Preliminary research

The first step of the development process is to define the specifications of the project and the best strategy to apply. One or more recommendations could be suggested in function of:

• Type of test: Homogeneous / Heterogeneous TR-FRET
• Target to be quantified/detected: antibody, protein, biomolecules, peptide, small molecule, etc.
• The expected amount of antigen present
• Matrix: serum, plasma, culture supernatant, etc.
• Choice of primary material: samples, antigen, antibodies, etc.

Optimization

Precise and optimal performance conditions are studied at each step of the test protocol. At this point, we evaluate and optimize the best parameters/conditions (best acceptor, saturation, antigen capture, detection, incubation time and temperature, etc.).

Validation

Validating the method ensures results are reproducible, reliable, robust, and accurate. Validation of the analytical method will be subject to the following experimental checks:

• Specificity (the method must differentiate the targeted analyte from all other matrix components)
• Linearity (range, LLOQ)
• Sensitivity (LOD)
• Accuracy (spiking recovery test)
• Precision (repeatability = intra assay, inter assay; reproducibility = inter laboratory assay, or on different days or different plates)
• Robustness (defined as the ability to remain unaffected by small variations in method parameters. These parameters could be incubation temperature, temperature of used components or varied incubation time due to time delay during pipetting).