title: Generation of VHH antibodies against the Arabidopsis thaliana seed storage proteins.
abstract: Antibodies and antibody derived fragments are excellent tools for the detection and purification of proteins. However, only few antibodies targeting Arabidopsis seed proteins are currently available. Here, we evaluate the process to make antibody libraries against crude protein extracts and more particularly to generate a VHH phage library against native Arabidopsis thaliana seed proteins. After immunising a dromedary with a crude Arabidopsis seed extract, we cloned the single-domain antigen-binding fragments from their heavy-chain only antibodies in a phage display vector and selected nanobodies (VHHs) against native Arabidopsis seed proteins. For 16 VHHs, the corresponding antigens were identified by affinity purification and MS/MS analysis. They were shown to bind the major Arabidopsis seed storage proteins albumin and globulin (14 to albumin and 2 to globulin). All 16 VHHs were suitable primary reagents for the detection of the Arabidopsis seed storage proteins by ELISA. Furthermore, several of the anti-albumin VHHs were used successfully for storage protein localisation via electron microscopy. The easy cloning, selection and production, together with the demonstrated functionality and applicability, strongly suggest that the VHH antibody format will play a more prominent role in future protein research, in particular for the study of native proteins.
authors: Department of Plant Systems Biology, VIB, 9052, Ghent, Belgium.
authors: Thomas
authors: T
authors: De Meyer
authors: Dominique
authors: D
authors: Eeckhout
authors: Riet
authors: R
authors: De Rycke
authors: Sylvie
authors: S
authors: De Buck
authors: Serge
authors: S
authors: Muyldermans
authors: Ann
authors: A
authors: Depicker
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000097:blood / Q000276:immunology* / Q000378:metabolism; D052179:Antigens, Plant / Q000276:immunology*; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000276:immunology* / Q000378:metabolism; D002162:Camelus / Q000097:blood; D008969:Molecular Sequence Data; D010802:Phylogeny; D055314:Seed Storage Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000906:Antibodies; D052179:Antigens, Plant; D029681:Arabidopsis Proteins; D055314:Seed Storage Proteins
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