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.