Improving Broad Specificity Hapten Recognition with Protein Engineering TEEMU KORPIMA¨ KI,* ,² JAANA ROSENBERG, ‡ PEKKA VIRTANEN, ² TUOMAS KARSKELA, ‡ URPO LAMMINMA¨ KI, ² MIKA TUOMOLA, ² MARKUS VEHNIA¨ INEN, ² AND PETRI SAVIRANTA ² Departments of Biotechnology and Bio-Organic Chemistry, University of Turku, FIN-20520 Turku, Finland Sulfa antibiotics (sulfonamides) are derivatives of p-aminobenzenesulfonamide that are widely used in veterinary medicine. Foods derived from treated animals may be contaminated with these drugs. However, current immunobased sulfonamide detection methods are unfit for screening of products because they are either too insensitive or specific for a few compounds only. An immunoassay capable of detecting all sulfas in a single reaction would be ideal for screening. For development of a binder capable of binding all sulfas, a protein engineering approach was chosen and the properties of monoclonal antibody 27G3 were improved with mutagenesis followed by selection with phage display. Several different mutant antibodies were isolated. The cross-reaction profile of the best mutant antibody was significantly improved over that of the wild-type antibody: it was capable of binding 9 of the tested 13 sulfonamides within a narrow concentration range and also bound the rest of the sulfas, albeit within a wider concentration range. KEYWORDS: Drug residues; group specificity; phage display; protein engineering; sulfonamides; time- resolved fluoroimmunoassay INTRODUCTION Sulfa antibiotics (sulfonamides) are a group of antimicrobial agents that are used in veterinary and human medicine for the treatment and prevention of bacterial infections and also as animal feed additives, because of their growth-promoting properties. Antimicrobial sulfonamides (Figure 1) are N 1 - substituted derivatives of p-aminobenzenesulfonamide (sulfa- nilamide), and they are thus structurally related. As a result of their use, foodstuffs (for example, meat and milk) derived from treated animals may be contaminated with sulfonamide drugs. These residues may cause adverse effects in some humans, as indicated by the fact that ∼5% of the patients receiving sulfonamide therapy experience some kind of unwanted effect (1). The maximum residue limit (MRL) for sulfonamides has been set to 100 μg/kg in the United States and the European Union, but, for example, in Japan it is as low as 20 μg/kg (2, 3). The current sulfonamide detection technologies are based on bacteriological growth inhibition (4, 5) or chromatographic methods (6-10). In addition, single sulfonamide drugs have been measured with immunochemical methods (11, 12). These methods are either laborious or slow for mass screening or, in the case of immunoassays, capable of detecting only a single analyte per each assay reaction. A rapid immunoassay capable of detecting nearly all sul- fonamides in one reaction would be very useful for the mass screening of foodstuffs. The development of this kind of immunoassay has, however, proven to be difficult; despite several efforts (1-3, 13, 14), a completely generic assay is still not available. The main obstacle is the development of an antibody capable of binding all different sulfonamides with an affinity yielding sufficient assay sensitivity. It seems that the obtained antibodies recognize the used immunogen and the * Address correspondence to this author at the Department of Biotech- nology, University of Turku, Tykisto¨katu 6, 6th Floor, FIN-20520 Turku, Finland (telephone +358-2-3338067; fax +358-2-3338050; e-mail teemu.korpimaki@utu.fi). ² Department of Biotechnology. ‡ Department of Bio-Organic Chemistry. Figure 1. Structures of selected sulfonamides. 4194 J. Agric. Food Chem. 2002, 50, 4194-4201 10.1021/jf0200624 CCC: $22.00 © 2002 American Chemical Society Published on Web 06/12/2002