Figure 2: The binding activity of TACI to BAFF and its domain mutants were tested with ELISA method. ELISA was performed using plates coated with 100nM of Trx 、rhBAFF 、rhBAFF mutants and human BAFF (PEPROTECH) respectively. TACI-Ig prepared with dilution buffer was then added to each well. The HRP-GAH was used as the secondary antibody. Binding signals were visualized using TMB substrate and analyzed at 450 nm. The OD value for recombinant BAFF was set as 100% binding activity. The binding capability of BAFF mutant was calculated by ODBAFF mutant/ODrhBAFF.
rhBAFF: recombinant human BAFF; Trx (Thioredoxin) was a BAFF-unrelated protein and used as the negative control. BAFF mutants (i.e. BAFFM1 (from Ile158 to Phe165 were replaced by alanines), BAFFM2 (from Asp203 to Leu211 were replaced by alanines), BAFFM3 (from Ser225 to Arg231 were replaced by alanines), BAFFM4 (from Ile233 to Glu238 were replaced by alanines)); human BAFF(peprotech): positive control.