Serum plaque neutralisation assay

Serum samples, diluted to 1 in 4 and 1 in 16 with serum free DMEM, were incubated with 200 pfu virus for 90 minutes before inoculation of a GMK monolayer in 96 well plates. After 1 hour with intermittent agitation, the inoculum was removed and an agarose-media plug layered onto the cells to prevent viral dissemination through the media. Assays were left for 48 hours for plaques to develop, before harsh fixation with Carnoy’s fix, washing, plug removal and staining with crystal violet. Plates were imaged using a Bio-sys Bioreader and restriction of virus-plaque formation quantified against virus-only inoculation controls. Virus strains used were: Coxsackievirus B1 (ATCC^® VR-28^™)–Conn strain; Coxsackievirus B 2 (ATCC^® VR-29^™)–Ohio strain; Coxsackievirus B3 (ATCC^® VR-30^™)–Nancy strain; Coxsackievirus B4 (ATCC^® VR-184^™)–J.V.B strain; Coxsackievirus B 5 (ATCC^® VR-185^™)–Kentucky strain and Coxsackievirus B 6 (ATCC^® VR-155^™)–Schmitt strain.