Time-resolved fluorescent microspheres are rare ions encapsulated within polystyrene, featuring high fluorescence intensity, large Stokes shift, small batch-to-batch variation, good repeatability, and are suitable for the research and large-scale production of lateral chromatography reagents. 

Technical Specifications: 

Excitation wavelength: 365nm 

2. Emission wavelength: 615nm 

3. Functional group: Carboxyl group (COOH) (Customizable SA) 

3. Particle size range: 200nm - 400nm 

4. Uniformity Index: CV < 5% 

5. Storage conditions: Store at 2-8℃. Do not freeze.

Labeling Process

1. Take 100 µL of time-resolved fluorescent microsphere solution (1 wt%), add 500 µL of activation buffer to wash once, and centrifuge to remove the supernatant.

2. Add 500 µL of activation buffer to resuspend the microspheres. Then add 5 µL EDC and 10 µL NHS (stock concentrations of both are 100 mg/mL, diluted in activation buffer, prepared fresh before use). Rotate and shake in the dark for 30 min, then centrifuge to remove the supernatant.

3. Wash once with 500 µL of coupling buffer. Resuspend the microspheres in 500 µL of coupling buffer, add 10–100 µg of antibody (adjust based on project requirements), rotate and shake in the dark for 2 h, then centrifuge to remove the supernatant.

4. Add 500 µL of blocking solution to resuspend, rotate and shake in the dark for 1 h, then centrifuge to remove the supernatant.

5. Wash once with 500 µL of storage solution and centrifuge to remove the supernatant.

6. Resuspend in 100 µL of storage solution and store at 2–8 °C.

Note: Recommended centrifugation speed is 15,000 rpm (22,000 × g); centrifugation time is 15–20 min.

Precautions

1. Time-resolved fluorescent microspheres should be vortexed or sonicated to homogeneity before use; probe sonication is the most effective method for resuspension.

2. EDC is highly sensitive to moisture and cannot be used if damp or caked. EDC solution should be prepared fresh before use; NHS should also be prepared fresh.

3. To achieve optimal coupling efficiency, the coupling reaction buffer system must be free of free amino groups; Tris, glycine, and acetate buffers must not be used.

4. Adding trace surfactants (e.g., Tween-20, not exceeding 0.01%) to the reaction buffer can effectively prevent microsphere aggregation.

5. The concentration of the microsphere solution can be adjusted based on the signal intensity of the project; the recommended dosage for T-line labeling is 3%–8%, and for C-line labeling, 1%–2%.

6. Pad material, pad pretreatment buffer, microsphere diluent, activator dosage, and antibody ratio can be optimized based on project performance.

Disclaimer: This protocol is intended for preliminary research reference only. Researchers should optimize specific experimental conditions according to their own project requirements.