Analytical Methods Characterisation of monoclonal antibody against aflatoxin B 1 produced in hybridoma 2C12 and its single-chain variable fragment expressed in recombinant Escherichia coli Won-Ki Min a , Dae-Hyuk Kweon b , Kyungmoon Park c , Yong-Cheol Park d,⇑⇑ , Jin-Ho Seo a,⇑ a Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, South Korea b Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, South Korea c Department of Biological and Chemical Engineering, Hongik University, Jochiwon, Chungnam 339-701, South Korea d Department of Advanced Fermentation Fusion Science and Technology, Kookmin University, Seoul 136-702, South Korea article info Article history: Received 19 February 2010 Received in revised form 5 September 2010 Accepted 14 November 2010 Available online 20 November 2010 Keywords: Aflatoxin B 1 Monoclonal antibody Single-chain variable fragment Hybridoma cell Escherichia coli abstract An anti-aflatoxin B 1 monoclonal antibody (anti-AFB 1 mAb) from the hybridoma 2C12 was established and its inhibition concentration fifty (IC 50 ) for AFB 1 and relative cross-reactivities (CRs) to other mycotoxins were estimated to be 8 ng/mL and less than 4% compared with AFB 1 by a competitive direct enzyme- linked immunosorbent assay. For production of anti-AFB 1 single-chain variable fragment (anti-AFB 1 scFv) in recombinant Escherichia coli, its scFv-coding genes were cloned from the hybridoma 2C12. The anti- AFB 1 scFv formed inclusion bodies in the cytoplasm of E. coli required in vitro refolding process and hence recovered to retain binding activity successfully. Surface plasmon resonance analysis resulted that anti- AFB 1 scFv possessed 1.16 Â 10 À7 M of equilibrium dissociation constant (K D ), which was about 17 times higher than the parental anti-AFB 1 mAb of 6.95 Â 10 À9 M. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Aflatoxin B 1 (AFB 1 ) is a fungal metabolite produced by Aspergil- lus flavus and Aspergillus parasiticus. It possesses hepatotoxic, teratogenic and mutagenic properties and causes toxic hepatitis, hemorrhage, immunosuppression and hepatic carcinoma (Reddy, Reddy, & Muralidharan, 2009). Many reports have warned severe human aflatoxicosis caused by international consumption of heavily contaminated foods such as maize, cacao, and peanuts (Asis, Di Paola, & Aldao, 2002; Fernández-Ibañeza, Soldado, Martínez-Fernándeza, & de la Roza-Delgado, 2009; Var, Kabak, & Gök, 2007). Especially, International Agency of Research on Cancer (IARC) categorised AFB 1 into a group I carcinogen for humans (IARC (International Agency for Research on Cancer)). European Union established the action levels between 2 and 50 ppb for AFB 1 pres- ent in all feed materials (Zheng, Richard, & Binder, 2006). AFB 1 -contaminated raw and processed foods are monitored through several screening and analytical methods which are based on chromatography or antibody platforms. Liquid chromatography (LC) is generally used for quantification of AFB 1 , and LC-tandem mass spectrometry and gas chromatography were introduced to analyse AFB 1 (Akiyama, Goda, Tanaka, & Toyoda, 2001; Monbaliu et al., 2009). Chromatographic technologies are generally improper for real-time assay in actual fields and incompatible with demands of most of food industries because they want to manage the large quantity of raw materials and products in a short period. For easy and rapid screening of AFB 1 , antibody-based techniques including enzyme-linked immunosorbent assay (ELISA) and immuno-chro- matographic test have been used in place of acceptances. (Lee & Rachmawati, 2006; Li et al., 2009). ELISA technology requires high sensitive and specific polyclonal antibody, monoclonal antibody (mAb) or recombinant antibody against target antigens for their accurate detection. In spite of complexity in the construction of hybridoma cell line, once hybridoma is constructed, mAb can be produced stably and consistently. Recombinant antibodies have wide applications to therapeutics and diagnostics agents and have been developed for analysing foods contaminants such as ampicil- lin, domoic acid and sulfamethazine (Burmester et al., 2001; Finlay, Shaw, Reilly, & Kane, 2006; Yang et al., 2007). Single-chain variable fragment (scFv) is one of small recombi- nant antibody (approximately one-fifth size of full-length immunoglobulin G (IgG) 630 kD) and constructed by fusing two variable regions of the heavy and light chains (V H and V L ) of antibodies via a short peptide linker. It can be produced highly in recombinant microorganisms such as Escherichia coli (E. coli), 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.11.088 ⇑ Corresponding author. Tel.: +82 2 880 4855; fax: +82 2 873 5095. ⇑⇑ Co-Corresponding author. Tel.: +82 2 910 5462; fax: +82 2 910 5739. E-mail addresses: ycpark@kookmin.ac.kr (Y.-C. Park), jhseo94@snu.ac.kr (J.-H. Seo). Food Chemistry 126 (2011) 1316–1323 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem