ELBA and ELISA

Binding of virions and overexpressed monobodies was monitored in microtiter plate wells, as described previously [43]. An ELBA was performed, with published protocols [31,43], to evaluate the specificities of clones isolated from the primary library. This was accomplished through several steps, which are briefly described here. First, GST fusions to the SH3 domains of Lyn, Hck, Src, and Yes were diluted in phosphate buffered saline (PBS; 137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄) to 5 μg/mL and distributed to triplicate wells of a Nunc Maxisorp microtiter plate (Thermo Fisher Scientific, # 44240421), for overnight incubation at 4°C. As controls, bovine serum albumin (BSA) and the anti-FLAG antibody (Sigma-Aldrich) were added to separate wells. The next day, the non-specific binding sites in the wells were blocked with casein (Thermo Fisher Scientific, # PI-37528) for 1 h, followed by addition of FN3-alkaline phosphatase fusion protein of TA1, TA7 and TA8 that had been diluted to 5 μg/mL in PBS containing 0.1% Tween 20. After 1 h incubation shaken at 200 rpm, the microtiter plate was washed five times with PBS + 0.1% Tween 20 and loaded with a chromogenic substrate, para-nitrophenyl phosphate (pNPP; Sigma-Aldrich, # N9389). The resulting absorbance was measured at 405 nm with a microtiter plate spectrophotometer (BMG Labtech; Cary, NC).

For assessing the specificities of three TA8 variants, 2H7, 2H10 and 3C12, phage ELISA was performed. First, a Nunc microtiter plate was first coated with SUMO-SH3 domains of Lyn, Hck and Btk at 5 μg/mL. The blocking reagent of casein served as the negative control. The ELISA assay was performed as described [43], and it was run in triplicate. Similar phage ELISA was performed for evaluating the specificity of 2H7 to the SH3 domains of all SFKs. In this assay, 1F11, a monobody that binds to several SFKs SH3 domains [31], was included to assess the quality of the immobilized SH3 domains. An anti His₆-tag antibody conjugated to horseradish peroxidase (HRP) (Sigma-Aldrich, # A7058) was also included to normalize the amount of SH3 domain proteins immobilized in the microtiter plate wells.