Journal of General Virology 2008, Vol. 89:pp 177–187This study examined whether antigenic differences among porcine circovirus type 2 (PCV-2) strains could be detected using monoclonal antibodies (mAbs). A subtractive immunization protocol was used for the genotype 2 post-weaning multisystemic wasting syndrome (PMWS)-associated PCV-2 strain Stoon-1010. Sixteen stable hybridomas that produced mAbs with an immunoperoxidase monolayer assay (IPMA) titre of 1000 or more to Stoon-1010 were obtained. Staining of recombinant PCV-2 virus-like particles demonstrated that all mAbs were directed against the PCV-2 capsid protein. Cross-reactivity of mAbs was tested by IPMA and neutralization assay for genotype 1 strains 48285, 1206, VC2002 and 1147, and genotype 2 strains 1121 and 1103. Eleven mAbs (9C3, 16G12, 21C12, 38C1, 43E10, 55B1, 63H3, 70A7, 94H8, 103H7 and 114C8) recognized all strains in the IPMA and demonstrated neutralization of Stoon-1010, 48285, 1206 and 1103, but not VC2002, 1147 and 1121. mAbs 31D5, 48B5, 59C6 and 108E8 did not react with genotype 1 strains or had a reduced affinity compared with genotype 2 strains in the IPMA and neutralization assay. mAb 13H4 reacted in the IPMA with PMWS-associated strains Stoon-1010, 48285, 1206 and VC2002, and the porcine dermatitis and nephropathy syndrome-associated strain 1147, but not with reproductive failure-associated strains 1121 and 1103. mAb 13H4 did not neutralize any of the tested strains. It was concluded that, despite the high amino acid identity of the capsid protein (¢91 %), antigenic differences at the capsid protein level are present among PCV-2 strains with a different genetic and clinical background.