Neutralizing Antibody Titer Assay for MP-12

Sera were heat inactivated at 56°C for 30 minutes and tested for nAb to the MP-12 virus as described previously.²⁰ (link) Briefly, equal volumes of 2-fold dilutions ranging from 1:5 or 1:10 through 1:1280 of each sample were prepared in minimal essential medium and incubated overnight at 4°C with an equal volume of 50 to 100 PFUs of the MP-12 virus. On the following day, 50 µL of the virus–sera mixture were inoculated in duplicate onto a confluent monolayer of Vero E6 cells grown in 24-well plates. Cultures and inocula were incubated for 1 h at 37°C and 5% CO₂. A mixture of 1% SeaKem agarose (VWR) with an equal volume of 2× Eagle’s basal medium with Earle’s salt, HEPES, sodium bicarbonate, 8% fetal bovine serum, and 1% penicillin/streptomycin and L-glutamine (Thermo Fisher Scientific) was prepared and 0.5 mL was overlaid onto each cell culture. The agarose overlay was allowed to solidify, and then the cultures were incubated for 3 days at 37°C and 5% CO₂. At 3 days post-infection, cultures were stained with a 0.33% neutral red solution and incubated for 4 to 6 hrs to identify PFUs. The PFUs were counted and the dilution of serum that reduced the number of plaques relative to the negative human serum control by 80% was considered to be the nAb titer.