Sensitive ELISA detection of amyloid‐β protofibrils in biological samples

Abstract

Amyloid-b (Ab) protofibrils are known intermediates of the in vitro Ab aggregation process and the protofibrillogenic Arctic mutation (APPE693G) provides clinical support for a pathogenic role of Ab protofibrils in Alzheimer's disease (AD). To verify their in vivo relevance and to establish a quantitative Ab protofibril immunoassay, Ab conformation dependent monoclonal antibodies were generated. One of these antibodies, mAb158 (IgG2a), was used in a sandwich ELISA to specifically detect picomolar concentrations of Ab protofibrils without interference from Ab monomers or the amyloid precursor protein (APP). The specificity and biological significance of this ELISA was demonstrated using cell cultures and transgenic mouse models expressing human APP containing the Swedish mutation (APPKN670/671ML), or the Swedish and Arctic mutation in combination. The mAb158 sandwich ELISA analysis revealed presence of Ab protofibrils in both cell and animal models, proving that Ab protofibrils are formed not only in vitro, but also in vivo. Furthermore, elevated Ab protofibril levels in the Arctic-Swedish samples emphasize the usefulness of the Arctic mutation as a model of enhanced protofibril formation. This assay provides a novel tool for investigating the role of Ab protofibrils in AD and has the potential of becoming an important diagnostic assay.

Stabilization of neurotoxic Alzheimer amyloid-β oligomers by protein engineering

“…We used two different conformation-specific antibodies that bind wild-type Aβ aggregates to examine if their binding epitopes are preserved in AβCC and to shed light on how the many AβCC aggregates are related. The mAb158 monoclonal antibody (22) was selected based on its affinity for protofibrils of Aβ 42 carrying the Arctic mutation (11). The mAb158 antibody also recognizes protofibrils of wild-type Aβ 42 , a protofibrillar form that is present in the medium of APP-expressing cells, and Aβ aggregates in brains of transgenic mice (22).…”

“…The mAb158 monoclonal antibody (22) was selected based on its affinity for protofibrils of Aβ 42 carrying the Arctic mutation (11). The mAb158 antibody also recognizes protofibrils of wild-type Aβ 42 , a protofibrillar form that is present in the medium of APP-expressing cells, and Aβ aggregates in brains of transgenic mice (22). ELISA experiments show that β-sheet oligomers/protofibrils of Aβ 42 CC bind mAb158 with the same affinity, or better, than wild-type protofibrils (Fig.…”

“…AβCC protofibrils are morphologically very similar to wild-type protofibrils. These protofibrils are also recognized in the mAb158 monoclonal antibody ELISA (22), which is conformation-specific for protofibrils of wild-type Aβ 42 .…”

Immunotherapeutic Approaches Targeting Amyloid-β, α-Synuclein, and Tau for the Treatment of Neurodegenerative Disorders

“…In this sense, the use of antibodies directed against specific epitopes or conformations of Aβ has yielded promising results. Passive immunization approaches using monoclonal antibodies against Aβ1-40 [103], Aβ1-42 [104], pyroglutamate Aβ [105], oligomers [106], or protofibrils [107][108][109] have been developed. Currently, clinical trials with the antibodies BAN2401 (recognizing protofibrils) [75], crenezumab (aggregated species) [76], gantenerumab (fibrils) [78][79][80], and solanezumab (Aβ mid-domain) [81,82] are ongoing (Table 1).…”

Multivalent foldamer-based affinity assay for selective recognition of Aβ oligomers

“…[19] Multiple approaches have been published for capturing/detecting soluble aggregated forms of Aβ: Aβ oligomer specific antibodies; [20][21][22][23][24] simultaneous application of multiple Nterminal specific antibodies; [25][26][27][28] a generic aggregation-sensitive peptide, [29] and Aβ self-recognition via seeded polymerization. [30,31] Advanced detection methods have also been utilized for Aβ oligomer sensing, [32] including DNA biobarcode amplification [21], localized surface plasmon resonance [20] and electrochemical techniques.…”