Branched Linkers for Site-Specific Fluorescent Labeling of Antibodies
Fluorescent antibodies have proved to be an invaluable tool for molecular biology and diagnostics. They are routinely produced by modification of lysine residues, which leads to high heterogeneity. As such, their affinity may be compromised if the antigen-binding site is affected, the probability of...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-01-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/28/1/425 |
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| author | Ksenia A. Sapozhnikova Evgeny L. Gulyak Vsevolod A. Misyurin Maria A. Simonova Ekaterina V. Ryabukhina Anastasiya V. Alexeeva Nataliya A. Tikhonova Natalia A. Lyzhko Galina P. Popova Andrey V. Misyurin Alexey V. Ustinov Vladimir A. Korshun Vera A. Alferova Dmitry Yu. Ryazantsev Vladimir A. Brylev |
| author_facet | Ksenia A. Sapozhnikova Evgeny L. Gulyak Vsevolod A. Misyurin Maria A. Simonova Ekaterina V. Ryabukhina Anastasiya V. Alexeeva Nataliya A. Tikhonova Natalia A. Lyzhko Galina P. Popova Andrey V. Misyurin Alexey V. Ustinov Vladimir A. Korshun Vera A. Alferova Dmitry Yu. Ryazantsev Vladimir A. Brylev |
| author_sort | Ksenia A. Sapozhnikova |
| collection | DOAJ |
| description | Fluorescent antibodies have proved to be an invaluable tool for molecular biology and diagnostics. They are routinely produced by modification of lysine residues, which leads to high heterogeneity. As such, their affinity may be compromised if the antigen-binding site is affected, the probability of which increases along with the degree of labeling. In this work, we propose a methodology for the synthesis of site-specific antibody-dye conjugates with a high degree of labeling. To this end, we synthesized two oxyamine-based branched triazide linkers and coupled them with a periodate-oxidized anti-PRAME antibody 6H8; two oxyamine-based linear monoazide linkers of similar structure were used as controls. The azide-labeled antibodies were subsequently conjugated with fluorescent dyes via SPAAC, a copper-free click reaction. Compared to their counterparts made with linear linkers, the branched conjugates possessed a higher degree of labeling. The utility of the methodology was demonstrated in the detection of the PRAME protein on the surface of the cell by flow cytometry. |
| first_indexed | 2024-03-09T03:29:50Z |
| format | Article |
| id | doaj.art-b462062bf3f444a6a6792430f6e3c587 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-09T03:29:50Z |
| publishDate | 2023-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-b462062bf3f444a6a6792430f6e3c5872023-12-03T14:58:01ZengMDPI AGMolecules1420-30492023-01-0128142510.3390/molecules28010425Branched Linkers for Site-Specific Fluorescent Labeling of AntibodiesKsenia A. Sapozhnikova0Evgeny L. Gulyak1Vsevolod A. Misyurin2Maria A. Simonova3Ekaterina V. Ryabukhina4Anastasiya V. Alexeeva5Nataliya A. Tikhonova6Natalia A. Lyzhko7Galina P. Popova8Andrey V. Misyurin9Alexey V. Ustinov10Vladimir A. Korshun11Vera A. Alferova12Dmitry Yu. Ryazantsev13Vladimir A. Brylev14Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, RussiaShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, RussiaN.N. Blokhin National Medical Cancer Research Center, Ministry of Health of Russia, Kashirskoye sh. 24, 115478 Moscow, RussiaShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, RussiaShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, RussiaGeneTechnology LLC, Profsoyuznaya 104, 117485 Moscow, RussiaGeneTechnology LLC, Profsoyuznaya 104, 117485 Moscow, RussiaGeneTechnology LLC, Profsoyuznaya 104, 117485 Moscow, RussiaShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, RussiaGeneTechnology LLC, Profsoyuznaya 104, 117485 Moscow, RussiaShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, RussiaShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, RussiaShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, RussiaShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, RussiaShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, RussiaFluorescent antibodies have proved to be an invaluable tool for molecular biology and diagnostics. They are routinely produced by modification of lysine residues, which leads to high heterogeneity. As such, their affinity may be compromised if the antigen-binding site is affected, the probability of which increases along with the degree of labeling. In this work, we propose a methodology for the synthesis of site-specific antibody-dye conjugates with a high degree of labeling. To this end, we synthesized two oxyamine-based branched triazide linkers and coupled them with a periodate-oxidized anti-PRAME antibody 6H8; two oxyamine-based linear monoazide linkers of similar structure were used as controls. The azide-labeled antibodies were subsequently conjugated with fluorescent dyes via SPAAC, a copper-free click reaction. Compared to their counterparts made with linear linkers, the branched conjugates possessed a higher degree of labeling. The utility of the methodology was demonstrated in the detection of the PRAME protein on the surface of the cell by flow cytometry.https://www.mdpi.com/1420-3049/28/1/425fluorescent antibodybranched linkersFRETdegree of labelingperiodate oxidationimmunoglobulin G |
| spellingShingle | Ksenia A. Sapozhnikova Evgeny L. Gulyak Vsevolod A. Misyurin Maria A. Simonova Ekaterina V. Ryabukhina Anastasiya V. Alexeeva Nataliya A. Tikhonova Natalia A. Lyzhko Galina P. Popova Andrey V. Misyurin Alexey V. Ustinov Vladimir A. Korshun Vera A. Alferova Dmitry Yu. Ryazantsev Vladimir A. Brylev Branched Linkers for Site-Specific Fluorescent Labeling of Antibodies Molecules fluorescent antibody branched linkers FRET degree of labeling periodate oxidation immunoglobulin G |
| title | Branched Linkers for Site-Specific Fluorescent Labeling of Antibodies |
| title_full | Branched Linkers for Site-Specific Fluorescent Labeling of Antibodies |
| title_fullStr | Branched Linkers for Site-Specific Fluorescent Labeling of Antibodies |
| title_full_unstemmed | Branched Linkers for Site-Specific Fluorescent Labeling of Antibodies |
| title_short | Branched Linkers for Site-Specific Fluorescent Labeling of Antibodies |
| title_sort | branched linkers for site specific fluorescent labeling of antibodies |
| topic | fluorescent antibody branched linkers FRET degree of labeling periodate oxidation immunoglobulin G |
| url | https://www.mdpi.com/1420-3049/28/1/425 |
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