Fig. 4. Ratiometric Plug-and-Play Immunodiagnostics (RAPPID) case. (A) System description.25 The antibodies A and B are both conjugated to a part of split NanoLuc luciferase, which emit light upon complex formation. The antibodies are specific for a certain analyte but target different epitopes. Upon antibody-analyte sandwich formation, the effective molarity for the split luciferases increases, resulting in increased complex formation and consequently increased signal. (B) Local parameter sensitivity analysis for the system using cardiac troponin I data (and corresponding concentrations) and a perturbation of +50%. The reference values are the point estimates determined by the framework. (C) Concentration plot generated with framework displaying how each concentration changes as the total titrate concentration increases. This analysis is performed without any experimental data with the fixed parameter values KD,N: 2.5 μM, KD,A: 10 nM KD,B: 15 nM, EM: 100 μM, [A]tot: 1 nM, [B]tot: 1 nM. The species BT and BTT have been omitted for clarity. (D) Simulation of shift in active ternary complex (ABTa) concentration for different combinations of antibody affinities and an EM value of 10 μM. Both antibodies have a total concentration of 1 nM.
