Enzyme and Microbial Technology 36 (2005) 510–513 Aldehyde–dextran–protein conjugates to immobilize amino-haptens: avoiding cross-reactions in the immunodetection Manuel Fuentes, Cesar Mateo, Roberto Fernandez-Lafuente ∗ , Jose M. Guis´ an 1 Departamento de Biocat´ alisis, Instituto de Cat´ alisis (CSIC), Campus UAM Cantoblanco, 28049 Madrid, Spain Received 22 March 2004; received in revised form 8 November 2004; accepted 17 November 2004 Abstract Proteins covered with aldehyde–dextran have been used as carrier molecules to detect the immuno-response against an aminated hapten. These conjugates were used in immunodetection after immobilization on ELISA plates and compared to hapten–protein conjugates produced by the same chemistry that was used to induce the immuno-response (by carbodiimide coupling method). It has been found that the use of dextran–protein carriers conjugates avoid the high cross-reaction found using the carbodiimide routes. The modification degree of the dextran by aminated haptens depended on the pH (the best results were achieved at alkaline pH values) and presence of organic solvents (the presence of dioxane improved the modification). © 2004 Elsevier Inc. All rights reserved. Keywords: Aldehyde–dextran; Proteins; Amino-haptens 1. Introduction The detection of antibodies produced against small com- pounds by ELISA assays in serum requires the hapten to be immobilized on microtiter plates. The direct adsorption of these small molecules on ELISA plates is complicated in a routine assay. Thus, the hapten is usually immobilized on microtiter plates via immobilization of hapten–protein car- rier conjugates [1–9]. Carbodiimide-mediated coupling is one of the most commonly used techniques to covalently link aminated or carboxylic haptens to carrier proteins for the production of anti-hapten antibodies [10–14]. This reagent may yield different side-modifications on the protein surface, together to the coupling reaction [10–14]. This makes that proteins treated with this reagent may give cross-immunoreaction, making the analysis of the anti-hapten antibodies serum more complicated [10–14]. ∗ Corresponding author. 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. Guis´ an). 1 Co-corresponding author. Therefore, the advantages promoted by the use of a dif- ferent chemistry in the immunogenization process and the evaluation of the antibodies title in serum by ELISA assays seems evident [15–17]. In this way, dextran seems a suit- able molecule for this purpose. These molecules are very flexible, with low immunogenecity, acts as a long spacer arm (permitting an easy hapten recognition by the antibody), and are very hydrophilic and inert, preventing any unde- sired protein adsorption on the plate surface or the protein [18–20]. A previous study [21], described a methodology employ- ing s-triazine–dextran conjugates to coat ELISA plates. Now, we propose the use of aldehyde–dextran–protein conjugates as a simpler carrier for immobilization of small ligands on standard ELISA plates surfaces. After checking the possibilities of this conjugate, the aldehyde-amino hapten reaction has been optimized. To simplify the studies, these have been performed using immobilized protein, and as a molecular model we have chosen octyldiamine, an primary amino group with a fairly unfavorable pK (over 10) and quite simple to quantify by using 2,4,6-trinitrobenzenesulfonic acid (TNBS) reaction [22]. 0141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2004.11.004