Reliable and controllable antibody fragment selections from Camelid non-immune libraries for target validation.
With the completion of the sequence of the human genome, emphasis is now switching to the human proteome. However, the number of proteins is not only larger than mRNAs in the transcriptome, proteins need often to be in complex with other proteins to be functional. A favourable option to study proteins in their natural context is with a combination of biochemical and microscopic techniques using specific antibodies. Therefore, we designed a fast, reliable and controllable selection and screening of single-domain antibody fragments (VHH) from a Camelid non-immune library. We isolated VHH for four muscle disease related proteins; emerin, actin, tropomyosin-1, and nuclear poly(A)-binding protein. Important features of antibodies for target validation studies are recognition of the antigen in natural conformations and biologically relevant complexes. We show that selected antibody fragments are functional in various immunological techniques and prove useful in diagnostic applications. Our selection strategy is amenable to automation and to the establishment of proteomics platforms. It opens the way to quickly and cost-effectively obtain multiple antibody fragments for many antigens that can detect changes in their localization, level, and modification as well as subtle changes in supramolecular structures, which often associate with disease.
Verheesen
Peter
P
University of Utrecht, Department of Molecular and Cellular Biology, Utrecht, The Netherlands. pverheesen@hotmail.com
Roussis
Andreas
A
de Haard
Hans J
HJ
Groot
Arjan J
AJ
Stam
Jord C
JC
den Dunnen
Johan T
JT
Frants
Rune R
RR
Verkleij
Arie J
AJ
Theo Verrips
C
C
van der Maarel
Silvère M
SM
D000199:Actins / Q000235:genetics / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics; D000915:Antibody Affinity; D002161:Camelids, New World / Q000235:genetics* / Q000276:immunology*; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000276:immunology; D039122:Poly(A)-Binding Protein I / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014335:Tropomyosin / Q000235:genetics / Q000276:immunology
D000199:Actins; D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D008565:Membrane Proteins; D009687:Nuclear Proteins; D039122:Poly(A)-Binding Protein I; D011994:Recombinant Proteins; C490775:TPM1 protein, human; D014335:Tropomyosin; C092710:emerin