TY - JOUR
T1 - Efficient propagation of variant Creutzfeldt-Jakob disease prion protein using the cell-protein misfolding cyclic amplification technique with samples containing plasma and heparin
AU - Oshita, Masatoshi
AU - Yokoyama, Takashi
AU - Takei, Yumiko
AU - Takeuchi, Atsuko
AU - Ironside, James W.
AU - Kitamoto, Tetsuyuki
AU - Morita, Masanori
N1 - Funding Information:
This study was supported by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (TK); a grant from the Ministry of Health, Labor and Welfare (TK); a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology (TK); and Japan Blood Products Organization (former the Benesis Corporation) and Mitsubishi Tanabe Pharmaceutical company (MO, TY, YT, and MM). We thank Ms M. Yamamoto of Tohoku University for technical assistance with the tissue culture. We thank Drs T. Imada and S. Uno of Japan Blood Products Organization for useful advice. We also thank Dr M. Head of the University of Edinburgh for reviewing the manuscript. The Edinburgh Brain and Tissue Bank is supported by theMedical Research Council. MO contributed most of the experiments and prepared the manuscript; TY, YT, and AT contributed a part of the experiments; JWI provided vCJD materials and reviewed the manuscript; TK supervised the study, MM designed and organized the study and edited the manuscript; and all contributed to the discussion. AT, TK, and JWI have disclosed no conflicts of interest. MO, TY, YT, and MM belong to Japan Blood Products Organization, a plasma fractionation company, which provided materials in this study. The authors declare that this fact did not affect study design and collection, analysis, and interpretation of data of this study.
Publisher Copyright:
© 2015 AABB.
PY - 2016/1/1
Y1 - 2016/1/1
N2 - BACKGROUND To prevent the iatrogenic spread of variant Creutzfeldt-Jakob disease (vCJD) between humans via blood products or transfusion, highly sensitive in vitro screening tests are necessary. Protein misfolding cyclic amplification (PMCA) is one such candidate test. However, plasma has been reported to inhibit the PMCA reaction. Therefore, we investigated the cell-PMCA conditions that permit vCJD prion amplification in the presence of plasma. STUDY DESIGN AND METHODS Cell-PMCA of vCJD samples was performed by adding various final concentrations of pooled plasma, citrate-phosphate-dextrose (CPD), albumin, globulin, or pooled plasma treated with ion exchangers. After heparin and plasma concentrations were optimized, multiround cell-PMCA was performed. RESULTS When 1% to 50% of pooled plasma was added to heparinized cell-PMCA, amplification efficiency showed a double-peaked profile at less than 1% and 40% final plasma concentrations, indicating that plasma contains not only PMCA inhibitors but also promoters. Intravenous globulin did not inhibit cell-PMCA, but the protein G-bound fraction did. CPD, albumin-depleted plasma, and the unbound fraction of anion-exchange chromatography inhibited cell-PMCA, but albumin and the unbound fraction of the cation-exchange chromatography did not. The detection limit of abnormal prion protein in multiround cell-PMCA, when maintaining the final plasma concentration at 40% at each round, was 10-10 dilutions of a vCJD brain specimen. CONCLUSION We have established a novel cell-PMCA format in the presence of plasma without any pretreatment, where vCJD prion protein was amplified at comparable levels to that found without plasma. Our data suggest the feasibility of cell-PMCA as a practical blood test for vCJD prions.
AB - BACKGROUND To prevent the iatrogenic spread of variant Creutzfeldt-Jakob disease (vCJD) between humans via blood products or transfusion, highly sensitive in vitro screening tests are necessary. Protein misfolding cyclic amplification (PMCA) is one such candidate test. However, plasma has been reported to inhibit the PMCA reaction. Therefore, we investigated the cell-PMCA conditions that permit vCJD prion amplification in the presence of plasma. STUDY DESIGN AND METHODS Cell-PMCA of vCJD samples was performed by adding various final concentrations of pooled plasma, citrate-phosphate-dextrose (CPD), albumin, globulin, or pooled plasma treated with ion exchangers. After heparin and plasma concentrations were optimized, multiround cell-PMCA was performed. RESULTS When 1% to 50% of pooled plasma was added to heparinized cell-PMCA, amplification efficiency showed a double-peaked profile at less than 1% and 40% final plasma concentrations, indicating that plasma contains not only PMCA inhibitors but also promoters. Intravenous globulin did not inhibit cell-PMCA, but the protein G-bound fraction did. CPD, albumin-depleted plasma, and the unbound fraction of anion-exchange chromatography inhibited cell-PMCA, but albumin and the unbound fraction of the cation-exchange chromatography did not. The detection limit of abnormal prion protein in multiround cell-PMCA, when maintaining the final plasma concentration at 40% at each round, was 10-10 dilutions of a vCJD brain specimen. CONCLUSION We have established a novel cell-PMCA format in the presence of plasma without any pretreatment, where vCJD prion protein was amplified at comparable levels to that found without plasma. Our data suggest the feasibility of cell-PMCA as a practical blood test for vCJD prions.
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U2 - 10.1111/trf.13279
DO - 10.1111/trf.13279
M3 - Article
C2 - 26347231
AN - SCOPUS:84954392855
VL - 56
SP - 223
EP - 230
JO - Transfusion
JF - Transfusion
SN - 0041-1132
IS - 1
ER -