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Here, we present a modular, site-specific, and cost-efficient DNA tagging strategy \u2013 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 MaMBA-assisted immunosignal hybridization chain reaction (\n                    <jats:italic>mis<\/jats:italic>\n                    HCR) method for highly multiplexed in situ protein imaging via orthogonal HCR. Its cleavable variant,\n                    <jats:italic>mis<\/jats:italic>\n                    HCR\n                    <jats:sup>n<\/jats:sup>\n                    , 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 hepatitis B virus (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.\n                  <\/jats:p>","DOI":"10.7554\/elife.105225","type":"journal-article","created":{"date-parts":[[2025,5,14]],"date-time":"2025-05-14T11:30:06Z","timestamp":1747222206000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.105225","source":"Crossref","is-referenced-by-count":3,"title":["Modular DNA barcoding of nanobodies enables multiplexed in situ protein imaging and high-throughput biomolecule detection"],"prefix":"10.7554","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0295-1294","authenticated-orcid":true,"given":"Shilin","family":"Zhong","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02v51f717","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Life Sciences, Peking University","place":["Beijing, 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