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  • br Introduction Quenchbody Q body immunoassay is a novel bio

    2024-04-17


    Introduction Quenchbody (Q-body) immunoassay is a novel biosensing technology that uses the quenching of fluorescence by intrinsic tryptophan (Trp) residues in antibody variable regions when dye(s) are conjugated to an antibody or antibody fragments in appropriate position, and de-quenching while the antibody binds to the antigen. Compared with conventional fluoroimmunoassays, the Q-body assay is simple, and requires no additional reagents to perform the detection [1,2]. We also found that Q-bodies prepared with antigen binding fragments (Fab) of an antibody (ultra Q-body, UQ-body) generally produce a larger fluorescence increase when bound to the antigen compared with Q-bodies comprising a single chain variable region Fv (scFv) [3]. To date, Q-bodies to detect small chemicals, including illegal drugs [1], pesticides [4], peptides [3], and also larger proteins including cancer-related membrane protein claudin [5], have been developed. Alzheimer's disease (AD) is a serious disease of the elderly and has been studied for many years. Progressive dementia in AD is associated with selective neuronal degeneration and death [6]. AD is characterized by two pathological features in the brain: Extracellular senile plaques and intracellular neurofibrillary tangles. Senile plaques consist of amyloid-β peptide (Aβ), which is generated from amyloid precursor protein (APP) through sequential proteolytic processing by β-secretase and γ-secretase. Aβ42, one of the major peptide forms, apomorphine faster than other peptides, and results in higher neurotoxicity [7]. Historically, Aβ fibrils have been considered to be primarily responsible for neuronal dysfunction and death, because fibrils were detectable in diseased brains and led to neuronal dysfunction, dystrophy, and synaptic loss [8,9]. Busciglio et al. demonstrated that amyloid fibril formation alters the phosphorylation of tau, which leads to loss of its microtubule binding capacity [10]. Until now, the post mortem identification of amyloid plaques consisting of the Aβ peptide, and neurofibrillary tangles in the brains of the patients are the most reliable diagnosis of AD. Great efforts are being made to identify reliable biomarkers that are suitable for minimal invasive early diagnosis and prognosis of AD. During the past years, body fluids of AD patients were assayed for their content of total or soluble Aβ40 or Aβ42 using classical methods, such as enzyme-linked immunosorbent assay (ELISA) or non-classical readout [11,12]. The presence of Aβ aggregates seems to be the most direct disease biomarker for AD and increasing effort is being made to develop methods suitable to detect different Aβ aggregates in body fluids, such as cerebrospinal fluid (CSF) and plasma. Recent studies showed that soluble Aβ oligomers, such as Aβ-derived diffusible ligand (ADDL), are more toxic than fibrils [13,14]. ADDL binds to neurons and attacks memory-synapses, and has been identified in actual AD brains [15]. In this study, we aimed to develop UQ-bodies to detect the Aβ peptide and its oligomers.
    Materials and methods
    Results
    Discussions and conclusions In this study, we prepared three formats of Fab: Single-labeled Fab without a GS linker, single-labeled Fab with a GS linker, and a double-labeled Fab with a GS linker on both chains of Fab. Although the antigen-dependent fluorescence was not significant for the single-labeled UQ-bodies, the overall length of the linker between the dye and the N-terminus of antibody VH is still considered important. As shown in Table 2, the UQ-bodies with a GS linker between the Cys-tag and the antibody VH showed higher de-quenching when they were chemically denatured, especially for the TAMRA-labeled UQ-bodies. In addition, the TAMRA-C6 labeled UQ-bodies showed greater de-quenching than the TAMRA-C5 labeled ones. The longer linker might have allowed the dye to move closer to the Trp residues in the variable region, which would increase the quenching of the UQ-body in the native state without the antigen.