Eia ria high binding 96 well plates

Example 3

Affinity maturation of DNV3 was conducted according to the previously reported protocol (Zhu et al., J Infect Dis 2008, 197: 846-853). Briefly, a phage-display light-chain shuffling Fab library was constructed, panned and screened for DNV3 variants with higher binding to recombinant human LAG3. A total of 14 DNV3 variants were identified which had the same heavy chain as DNV3 but different light chains. ELISA was performed to measure the binding activity of the selected DNV3 variants. Briefly, recombinant human LAG3 (Sino Biological Inc.) was coated on Corning EIA/RIA high-binding 96-well plates (Corning Inc.) at 50 ng per well overnight at 4° C. and blocked with 3% nonfat milk in PBS (pH7.4). Fivefold serially diluted antibodies were added and incubated at room temperature for 2 h. The plates were washed with PBS containing 0.05% Tween 20. Bound antibodies were detected by HRP-conjugated anti-FLAG tag antibody (Sigma-Aldrich). The assay was developed at room temperature with TMB substrate (Sigma-Aldrich) and monitored at 450 nm with a microplate reader. The result showed that all purified DNV3 variants had higher binding (EC₅₀s, 0.6-4 nM) than DNV3 (EC₅₀, 6 nM) (FIG. 6).