Enhancing Western Blot Detection of DRD2 in Human Adipose Tissue via Oligonucleotide-based Signal Amplification

Sammanfattning

The global prevalence of obesity and type 2 diabetes is increasing and these chronic metabolic disorders are associated with dysregulation of multiple signaling pathways, including those involving dopamine. Dopamine receptor D2 (DRD2) is a low-abundance G protein-coupled receptor expressed in adipose tissue and involved in the regulation of energy metabolism, insulin secretion and reward-related eating behavior, making its reliable detection and quantification in biological samples relevant to metabolic disease research. Western blot is a widely used technique for protein detection, but its sensitivity is limited by the signal generation capacity of conventional detection systems, which may be insufficient for low-abundance targets such as DRD2. Binding Oligo Ladder Detection (BOLD) is a signal amplification strategy based on polymerization and incorporation of bromodeoxyuridine triphosphate (BrdUTP) into a DNA/RNA strand conjugated to secondary antibody, enabling the detection of the DNA/RNA strand by anti-BrdUTP tertiary antibody. This study investigated the feasibility of integrating BOLD into a Western blot workflow for DRD2 detection in adipose tissue cell lysate. Two goat anti-rabbit secondary antibodies were conjugated to an oligonucleotide primer using click chemistry and their biotinylated variants were prepared for comparison. Functionality of conjugates was verified by dot blotting, which also demonstrated that BOLD produced signal amplification superior to that of biotinylated antibody-based detection at low antigen concentrations, providing proof of concept for the detection system. Western blot experiments were performed to evaluate detection performance, investigate background signal, optimize conditions and benchmark BOLD against conventional methods. In the initial Western blot, one oligonucleotide-conjugated secondary antibody achieved signal-to-noise ratio comparable to standard detection with an HRP-conjugated secondary antibody, while the other underperformed, suggesting that click chemistry configuration affects conjugate performance. Persistent background signal was observed across experiments and was attributed primarily to non-specific binding of HRP-conjugated anti-BrdUTP tertiary antibody (with residual primer as a possible additional contributing factor). Reducing the tertiary antibody concentration substantially improved signal-to-noise ratio. EveryBlot Blocking Buffer was identified as the most favorable blocking strategy for oligo-dT-conjugated antibodies, while PVDF membranes were incompatible with BOLD under the tested conditions. In benchmarking, a standard HRP-conjugated secondary antibody outperformed both oligonucleotide-conjugated and biotinylated secondary antibodies in signal intensity, signal-to-noise ratio and detection sensitivity. However, as the HRP-conjugated secondary antibody was of a different clone than the oligo-dT-conjugated and biotinylated antibodies, differences in binding affinity may have contributed to the observed performance difference. These findings demonstrate that BOLD is feasible in a Western blot workflow and shows promising signal amplification potential. Repeating the benchmarking experiment at optimized conditions for BOLD using the existing antibodies is recommended as an immediate next step, alongside obtaining an unmodified variant of the HRP-conjugated goat anti-rabbit antibody to enable comparison of HRP- and oligo-dT-conjugated variants of this antibody clone.