Abstract
Monoclonal antibodies (mAbs) against human proteins are the primary protein capture reagents for basic research, diagnosis, and molecular therapeutics. The 2 most important attributes of mAbs used in all of these applications are their specificity and avidity. While specificity of a mAb raised against a human protein can be readily defined based on its binding profile on a human proteome microarray, it has been a challenge to determine avidity values for mAbs in a high-throughput and cost-effective fashion. To undertake this challenge, we employed the oblique-incidence reflectivity difference (OIRD) platform to characterize mAbs in a protein microarray format. We first systematically determined the Kon and Koff values of 50 mAbs measured with the OIRD method and deduced the avidity values. Second, we established a multiplexed approach that simultaneously measured avidity values of a mixture of 9 monospecific mAbs that do not cross-react to the antigens. Third, we demonstrated that avidity values of a group of mAbs could be sequentially determined using a flow-cell device. Finally, we implemented a sequential competition assay that allowed us to bin multiple mAbs that recognize the same antigens. Our study demonstrated that OIRD offers a high-throughput and cost-effective platform for characterization of the binding kinetics of mAbs.
Original language | English (US) |
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Pages (from-to) | 110-119 |
Number of pages | 10 |
Journal | mAbs |
Volume | 7 |
Issue number | 1 |
DOIs | |
State | Published - Jan 1 2015 |
Keywords
- Affinity
- Avidity
- KD
- Kinetics
- Monoclonal antibodies
- OIRD
- Protein microarrays
ASJC Scopus subject areas
- Immunology and Allergy
- Immunology