Mxra8-Fc Binding Assay for CHIKV

A fragment of cDNA encoding the Mouse-Mxra8 extracellular domain (residues 23–336, GenBank accession number NM_024263.4) or Human-Mxra8 extracellular domain (residues 24–337, GenBank accession number NM_032348.3) was appended with a TEV enzyme site and a human IgG1 Fc at the C-terminus as well as the IL-2 signal peptide at the N-terminus in pcDNA 3.4 expression vectors and transiently transfected into HEK293 cells followed by media collection and purification using protein A sepharose. The Mxra8-Fc binding assay was adapted from the previously described [27 (link)]. MaxiSorp ELISA plates (Corning) were coated with 2 µg/mL anti-mouse CHKV E2 monoclonal antibodies in CBS (pH9.6) overnight at 4 °C. The wells were washed four times with PBS and blocked with 4% BSA(Sigma-Aldrich) for 1 h at room temperature (RT). CHIKV virions (1 µg/ml) were diluted and added for 1 h at RT. After washing, MoNb-2E8 and MoNb-3C5 or Mouse Mxra8-Fc fusion protein (all at 10 µg/ml) were incubated for 30 min. Plates were washed and Human Mxra8-Fc (tenfold serial dilutions) were added to the plates and incubated for 1 h at RT. Plates were washed again and incubated with secondary rabbit anti-human IgG-Fc (1:5000, Bioss). After washing, the plates were developed with TMB substrate (TIANGEN) and 2 N H₂SO₄. Absorbance was measured at 450 nm.