Generation of a recombinant Fab antibody reactive with the Alzheimer's disease-related Abeta peptide.
A recombinant Fab antibody, designated 1E8-4b, which reacts with the Alzheimer's disease (AD)-related Abeta peptides, Abeta[1-40], Abeta[1-42] and Abeta[1-43] has been developed. The 1E8-4b Fab was constructed by cloning the V(H)C(H1) and V(L)C(L) domains from the parent hybridoma 1E8 antibody, reported previously to recognize these Abeta peptides. Briefly, a C-terminal Flag tag sequence was incorporated into this construct, which was ligated into the vector pHFA2 and expressed in Escherichia coli. Following purification on an M2 anti-Flag affinity column, the 1E8-4b recombinant Fab antibody was shown to bind plaques within sections of brain tissue from CERAD-defined AD patients by immunohistochemistry. ELISA, epitope mapping and immunoblotting confirmed the recognition of the Abeta1-40/42/43] peptides by the 1E8-4b Fab. The 1E8-4b Fab did not recognize APP695 or APP770 which contain the Abeta sequence. The Abeta specificity of the recombinant 1E8-4b Fab antibody was identical to the parent 1E8 monoclonal antibody.
Department of Pathology, Melbourne University, Parkville, Victoria, Australia.
A H
AH
Tammer
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G
Coia
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R
Cappai
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S
Fuller
C L
CL
Masters
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Hudson
J R
JR
Underwood
D000544:Alzheimer Disease; D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001483:Base Sequence; D001923:Brain Chemistry; D004306:Dose-Response Relationship, Immunologic; D018604:Epitope Mapping; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
C465643:1E8-4b Fab; D016229:Amyloid beta-Peptides; D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D010446:Peptide Fragments; D011994:Recombinant Proteins; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42); C098994:amyloid beta-protein (1-43)