Antibodies, A Laboratory Manual, Second Edition, Edited by Edward A. Greenfield

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Modification of Antibody Function by Mutagenesis

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After immunoglobulin heavy and light chains for a particular antibody have been cloned, the binding sitenamely, the CDR regionscan be manipulated by mutagenesis to obtain antibody variants with improved properties. A wide number of mutagenic approaches have been described. Many of the mutagenic approaches are based on the seminal work of Kunkel (1985) and thus have been referred to as Kunkel mutagenesis. Several improvements to the Kunkel mutagenesis approach have been described. A recent paper by Huovinen et al. (2012) improved the number of mutant transformants by 300-fold over the Kunkel method by using rolling circle amplification. This work, like many papers in this field, is the subject of an international patent application (PCT/FI2010/051068). For this chapter, an approach described by Kelley and Momany (2003) is included. This method is relatively simple, uses commercially available reagents, and is effective. Phage pComb3H, used by Kelley and Momany, is available from Scripps Research Institute, after execution of a Material Transfer agreement ( The sequence of pComb3H is available at GenBank (AY254174). Using the pComb3H vector, a commercial mutagenesis kit, PfuTurbo Polymerase (Agilent), and two mutagenic primers, a library of phage with mutagenized heavy and light CDR3 can be obtained.

Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition

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