ORE Open Research Exeter TITLE Detection of the sour-rot pathogen Geotrichum candidum in tomato fruit and juice by using a highly specific monoclonal antibody-based ELISA AUTHORS Thornton, CR; Slaughter, DC; Davis, Richard M. JOURNAL International Journal of Food Microbiology DEPOSITED IN ORE 20 November 2013 This version available at http://hdl.handle.net/10871/13974 COPYRIGHT AND REUSE Open Research Exeter makes this work available in accordance with publisher policies. A NOTE ON VERSIONS The version presented here may differ from the published version. If citing, you are advised to consult the published version for pagination, volume/issue and date of publication !!!!!!!!!!!!!!!!!!!!!!!!!!!!!"#$%&'%(!")'*+(',#!-.$*%/0*/1!2+(!34*%(4,*'+4,#!5+6(4,#!+2!7++)!8'9(+:'+#+;.! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!8,46$9('<*!=(,2*! ! ! 8,46$9('<*!>6/:%(?!7@@=A=ABCACCDEEFB! ! G'*#%?!=%*%9*'+4!+2!*H%!$+6(A(+*!<,*H+;%4!I%+*('9H6/!9,4)')6/!'4!*+/,*+!2(6'*!,4)!J6'9%!:.!6$'4;!,! 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E-mail address: 8 [email protected] 9 10 Suggestion for an abbreviated title: Geotrichum candidum enzyme-linked immunosorbent assay 11 12 13 14 15 16 17 18 19 20 21 22 1 23 Abstract 24 Geotrichum candidum is a common soil-borne fungus that causes sour-rot of tomatoes, citrus 25 fruits and vegetables, and is a major contaminant on tomato processing equipment. The aim of 26 this work was to produce a monoclonal antibody and diagnostic assay for its detection in tomato 27 fruit and juice. Using hybridoma technology, a cell line (FE10) was generated that produced a 28 monoclonal antibody belonging to the immunoglobulin class M (IgM) that was specific to G. 29 candidum and the closely related teleomorphic species Galactomyces geotrichum and 30 anamorphic species Geotrichum europaeum and Geotrichum pseudocandidum in the 31 Galactomyces geotrichum/G. candidum complex. The MAb did not cross-react with a wide 32 range of unrelated fungi, including some likely to be encountered during crop production and 33 processing. The MAb binds to an immunodominant high molecular mass (>200 kDa) 34 extracellular polysaccharide antigen that is present on the surface of arthroconidia and hyphae of 35 G. candidum. The MAb was used in a highly specific Enzyme-Linked Immunosorbent Assay 36 (ELISA) to accurately detect the fungus in infected tomato fruit and juice. Specificity of the 37 ELISA was confirmed by sequencing of the internally transcribed spacer (ITS) 1-5.8S-ITS2 38 rRNA-encoding regions of fungi isolated from naturally-infected tomatoes. 39 40 Keywords: Geotrichum candidum, hybridoma, monoclonal antibody, enzyme-linked 41 immunosorbent assay 42 43 44 45 2 46 1. Introduction 47 Geotrichum candidum is a filamentous yeast-like fungus. It is a ubiquitous organism 48 found in a range of habitats including air, water, silage and soil (Butler and Eckert, 1962; 49 O’Brien et al., 2005) and also in foodstuffs such as milk, cheese and fermented milk products 50 (Boutrou and Guégen, 2005; Marcellino et al., 2001; Mistry, 2004; Pottier et al., 2008; Ruas- 51 Madiedo et al., 2006; Wouters et al., 2002). As a component of the normal microbiota of the 52 human digestive tract, it has been reported, albeit infrequently, as a cause of infection in 53 immuno-compromised patients (Farina et al., 1999; Vasei and Imanieh, 1999; Verghese and 54 Ravichandran, 2003; Andre et al., 2004; Sfakianakis et al., 2007; Henrich et al., 2009). 55 As a plant pathogen, G. candidum causes sour-rot of citrus fruit, tomatoes, carrot, and 56 other fruits and vegetables (Pitt and Hocking, 2009). Most susceptible to infection are ripe or 57 overripe fruits and vegetables, particularly those kept in moisture-holding packaging (Skavia et 58 al., 2004) and wounded tissue (Moline, 1984). Decay spreads rapidly, resulting in a sour- 59 smelling watery mass. While the fungus prefers high temperatures and humidity, it is active at 60 temperatures as low as 2oC. Consequently, the fungus is an important post-harvest storage 61 pathogen (Moline, 1984; Oladiran and Iwu, 1992; Skavia, 2004; Tournas, 2005). In addition, G. 62 candidum is the predominant contaminant on tomato processing equipment and is referred to as 63 “machinery mould” (Splittstoesser et al., 2006). As a consequence, the fungus is used by 64 regulatory inspectors as a microbiological criterion to assess sanitation of equipment in fruit and 65 vegetable processing plants and in bottling works (Xu and Hang, 1988). 66 While microscope and culture-based mould counts and a lectin-mediated chitin-binding 67 assay can be used to assess the total fungal burden in raw tomato juice (Potts et al., 2000, 2001; 68 AOAC, 2010), these tests are not sufficiently specific to discriminate between different 3 69 contaminating organisms. As an alternative to mould counts, an enzyme immunoassay using 70 polyclonal antiserum was developed to quantify mould content of tomato paste, but the 71 antibodies cross-reacted with all of the major tomato spoilage organisms (Robertson and Patel, 72 1989). Because of this, there is a pressing need for the development of a test that allows the 73 specific detection of G. candidum during the production, harvesting, post-harvest handling and 74 processing of tomatoes. 75 Hybridoma technology allows the production of monoclonal antibodies (MAbs) that are 76 specific to individual genera, species or even isolates of fungi (Thornton et al., 2002; Thornton, 77 2008; Thornton, 2009). The aim of this paper is to describe the development of a MAb specific 78 to G. candidum and its use to develop a highly specific and sensitive diagnostic assay (ELISA) 79 for detection of the fungus in tomato fruit and juice. 80 81 82 83 84 85 86 87 88 89 90 91 4 92 2. Materials and methods 93 2.1. Fungal culture 94 All fungi (Table 1) were routinely cultured on malt yeast agar (MYA) slants under a 16 h 95 fluorescent light regime at 26 oC. MYA agar consisted of malt yeast (MY) medium 96 (bactopeptone, 0.5% w/v; malt extract, 1.0% w/v; glucose, 0.4% w/v; yeast extract, 0.4% w/v) 97 and agar, 2.0% w/v; and was adjusted to pH 7.3 with 1M NaOH prior to autoclaving at 121 oC 98 for 15 min. 99 100 2.2. Development of MAbs, preparation of immunogen, and immunization regime 101 Balb/c mice were immunized with soluble antigens prepared from lyophilized mycelium of 102 G. candidum (CBS strain 115.23). Arthroconidia were suspended in water after 5-d-old MYA 103 slant cultures were flooded with 5 mL dH2O and gently agitated with an inoculation loop. 104 Conidial suspensions were then filtered through Miracloth to remove mycelium and transferred to 105 1.5 mL micro-centrifuge tubes. The conidia were washed three times with dH2O by repeated 106 vortexing and centrifugation at 14,462 g for 5 min and finally suspended in dH2O to give a 107 concentration of 106 conidia/mL solution. Flasks containing 150 mL of MY medium were 108 inoculated with 200 mL of the conidial suspension and incubated with shaking (150 rpm) for 48 h o 109 at 26 C. The contents of the flasks were snap frozen in liquid N2, and lyophilized. Lyophilized 110 material was suspended in phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 8 mM 111 Na2HPO4, and 1.5 mM KH2PO4 [pH7.2]) and the resultant suspension centrifuged for 5 min at 112 14,462 g. The supernatant, containing solubilized antigens, was used as the immunogen and as a 113 source of antigens for hybridoma screening assays. For immunization, 6-wk-old BALB/c female 114 white mice were given four intraperitoneal injections (300 µL per injection) of antigen extract 5 115 containing 2.3 mg protein per mL PBS at 2-wk intervals and a single booster injection five days 116 before fusion. 117 118 2.3. Production and screening of hybridomas and determination of antibody specificity 119 Hybridoma cells were produced by the method described elsewhere (Thornton, 2001) and 120 the supernatants were screened by enzyme-linked immunosorbent assay (ELISA) against 121 antigens immobilized to the wells of Maxisorp microtitre plates (Nunc; 442404)(50 µL per 122 well). For antibody specificity tests, fungi were grown on MYA slopes and surface washings 123 prepared as described elsewhere (Thornton, 2009). Protein concentrations, determined 124 spectrophotometrically at 280 nm (Nanodrop, Agilent Technologies Limited, Berkshire, UK), 125 were adjusted to 60 µg/mL buffer. Fifty µL volumes were then used to coat the wells of 126 microtitre plates. After incubating overnight at 4 oC, wells were washed four times with PBST 127 (PBS containing Tween 20, 0.05% v/v) and once each with PBS and dH2O and air-dried at 23 128 oC in a laminar flow hood.