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Construction of scFv fragments from hybridoma or spleen cells by PCR assembly


Schaefer, J V; Honegger, A; Plückthun, A (2010). Construction of scFv fragments from hybridoma or spleen cells by PCR assembly. In: Kontermann, R; Dübel, S. Antibody Engineering. Berlin Heidelberg: Springern-Verlag, 21-44.

Abstract

Today, antibodies can be obtained from naive repertoires (Winter et al. 1994; Vaughan et al. 1996) or libraries of fully synthetic genes (Knappik et al. 2000),
and in the last decade, numerous libraries have been described (reviewed in Mondon et al. 2008). Nonetheless, hybridomas have remained the predominant
source of antibodies, and a wealth of well characterized and even unique clones exist and are continuing to be generated. There is, thus, great interest in immortalizing
these clones, in the extreme case, as a computer file of the sequences, as well as in accessing the antibody in a variety of new formats. To obtain enough material for detailed biochemical and biophysical analyses of the deduced antibodies after immunization, their cloning into formats compatible with recombinant expression is beneficial, if not essential. For this purpose, the antibody genes must be cloned, and the binding properties of the recombinant protein have to be verified. In addition to existing hybridomas, the immune response of an animal upon exposure to various antigens may often be of particular scientific interest in itself and also lead to the discovery of new and potent binders. Therefore, there is merit in immortalizing the results from new immunizations as well. In this case, it is not necessary to take the detour of first making hybridomas, but instead, mRNA isolated from spleen can be directly used for the creation of an immune library, from which binders can be subsequently isolated by phage display and their sequences determined.

Abstract

Today, antibodies can be obtained from naive repertoires (Winter et al. 1994; Vaughan et al. 1996) or libraries of fully synthetic genes (Knappik et al. 2000),
and in the last decade, numerous libraries have been described (reviewed in Mondon et al. 2008). Nonetheless, hybridomas have remained the predominant
source of antibodies, and a wealth of well characterized and even unique clones exist and are continuing to be generated. There is, thus, great interest in immortalizing
these clones, in the extreme case, as a computer file of the sequences, as well as in accessing the antibody in a variety of new formats. To obtain enough material for detailed biochemical and biophysical analyses of the deduced antibodies after immunization, their cloning into formats compatible with recombinant expression is beneficial, if not essential. For this purpose, the antibody genes must be cloned, and the binding properties of the recombinant protein have to be verified. In addition to existing hybridomas, the immune response of an animal upon exposure to various antigens may often be of particular scientific interest in itself and also lead to the discovery of new and potent binders. Therefore, there is merit in immortalizing the results from new immunizations as well. In this case, it is not necessary to take the detour of first making hybridomas, but instead, mRNA isolated from spleen can be directly used for the creation of an immune library, from which binders can be subsequently isolated by phage display and their sequences determined.

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Additional indexing

Item Type:Book Section, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2010
Deposited On:26 Jan 2011 11:44
Last Modified:07 Dec 2017 06:15
Publisher:Springern-Verlag
Number:2
ISBN:978-3-642-01143-6
Publisher DOI:https://doi.org/10.1007/978-3-642-01144-3_3
Official URL:http://www.springer.com/new+%26+forthcoming+titles+(default)/book/978-3-642-01143-6

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