CloneID: iMab
Antigen Long Description: The original antibody was selected from the Garvan-2 human scFv library via phage display.
Buffer Composition: PBS only.
Chimeric Use Statement: This chimeric mouse IgG2a antibody was made using the variable domain sequences of the original scFv format, for improved compatibility with existing reagents, assays and techniques.
Specificity Statement: This antibody is specific for the DNA i-motif, a four-stranded DNA structure that forms in cytosine-rich regions and is potentially involved in regulating DNA function.
Application Notes (Clone): The original human scFv version of this antibody was validated for its specificity towards various i-motif types through ELISA and bio-layer interferometry (BLI), with a dissociation constant (KD) of 59 nM. It selectively binds to i-motifs, including those in 1G22, 1A83, c-MYC, RET, and VEGF sequences, located in regions such as telomeres, centromeres, and proto-oncogene promoters. Importantly, this antibody does not interact with structures resembling i-motif DNA, such as DNA G4 quadruplex forms, double-stranded DNA (dsDNA), hairpin DNA, miRNA, or mutant human telomeres lacking i-motifs (Zeraati et al., 2018; PMID: 29686376). Notably, it does not bind to various DNA G4 quadruplex forms, including promoter G4s c-MYC44, BCL245, VEGF, telomeric 2JSM47, 2GKU, or aptamer G4 TBA. Additionally, the antibody has proven effective for immunofluorescence detection of i-motifs in multiple cell lines (MCF7, U2OS, and HeLa), confirming the presence of i-motif structures in human nuclei and their dependence on pH and cell-cycle stage (Zeraati et al., 2018; PMID: 29686376). This antibody selectively binds to both intramolecular and intermolecular i-motif structures. The composition of buffers used during binding and washing steps significantly influences the antibody's selectivity, emphasizing the importance of optimizing DNA concentrations, blocking conditions, and washing protocols. These factors are crucial for confirming the antibody's selectivity for i-motifs and ensuring accurate detection of both intramolecular and intermolecular i-motifs (Ruggiero et al., 2024; DOI: 10.1101/2024.06.22.600195v1).
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