bei was added to the supernatant to a final concentration of 5 mm the samples were then stirred continuously for 72 to 96 hrs a 1 0 m sodium thiosulfate solution to give a final minimum concentration of 5 mm was added to neutralize any residual bei after inactivation pcv2 orf 2 buffered with phosphate buffer and carbopol was added to about 0 5 to 2 5 mg dose the final dose comprises about 16 u03bcg pcv2 orf 2 antigen pk15 e g atcc ccl 33 or vido r1 cells described in wo 02 07721 are seeded onto a 96 well plate about 20 000 to 60 000 cells per wells cells are infected with a pcv2 isolate when monolayers are approximately 65 to 85 confluent infected cells are incubated for 48 hours medium is removed and wells are washed 2 times with pbs the wash buffer is discarded and cells are treated with cold 50 50 methanol acetone fixative u02dc 100 u03bcl well for about 15 min at about u2212 20 u00b0 c the fixative is discarded and the plates are air tried serial dilutions of porcine serum samples are prepared in pbs added to the plates and incubated to allow antibodies to bind if present in the serum samples for about 1 hr at 36 5 u00b1 1 u00b0 c in addition serial dilutions of an anti pcv2 positive and negative control sample positive control and negative control samples are run in parallel the plates are then washed three times with pbs the pbs is discarded plates are then stained with a commercial goat anti swine fitc conjugate diluted 1 100 in pbs and incubated for about 1 hr at 36 5 u00b1 1 u00b0 c which allows detection of antibodies bound to infected cells after incubation.