Specific interchain cross-linking of antibodies using bismaleimides. Repression of ligand leakage in immunoaffinity chromatography
| Autor: | Kaja L. Knudsen, Michel Goldberg, Edward A. Bayer, David Platt, Meir Wilchek, Fortune Kohen |
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| Rok vydání: | 1991 |
| Předmět: |
Biomedical Engineering
Pharmaceutical Science Biotin Bioengineering Immunoglobulin light chain Antibodies Chromatography Affinity Sepharose Maleimides chemistry.chemical_compound Affinity chromatography Sulfhydryl Compounds Bifunctional Pharmacology chemistry.chemical_classification Chromatography Elution Organic Chemistry Proteins Cross-Linking Reagents Chaotropic agent chemistry Thiol Biotechnology |
| Zdroj: | Bioconjugate chemistry. 2(4) |
| ISSN: | 1043-1802 |
| Popis: | The extensive use of antibody-containing affinity columns in the purification of biologically active compounds (e.g., genetically engineered proteins) is severely hampered by the leaching of antibody (or portions thereof) from the immunoaffinity resin during elution of the target antigen. One of the major problems in this context is the combined use of reducing (i.e., thiols) and chaotropic (e.g., detergents and denaturants) agents in the elution step, which causes the disassociation of heavy and/or light chains from the immobilized antibody, thereby contaminating the resultant product. In order to overcome this problem, we have cross-linked the four antibody chains at their sites of disulfide interlinkage, thus producing a single antibody chain. To accomplish this, interchain disulfide bonds were reduced, and the resultant thiol groups were cross-linked by using bifunctional SH-specific reagents (particularly bismaleimides). Cross-linking of up to 95% of the available SH groups produced was achieved with concomitant retention of antigen-binding activity. The cross-linked antibody was immobilized onto CNBr-activated Sepharose, and the resultant column was found to be substantially more stable to harsh elution conditions than similar columns which contain the un-cross-linked antibody. |
| Databáze: | OpenAIRE |
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