Archives of Insect Biochemistry and Physiology 52:17–34 (2003) © 2003 Wiley-Liss, Inc. DOI: 10.1002/arch.10061 Published online in Wiley InterScience (www.interscience.wiley.com) Monoclonal Antibodies to Elongation Factor-1 a Inhibit In Vitro Translation in Lysates of Sf21 Cells M.K. Stuart* and N.R. Chamberlain Elongation factor-1a (EF-1a) is an enzyme that is essential for protein synthesis. Although EF-1a offers an excellent target for the disruption of insect metabolism, agents known to interfere with EF-1a activity are toxic to humans. In this article, we describe the development of monoclonal antibodies (MAbs) that can disrupt the activity of insect EF-1a without cross-reacting with the human enzyme. MAbs were generated to EF-1a from Sf21 cells derived from the fall armyworm, Spodoptera frugiperda, by immunizing mice with EF-1a eluted from SDS-PAGE gels. The MAbs reacted with EF-1a in eggs and first through fifth instars of the fall armyworm in immunoblots of SDS-PAGE gels, but did not recognize EF-1a in human carci- noma cells and normal tissues. MAbs with the ability to recognize EF-1a in its native conformation, identified through immunoprecipitation experiments, were added to Sf21 cell lysates to determine whether the antibodies could inhibit incorpo- ration of [ 35 S]methionine into newly synthesized in vitro translation products. Of the four EF-1a-specific MAbs tested, three significantly inhibited protein synthesis when compared to the negative control antibody (P < 0.001, one-way ANOVA; followed by Dunnett’s test, P < 0.05). Arch. Insect Biochem. Physiol. 52:17–34, 2003. © 2003 Wiley-Liss, Inc. KEYWORDS: elongation factor-1a; Spodoptera frugiperda; protein translation; monoclonal antibodies Department of Microbiology/Immunology, Kirksville College of Osteopathic Medicine, Kirksville, Missouri 63501 Contract grant sponsor: U.S. Department of Agriculture, National Research Initiative Competitive Grants Program; Contract grant number: 98-35311-6688. Abbreviations used: ANOVA = analysis of variance; cpm = counts per minute; DTT = dithiothreitol; EDTA = ethylenediaminetetraacetic acid; EF-1a = elongation factor-1 alpha; kDa = kilodaltons; MAbs = monoclonal antibodies; PBS = phosphate-buffered saline; PMSF = phenylmethylsulfonyl fluoride; SDS-PAGE = sodium dodecylsulfate-polyacrylamide gel electrophoresis. *Correspondence to: Melissa K. Stuart, Dept. of Microbiology/Immunology, Kirksville College of Osteopathic Medicine, 800 W. Jefferson Street, Kirksville, MO 63501. E-mail: MStuart@kcom.edu Received 27 November 2001; Accepted 12 August 2002 INTRODUCTION Elongation factor-1a (EF-1a) is an abundant cytoplasmic enzyme that promotes GTP-dependent binding of aminoacyl-tRNA to the acceptor A site on ribosomes during eukaryotic protein chain elon- gation. It is part of an enzymatic complex that also contains b, g, and d subunits, which provide the guanine nucleotide exchange activity needed to re- generate EF-1a·GTP from EF-1a·GDP (Janssen et al., 1994). In addition, EF-1a has been shown to control the aminoacylation reaction catalyzed by valyl-tRNA synthetase and has been implicated in the regulation of aspartyl- and phenylalanyl-tRNA synthetases (Negrutskii et al., 1999). Besides its di- rect involvement in protein synthesis, the enzyme coordinates protein synthetic activity with other cellular events, through functions that include in- teracting with the mitotic apparatus (Ohta et al., 1990), binding and bundling actin (Kurasawa et al., 1996), severing microtubules (Shiina et al., 1994), activating membrane phosphatidylinositol 4-kinase (Yang and Boss, 1994), and playing a role in ubiquitin-dependent degradation of N-a-acety- lated proteins (Gonen et al., 1994). EF-1a offers an excellent target for perturbing normal metabolism, a hypothesis that is supported by studies employing didemnin B, a 7–amino acid