Generation of a recombinant Fab antibody reactive with the Alzheimer's disease-related Aβ peptide

Abstract

SUMMARY A recombinant Fab antibody, designated 1E8–4b, which reacts with the Alzheimer's disease (AD)-related Aβ peptides, Aβ[1–40], Aβ[1–42] and Aβ[1–43] has been developed. The 1E8–4b Fab was constructed by cloning the VHCH1 and VLCL domains from the parent hybridoma 1E8 antibody, reported previously to recognize these Aβ 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 Aβ1–40/42/43] peptides by the 1E8–4b Fab. The 1E8–4b Fab did not recognize APP695 or APP770 which contain the Aβ sequence. The Aβ specificity of the recombinant 1E8–4b Fab antibody was identical to the parent 1E8 monoclonal antibody.