Llama VHH antibody fragments against GFAP: better diffusion in fixed tissues than classical monoclonal antibodies.
Camelids produce antibodies made of homodimeric heavy chains, and the antigen-binding region being composed of a single domain called VHH. These VHHs are much smaller than complete IgG. They are also more thermostable and more soluble in water; they should, therefore, diffuse more readily in the tissues. VHHs, expressed in bacteria, are easier to produce than conventional monoclonal antibodies. Because of these special characteristics, these antibody fragments could have interesting developments in immunohistochemistry and in the development of biomarkers. To test the possibility of their use in immunohistochemistry (IHC), we selected the glial fibrillary acidic protein (GFAP), a well-known marker of astrocytes. One alpaca (Lama pacos) was immunized against GFAP. Lymphocytes were isolated; the DNA was extracted; the VHH-coding sequences were selectively amplified. Three VHHs with a high affinity for GFAP and their corresponding mRNA were selected by ribosome display. Large quantities of the recombinant VHHs coupled with different tags were harvested from transfected bacteria. One of them was shown to immunolabel strongly and specifically to GFAP of human astrocytes in tissue sections. The quality of the IHC was comparable or, in some aspects, superior to the quality obtained with conventional IgG. The VHH was shown to diffuse on a longer distance than conventional monoclonal antibodies in fixed cortical tissue: a property that may be useful in immunolabeling of thick sections.
Perruchini
Claire
C
INSERM U289, Hôpital de la Salpêtrière, Paris, France.
Pecorari
Frederic
F
Bourgeois
Jean-Pierre
JP
Duyckaerts
Charles
C
Rougeon
François
F
Lafaye
Pierre
P
D000544:Alzheimer Disease / Q000378:metabolism / Q000473:pathology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001932:Brain Neoplasms / Q000378:metabolism / Q000473:pathology; D002161:Camelids, New World / Q000276:immunology*; D002540:Cerebral Cortex / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015870:Gene Expression; D015723:Gene Library; D005904:Glial Fibrillary Acidic Protein / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology*; D008297:Male; D008970:Molecular Weight; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D005904:Glial Fibrillary Acidic Protein; D007143:Immunoglobulin Heavy Chains; D012333:RNA, Messenger; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies