Preparation of immunogen

Immunogens for antibody production were prepared as either whole cell antigens (WCA) or ethanol extracted antigens (EEA) from MAP strain B4 (a Northern Ireland bovine field isolate) which had been grown to stationary phase in Middlebrook 7H9 broth supplemented with 10% OADC (both from Difco™), 0.005% Tween 80 and 2 μg/ml mycobactin J (Synbiotics Europe SAS, Lyon, France). The culture was centrifuged, washed twice in phosphate buffered saline (PBS, pH 7.4) and re-suspended in an equal volume of PBS. The MAP cell concentration was calculated to be ~10⁸ cfu/ml; quantified using the phage amplification assay prior to gamma irradiation to a dose of 10 kGy using a Gammabeam 650 irradiator. The irradiated suspension served as WCA. EEA was generated from the WCA as described by Eda et al. [20]. Briefly, irradiated MAP cells (WCA) were vortexed at high speed in 80% (v/v) ethanol solution for approx. 30 sec. The cells were removed through centrifugation, and the supernatant was transferred to a fresh tube. The ethanol was aspirated under nitrogen gas and the protein suspension freeze-dried to remove any excess water before final resuspension in sterile water at a concentration of 1 mg/ml. Both immunogens were stored at -80°C until required.