Abstract
We describe a novel approach to produce conformational monoclonal antibodies selected to specifically react with the β-sheet secondary structure of pathological oligomeric conformers, characteristic of many neurodegenerative diseases. Contrary to past and current efforts, we utilize a mammalian non-self-antigen as an immunogen. The small, non-self peptide selected was covalently polymerized with glutaraldehyde until it reached a high β-sheet secondary structure content, and species between 10–100kDa that are immunogenic, stable and soluble (p13Bri). Inoculation of p13Bri in mice elicited antibodies to the peptide and the β-sheet secondary structure conformation. Hybridomas were produced and clones selected for their reactivity with at least two different oligomeric conformers from Alzheimer’s, Parkinson and/or Prion diseases. The resulting conformational monoclonals are able to detect pathological oligomeric forms in different human neurodegenerative diseases by ELISA, immunohistochemistry and immunoblots. This technological approach may be useful to develop tools for detection, monitoring and treatment of multiple misfolding disorders.
Highlights
Immunogens for active immunization or for the production and selection of anti-conformation monoclonal antibodies, with the latent possibility of autoimmune toxicity
We selected a mild alkaline pH for the reaction to stabilize the Lysines net charges and maximize separation by charge repulsion, a high temperature and high rpm shaking to avoid stabilizing a blocked monomeric structure during the chemical reaction, a buffer with no phosphate or Tris groups that could interfere with the progression of oligomerization, and a glutaraldehyde concentration to equalize the ratio of long self-polymerized glutaraldehyde chains versus the joining of two or
We have developed, using a novel methodology, anti-β-sheet secondary structure monoclonal antibodies to a dominant β-sheet structure that is present in pathology associated oligomers of misfolded protein/peptides of different NDD
Summary
Immunogens for active immunization or for the production and selection of anti-conformation monoclonal antibodies, with the latent possibility of autoimmune toxicity. The second is the restrictive specificity of the immunogen to a single or limited number of pathological conformers[18] (Fig. 1) To overcome these problems we developed a methodology to produce conformational anti-secondary structure β-sheet monoclonal antibodies. Hybridomas were produced and monoclonals selected by the novel approach of using as selector compounds, oligomeric conformers from different NDD with the only commonality being the shared β-sheet secondary structure (Fig. 1). These new monoclonals to β-sheet conformation in oligomers may more effectively detect, monitor and treat NDD in humans and other susceptible animals
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