Quantification of NP-Specific Antibodies by ELISA

ELISA was performed as previously described [45 (link)]. The wells of 96-well microplates were coated with NP-His protein (0.5 μg/mL). Then, the microplates were washed with PBST (PBS containing 0.1% Tween 20), followed by blocking with 1% bovine serum albumin (BSA). Serial dilutions of the indicated NP antibodies were added to the wells and incubated at room temperature (RT) for 1 h. After washing with PBST, horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) was incubated in the wells at RT for 1 h. Finally, the plates were incubated with chromogenic substrate 3,3′,5,5,’-tetramethylbenzidine (TMB; Sigma-Aldrich, St. Louis, MO, USA). The reactions were stopped with 3-N HCl, and the optical density was measured using a microplate reader at 450 nm.