Humoral immune response

The humoral response (serum immunoglobulin G [IgG] response) evoked by vaccination and infection was assessed by indirect enzyme-linked immunosorbent assay (ELISA) to evaluate the success of the affinity-purified fraction in achieving protective activity against cryptosporidiosis. Immunoglobulin G levels were measured in mice sera collected at weekly intervals along the experiment time, as previously described by Priest et al. [44] and Liu et al. [28]. Briefly, flat-bottom 96-well ELISA plates were coated with 4 μg/mL of purified fraction in carbonate buffer. Serum samples from mice in the three groups were diluted to 1:100 and added to the plates separately in triplicate. Then, we added antimouse IgG horseradish peroxidase-labeled conjugate (1:1000; Sigma-Aldrich) and ortho-phenylenediamine (Sigma-Aldrich) substrate buffer (1 mg/mL). The optimum antigen concentration and antibody and conjugate dilutions were determined by checkerboard titration. The plates were read spectrophotometrically at 450 nm using an ELx800UV microplate reader (BioTek Instruments, Inc., USA).