Nunc maxisorp 96 well

Example 9

The biochemical IC50 for each of the five main lead molecules was determined on both TACI and BCMA by serially diluting purified monomeric VNARs in blocking buffer (PBS-0.1% Tween+2.5% Milk) supplemented with 0.5 nM BAFF-Fc (Sino Biological). The pre-blocked proteins were then exposed to Nunc Maxisorp 96 well plates which were coated at 1 μg/mL with the recombinant human TACI or BCMA (Peprotech), preblocked in (PBS-0.1% Tween+2.5% Milk). After washing in PBS-0.1% Tween, BAFF bound to its receptor was detected via its Fc moiety using a peroxidase-conjugated anti-human Fc (Sigma). Absorbance at 450 nm was recorded using an Envision multiwell reader (Perkin Elmer) and IC50 values were calculated by fitting the curves (non-linear regression) using GraphPad Prism®. The results are shown in FIG. 15 and the fitted IC50 values are tabulated below. Results showed that for the selected leads, IC50s were generally lower on BCMA than on TACI (compare FIG. 15A, TACI coating, to FIG. 15B, BCMA coating).

Fitted IC50 Values

Example 9

The biochemical IC50 for each of the five main lead molecules was determined on both TACI and BCMA by serially diluting purified monomeric VNARs in blocking buffer (PBS-0.1% Tween+2.5% Milk) supplemented with 0.5 nM BAFF-Fc (Sino Biological). The pre-blocked proteins were then exposed to Nunc Maxisorp 96 well plates which were coated at 1 μg/mL with the recombinant human TACI or BCMA (Peprotech), preblocked in (PBS-0.1% Tween+2.5% Milk). After washing in PBS-0.1% Tween, BAFF bound to its receptor was detected via its Fc moiety using a peroxidase-conjugated anti-human Fc (Sigma). Absorbance at 450 nm was recorded using an Envision multiwell reader (Perkin Elmer) and IC50 values were calculated by fitting the curves (non-linear regression) using GraphPad Prism®. The results are shown in FIG. 15 and the fitted IC50 values are tabulated below. Results showed that for the selected leads, IC50s were generally lower on BCMA than on TACI (compare FIG. 15A, TACI coating, to FIG. 15B, BCMA coating).