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Generation of VHH antibodies against the Arabidopsis thaliana seed storage proteins

(2014) PLANT MOLECULAR BIOLOGY. 84(1-2). p.83-93
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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.
Keywords
MICROSCOPY, Arabidopsis thaliana, GENE, PHAGE, EXPRESSION, FRAGMENTS, NANOBODIES, Nanobody, Phage library, Seed storage proteins, 2S ALBUMIN, HUMAN PROTEOME, RECOMBINANT ANTIBODIES, HEAVY-CHAIN ANTIBODIES

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Chicago
De Meyer, Thomas, Dominique Eeckhout, Riet De Rycke, Sylvie De Buck, Serge Muyldermans, and Anna Depicker. 2014. “Generation of VHH Antibodies Against the Arabidopsis Thaliana Seed Storage Proteins.” Plant Molecular Biology 84 (1-2): 83–93.
APA
De Meyer, Thomas, Eeckhout, D., De Rycke, R., De Buck, S., Muyldermans, S., & Depicker, A. (2014). Generation of VHH antibodies against the Arabidopsis thaliana seed storage proteins. PLANT MOLECULAR BIOLOGY, 84(1-2), 83–93.
Vancouver
1.
De Meyer T, Eeckhout D, De Rycke R, De Buck S, Muyldermans S, Depicker A. Generation of VHH antibodies against the Arabidopsis thaliana seed storage proteins. PLANT MOLECULAR BIOLOGY. 2014;84(1-2):83–93.
MLA
De Meyer, Thomas, Dominique Eeckhout, Riet De Rycke, et al. “Generation of VHH Antibodies Against the Arabidopsis Thaliana Seed Storage Proteins.” PLANT MOLECULAR BIOLOGY 84.1-2 (2014): 83–93. Print.
@article{4223629,
  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.},
  author       = {De Meyer, Thomas and Eeckhout, Dominique and De Rycke, Riet and De Buck, Sylvie and Muyldermans, Serge and Depicker, Anna},
  issn         = {0167-4412},
  journal      = {PLANT MOLECULAR BIOLOGY},
  keyword      = {MICROSCOPY,Arabidopsis thaliana,GENE,PHAGE,EXPRESSION,FRAGMENTS,NANOBODIES,Nanobody,Phage library,Seed storage proteins,2S ALBUMIN,HUMAN PROTEOME,RECOMBINANT ANTIBODIES,HEAVY-CHAIN ANTIBODIES},
  language     = {eng},
  number       = {1-2},
  pages        = {83--93},
  title        = {Generation of VHH antibodies against the Arabidopsis thaliana seed storage proteins},
  url          = {http://dx.doi.org/10.1007/s11103-013-0118-0},
  volume       = {84},
  year         = {2014},
}

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