Elisa

Ferreira, Susana Carolina Martins, Torelli, Francesca, Klein, Sandra, Fyumagwa, Robert, Karesh, William B., Hofer, Heribert, Seeber, Frank & East, Marion L., 2019, Evidence of high exposure to Toxoplasma gondii in free-ranging and captive African carnivores, International Journal for Parasitology: Parasites and Wildlife 8, pp. 111-117 : 113

2.3. In-house ELISA

We applied an in-house ELISA to all lion, spotted hyena and stripped hyena samples, including the samples from European zoos. Expression and purification of recombinant SAG1 (rSAG1-6H) was done as follows. Briefly, C-terminally 6His-tagged SAG1 (amino acids 31–289) from pSAG1-GPI ( Seeber et al., 1998) was cloned into plasmid pASG-IBA33 (IBA, Göttingen, Germany) according to the manufacturer's instructions. For expression the resulting plasmid pASG33-SAG1 was transformed into Escherichia coli SHuffle ª T7 Express cells (New England Biolabs, Frankfurt am Main, Germany) together with plasmid pMJS9 ( Nguyen et al., 2011) to aid in proper disulphide bonding of rSAG1-6H. After induction of expression with 0.5% arabinose and 200 ng /ml anhydrotetracycline for 18 h at 30 ̊C, rSAG1-6H protein was purified using a HisTrap FF 1 ml column on an ÄktaPurifier FPLC system essentially as described by the manufacturer (GE Healthcare, Chicago, USA). Finally, buffer was exchanged to PBS on a PD10 column (GE Healthcare) before the protein was stored at −20 ̊C until further use. Protein concentration was determined using the BCA assay (Thermo Fisher, Darmstadt, Germany). Protein purity was assessed by SDSpolyacrylamide gel electrophoresis, silver staining and immunoblot using anti-His tag antibodies and found to be ∼95% pure.

For the ELISA MaxiSorp ª plates (Thermo Fisher) were coated overnight at 4 ̊C with 100 ng of rSAG1-6H per well or PBS as control. All further steps were performed at room temperature. Unspecific binding of serum to the plate was blocked by incubation with 5% soluble milk powder in PBS for 1 h. Then sera from lions and hyenas were serially diluted 1:100, 1:200 and 1:400, added in duplicates to wells and incubated for 90 min. As positive and negative controls we used seropositive plasma and seronegative serum from domestic cats (kindly provided by G. Schares; Friedrich-Loeffler-Institut, Riems, Germany). Controls were used at a dilution of 1:2000. As secondary antibody we used a peroxidase-conjugated goat anti-cat IgG (H + L) at a dilution 1:4000 (KPL, Gaithersburg, MD, USA). SureBlue ª TMB Peroxidase substrate (KPL, Gaithersburg, MD, USA) was added and the reaction stopped after 10 min by adding sulphuric acid. The resulting colour signal measured at 450 nm ( 650 nm reference) at a Tecan Infinite M200 PRO reader. Samples were considered positive when the value was higher than the mean from two independent experiments +3 standard deviations of negative cat or hyena sera of the same dilution.