Alexa fluor 564 conjugated goat anti rabbit igg

Infection of Huh7 cells for IFA was done as in FFA (see above) using 0.2 FFU/cell of OC43 or 229E in infection medium. The cells were fixed and permeabilized as in FFA. Serum samples were diluted 1:25, 1:100, 1:400 and 1:1600 into PBS supplemented with 3% bovine serum albumin (BSA). Dilution series of a serum sample was added to cells in duplicates, and incubated for 30 min, washed and 1:1000 dilution of polyclonal rabbit 229E anti-N-GST or rabbit OC43 anti-N-GST antibodies (Huttunen et al., manuscript) were added for another 30 min. The detection of bound antibodies was done as in FFA using Alexa Fluor 488 conjugated goat anti-human IgG, Alexa Fluor 564 conjugated goat anti-rabbit IgG and DAPI (Thermo Scientific). Binding of serum IgG antibodies to virus antigens in infected cells was visualized using EVOS FL Auto Fluorescence Inverted Microscope (Life Technologies). Serum samples that were positive in IFA with a dilution of 1:25 or higher were considered IFA-positive. IgG antibody titer was defined as the highest dilution with visually detectable antibodies.

Infection of Huh7 cells for IFA was done as in FFA (see above) using 0.2 FFU/cell of OC43 or 229E in infection medium. The cells were fixed and permeabilized as in FFA. Serum samples were diluted 1:25, 1:100, 1:400 and 1:1600 into PBS supplemented with 3% bovine serum albumin (BSA). Dilution series of a serum sample was added to cells in duplicates, and incubated for 30 min, washed and 1:1000 dilution of polyclonal rabbit 229E anti-N-GST or rabbit OC43 anti-N-GST antibodies (Huttunen et al., manuscript) were added for another 30 min. The detection of bound antibodies was done as in FFA using Alexa Fluor 488 conjugated goat anti-human IgG, Alexa Fluor 564 conjugated goat anti-rabbit IgG and DAPI (Thermo Scientific). Binding of serum IgG antibodies to virus antigens in infected cells was visualized using EVOS FL Auto Fluorescence Inverted Microscope (Life Technologies). Serum samples that were positive in IFA with a dilution of 1:25 or higher were considered IFA-positive. IgG antibody titer was defined as the highest dilution with visually detectable antibodies.