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 .
