ELISA for Detecting HSV-1 Antibodies in Rhesus Macaques

ELISA plates (Corning, Shanghai, China) were coated with whole HSV-1 (F strain) as the antigen for serum IgG antibody detection. Briefly, Vero cells were infected with HSV-1 for 36 h, the cell suspension was harvested and sonicated for 30 s and 45 μg of cell lysate/well was coated on the ELISA plates at 4 °C overnight. The plates were washed 5 times with PBS/0.05% Tween 20 buffer and then blocked with PBS/5% BSA at 4 °C overnight. Each sample of the rhesus macaque serum was diluted at 1:500 in PBS/0.5% BSA, added to two wells, and then incubated at room temperature for 2 h. Next, the plates were washed as described above and incubated with HRP-conjugated mouse anti-rhesus IgG (Bioss, Shanghai, China) diluted at a ratio of 1:5000 in PBS /0.5% BSA at 37 °C for 1 h. TMB Single-Component Substrate solution (Solarbio, Beijing, China) was added to each well after 5 washes. The color reaction was stopped using ELISA Stop Solution (Solarbio, Beijing, China). Subsequently, the wells were read at an absorbance of 450 nm. Similarly, the ELISA plates coated with Vero cells were mock-infected as the control and the procedures were repeated as described above. The resulting values were determined by subtracting the values obtained for uninfected cell lysates from the values obtained with infected cell lysates [29 (link)].