Modular DNA Barcoding of Nanobodies Enables Multiplexed in situ Protein Imaging and High-throughput Biomolecule Detection

Immunodetection methods based on antibody-antigen interactions are routinely used in biological and clinical laboratories. DNA-labeled antibodies offer the advantage of simultaneous detection of multiple target molecules via orthogonal DNA barcode sequences. However, current methods for antibody-DNA conjugation typically involve non-site-specific modifications tailored for each specific application, a process that is labor-intensive and often compromises antibody affinity and specificity. To overcome these limitations, here we present a modular, site-specific, and cost-efficient DNA tagging strategy — multiplexed and modular barcoding of antibodies (MaMBA). Utilizing nanobodies as modular adaptors, MaMBA enables direct site-specific labeling of off-the-shelf IgG antibodies with a one-component design. We first applied MaMBA to develop the mis HCR method for highly multiplexed in situ protein imaging via orthogonal hybridization chain reaction (HCR). We then introduced a cleavable version of MaMBA, which allows iterative use of the same HCR probes sets, thereby enhancing the multiplexity of mis HCR ( mis HCR ⁿ ). mis HCR ⁿ achieves simultaneous labeling of 12 different target molecules within the same mouse brain sections. We further extended the cleavable MaMBA to develop the barcode-linked immunosorbent assay (BLISA) for multiplexed and high-throughput biomolecule detections. By combining BLISA with next-generation sequencing, we successfully measured SARS-CoV-2 IgG and HBV-associated antigens in a large number of human serum samples. Additionally, we demonstrated a small-scale drug screen by using BLISA to simultaneously detect eight protein targets. In conclusion, MaMBA offers a highly modular and easily adaptable approach for antibody DNA-barcoding, which can be broadly applied in basic research and clinical diagnostics.