Table 2. Results of Antibody Generation for the Selections Using the Biotinylated Peptides in the Two RapMAT Panning Roundsa.
gene symbol | protein description | peptide sequence | clones screened in ELISA | ELISA positive | binding to free peptide | clones sequenced | unique antibody sequences | affinity range (nM) |
---|---|---|---|---|---|---|---|---|
Spp1 | osteopontin | GDSLAYGLR | 1472 | 408 (28%) | 81 (6%) | 40 | 9 | 0.6–380 |
SELE | E-Selectin | NWAPGEPNNR | 1472 | 325 (22%) | 192 (13%) | 42 | 20 | 0.4–243 |
ADA17 | ADAM17 metalloprotease | VDNEELLPK | 1472 | 577 (39%) | 40 (3%) | 33 | 17 | 33–91 |
Clones with a signal at least five times above the background in ELISA on the respective biotinylated peptide captured by coated streptavidin were considered positive. The clones which in addition showed an at least five times lower signal on the biotinylated peptide in the presence of 10μM peptide with free amino- and C-termini were considered positive for binding to free peptide. For Spp1 and ADA17, also a few clones with less than five-fold signal reduction in the competition screening were sequenced. The affinity to the free peptide was determined for 5 (Spp1), 17 (SELE), and 5 antibodies (ADA17).