Hapten Syntheses and Antibody Generation for the Development of a Polybrominated Flame Retardant ELISA | WEILIN L. SHELVER,* ,† YOUNG-SOO KEUM, ‡ HEE-JOO KIM, ‡,# DREW RUTHERFORD, § HELDUR H. HAKK, † ÅKE BERGMAN, ⊥ AND QING X. LI ‡ USDA-ARS Biosciences Research Laboratory, 1605 Albrecht Boulevard, Fargo, North Dakota 58105, Department of Molecular Biosciences and Biosystems Engineering, University of Hawaii, 1955 East-West Road, Honolulu, Hawaii 96822, Department of Chemistry, Concordia College, 901 South 8th Street, Moorhead, Minnesota 56562, and Department of Environmental Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants that are increasingly an environmental concern. Several antibodies were developed for the polybrominated diphenyl ether flame retardant BDE-47 (1), often found in the highest concentration in human milk, plasma, and adipose tissue. Four haptens with different bromine and linker substitution patterns were synthesized and utilized to generate five polyclonal antibodies from goats and two polyclonal antibodies from rabbits. Competition was assessed using four different coating antigens for all seven antibodies. The coating antigen showed marked effects on competition. When the same hapten was used for antibody and the coating antigen less competition was observed. The effect of BDE structure on competition was evaluated by using BDE-47 (1), BDE-99 (2), BDE-100 (3), BDE-153 (4), and BDE-183 (5). None of the compounds showed high competition with antibody I-KLH, presumably because steric hindrance prevented formation of an efficient binding site. As predicted from structural considerations, BDE-47 (1) competed well with the remaining antibodies, whereas BDE-100 (3) competed well with only II-KLH. The remaining congeners (BDE-99 (2), BDE-153 (4), and BDE-183 (5)) contain bromines that cannot be positioned in binding sites and thus cross-react poorly. The competition study demonstrated that a bromine substitution on the congener could occupy a position analogous to the linker’s position. KEYWORDS: ELISA; immunoassay; analysis; brominated flame retardants; PBDEs; polybrominated diphenyl ether INTRODUCTION Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants consisting of a mixture of congeners. Extensive PBDE use has resulted in its increasingly widespread presence in the environment (1). Ingestion of PBDEs in foodstuffs and exposure to PBDEs in the environment has resulted in their bioaccumulation in fatty tissues and the milk of humans (2). In Sweden and Japan, decreased use of PBDEs, beginning in 1998, has apparently decreased PBDE burdens in human. While Swedish and Japanese women have similar levels of PBDEs in milk (3, 4), averages in American women are nearly 10-fold higher (1), although there is considerable variability between individuals. The increase in PBDE levels in North America may be attributed to extensive usage of commercial pentaBDE formulations (5). Environmental contamination with PBDE and the observation of toxic responses to PBDEs (6, 7) have resulted in proposals to ban their usage. The European Union banned use of pentaBDE and octaBDE in 2004 (8). Commercial preparations of PBDEs contain a mixture of congeners; the structures of the ones used in this study are shown in Figure 1. In clinical samples of 47 nursing mothers (9) the percent of the congener in milk was 54% BDE-47, 16.8% BDE- 99, 8.5% BDE-100, and 5.9% BDE-153 with other congeners making up the remainder. In plasma (10) the percent of the congener for the 2000-2002 sample group was 56% BDE-47, 18% BDE-99, 10% BDE-100, and 12% BDE-153. More alarming, these workers observed significant increases of PBDE levels over time. Other sample matrices have different congener distributions: for example BDE-99 is the most abundant in meat and poultry closely followed by BDE-47, (11, 12); these are also the dominant congeners present in the commercial penta- * Corresponding author. Telephone: +1-701-239-1425. Fax: +1-701- 239-1430. E-mail: shelverw@fargo.ars.usda.gov. | Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable. † USDA-ARS Biosciences Research Laboratory. ‡ University of Hawaii. # Current Address: Department of Entomology, University of California, Davis, CA 95616. § Concordia College. ⊥ Stockholm University. 3840 J. Agric. Food Chem. 2005, 53, 3840-3847 10.1021/jf047863m CCC: $30.25 © 2005 American Chemical Society Published on Web 04/15/2005