Subject to the usual caveats inherent in studies with small sample size, this pilot phase 2 study supports further investigation of this novel treatment strategy using a metal-protein-attenuating compound.
SUMMARYA recombinant Fab antibody, designated 1E8-4b, which reacts with the Alzheimer's disease (AD)-related Ab peptides, Ab , Ab and Ab 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 Ab peptides. Briefly, a C-terminal Flag tag sequence was incorporated into this construct, which was ligated into the vector pHFA 2 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 Ab[1-40/42/43] peptides by the 1E8-4b Fab. The 1E8-4b Fab did not recognize APP695 or APP770 which contain the Ab sequence. The Ab specificity of the recombinant 1E8-4b Fab antibody was identical to the parent 1E8 monoclonal antibody.
A hybridoma, F31P46B, secreting monoclonal antibodies (mAbs) comprised of mu and gamma heavy chains in association with a single kappa light chain, has been characterized. This hybridoma was prepared by fusing splenocytes, derived from a BALB/c mouse immunized with Vibrio vulnificus and SP2/O-Ag-14 mouse myeloma cells. The specificity of this hybridoma was determined by ELISA screening on a large number of bacterial strains. Hybridoma cells of F31P46B were cloned by limiting dilution to an average cell density of 0.1 cells/well and repeated 3 times to ensure monoclonality. Isotyping of 7 subclones was then performed by Ouchterlony gel double diffusion, as well as a Bio-Rad isotyping kit, and both methods showed that both IgM and IgG2b were secreted. PAGE and immunoblotting showed the presence of mu, gamma, and kappa chains with respective molecular weights of 80, 50, and 25kDa. A series of fractions, collected from F31P46B ascites during Superose 12 gel chromatography, were tested by the two isotyping methods and each confirmed the presence of two immunoglobulin products. These data indicated that the hybridoma secreted two separate immunoglobulins, IgM/kappa and IgG2b/kappa.
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