Application of Recombinant Histidine-Tagged Antigens in the Diagnosis of Viral Infections

Histidine-tagged proteins are frequently detected with antibodiesHistidine-tagged proteins are frequently detected with antibodiesspecific for the histidine tag; these are useful in immunologicalstudies. A penta-His monoclonal antibody (mAb) was used toestimate the relative amount of recombinant protein attachedto each microsphere bead set in a fluorescent microsphereimmunoassay (FMIA). In this study, the use of the anti-His mAbfor the detection of recombinant antigens from seven differentanimal viruses: porcine reproductive and respiratory syndromevirus (PRRSV), porcine circovirus type 2 (PCV2), swine influenzavirus (SIV), African swine fever virus (ASFV), classical swine fevervirus (CSFV), bovine viral diarrhea virus (BVDV), and Rift Valleyfever virus (RVFV) bound to microsphere beads after coupling weretested. Proteins were produced as 6xHis-ubiquitin fusion protein andcovalently coupled to Luminex MagPlex® polystyrene, carboxylatedmicrosphere beads. The target antigens were assembled into a singlemultiplex and tested in the presence of anti His-tag mAb. The resultswere reported as mean fluorescent intensity (MFI). The interactionbetween His-tagged recombinant antigens and anti-His tag mAbwere tested using His-tag blocking peptide. The results of the His-tagblocking assay showed that 1 μg/ml of His-tag blocking peptide cancompletely inhibit the interaction of His-tagged antigens and anti-HismAb. The MFI values for the negative serum samples showed 10-40fold reduction compared to PRRSV-N, PCV2, SIV, ASFV, CSFV, andBVDV positive sera. Therefore, the use of the anti-His mAb provides aconvenient means for assessing antigen binding to beads.