Short communication Immobilization of antibodies through the surface regions having the highest density in lysine groups on finally inert support surfaces Pilar Batalla, Cesar Mateo, Valeria Grazu, Roberto Fernandez-Lafuente *, Jose M. Guisan * Departamento de Biocata ´lisis, Instituto de Cata ´lisis (CSIC), Campus UAM Cantoblanco, 28049 Madrid, Spain 1. Introduction Immobilized antibodies have a great potential application in areas as biosensor design [1–3] or immunoaffinity chromatogra- phy [4–7]. However, the immobilization step is critical in the design of the final system. The immobilization system must fulfill some requirements. First, the support surface needs to be fully inert to prevent unspecific adsorptions of other components of the mixture during use, after the antibody immobilization. The unspecific adsorption may greatly reduce the sensitivity of the biosensor (by increasing the blank signal) or the purification degree achieved in immunoaffinity chromatography. Moreover, the antibody must keep its capability of recognize the antigen. This means that the antibody structure should be maintained and also the Fab region needs to be sufficiently exposed to the medium. One of the solutions to get this right orientation is to immobilize it by the Fc regions, in an area opposite to the Fab region. This orientation has been achieved using different immobilization protocols: using immobilized protein A [8,9] or protein G [10,11], via covalent immobilization through the oxidized sugar chains of the antibody [12,13] by site directed biotinylation of the Fc region to get site direct immobilization on avidin or streptavidin supports [14,15], etc. However, for some applications, may be more convenient to have the Fab region of the antibody very near to the supports surface. That may be the case of the use of antibodies immobilized on carbon nanotubes, if the capture of the antigen is to be detected by nanotube field effect transistors technique [16–18]. Fig. 1 shows the distribution of amino residues (e-amino of Lys and terminal amino group) in an IgG. The four terminal amino groups of the antibody (that are usually the fully exposed amino groups presenting the highest reactivity in a protein) are near to the recognition place, thus immobilization via these groups do not seem to be desirable. Consequently, immobilization of antibodies using supports that directly immobilize proteins via the most reactive amine should not be a suitable one [19]. In this way, apparently the immobilization of antibodies using their amino groups did not seem to be an appropriate one. The immobilization of the proteins on glyoxyl supports involves the amino groups of the protein, but occurs via a multipoint process [20]. This makes that a protein become immobilized on these supports by the richest area in amino groups very rapidly [21], no by the most reactive amino group like in most of the amino-reactive supports. This feature has permitted that glyoxyl supports have been used for immobilization and stabilization of many enzymes [22]. After the immobilization of proteins in these supports, a last reduction step (e.g., using sodium borohydride) finally gives a physically and chemically inert surface [23]. Process Biochemistry 44 (2009) 365–368 ARTICLE INFO Article history: Received 28 October 2008 Accepted 25 November 2008 Keywords: Immobilized antibodies Glyoxyl supports Oriented immobilization Inert surfaces Biosensor Immuno-chromatography matrices ABSTRACT Immobilization of anti-horseradish peroxidase on glyoxyl-agarose proceeds rapidly, and after the immobilization, it was found that the antibody captured almost the same amount of peroxidase than the free antibody. After boiling the antibodies in the presence of SDS and mercaptoethanol, more than 95% of the immobilized antibodies presented the four subunits attached to the support. The reduction of the preparation converts the glyoxyl groups into very hydrophilic and inert hydroxyl-groups. That way, the final support was fully unable to adsorb any protein under any condition, and the only adsorbed proteins on the immobilized antibody are these recognized by the antibody. The immobilized antibody maintained intact their capacity to capture peroxidase after 20 weeks of storage at 4 8C. The high functionality of the immobilized antibody and the fully inert surface suggest that this technique may be a very suitable one to immobilize antibodies for biosensor design or immuno- chromatographic matrices. ß 2008 Elsevier Ltd. All rights reserved. * Corresponding authors. Tel.: +34 91 585 4809; fax: +34 91 585 4760. E-mail addresses: rfl@icp.csic.es (R. Fernandez-Lafuente), jmguisan@icp.csic.es (J.M. Guisan). Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio 1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.11.017