Binding Assay for Nectin-1 and CD96 Interaction

Nectin-1(143t)-MPB was purified from bacterial extract as described previously [18 (link)]. Purified Nectin-1(143t)-MPB was diluted to 10 μg/ml in PBS and used to coat 96-well ELISA plates overnight at 4°C. Control wells included only milk proteins. Plates were washed with 0.1% Tween 20 in PBS (PBS-Tween) and incubated in blocking solution (PBS-Tween with 5% milk) for 1 h at room temperature (RT). Plates were washed with PBS-Tween and incubated with various concentrations of hCD96(516t) in blocking solution for 3 h at RT. Plates were washed with PBS-Tween and incubated in blocking solution containing anti-tetra-His antibody (QIAgen) at 0.2 μg/ml for 1 h at RT. After being washed with PBS-Tween, the plates were incubated with horseradish peroxidase-conjugated secondary anti-mouse Ig antibody at 0.2 μg/ml in blocking solution for 30 min at RT. Plates were then washed with PBS-Tween and with 20 mM citrate buffer (pH 4.5). The horseradish peroxidase substrate [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid); Moss, Inc.] in citrate buffer (pH 4.5) was added, and the absorbance at 405 nm was read with a microtiter plate reader (Bio-Tek).