CloneID: Nb484
Heavy Chain modification: His-tagged
Antigen Long Description: The original antibody was generated by immunizing a llama with a segment of the mouse prion protein (MoPrP(23-230)).
Origin Pub PMID: 24400836
Buffer Composition: PBS only.
Uniprot Accession No.: P04156; P04925
Specificity Statement: This antibody is specific for major prion proteins, with a demonstrated specificity for a discontinuous epitope on the human prion protein (HuPrP). It binds to specific structural elements and loops within HuPrP, particularly the β0-β1 loop (residues 123−125), the β2-α2 loop (residues 164−170), and the α2-helix (residues 174−185). It has also been shown to bind mouse prion protein (MoPrP), where it binds residues 123 and 125 of the β0-β1 loop; residue 128 of the β1 strand; residues 164, 167, 168, and 169 of the β2-α2 loop; and residues 173, 174, 177, 178, 182, 185 and 189 of the α2-helix.
Application Notes (Clone): The original version of this antibody (alpaca VHH) bound mouse and human prion proteins (MoPrP and HuPrP, respectively) as demonstrated by SPR binding assays with a Kd of 40 nM, 50 nM, 9.54 nM, and 7 µM for MoPrP(23-230), MoPrP(89-230), HuPrP(90-231), and HuPrP(23-144), respectively. The addition of this antibody to MoPrP(23−230) extended the lag phase of fibrillization by about 40 hours in an amyloid seeding assay (ASA), indicating that the interaction of this antibody with the full-length MoPrP inhibited the formation of prion protein infectious scrapie agent (PrPSc)-like aggregates. This antibody's ability to inhibit prion propagation was further confirmed by treating scrapie-infected murine cells (ScGT1) with different antibody concentrations. The treatment resulted in a dramatic, dose-dependent reduction in PrPSc levels, as measured by PK assay and Western blotting. At a concentration of 3.5 μM, PrPSc levels were no longer detectable, and this effect persisted even after removing the antibody from the medium. This antibody was complexed with HuPrP(23-231) and HuPrP(90-231), and their crystal structure was examined. The binding of this antibody stabilizes the conformation of the human prion protein, particularly the β2-α2 loop, which is known to be flexible in the native structure but becomes rigid upon binding (Abskharon et al., 2014; PMID: 24400836). The binding of this antibody to recombinant PrP (recPrP) and recPrPSc was evaluated using ELISA. It was found to have a high affinity for recPrP, as previously reported, but its binding to the infectious recPrPSc is minimal, indicating that the antibody exerts its inhibitory effect on prion propagation through binding to the non-infectious recPrP. The binding of this antibody to recPrP was further supported by the results of the discontinuous iodixanol density gradient floatation assay. This antibody was crystallized with MoPrP(89–230) at pH 6.0 and 8.0 and alone (free form). This antibody inhibited prion conversion by competitively binding to the hydrophobic region of MoPrP, thereby blocking the interaction with cofactors essential for prion propagation, as evidenced by Protein Misfolding Cyclic Amplification (PMCA) assays and lipid binding studies (Abskharon et al., 2019; PMID: 31815959). This antibody recognizes and binds the β2−α2 loop epitope across various HuPrP structures; the binding mechanisms of this antibody to different variants of HuPrP, including wild-type, E219K polymorphism, and V210I mutation, were studied using molecular dynamics simulations and experimental data. It was found to recognize flexible epitopes within the β2−α2 loop of HuPrP through a conformational selection model, stabilizing diverse conformations present in these variants. Despite structural differences among HuPrP variants, the antibody exhibited high-affinity binding, emphasizing its ability to interact with disordered regions crucial for amyloidogenic protein recognition (Mollica & Giachin, 2023; PMID: 36580661).
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