Carta, Franco and Lobina, Omar and Mannu, Franca Rita and Ferru, Emanuela and Pantaleo, Antonella and Orrù, Marcella and Turrini, Francesco Michelangelo (2012) A 2DE approach for high-throughput antigen separation applicable to mAb production. Electrophoresis, Vol. 33 (16), p. 2546-2552. eISSN 1522-2683. Article.
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A constantly increasing number of mABs are required for the validation of a large proportion of proteomic and protein–protein interaction data. The development of new robotic platforms has greatly enhanced the throughput of monoclonal antibody production; however, the availability of highly purified proteins to use as antigens currently represents the major bottleneck of the process. In this article, we describe a new 2DE approach to purify hundreds of proteins from cellular extracts in a very cost-effective and time-efficient way. The accuracy of the new purification method is shown to be comparable to high-resolution analytical 2DE. The effectiveness and the throughput of the method to purify proteins suitable for the development ofmAbs are then assessed. Using this methodology, we were able to separate 447 proteins starting from 50 mg of proteins extracted from HT29 cells. Fractions containing more than 30 g of protein constantly induced immunization in mice. Using a high-throughput process for monoclonal antibody production, we obtained an average of 3.5 mAbs for each protein. According to pilot experiments, we can predict that starting from an unfractionated cellular extract it is possible to obtain approximately 200 proteins usable for monoclonal antibody development. Our results indicate that the number of antigens available formonoclonal antibody production can be further increased by running parallel separations. The proposed methodology will then facilitate the highthroughput monoclonal antibody process providing a vast array of high quality antigens at very low cost.
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