Hi Chatterjee, I hope you're well.

• Yes, you can assess the interaction of your conjugate with the ligand using the microplate fluorescence assay. To do this, you need to take a few precautions, including using a dark microplate. This is because, unlike the absorbance assay in microplates, it is not interesting to detect the light transmitted by the sample, but only the light emitted by the wells. In the parameters of your equipment, you must have compatible microplate models. if you don't already have one, I recommend the perkin elmer ones
• Care must also be taken when preparing the experiment. For example, you should place wells only for your fluorophore, only for your recognition molecule and wells for the test (your conjugate). You should also wash with saline or phosphate solution after incubating the conjugate with the protein, to remove material that has not bound to the immobilized protein.
• Another important point is to make sure that the conjugate has no unpaired electrons, which could result in a non-specific response. In the following work, developed by a colleague in the laboratory, you can use the methodology employed as a basis.

Article Quantum dots-based fluoroimmunoassay for anti-Zika virus IgG...

I hope I've helped,

best regards,

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MS.c João V. A. Lima

Regarding the plate type, solid black plates are preferred for organic fluorophores. Top reading should be selected, if the plate reader allows a choice of top and bottom reading. Using black plates prevents cross-talk between wells.

For lanthanide-chelate fluorophores (europium, terbium), solid white plates are preferred with time-gated measurement. The plates themselves are fluorescent, but time-gating allows the short-lifetime plate fluorescence to decay, whereas the long-lifetime lanthanide fluorescence continues. Black plates can also be used, but the measurement is more sensitive with white plates.

Hi,

We develop advanced FLISA (Fluorescence-Linked Immunosorbent Assays) using ultra-bright lanthanide-based fluorescent nanoparticles. As mentioned by Adam B Shapiro, FLISA with lanthanide nanoparticles offers several key advantages over conventional fluorophores or ELISA:

• High Brightness: The unique optical properties of nanoparticles provide exceptionally strong signals, enabling the detection of low-abundance targets.

• Background Noise Elimination: The long fluorescence lifetime allows for time-resolved fluorescence (TRF), effectively reducing background autofluorescence and non-specific signals.

• Flexible Plate Compatibility: Our assays work with both black and white 96-well plates, offering flexibility based on experimental needs.

• Versatile Reader Compatibility: Compatible with any plate reader equipped with TRF or HTRF modules, ensuring easy integration into existing lab setups.

Thank you all Joan Goetz Adam B Shapiro João Victor Araújo de Lima for your kind guidance.