Fig. 3. Setting up the SIMPL ELISA platform and its evaluation with reference PPIs.

a An extra HA tag is introduced into the bait construct. The spliced proteins are captured by immobilized α-FLAG antibody and measured with α-HA antibody conjugated with HRP. All four SIMPL formats described in Fig. 2 are compatible with ELISA. b The bait proteins can be measured similarly by ELISA using immobilized α-V5 antibody and HRP-conjugated α-HA antibody probe. c The ELISA platform was assessed with the rapamycin-induced FRB/FKBP1A interaction. HEK 293 cells expressing FRB and FKBP1A in different formats were treated with different doses of rapamycin as indicated for 30 min followed by lysis and ELISA analysis. The experiment was performed with four technical replicates and each replicate is presented as a single dot. d Benchmarking analysis of the overall performance of SIMPL ELISA platform. Eighty-eight PPIs well documented in literature were chosen as positive reference set (PRS). Eighty-eight pairs of bait/prey combinations with the least possibility of interaction were selected from the bait and preys of the PRS to form the random reference set (RRS). Both sets were then screened using SIMPL ELISA analysis in both IN/IC and IN/CIC formats. The spliced signal was normalized to bait expression. Receiver operating characteristic (ROC) analysis was performed as presented. Data shown here are a representative result of three experiments. e Performance of the SIMPL assay in terms of sensitivity (true-positive rate) and false-positive rate (1-specificity). The threshold values for positive detection were determined from ROC analysis as in d. Results are averages of three independent experiments showing mean recovery rate ± SEM. f Comparison of SIMPL detection of individual PPIs in the PRS to results from seven different PPI methods obtained from the literature^16–18. Source data are available in the Source Data file.