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

Abstract Current immunodetection methods using antibody-DNA conjugates enable multiplexed target detection through orthogonal DNA barcodes, but existing conjugation approaches are labor-intensive and often compromise antibody function. 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 misHCR method for highly multiplexed in situ protein imaging via orthogonal hybridization chain reaction (HCR). Its cleavable variant, misHCRn, achieves simultaneous visualization of 12 different targets within the same mouse brain sections through iterative probe use. 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.