Production and Characterization of Monoclonal Antibodies against the Antibiotic Tilmicosin ROSS C. BEIER,* ,† LAWRENCE C. CREEMER, ‡ RICHARD L. ZIPRIN, † AND DAVID J. NISBET † Southern Plains Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, 2881 F&B Road, College Station, Texas 77845-4998, and Elanco Animal Health, A Division of Eli Lilly and Company, 2001 West Main Street, Greenfield, Indiana 46140 Monoclonal antibodies (Mabs) were developed that specifically bind tilmicosin. Keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) conjugates were used for the immunogen and plate coating antigen, respectively. The conjugates were synthesized by different methods, resulting in different linkages. Six hybridoma cell lines were isolated that produced Mabs that competed with tilmicosin, and have IgG1 isotype. The Til-1 and Til-5 Mabs had IC 50 values for tilmicosin of 9.6 and 6.4 ng/well (48 and 32 ng/mL), respectively, and limits of detection at IC 20 of 1.84 and 0.89 ng/well (9.2 and 4.45 ng/mL), respectively. The Mabs demonstrated high cross-reactivity to the macrolides containing 3,5-dimethylpiperidine at C20 and the amino sugar at C5. No cross-reactivity was observed for tylosin and other macrolides that did not contain 3,5-dimethylpiperidine. A competitive enzyme- linked immunosorbent assay (ELISA) was developed for the antibiotic tilmicosin by use of the developed Mabs. These Mabs may be excellent candidates for the determination and immunolocal- ization of tilmicosin. KEYWORDS: Antibiotic; ELISA; immunoassay; Mabs; macrolide antibiotics; monoclonal antibodies; tilmicosin; tylosin INTRODUCTION 20-Deoxo-20-(3,5-dimethylpiperidin-1-yl)desmycosin (tilmi- cosin) is a semisynthetic macrolide broad-spectrum antibiotic derived from the macrolide antibiotic tylosin (1, 2)(Figure 1), which is produced by fermentation of Streptomyces fradiae. As a result of its long half-life, it is effective for the treatment of respiratory diseases in cattle (3-5). A single subcutaneous injection in cattle of 10 mg/kg tilmicosin can result in pulmonary tissue tilmicosin levels above the minimum inhibitory concen- tration (MIC) for Pasteurella haemolytica (6), a cause of pneumonia in cattle (3). Most antimicrobial agents have limited ability for cellular penetration, but tilmicosin accumulates in polymorphonuclear neutrophilic leukocytes (PMN) and mac- rophage cells in bovine lung tissue (7). Tilmicosin is effective for treating cattle with leptospirosis caused by Leptospira borgpetersenii serovar Hardjo (8), which can cause mastitis, decreased milk production, weak calves, abortion, and infertility (9). Tilmicosin is approved for veterinary use in swine (10) and poultry (11) to combat respiratory diseases and also is used for treating sheep in various areas of the world for mastitis and respiratory disease (12-14). Attempts have been made to produce antibodies to tylosin- related macrolides for use in determining residues of tilmicosin and tylosin in feeds and tissues. Jackman et al. (15) reported preparation of polyclonal antibodies against desmycosin, con- jugated through the C20 aldehyde group; however, such * To whom correspondence should be addressed: phone 979-260-9411; fax 979-260-9332; e-mail rcbeier@ffsru.tamu.edu. † Southern Plains Agricultural Research Center. ‡ Elanco Animal Health. Figure 1. Chemical structures of the macrolide antibiotics tilmicosin and tylosin. J. Agric. Food Chem. 2005, 53, 9679-9688 9679 10.1021/jf051987x CCC: $30.25 © 2005 American Chemical Society Published on Web 11/15/2005