A potential blood test for transmissible spongiform encephalopathies by detecting carbohydrate-dependent aggregates of PrPres-like proteins in scrapie-Infected hamster plasma.
PrPres has rarely been detected in blood (except in leukocytes) even in diseased animal models that are known to contain a large amount of PrPres in infected tissues. It seems likely that PrPres detection in blood is difficult because of the low titer of infectious material within the blood. Here, we demonstrate the detection of proteinase K-resistant 3F4-reactive protein in the plasma of scrapie-infected hamsters but not in the plasma of mock-infected hamsters by partial purification using a novel method termed "acidic SDS precipitation," in conjunction with a highly sensitive chemiluminescence detection system used to show the presence of PrP at a concentration equivalent to 1.4x10(-9) g of brain homogenate or 1.5x10(-12) g (6.5x10(-17) mol) of rPrP by conventional Western blotting. The 3F4-reactive proteins in scrapie-infected hamster plasma often resulted in multiple Mw protein bands occurring at higher Mw positions than the position of the di-glycosyl PrP molecule. Mixing scrapie-infected hamster brain homogenate with mock-infected hamster plasma resulted in the formation of similar Mw positions for multiple 3F4-reactive proteins. Predigestion of carbohydrate side chains from the proteins in the plasma or brain homogenate before mixing resulted in failure to obtain these multiple 3F4-reactive proteins. These observations indicate that PrPres aggregated with other proteins in the plasma through carbohydrate side chains and was successfully detected in the plasma of scrapie-infected hamsters. Counterparts in these aggregates with PrPres-like proteins in scHaPl are not known but any that exist should resist the PK digestion.
Tsukui
Kazuo
K
Central Blood Institute, The Japanese Red Cross Society, Koto-ku, Tokyo, Japan. k-tsukui@bs.jrc.or.jp
Takata
Masuhiro
M
Tadokoro
Kenji
K
D000818:Animals; D000911:Antibodies, Monoclonal; D006224:Cricetinae; D005590:Fractional Precipitation; D006403:Hematologic Tests / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008647:Mesocricetus; D018620:PrPSc Proteins / Q000097:blood*; D017096:Prion Diseases / Q000175:diagnosis*; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018620:PrPSc Proteins; D012967:Sodium Dodecyl Sulfate