Neutralizing Antibody Titration for LOM Virus

Anti-CSF-neutralizing antibodies were detected by a neutralizing peroxidase-linked assay (NPLA) according to the standards manual of the World Organization for Animal Health (WOAH) [26 (link)]. Heat-inactivated serum samples (56 °C, 30 min) were serially diluted 2-fold (1:2 to 1:2048) and neutralized by adding 200 TCID₅₀ of the LOM virus for 1 h at 37 °C. The neutralized serum containing virus was inoculated into PK-15 cells (porcine kidney cells) and cultured for 3 days in a 37 °C incubator. The cells were then fixed in chilled 80% acetone and reacted with a commercial anti-LOM mAb (MEDIAN diagnostic Co., Cat No. 9013, Chuncheon, Republic of Korea). The cells were then stained using a VECTASTAIN^® ABC-HRP kit (Vector Laboratories Inc., Cat no. PK-4000, Newark, CA, USA) and an ImmPACT DAB Peroxidase (HRP) substrate (Vector Laboratories Inc., Cat no. PK-4100, USA). Neutralizing antibody titers were expressed as the reciprocal of the highest dilution that yielded 50% neutralization and determined to ≥10-fold positive antibodies according to the WOAH criteria.