Liu S.,CAS Institute of Physics |
Zhang H.,CAS Technical Institute of Physics and Chemistry |
Dai J.,CAS Institute of Physics |
Hu S.,Guanajibo Research and Innovation Park |
And 4 more authors.
mAbs | Year: 2015
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. © Shuang Liu, Hongyan Zhang, Jun Dai, Shaohu Hu, Ignacio Pino, Daniel J Eichinger, Huibin Lyu, and Heng Zhu.
Jeong J.S.,Johns Hopkins University |
Jiang L.,Johns Hopkins University |
Albino E.,Guanajibo Research and Innovation Park |
Marrero J.,Guanajibo Research and Innovation Park |
And 16 more authors.
Molecular and Cellular Proteomics | Year: 2012
To broaden the range of tools available for proteomic research, we generated a library of 16,368 unique full-length human ORFs that are expressible as N-terminal GST-His6 fusion proteins. Following expression in yeast, these proteins were then individually purified and used to construct a human proteome microarray. To demonstrate the usefulness of this reagent, we developed a streamlined strategy for the production of monospecific monoclonal antibodies that used immunization with live human cells and microarray-based analysis of antibody specificity as its central components. We showed that microarray-based analysis of antibody specificity can be performed efficiently using a two-dimensional pooling strategy. We also demonstrated that our immunization and selection strategies result in a large fraction of monospecific monoclonal antibodies that are both immunoblot and immunoprecipitation grade. Our data indicate that the pipeline provides a robust platform for the generation of monoclonal antibodies of exceptional specificity. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.