BTBCT

BTBCT is mainly used as a label in time-resolved fluorescence immunoassays (TRFIA). The lower limit of detection for TSH TR-IFMA is 0.011 mIU/L in a 10 μl sample volume. The high fluorescence intensity and stability of BTBCT improves the sensitivity of the assay^[1]. In Vitro: Protein labeling with BTBCT
1：Dissolve BTBCT at a concentration of 10 mg/mL in dry ethanol.
2：Dissolve the target protein (e.g., BSA or streptavidin) in 0.1 M sodium carbonate buffer to the desired concentration, typically 1 mg/mL.
3：Gradually add the BTBCT solution to the protein solution while stirring.
4：Maintain stirring at room temperature for 2 hours.
5：Filter the mixture using an appropriate size filter (usually 0.2 µm).
6:If necessary, further purify the labeled protein via affinity chromatography to ensure that only functionalized protein is collected.
7:Dialyze the purified protein against a suitable storage buffer (commonly containing 0.05% NaN3).
8:Store at 4°C or freeze as needed.

Indirect Serum TSH TR-IFMA Procedure:
1: Coating the Microplate: Add 30 µL of coating buffer containing 15 µg/mL of anti-TSH McAb-05 to each well. Incubate at room temperature for 24 hours. Wash twice with wash buffer.
2: Blocking: Add 40 µL of blocking buffer (typically containing BSA or another protein) and incubate at room temperature for 6 hours. Wash and air dry.
3:Adding Samples and Standards: Add 10 µL of TSH standard or serum sample to be tested to each well. Add 10 µL of a mixture containing biotinylated anti-TSH McAb-04 and McAb-03 at a concentration of about 30 ng/µL. Incubate with gentle shaking at room temperature for 1 hour.
4:Washing: Thoroughly wash the microplate with wash buffer.
Adding Signal Generation Reagent: Add 20 µL of TSH assay buffer containing streptavidin-BSA-BTBCT-Eu complex to each well. Gently shake and incubate for another 20 minutes.
5: Final Washing: Wash four times and rinse twice with distilled water.
6: Fluorescence Measurement: Measure the fluorescence intensity of each well using a time-resolved fluorometer.
Direct Serum T4 TRFIA Procedure: 1: Microplate Preparation: Use a microplate coated with anti-T4 antibody, typically at a concentration of 10 µg/mL. Incubate the coated antibody at 4°C overnight.
2: Blocking Non-Specific Sites: Block the microplate with blocking buffer (usually containing 1% BSA) at room temperature for 1-2 hours.
3: Adding Samples and Label: Add a predetermined amount of the labeled T4-BSA-BTBCT-Eu complex along with the serum sample to be tested or T4 standard to each well.
4: Competition Reaction: Incubate the plate at room temperature for 1-2 hours.
5: Washing: Thoroughly wash the microplate with wash buffer.
6: Fluorescence Measurement: Measure the fluorescence signal of each well using a time-resolved fluorometer.