Gram, Hermann and Marconi, Lori-Anne and Barbas III, Carlos F. and Collet, Thomas A. and Lerner, Richard A. and Kang, Angray S. (1992) In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library. Proceedings of the National Academy of Sciences, 89 (8). pp. 3576-3580. ISSN 0027-8424
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Official URL: http://www.pnas.org/cgi/reprint/89/8/3576
We have used a combinatorial immunoglobulin library approach to obtain monoclonal antibodies from nonimmune adult mice, thereby establishing the principles of (i) accessing naive combinatorial antibody libraries for predetermined specificities and (ii) increasing the affinity of the selected antibody binding sites by random mutagenesis. A combinatorial Fab library expressing immunoglobulin Ip and it light-chain fragments on the surface of filamentous phage was prepared from bone marrow of nonimmunized, adult BALB/c mice with the multivalent display vector pComb8. Phage displaying low affinity Fabs (binding constants, 104-1O M-') binding to a progesterone-bovine serum albumin conjugate were isolated from the library. Random mutagenesis of the heavy- and light-chain variable regions expressed in the monovalent phage display vector pComb3 was performed by errorprone PCR, and subsequently clones with improved affinity for the hapten conjugate were selected. We demonstrate that antibodies with desirable characteristics from a nonimmune source may be selected and affinity maturation may be achieved by using the twin vectors pComb8 and pComb3, thus opening the route to obtaining specific antibodies from a generic library and bypassing immunization.
|Research Community:||University of Westminster > Life Sciences, School of|
|Deposited On:||02 Jan 2008|
|Last Modified:||23 Dec 2009 14:26|
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