Capture optimization of ovine brain PrP^TSE.

<p><b>3A:</b> 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 <b>3B</b>: 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.</p

Evaluation of the PrP^TSE detection assay on human blinded panel (NIBSC).

<p>All the controls were collected from individuals showing no sign of vCJD (brain, spleen and plasma).</p

Evaluation of “standard” and commercially available decontamination procedures on human vCJD prions.

<p>Steel wires contaminated with 10% human vCJD brain homogenate were treated with “standard” (A) and commercially available (B) prion decontamination procedures and subjected to 4 serial PMCA rounds. Two independent series of PMCA were performed on 4 wires per treatment (Black cross (<b>Χ)</b> Series 1 and Red square (■) Series 2). PAA: Per acetic acid 1.2%—60 min; NaOH: sodium hydroxide 0.1N—15 min or 1N—60 min; Auto: steam sterilization 121°C -20 min or 134°C -20 min; NaOCl: Sodium hypochlorite 0.2% (2000ppm)– 15 min or 2% (20000ppm)– 60 min; H₂O: untreated positive control; NEG: negative control (wire mock-contaminated with normal brain homogenate). (C) Representative Surf-PMCA results from two individual wires after both reference and marketed decontamination procedures (3F4 monoclonal antibody). Bars to the left of Western blot panels indicate the 30 and 20 kilo Dalton marker positions.</p

Efficacy of PrP^TSE capture.

<p>Ten microliters of 10% 127S (lanes 1 and 2) and vCJD IBH (lanes 3 and 4) were diluted in 500 µl of plasma and incubated 2 h with plasminogen-coated beads. PrP^TSE bound to the beads were PK-digested and denatured in sample buffer for western blot analysis with 6D11 and 3F4 anti-PrP MAbs. Percentage yield was quantified with Genetools software after acquisition of the chemioluminescent western blot signals with the Genegnome digital imager (Syngene, US) Lanes 1 and 2∶127S IBH capture Lanes 3 and 4: vCJD IBH capture.</p

Sensitivity of the ovine PrP^TSE optimized test.

<p>Tg338 127S IBH dilutions (10⁻⁵, 10⁻⁶) in 500 µl of human plasma were captured with 10 µl of coated nanobeads at 10 µg of plasminogen/mg of beads, amplified by PMCA (one round of 80 cycles) and detection was performed on PK-digested and amplified products using western blot analysis with 6D11 as the primary antibody. −2F: 10⁻² IBH dilution without PMCA (Frozen) C10^−6: IBH dilution amplified directly by PMCA without capture step NC: negative control analyzed along with samples.</p

Optimization of steel wire contamination in 96-well microplates.

<p>Wires were contaminated with either 1% 127S-scrapie or 10% human vCJD brain homogenates at different times (1 min, 10 min or 1 hr) and subjected to one Surf-PMCA round. Protease-resistant prion protein was detected with 6D11 (127S-scrapie) or 3F4 (human vCJD) monoclonal antibodies. NEG: Negative control (wires mock-contaminated in normal brain homogenate). M: molecular mass marker.</p

Effect of PrP^C level on PMCA efficacy.

<p>Tg338 127S infected brain homogenate dilutions (10⁻⁵ and 10⁻⁶) were PMCA amplified (one round) using 10% tg338 NBH (Undil.), 10% tg338NBH diluted 1/6 in 10% tg-PrP^0/0NBH and 10% tg338NBH diluted 1/8 in 10% tg-PrP^0/0NBH as PMCA substrates. The detection was performed on PK-digested amplified products using western blot analysis with 6D11 as the primary antibody. Undil.: 10% tg338 NBH without dilution in 10% tg-PrP^0/0NBH. NC: negative control, NBH only.</p

Detection of PrP^TSE from SWBC of scrapie-infected sheep.

<p>Four infected and healthy SWBC (sheep white blood cells) samples underwent the capture step with 10 µl of coated beads at 10 µg of plasminogen/mg of beads. After three rounds of PMCA, PrP^res detection was performed after PK digestion of the amplified products, using western blot analysis with 6D11 as the primary antibody.</p

Evaluation of “standard” decontamination procedures on 127S scrapie prions.

<p>Steel wires contaminated with 1% 127S scrapie brain homogenate were treated with “standard” prion decontamination procedures and subjected to three serial PMCA rounds. Three independent series of PMCA were performed on 4 wires per treatment (Black cross (<b>Χ)</b> Series 1, Red square (■) Series 2 and Green circle (●) Series 3). PAA: Per acetic acid 1.2%—60 min; NaOH: sodium hydroxide 0.1N—15 min or 1N—60 min; Auto: steam sterilization 121°C -20 min or 134°C -20 min; NaOCl: Sodium hypochlorite 0.2% (2000ppm)– 15 min or 2% (20000ppm)– 60 min; H₂O: untreated positive control; NEG: negative control (wire mock-contaminated with normal brain homogenate).</p

Ovine PMCA optimization.

<p>Tg338 127S infected brain homogenate dilutions (10⁻⁴ to 10⁻¹⁰) were tested using two rounds (one round = 80 cycles) of PMCA and detection was performed on PK-digested amplified products using western blot analysis with 6D11 as the primary antibody. Molecular weight markers are shown on the right. NC: negative control, NBH only. F10⁻²∶10⁻² IBH dilution without PMCA (Frozen).</p