Establishment of a chicken monoclonal antibody panel against mammalian prion protein.
A panel of chicken monoclonal antibodies (mAbs) was developed against prion protein (PrP), the sequence of which is a highly conserved molecule among mammals. A portion of the splenocytes from chickens immunized with recombinant mouse PrP was fused with the chicken B cell line, MuH1. The remaining splenocytes were used to generate the recombinant mAbs by phage display. A total of 36 anti-PrP mAbs, 2 from cell fusion and 34 from phage display were established. The specificity of these mAbs was determined by Western blot and ELISA using various PrP antigens including recombinant PrPs, synthetic PrP peptides and PrPs from brains or scrapie-infected neuroblastoma cell line. These mAbs were classified into three main groups, protease K (PK)-sensitive (Group I), PK cleavage site proximal (Group II) and PK-resistant (Group III), based on their abilities to recognize PrP following PK-treatment. Some mAbs were found to selectively recognize different glycoforms of PrP as well as the metabolic fragments of PrP. Furthermore, we found that PrP recognition by chickens differed from that by PrP-knockout mouse. These results indicate that these newly generated PrP antibodies from chickens will help to research the PrP and to establish the diagnosis of prion disease.
Nakamura
Naoto
N
Laboratory of Immunobiology, Department of Molecular and Applied Bioscience, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Japan.
Shuyama
Aki
A
Hojyo
Shintaro
S
Shimokawa
Mariko
M
Miyamoto
Kazuyoshi
K
Kawashima
Tsuyoshi
T
Aosasa
Masayoshi
M
Horiuchi
Hiroyuki
H
Furusawa
Shuichi
S
Matsuda
Haruo
H
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D000918:Antibody Specificity; D015153:Blotting, Western / Q000662:veterinary; D001921:Brain / Q000276:immunology; D002417:Cattle; D002459:Cell Fusion / Q000662:veterinary; D045744:Cell Line, Tumor / Q000378:metabolism; D002645:Chickens / Q000276:immunology*; D003429:Cross Reactions; D019286:Endopeptidase K / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D019151:Peptide Library; D018620:PrPSc Proteins / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D012756:Sheep
D000911:Antibodies, Monoclonal; D019151:Peptide Library; D018620:PrPSc Proteins; D011994:Recombinant Proteins; D019286:Endopeptidase K