Modular DNA barcoding of nanobodies enables multiplexed in situ protein imaging and high-throughput biomolecule detection
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...
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eLife Sciences Publications Ltd
2025-07-01
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| Online Access: | https://elifesciences.org/articles/105225 |
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| author | Shilin Zhong Ruiyu Wang Xinwei Gao Qingchun Guo Rui Lin Minmin Luo |
| author_facet | Shilin Zhong Ruiyu Wang Xinwei Gao Qingchun Guo Rui Lin Minmin Luo |
| author_sort | Shilin Zhong |
| collection | DOAJ |
| description | 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 MaMBA-assisted immunosignal hybridization chain reaction (misHCR) method for highly multiplexed in situ protein imaging via orthogonal 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 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. |
| format | Article |
| id | doaj-art-209fa16557e04df5b1ca9bacdfe3a594 |
| institution | DOAJ |
| issn | 2050-084X |
| language | English |
| publishDate | 2025-07-01 |
| publisher | eLife Sciences Publications Ltd |
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| spelling | doaj-art-209fa16557e04df5b1ca9bacdfe3a5942025-08-20T03:08:20ZengeLife Sciences Publications LtdeLife2050-084X2025-07-011410.7554/eLife.105225Modular DNA barcoding of nanobodies enables multiplexed in situ protein imaging and high-throughput biomolecule detectionShilin Zhong0https://orcid.org/0000-0002-0295-1294Ruiyu Wang1https://orcid.org/0000-0002-7133-7693Xinwei Gao2Qingchun Guo3Rui Lin4https://orcid.org/0000-0003-1044-883XMinmin Luo5https://orcid.org/0000-0003-3535-6624School of Life Sciences, Peking University, Beijing, China; National Institute of Biological Sciences (NIBS), Beijing, China; Peking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program, NIBS, Beijing, ChinaSchool of Life Sciences, Peking University, Beijing, China; National Institute of Biological Sciences (NIBS), Beijing, China; Peking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program, NIBS, Beijing, China; Chinese Institute for Brain Research, Beijing, ChinaChinese Institute for Brain Research, Beijing, ChinaChinese Institute for Brain Research, Beijing, China; School of Biomedical Engineering, Capital Medical University, Beijing, ChinaNational Institute of Biological Sciences (NIBS), Beijing, China; Tsinghua Institute of Multidisciplinary Biomedical Research (TIMBR), Beijing, ChinaChinese Institute for Brain Research, Beijing, China; New Cornerstone Science Laboratory, Shenzhen, China; Research Unit of Medical Neurobiology, Chinese Academy of Medical Sciences, Beijing, China; Beijing Institute for Brain Research, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, ChinaCurrent 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 MaMBA-assisted immunosignal hybridization chain reaction (misHCR) method for highly multiplexed in situ protein imaging via orthogonal 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 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.https://elifesciences.org/articles/105225DNA barcodingmultiplexed detectionnanobodyimmunostainingimmunoassayHCR |
| spellingShingle | Shilin Zhong Ruiyu Wang Xinwei Gao Qingchun Guo Rui Lin Minmin Luo Modular DNA barcoding of nanobodies enables multiplexed in situ protein imaging and high-throughput biomolecule detection eLife DNA barcoding multiplexed detection nanobody immunostaining immunoassay HCR |
| title | Modular DNA barcoding of nanobodies enables multiplexed in situ protein imaging and high-throughput biomolecule detection |
| title_full | Modular DNA barcoding of nanobodies enables multiplexed in situ protein imaging and high-throughput biomolecule detection |
| title_fullStr | Modular DNA barcoding of nanobodies enables multiplexed in situ protein imaging and high-throughput biomolecule detection |
| title_full_unstemmed | Modular DNA barcoding of nanobodies enables multiplexed in situ protein imaging and high-throughput biomolecule detection |
| title_short | Modular DNA barcoding of nanobodies enables multiplexed in situ protein imaging and high-throughput biomolecule detection |
| title_sort | modular dna barcoding of nanobodies enables multiplexed in situ protein imaging and high throughput biomolecule detection |
| topic | DNA barcoding multiplexed detection nanobody immunostaining immunoassay HCR |
| url | https://elifesciences.org/articles/105225 |
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