Aptamer-based ELISA for Detecting Mouse Antibodies

ELISA plates were coated with 50 μl of plasma (1/200 dilution in PBS) from non-infected or infected mice at different times (21 to 124 dpi). After blocking with 1% bovine serum albumin (BSA), biotinylated Aptamer-29 (Apt-29) was added to each well and incubated for 1 hour at room temperature. Next, plates were washed three times with PBS and a Streptavidin-Alkaline phosphatase conjugate was added. Following a 30-minute incubation, plates were washed with PBS and the bound aptamers were detected using 4-Methyliumbeliferyl Phosphate (4-MUP, Sigma, MO). Fluorescence was read at 340 nm and emission was recorded at 440 nm, using a Spectra Max, M5 microplate reader (Molecular Devices, PA) [31 (link)].