Immunology Letters 106 (2006) 57–62 A novel monoclonal antibody with catalytic activity against beta human chorionic gonadotropin Manouchehr Mirshahi a,b,∗ , Freshteh Shamsipour a , Tooran Mirshahi b , Khosro Khajeh a , Hossein Naderi-Manesh a a Department of Biochemistry, Faculty of Science, Tarbiat Modares University, Tehran, Iran b Monoclonal Antibodies Research Centre, No. 1855, 13th St., Shahrak Gharb, Tehran, Iran Received 13 December 2005; received in revised form 19 April 2006; accepted 25 April 2006 Available online 15 May 2006 Abstract In this study, we report for the first time, production of monoclonal antibody (MAb) against beta human chorionic gonadotropin (hCG) with proteolytic activity. MAb “7D9” was raised in Balb/C mice using purified human chorionic gonadotropin. Immunoblot analysis and enzyme-linked immunoabsorbent assay (ELISA) showed that this MAb reacts with beta hCG. The epitope for this antibody appears to be located in the C-terminal of beta chain as suggested by the absence of cross-reaction with other glycoprotein hormones such as FSH, TSH and LH. Our data reveal that this MAb is very unstable and has autodegradation characteristics. Zymogram analyses also show that 7D9 MAb has a high level of hydrolytic activity against different substrates such as casein and gelatin. This proteolytic activity can be inhibited by EDTA. These findings demonstrate the proteolytic character of 7D9 MAb and consequently explain its instability. © 2006 Published by Elsevier B.V. Keywords: Catalytic antibody; Anti h-CG; Abzyme; Proteolytic activity; Super catalytic antibody 1. Introduction Since the introduction of hybridoma technology in 1975 by Kohler and Milstein [1], monoclonal antibodies have been obtained against a wide spectrum of antigens and have become a major tool in most areas of biology and medicine. Antibodies are large proteins (150 kDa in the case of immunoglobulin G) with four polypeptide chains, two identical heavy chains and two identical light chains. The binding site to antigenic determinant consists of approximately the first 110 amino acids of the heavy and light chains and is termed variable region. These domains display a high degree of sequence variability and provide the basis for the diversity of antibodies molecules [2]. Based on this diversity, immunoglobulin can bind virtually to any natural and synthetic molecules with high specificity. Selective recognition is achieved through a large number of weak binding interactions involving hydrogen bonds, Van der Waals and electrostatic inter- actions. Antibodies bind to molecules with association constants that range from 10 -4 to 10 -14 M. The specificity of antibodies ∗ Corresponding author. Tel.: +98 2188009730; fax: +98 2188009730. E-mail address: mirshahi@modares.ac.ir (M. Mirshahi). for their ligands can exceed that of enzymes for substrates. In fact, Paul [3] first pointed out more than 50 years ago that the fundamental difference between enzyme and antibodies is that the former selectively binds to transition states and the latter bind to the ground states. In 1986, the first monoclonal catalytic anti- bodies against chemically stable analog of the transition state of reaction were obtained and termed abzyme. At present, there are more than 100 artificial monoclonal abzymes that are capable to catalyze different distinct chemical reactions are introduced. Recently, natural abzymes with protease activity [4–6], RNase activity [7,8], DNase activity [9], nucleotidase activities [10,11], protein kinase [12] and lipid kinase [13] have been reported in autoimmune disease, pregnancy, and in the milk of normal human mothers. In this report, for the first time we show that a MAb against beta hCG has a proteolytic activity. 2. Materials and methods 2.1. Chemical Protein G affinity column chromatography, DEAE-sepharose and Superdex 200 HR 10/30 were provided by Amersham Phar- 0165-2478/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.imlet.2006.04.008