Production of Monoclonal Antibodies to Pathologic β-sheet Oligomeric Conformers in Neurodegenerative Diseases

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.

Anti-β-sheet conformation monoclonal antibody reduces tau and Aβ oligomer pathology in an Alzheimer’s disease model

“…The availability of only a small volume of cell supernatant from the initially viable hybridoma cells allowed only one determination per sample per each antigen and detection of all the main classes of immunoglobulins together by total goat antimouse immunoglobulins antisera. The selection process was previously described and involved the measurement of the differential reactivity to Aβ 40 , Aβ 42 , and PHF measured at least three times over the background optical density readings compared with an irrelevant hybridoma clone from the same fusion (Additional file 1 : Figure S1b) [ 17 ]. The 23B clone passed three rounds of selection before being classified as GW-23B7 aβComAb.…”

“…The aβComAb GW-23B7 was obtained after immunization of a CD-1 mouse with the p13Bri immunogen and subsequent hybridoma production performed at the bi-institutional Antibody and Bioresource Core Facility of Memorial Sloan Kettering Cancer Center and The Rockefeller University as previously described [ 17 ]. All procedures were approved by the institutional animal care and use committee (protocol 97-03-009) and were carried out in accordance with National Institutes of Health (NIH) standards.…”

“…Selection of a conformational mAb with reactivity to oligomers presenting with a dominant β-sheet secondary structure was done by enzyme-linked immunosorbent assay (ELISA) analysis as previously described [ 17 – 19 ]. Plates were coated overnight at 4 °C with either Aβ 1–40 or Aβ 1–42 in 50 mM ammonium bicarbonate, pH 9.6, maximizing the oligomeric content difference between both Aβ 40 /Aβ 42 peptides (Additional file 1 : Figure S1), paired helical filaments (PHFs) purified from a human AD brain, or protease-resistant prion protein (PrP Res ) as previously described [ 17 , 19 ]. Approximately 125 μl of cell supernatant was diluted 1:1 with 50 mM Tris-buffered saline, pH 7.2, with 0.1% Tween 20 (TBS-T), and 50 μl/well were applied to each one of the four Immulon 2 HB (Thermo Fisher Scientific, Waltham, MA, USA) 96-well microtiter precoated plates.…”

“…So far, there has been a very high failure rate of AD trials targeting Aβ pathology, with more recent attempts addressing tau pathology, which correlates better with the cognitive deficits in AD [ 11 – 15 ]. We believe that addressing both of these pathologies concurrently might be a more optimal approach; however, until recently, this has not been feasible [ 16 , 17 ].…”

“…Previously, we demonstrated that immunizing mice with a synthetic non-self soluble antigen with a repetitive β-sheet secondary structure could induce sufficient numbers of B cells, which specifically recognize the defined secondary structure, to produce stable hybridomas by a selection process using pathological oligomeric forms of misfolded proteins/peptides of diverse NDDs [ 17 ]. One of the anti-β-sheet conformational monoclonal antibodies (aβComAb) generated, GW-23B7, showed affinity for both Aβ and tau oligomeric forms derived from human AD tissue.…”