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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Beyer, Natascha Helena Hansen, Morten Zoega Schou, Christian Højrup, Peter Heegaard, Niels H. H. |
| Description | Country affiliation: Denmark Author Affiliation: Beyer NH ( Department of Clinical Biochemistry and Immunology, Statens Serum Institute, Copenhagen S., Denmark.) |
| Abstract | The covalent immobilization of antibodies to solid supports for immunoaffinity (IA) purification is widely used in the biological sciences. However, relative immobilization yields, immobilization stability, and antigen-binding capacity vary significantly with the antibody and protocol used. A systematic study was conducted to determine the most versatile antibody immobilization method for use in on-line and off-line IA chromatography applications using commonly accessible immobilization methods. Four chemistries were examined using polyclonal and monoclonal antibodies and antibody fragments. We evaluated a method to survey optimal elution conditions and estimated immobilization yields, matrix stability, antigen binding capacities, and antigen recovery of different IA matrices. Some mAbs were sensitive to aminogroup-based immobilization, i.e., losing antigen binding capabilities after immobilization especially using epoxy chemistry. In general, the most optimal covalent antibody immobilization for on-line IA-LC-MS was achieved using aminogroup immobilization of intact antibodies by epoxy- or aldehyde-activated POROS R20-matrices and in some cases by chemical crosslinking to Protein G-POROS. Protein G-based matrices are very stable showing essentially no decline in performance after 50 application-wash-elution-reequilibration cycles and being easily prepared within 2-3 h of working time with a typical antibody coupling yield of above 80%. In off-line applications where constant flow conditions are not used, covalent crosslinking onto Protein G-POROS or Protein G-agarose is to be recommended. |
| File Format | HTM / HTML |
| ISSN | 16159306 |
| Issue Number | 10 |
| Volume Number | 32 |
| e-ISSN | 16159314 |
| Journal | Journal of Separation Science |
| Language | English |
| Publisher | Wiley-VCH |
| Publisher Date | 2009-05-01 |
| Publisher Place | Germany |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Discipline Analytical Discipline Chemistry Antibodies, Immobilized Chemistry Immunology Antigens Isolation & Purification Chromatography, Affinity Methods Animals Antigen-antibody Complex Epoxy Compounds Immunoglobulin Fab Fragments Nerve Tissue Proteins Journal Article |
| Content Type | Text |
| Resource Type | Article |
| Subject | Analytical Chemistry Filtration and Separation |
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