4.12. Calculation of HTRF Signals

The HTRF signals (ΔF) were calculated as normalized fluorescence transfer signals [63] using the following formula:

In the formula, the average fluorescence intensity of the sample acceptor (Ab-Alexa647) at 665 nm, i.e., "F(665 nm)Sample", was divided by the fluorescence intensity of the sample donor (Ab-Loisto615) at 612 nm i.e., "F(612 nm)Sample". The same ratio, calculated for the control, was subtracted from the sample ratio and the result was then divided by the control ratio. Two types of controls were used in this study: reference to blank (PBS) or reference to an internal control test (An additional test, where only the specific donor was applied, coupled with a non-specific acceptor). To evaluate the limit of detection (LOD), the average of background ΔF readings was calculated for each assay as the mean response of at least six “noise” (PBS) samples. The LOD was defined as five standard deviations (SDs) above the average background with a coefficient of variation (CV) < 15%. These values yielded a signal (noise + 5 SD)-to-noise (average noise), which was considered to be the LOD threshold. This value was lower than 0.5 for all tests. For simplicity in data presentation and comparison, a value of ΔF ≥ 0.5 was set as the positive threshold for all assays in accordance with ICH guidelines for validation of the analytical procedures [64]. This calculation enabled the normalization of multiple experiments and the determination of a universal threshold for positive samples.