Antibody Purification Using Spy Chemistry
Antibody purification is a fundamental technology for therapeutic and diagnostic applications. While traditional methods like ammonium sulfate precipitation and polyethylene glycol precipitation are cost-effective, they often result in low purity and require multiple purification steps. Protein A–ba...
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| Format: | Article |
| Language: | English |
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Bio-protocol LLC
2025-04-01
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| Series: | Bio-Protocol |
| Online Access: | https://bio-protocol.org/en/bpdetail?id=5286&type=0 |
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| author | Xiaofeng Yang Zhanglin Lin Ya Xiang Zisha Lao |
| author_facet | Xiaofeng Yang Zhanglin Lin Ya Xiang Zisha Lao |
| author_sort | Xiaofeng Yang |
| collection | DOAJ |
| description | Antibody purification is a fundamental technology for therapeutic and diagnostic applications. While traditional methods like ammonium sulfate precipitation and polyethylene glycol precipitation are cost-effective, they often result in low purity and require multiple purification steps. Protein A–based affinity chromatography, the gold standard for antibody purification, provides high specificity but can be further improved to increase loading capacity and reduce costs. In this protocol, we introduce a novel approach for purifying high-quality, high-purity antibodies from complex samples using SpyFixer/Z domain–modified resin. This method utilizes Spy chemistry for site-specific immobilization of the Z domain of Protein A, significantly enhancing antibody loading capacity up to 200 mg/mL resin and ensuring stable, durable immobilization. Using this protocol, we achieved >90% purity of human immunoglobulin G (hIgG) from diverse sources, including E. coli cell lysates, human serum, and industrial fermentation broth. The SpyFixer/Z domain–modified resin protocol is simple, cost-effective, and offers a robust, scalable solution for efficient antibody purification in bioengineering applications. This immobilization scheme based on Spy chemistry can also be extended to other immunoglobulin-binding proteins, such as Protein G and Protein L, to develop high-efficiency affinity resins. |
| format | Article |
| id | doaj-art-4f440a25fb9344e5b02ff023589ae97f |
| institution | OA Journals |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | Bio-protocol LLC |
| record_format | Article |
| series | Bio-Protocol |
| spelling | doaj-art-4f440a25fb9344e5b02ff023589ae97f2025-08-20T02:14:01ZengBio-protocol LLCBio-Protocol2331-83252025-04-0115810.21769/BioProtoc.5286Antibody Purification Using Spy ChemistryXiaofeng Yang0Zhanglin Lin1Ya Xiang2Zisha Lao3School of Biology and Biological Engineering, South China University of Technology, Guangzhou, Guangdong, ChinaSchool of Biology and Biological Engineering, South China University of Technology, Guangzhou, Guangdong, ChinaSchool of Biology and Biological Engineering, South China University of Technology, Guangzhou, Guangdong, ChinaSchool of Biology and Biological Engineering, South China University of Technology, Guangzhou, Guangdong, ChinaAntibody purification is a fundamental technology for therapeutic and diagnostic applications. While traditional methods like ammonium sulfate precipitation and polyethylene glycol precipitation are cost-effective, they often result in low purity and require multiple purification steps. Protein A–based affinity chromatography, the gold standard for antibody purification, provides high specificity but can be further improved to increase loading capacity and reduce costs. In this protocol, we introduce a novel approach for purifying high-quality, high-purity antibodies from complex samples using SpyFixer/Z domain–modified resin. This method utilizes Spy chemistry for site-specific immobilization of the Z domain of Protein A, significantly enhancing antibody loading capacity up to 200 mg/mL resin and ensuring stable, durable immobilization. Using this protocol, we achieved >90% purity of human immunoglobulin G (hIgG) from diverse sources, including E. coli cell lysates, human serum, and industrial fermentation broth. The SpyFixer/Z domain–modified resin protocol is simple, cost-effective, and offers a robust, scalable solution for efficient antibody purification in bioengineering applications. This immobilization scheme based on Spy chemistry can also be extended to other immunoglobulin-binding proteins, such as Protein G and Protein L, to develop high-efficiency affinity resins.https://bio-protocol.org/en/bpdetail?id=5286&type=0 |
| spellingShingle | Xiaofeng Yang Zhanglin Lin Ya Xiang Zisha Lao Antibody Purification Using Spy Chemistry Bio-Protocol |
| title | Antibody Purification Using Spy Chemistry |
| title_full | Antibody Purification Using Spy Chemistry |
| title_fullStr | Antibody Purification Using Spy Chemistry |
| title_full_unstemmed | Antibody Purification Using Spy Chemistry |
| title_short | Antibody Purification Using Spy Chemistry |
| title_sort | antibody purification using spy chemistry |
| url | https://bio-protocol.org/en/bpdetail?id=5286&type=0 |
| work_keys_str_mv | AT xiaofengyang antibodypurificationusingspychemistry AT zhanglinlin antibodypurificationusingspychemistry AT yaxiang antibodypurificationusingspychemistry AT zishalao antibodypurificationusingspychemistry |