, when PrPTSE was captured from 10 µl Tg338 127S IBH or 10 µl vCJD IBH diluted in 500 µl of plasma and analyzed directly by western blot without PMCA amplification, a 95% yield (percentage of recovery calculated from PrPres signal) was observed (Fig. 4). In the same way, the PrPTSE signal obtained after one PMCA round from the 10−6 plasma dilution appeared almost similar to that obtained when a control 10−6 IBH dilution (Fig. 5 C10−6) was used directly to seed the PMCA substrate suggesting that the association of the capture by plasminogen-coated nanobeads and PMCA reached almost 100% efficiency (Fig. 5, lanes 10−6 and C10−6). Both negative controls of 500 µl of human plasma mixed with plasminogen-coated nanobeads prior to PMCA remained negative (Fig. 5 lanes NC).


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)

Capture optimization of ovine brain PrPTSE.3A: Batches of nanobeads (1% suspension) coated with 10, 20, and 30 µg of plasminogen/mg of beads were used to test prion capture efficacy using 30, 60 and 90 µl of plasminogen-coupled beads per 500 µl of human plasma spiked with a 10−3 dilution of 127S IBH. After one round of PMCA, detection was performed on PK-digested and amplified products using western blot analysis with 6D11 as the primary antibody. Lane 1: NBH: normal brain homogenate without PK digestion Lane 2: F10−2 IBH dilution without PMCA (Frozen) Lane 6: negative control: plasma only 3B: Tg338 127S IBH dilution (10−4) in plasma was tested using different volumes of coated beads at 10 µg of plasminogen/mg of beads (1% suspension) for the prion capture. After one round of PMCA, detection was performed on PK-digested and amplified product using western blot analysis with 6D11 as the primary antibody. Lane 1: NBH: normal brain homogenate without PK digestion Lane 5: negative control: plasma only.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3722129&req=5

pone-0069632-g003: Capture optimization of ovine brain PrPTSE.3A: Batches of nanobeads (1% suspension) coated with 10, 20, and 30 µg of plasminogen/mg of beads were used to test prion capture efficacy using 30, 60 and 90 µl of plasminogen-coupled beads per 500 µl of human plasma spiked with a 10−3 dilution of 127S IBH. After one round of PMCA, detection was performed on PK-digested and amplified products using western blot analysis with 6D11 as the primary antibody. Lane 1: NBH: normal brain homogenate without PK digestion Lane 2: F10−2 IBH dilution without PMCA (Frozen) Lane 6: negative control: plasma only 3B: Tg338 127S IBH dilution (10−4) in plasma was tested using different volumes of coated beads at 10 µg of plasminogen/mg of beads (1% suspension) for the prion capture. After one round of PMCA, detection was performed on PK-digested and amplified product using western blot analysis with 6D11 as the primary antibody. Lane 1: NBH: normal brain homogenate without PK digestion Lane 5: negative control: plasma only.
Mentions: Prior to PMCA, PrPTSE needs to be captured from blood samples and concentrated. The optimum plasminogen concentration and bead volume for PrPTSE capture were firstly calculated using a series of plasminogen-coated nanobeads. Scrapie IBH at 10−3 and 10−4 dilutions was diluted in human plasma (500 µl) and mixed with various volumes of beads with different plasminogen concentrations before PMCA amplification. Representative results are shown in Fig. 3A and B. It is worth noting that lower signals were observed with increasing bead volume (Fig. 3 B lanes 6–8) and plasminogen concentration (Fig. 3A lanes 3, 4, 5, 7, 8, 9, 10, 11, 12). The strongest PrPTSE signal was observed using a plasminogen (Plg) concentration of 10 µg Plg/mg of beads (Fig. 3A lanes 3) and a volume of 10 µl of the 1% bead suspension per 500 µl of spiked plasma (Fig. 3B lanes 4, 6). Under these conditions, when PrPTSE was captured from 10 µl Tg338 127S IBH or 10 µl vCJD IBH diluted in 500 µl of plasma and analyzed directly by western blot without PMCA amplification, a 95% yield (percentage of recovery calculated from PrPres signal) was observed (Fig. 4). In the same way, the PrPTSE signal obtained after one PMCA round from the 10−6 plasma dilution appeared almost similar to that obtained when a control 10−6 IBH dilution (Fig. 5 C10−6) was used directly to seed the PMCA substrate suggesting that the association of the capture by plasminogen-coated nanobeads and PMCA reached almost 100% efficiency (Fig. 5, lanes 10−6 and C10−6). Both negative controls of 500 µl of human plasma mixed with plasminogen-coated nanobeads prior to PMCA remained negative (Fig. 5 lanes NC).

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