Serial passaging of PMCAb-derived 263K^MH, 263K^(M)H and 263K^(MH) in Syrian hamsters.

<p>(A) Western blot analysis of brain material from hamsters inoculated with PMCAb-derived 263K^MH, 263K^(M)H or 263K^(MH). Western blots were overexposed for detecting minor amounts of PK-resistant material. Brains marked by asterisks were used to prepare inocula for the second passage. (B) Western blot analysis of brain material from the animals of the 2^nd passage of 263K^MH, 263K^(M)H or 263K^(MH). Brain materials used to prepare inocula for the second and third passages are marked by asterisks and hash signs, respectively. Rectangles show animals in 263K^(M)H and 263K^(MH) groups euthanized at 347 days postinoculation. (C) Western blot analysis of brain material from the animals of the 3^rd passage of 263K^MH, 263K^(M)H or 263K^(MH). Brain materials used to prepare inocula for the second and third passages are marked by asterisks and hash signs, respectively, and are shown as references. In all panels, Western blots were stained with 3F4 (top panels) or SAF-84 antibody (bottom panels). 1% 263K brain homogenate was used as a cross-reference between panels. Brains used for histopathology analysis are marked by “hp”. For the animals that developed clinical diseases, the incubation times to the terminal stage are indicated. (D) Comparison of glycoform composition and molecular weight of PK-resistant products in brain material from the animals of 263K group (#1, black lines), the 3^rd passages of 263K^MH (#2, green lines), 263K^(M)H (#3, red lines) or 263K^(MH) (#4, blue lines) by Western blot (left panel); n = 3 individual animals per group. For 263K, 263K^MH and 263K^(M)H 1% brain homogenates (BH) were loaded onto SDS-PAGE, whereas 10% brain homogenates were loaded for 263K^(MH). Right panel shows intensity profiles for 263K, 263K^MH, 263K^(M)H and 263K^(MH) group normalized by the intensity of diglycosylated isoform within individual samples. Vertical black lines identify positions of di-, mono- and unglycosylated isoforms for 263K group. With the exception of the brain materials shown on the first lanes (-PK), all brain materials were treated with 20 μg/ml PK.