Anti-Neu5Gc Antibody Kit Protocol - ELISA
Coat the 96-well ELISA plate with the target molecule in 50 mM sodium carbonate-bicarbonate buffer, pH 9.5 (up to 1 μg/well in 100 μl carbonate buffer, in triplicate) for at least 2 hours at ambient temperature or overnight at 4°C.
Wash the wells 3 to 5 times with PBS.
Block wells with PBS-T (200 μl/well) for 1 hour at room temperature.
Add the anti-Neu5Gc antibody (or the negative control antibody), diluted in PBS-T, to the appropriate well(s) and incubate for 2 hours at room temperature.
Wash wells as before with PBS-T. 
Add enzyme-conjugated secondary antibody (donkey-anti-chicken antibody, 1:10,000 in PBS) to each well and incubate for 1 hour at room temperature.
Wash wells again with PBS-T. 
Add enzyme substrate, allow the wells to develop; quench the reaction if necessary.
Read absorbance on an ELISA plate reader at the appropriate wavelength.
