Anti-CD0873 Antibody Detection by ELISA

Anti-CD0873 IgG and IgA were detected in serum and feces, respectively, by indirect ELISA as described previously (38 (link)). Briefly, 96-well microtiter plates (MaxiSorp, Nunc) were coated with 5 μg of recombinant purified CD0873 protein. Fecal supernatants were tested at a dilution of 1:2 and mouse sera at a dilution of 1:20. After washing, anti-CD0873 antibodies were detected by successive incubations for 30 min at 37 °C with a goat anti-mouse IgG or IgA conjugated to biotin (1:20,000 and 1:10,000 dilution, respectively; Sigma) and for 30 min at 37 °C with a streptavidin-horseradish peroxidase conjugate (1:5,000 dilution; Thermo Scientific). All samples were treated simultaneously and tested in duplicate to avoid inter-assay variation. Assays with antigen in the absence of sera served as negative controls.