Session: Current Topics in Biological Chemistry:

Diagnostic immunoassays are critical for early diagnosis of diseases for which early intervention is considered the only viable therapeutic treatment. Typically, disease-relevant antibodies are detected in an ELISA format, which require immobilization of the cognate antigen within the well of a polystyrene microtiter plate. Site-specific modifications have been shown to provide more robust results, especially in diagnostic applications where sensitivity is critical. Chemoselective bioconjugation techniques typically modify functional groups that are abundantly featured on protein side chains. Consequently, these modifications are likely to alternative epitopes recognized by IgG antibodies, particularly epitopes found on smaller proteins and peptides such as amyloidogenic proteins amyloid-β and α-synuclein. A chemoselective bioconjugation based on the bio-orthogonal redox transformation between methionine and oxaziridine was investigated for the specific detection of IgG antibodies recognizing amyloidogenic proteins. We envisioned that such an assay, when applied as an ELISA-like platform would form the basis for biochemical diagnostics that distinguish patients with Alzheimer's disease or Parkinson's disease (AD or PD). PEGylated TentaGel microspheres were selected as a support to reduce nonspecific binding compared to polystyrene supports and prevent amyloid aggregation. Additionally, the fluorescence intensity of TentaGel microspheres resulting from tightly bound fluorescently labeled targets can be measured by flow cytometry, enhancing the throughput of analysis. The amyloidogenic proteins α-synuclein were labeled at methionine side chains using an oxaziridine linker containing an azide. The resultant modified protein was covalently bound to TentaGel microspheres via strain-promoted alkyne-azide cycloaddition. Following the validation of ELISA-like assay, α-synuclein autoantibody (IgG) levels in plasma samples derived from donors with or without PD to determine whether titers to α-synuclein oligomers would be significantly higher in PD patients compared to healthy control donors. Overall, the results presented here demonstrate proof-of-concept for immobilizing target biomolecules to a PEGylated scaffold for use in detecting protein-protein interactions by measuring using methionine-specific solid-phase conjugation immunoassay as a novel ELISA-like assay platform for amyloidogenic and more hydrophobic proteins.

Alexander K Hurben

Graduate Student, University of Minnesota

Appearances