Isolation, expression and characterization of two single-chain variable fragment antibodies against an endo-polygalacturonase secreted by Sclerotinia sclerotiorum Bo Yang a , William Yajima a , Dipankar Das b , Mavanur R. Suresh b , Nat N.V. Kav a, * a Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5 b Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8 article info Article history: Received 28 October 2008 and in revised form 3 December 2008 Available online 16 December 2008 Keywords: Endo-polygalacturonase Phage display Single-chain variable fragment Sclerotinia sclerotiorum Surface plasmon resonance abstract Canola is a very important economic crop in the world and canola stem rot caused by Sclerotinia scle- rotiorum (Lib.) de Bary, a necrotrophic, highly destructive and non-host-specific fungus, can reduce yield significantly. This fungus secretes numerous cell wall degrading enzymes including an endo-pol- ygalacturonase, SSPG1d, which has been detected at early stages of infection. In this report we describe the isolation of two recombinant antibodies of the single-chain variable fragment (ScFv) for- mat from RNA of mice immunized with recombinant SSPG1d (rSSPG1d) or a peptide derived from SSPG1d (peptide 3796) that was predicted to be antigenic. The ScFvs were isolated using the estab- lished phage display technology. These recombinant antibodies were expressed, purified and refolded to functional antibodies with a yield of 120–500 lg per liter of cell culture. Recombinant antibodies were characterized using various techniques including enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Of the two ScFvs, it appears that only ScFv-rSSPG1d is able to detect whole SSPG1d produced by the fungus. Thus our results indicate that this ScFv may have utility in the detection of the SSPG1d enzyme in an antibody-based diagnostic test for S. sclerotiorum infection. Ó 2008 Elsevier Inc. All rights reserved. Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic, highly destructive and non-host-specific fungus which affects more than 400 plant species [1–3] and is prevalent in temperate regions of North America, Europe and New Zealand [4]. Sclerotinia stem rot is a very serious disease in canola, which is an important North American crop with 8.7 million tonnes harvested in Canada in 2007 [5]. It is very difficult to control this fungus due to long sur- vival of sclerotia (an overwintering structure), ascospore dispersal potential, limited source of resistance in canola and related species as well as the fact that its host range is so broad, which can limit the effectiveness of crop rotations [2]. There are several fungicides effective in controlling S. sclerotiorum, but it is important to deter- mine the need for fungicide application early using risk assessment tools [6–9]. Secreted virulence factors are essential for S. sclerotiorum path- ogenesis and include oxalic acid [10–16], hydrolytic enzymes [17– 20] and several other factors such as oleic acid and sclerin [21–24]. Full virulence of S. sclerotiorum is thought to be due to the synergis- tic activities of oxalic acid and endo-polygalacturonases [23]. Polygalacturonases (PGs) 1 , encoded by multigene families, are pro- duced in several molecular forms with different specificities and are subject to differential regulation. This multiplicity can grant adaptive flexibility to pathogens when infecting different hosts or plant organs [25]. Internal a(1–4) glycosidic bonds in deesterified regions of the middle lamella and primary cell wall homogalactu- ronans are hydrolyzed by PGs, and when produced in sufficient amounts, determine the degree of maceration of infected tissues [26]. Among the PGs, one of the endo-polygalacturonases, SSPG1d, is detected in the early stages of S. sclerotiorum pathogenesis and its expression is optimal in low pH environments [19,27]. SSPG1d is highly homologous at the nucleotide level to other S. sclerotiorum endo-polygalacturonases such as SSPG1a-c [28]. The detection of these polygalacturonases using antibody-based tests may have utility in confirming the presence of this fungus, especially when used in a panel format assay with other Scleroti- 1046-5928/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2008.12.007 * Corresponding author. Fax: +1 780 492 4265. E-mail address: nat@ualberta.ca (N.N.V. Kav). 1 Abbreviations used: ScFv, single-chain variable fragment; ELISA, enzyme-linked immunosorbent assay; SPR, surface plasmon resonance; RU, resonance units; PGs, polygalacturonases; PDA, potato dextrose agar, RT, room temperature; IPTG, Isopro- pyl b-D-1-thiogalactopyranoside; API, Alberta Peptide Institute; FCA, Freund’s com- plete adjuvant; FIA, Freund’s incomplete adjuvant; HRP, horseradish peroxidase. Protein Expression and Purification 64 (2009) 237–243 Contents lists available at ScienceDirect Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep