Envision multiwell reader

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).

Example 8

In order to group the blocking clones into different categories based on the epitope that each one recognizes on the BAFF proteins, Nunc Maxisorp 96 well plates were coated at 1 μg/mL with recombinant BAFF (Sino biologicals). Clones were grown in a 96 well format and periplasmic fraction was extracted by osmotic shock as previously described. The periplasmic fraction was then pre-blocked in PBS-0.1% Tween+2.5% Milk in the presence of a competitor VNAR-Fc molecule at 2 μM final concentration. Two anti-BAFF VNAR-Fcs were used in this assay (A07, B07) as well as a negative control lysozyme-binding VNAR (5A7). The pre-blocked fraction was then exposed to the coated surface, and after washing in PBS-0.1% Tween, bound VNARs were detected using a peroxidase-conjugated anti-FLAG antibody (Sigma). Absorbance at 450 nm was recorded using an Envision multiwell reader (Perkin Elmer). The results are shown in FIG. 14. Results showed that the binding of each clone to BAFF was competed by both A07 and B07, revealing that all the clones of the selected panel of blockers target the same (or at least an overlapping) epitope on BAFF.

Example 6

The ability of BAFF/BR3 (BAFFr) blocking clones to also block the interaction between BAFF and its other two receptors (TACI and BCMA) were tested in a blocking ELISA format. Briefly, selected clones were grown in a 96 deep-well format and periplasmic fractions were extracted as previously described. Nunc Maxisorp 96 well plates were coated at 1 μg/mL with either TACI or BCMA (Peprotech) and blocked in PBS-0.1% Tween+2.5% Milk. The periplasmic fraction was also pre-blocked in PBS-0.1% Tween+2.5% Milk in the presence of 0.5 nM BAFF-Fc (Sino Biological) before being exposed to the receptor-coated surface. 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). The results are shown in FIG. 12. Results showed that as compared to a negative control VNAR anti-Lysosyme (a-Lys), the thirteen tested clones appeared to block the interaction between BAFF and both TACI and BCMA.

Example 7

Clones blocking the interaction between BAFF and its three receptors were tested for their ability to cross-react with BAFF's closest related protein, APRIL. Clones were grown in a 96 well format, periplasmic fraction was extracted by osmotic shock as previously described and directly used in a binding ELISA. Nunc Maxisorp 96 well plates were coated at 1 μg/mL with either BAFF-Fc (Sino Biological), HSA (Sigma), or APRIL (Sigma) and periplasmic fraction, pre-blocked in PBS-0.1% Tween+2.5% Milk, was exposed to the coated surface. After washing in PBS-0.1% Tween, bound VNARs were detected using a peroxidase-conjugated anti-FLAG antibody (Sigma). Absorbance at 450 nm was recorded using an Envision multiwell reader (Perkin Elmer). As shown in FIG. 13, with the exception of B07 which showed a slight binding to APRIL, all the blocker clones appeared specific to BAFF.

Example 6

The ability of BAFF/BR3 (BAFFr) blocking clones to also block the interaction between BAFF and its other two receptors (TACI and BCMA) were tested in a blocking ELISA format. Briefly, selected clones were grown in a 96 deep-well format and periplasmic fractions were extracted as previously described. Nunc Maxisorp 96 well plates were coated at 1 μg/mL with either TACI or BCMA (Peprotech) and blocked in PBS-0.1% Tween+2.5% Milk. The periplasmic fraction was also pre-blocked in PBS-0.1% Tween+2.5% Milk in the presence of 0.5 nM BAFF-Fc (Sino Biological) before being exposed to the receptor-coated surface. 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). The results are shown in FIG. 12. Results showed that as compared to a negative control VNAR anti-Lysosyme (a-Lys), the thirteen tested clones appeared to block the interaction between BAFF and both TACI and BCMA.