Preparation and characterization of a monoclonal antibody with high affinity for soluble Abeta oligomers.
Amyloid beta-protein (Abeta) has been causally implicated in the neurodegenerative processes that accompany Alzheimer's disease. Soluble oligomers of the Abeta(1-42) fragment are thought to be significantly more neurotoxic than higher molecular weight aggregates. We report the isolation and characterization of a mouse monoclonal antibody (MAb) directed against soluble Abeta(1-42) oligomers. Synthetic Abeta(1-42) oligomers were assembled in vitro; these were used to immunize mice, and hybridomas were isolated following myeloma fusion of splenocytes from immunized animals. Screening for reactivity against Abeta(1-42) resulted in the identification of MAb A8 with high affinity for soluble oligomers. The isotype of A8 was found to be IgG(2b). Experiments using sub-peptides of Abeta(1-42) revealed that the epitope identified by A8 lies within the 1-6 region of Abeta. The antibody displays high affinity for soluble Abeta(1-42) oligomers in the molecular weight range of 16.5-25 kDa, and detected target antigen in brain sections from senescence-accelerated SAMP 8 mice. The sensitivity and optimal titers for the detection of soluble Abeta(1-42) oligomers were determined to be 0.625 microg/mL in indirect ELISA, and 1:10(6), 1:4000, and 1:150 for ELISA, Western blotting, and immunohistochemistry, respectively. The A8 antibody specific for soluble Abeta(1-42) oligomers will provide a valuable tool for Alzheimer's disease research.
Ying
Zhang
Z
College of Life Sciences and Bioengineering, School of Science, Beijing Jiaotong University, Beijing, China.
Xin
Wang
W
Jin-Sheng
He
H
Fu-Xiang
Bao
B
Wei-Min
Sun
S
Xin-Xian
Dai
D
Xiao-Bo
Wang
W
Yi-Qin
Li
L
Xian-Xian
Zhen
Z
Hong-Gang
Hu
H
Xiang-Lei
Peng
P
Yan-Peng
Zheng
Z
Ling-Ling
Hou
H
Tao
Hong
H
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D000915:Antibody Affinity; D000918:Antibody Specificity; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data
D016229:Amyloid beta-Peptides; D000911:Antibodies, Monoclonal