ORIGINAL PAPER Llama V H H antibody fragments against GFAP: better diffusion in fixed tissues than classical monoclonal antibodies Claire Perruchini Æ Frederic Pecorari Æ Jean-Pierre Bourgeois Æ Charles Duyckaerts Æ Franc ¸ois Rougeon Æ Pierre Lafaye Received: 5 June 2009 / Revised: 2 July 2009 / Accepted: 4 July 2009 / Published online: 14 July 2009 Ó Springer-Verlag 2009 Abstract Camelids produce antibodies made of homodimeric heavy chains, and the antigen-binding region being composed of a single domain called V H H. These V H Hs 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. V H Hs, expressed in bacteria, are easier to produce than conven- tional 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 V H H-coding sequences were selectively amplified. Three V H Hs with a high affinity for GFAP and their corresponding mRNA were selected by ribosome display. Large quantities of the recombinant V H Hs coupled with different tags were harvested from transfected bacte- ria. One of them was shown to immunolabel strongly and specifically to GFAP of human astrocytes in tissue sec- tions. The quality of the IHC was comparable or, in some aspects, superior to the quality obtained with conventional IgG. The V H H 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. Keywords Homodimeric antibodies Á Camel Á GFAP Á V H H Á Immunohistochemistry Introduction Hamers-Casterman et al. [15] have serendipitously found that about 50% of the IgG antibodies from Camelidae species (camels, dromedaries, llamas and alpacas) are ‘‘heavy chain antibodies’’, devoid of light chain. These antibodies form homodimers and interact with the antigens by the virtue of only one single variable domain of the heavy chain, referred to as V H H (VH domain of heavy- chain antibodies) to distinguish it from conventional VH [1, 2, 5, 26] (Fig. 1). Despite the absence of light chain, the heavy-chain antibodies exhibit a broad antigen-binding repertoire, their hypervariable region being expanded and exhibiting special characteristics, such as the substitution of three to four hydrophobic residues (which interact with the VL in common antibodies) by more hydrophilic amino acids (for reviews, see [6, 17, 25]). Recombinant V H Hs have a number of advantages compared with the conven- tional antibody fragments (Fab or Fv) derived from other mammals: they are highly soluble in aqueous environments and are stable at high temperature [29, 35]. V H H have also been shown to be plastic: after denaturation by heat, most of them are able to refold and to recover their initial C. Perruchini Á C. Duyckaerts INSERM U289, Ho ˆpital de la Salpe ˆtrie `re, Paris, France F. Pecorari CNRS UMR 6204, Universite ´ de Nantes, Nantes, France J.-P. Bourgeois Institut Pasteur, Unite ´ de Ge ´ne ´tique Humaine et Fonctions Cognitives, CNRS URA 2182, Paris Cedex, France F. Rougeon Á P. Lafaye (&) Institut Pasteur, Unite ´ de Ge ´ne ´tique et Biochimie du De ´veloppement, CNRS U2581, Rue du Dr Roux, 75724 Paris Cedex 15, France e-mail: plafaye@pasteur.fr 123 Acta Neuropathol (2009) 118:685–695 DOI 10.1007/s00401-009-0572-6