Received: 15 December 2009, Revised: 6 August 2010, Accepted: 23 August 2010, Published online in Wiley Online Library: 2010 Solid-phase preparation of protein complexes z Paolo Pengo a * , Gianluca Veggiani a , Kwanchai Rattanamanee a,by , Andrea Gallotta a , Luca Beneduce a and Giorgio Fassina a Protein–protein conjugation is usually achieved by solution phase methods requiring concentrated protein solution and post-synthetic purification steps. In this report we describe a novel continuous-flow solid-phase approach enabling the assembly of protein complexes minimizing the amount of material needed and allowing the repeated use of the same solid phase. The method exploits an immunoaffinity matrix as solid support; the matrix reversibly binds the first of the complex components while the other components are sequentially introduced, thus allowing the complex to grow while immobilized. The tethering technique employed relies on the use of the very mild synthetic conditions and fast association rates allowed by the avidin–biotin system. At the end of the assembly, the immobilized complexes can be removed from the solid support and recovered by lowering the pH of the medium. Under the conditions used for the sequential complexation and recovery, the solid phase was not damaged or irreversibly modified and could be reused without loss of binding capacity. The method was specifically designed to prepare protein complexes to be used in immunometric methods of analysis, where the immunoreactivity of each component needs to be preserved. The approach was successfully exploited for the preparation of two different immunoaffinity reagents with immunoreactivity mimicking native squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) and alphafetoprotein-immunoglobulin M (AFP-IgM) immune complexes, which were characterized by dedicated sandwich enzyme-linked immunosorbent assay (ELISA) and immunoblot. Besides the specific application described in the paper, the method is sufficiently general to be used for the preparation of a broad range of protein assemblies. Copyright ß 2010 John Wiley & Sons, Ltd. Keywords: protein complexes; solid-phase complex preparation; immune complexes INTRODUCTION Chemical conjugation is the simplest approach to protein tagging or to the implementation of different biochemical properties in the same protein complex. Though rather simplistic, this approach has allowed the development of the large range of conjugates routinely used by virtually any immunometric methods of analysis. Conjugation usually relies on the formation of covalent bonds between the different partners by exploiting homo- or hetero-bifunctional reagents reacting with aminoacid side chains, usually lysine, cysteine or carboxylic acids con- veniently found on the protein surface. Alternatively, a reactive group, such as an aldehyde, can be introduced by oxidation of the sugar moieties at the protein glycosylation sites and then made to react with the free lysine side chains of another protein species forming an imine or a secondary amine if a suitable reducing agent is present. These conjugation procedures are well described in the literature and are routinely used in the practice (Wong, 1991); other approaches, such as the click-chemistry and the Staudinger ligation, are increasingly being pursued (Brennan et al., 2006; Gauthier and Klok, 2008; Best, 2009). Despite their widespread use, standard conjugation protocols present major drawbacks related to the need of concentrated protein solutions, usually in the range of 1–10 mg/ml or more, (Wong, 1991) to ensure an efficient conjugation and the need of post-synthetic purification steps to remove large polymeric species or unreacted materials. This feature limits the applicability of the approach to cheap proteins stable in concentrated solutions and readily available in large amounts. Conversely, the conjugation of proteins available in small amounts or prone to the formation of aggregates in concentrated solutions would be difficult to achieve by this method. An approach which may, in principle, overcome some of these limitations is the development of efficient solid-phase protocols allowing the preservation of protein structure and functional activity reducing the need of post-synthetic purification processes. Some reports in the literature have described the effort to devise solid-phase approaches to protein conjugation and functionalization indi- cating a growing attention towards these alternative protocols. (wileyonlinelibrary.com) DOI:10.1002/jmr.1095 Research Article * Correspondence to: P. Pengo, Xeptagen SpA, Via delle Industrie 9, Margher- a-Venezia I-30175, Italy. E-mail: pengo@xeptagen.com a P. Pengo, G. Veggiani, K. Rattanamanee, A. Gallotta, L. Beneduce, G. Fassina Xeptagen SpA, Via delle Industrie 9, Marghera-Venezia I-30175, Italy b K. Rattanamanee Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Naresuan University, Pitsanulok, Thailand y Supported by Naresuan and Chulalongkorn Universities within the ICS-UNIDO collaborative framework of activities. z This article is published in Journal of Molecular Recognition as a special issue on Affinity 2009, edited by Gideon Fleminger, Tel-Aviv University, Tel-Aviv, Israel and George Ehrlich, Hoffmann-La Roche, Nutley, NJ, USA. Abbreviations: SCCA, Squamous Cell Carcinoma Antigen; AFP, alphafetopro- tein; IgM, Immunoglobulin M; ELISA, Enzyme-linked Immunosorbent Assay; BSA, Bovine Serum Albumin; IgG, immunoglobulin G; NHS, N-hydroxysuccinimide; HRP, Horseradish Peroxidase; ABTS, 2,2 0 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid); SDS–PAGE, Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis. J. Mol. Recognit. 2010; 23: 551–558 Copyright ß 2010 John Wiley & Sons, Ltd. 551