Example 5
Determination of Serum Titers (ELISA) from Immunized Rabbits
Human recombinant soluble TPBG extracellular domain was immobilized on a 96-well NUNC Maxisorp plate at 2 μg/ml, 100 μl/well, in PBS, followed by: blocking of the plate with 2% Crotein C in PBS, 200 μl/well; application of serial dilutions of antisera, in duplicates, in 0.5% Crotein C in PBS, 100 μl/well; detection with either (1) HRP-conjugated donkey anti-rabbit IgG antibody (Jackson Immunoresearch/Dianova), or (2) HRP-conjugated rabbit anti-human IgG antibody (Pierce/Thermo Scientific; 1/5000), or (3) biotinylated goat anti-human kappa antibody (Southern Biotech/Biozol; 1/5000) and streptavidin-HRP; each diluted in 0.5% Crotein C in PBS, 100 μl/well. For all steps, plates were incubated for 1 h at 37° C. Between all steps plates were washed 3 times with 0.05% Tween 20 in PBS. Signal was developed by addition of BM Blue POD Substrate soluble (Roche), 100 μl/well; and stopped by addition of 1 M HCl, 100 μl/well. Absorbance was read out at 450 nm, against 690 nm as reference. Titer was defined as dilution of antisera resulting in half-maximal signal.