CTGF Assay Principle

CTGF is measured using a sandwich immunoassay involving two specific anti-CTGF antibodies, respectively labeled with HTRF donor and acceptor dyes. The intensity of the signal is directly proportional to the concentration of CTGF present in the sample.

CTGF Assay Protocol

Cell supernatant, sample, or standard is dispensed directly into the assay plate for CTGF detection by HTRF reagents (384-well low-volume white plate in 20 µL). The antibodies labeled with the HTRF donor and acceptor are pre-mixed and added in a single dispensing step, to further streamline the assay procedure

Intra / Inter assay

Intra-assay (n=24)

Each of the 3 samples was measured 24 times, and the %CV for the concentration was calculated for each sample

Inter-assay (n=4)

Each of the samples was measured in 4 different experiments, and the %CV for the concentration was calculated for each sample.

CTGF secretion in MRC5 cell line stimulated with TGFbeta

Human MRC5 cells were plated at 400,000 cells/well in a 12-well plate and cultured overnight with 600 µL of complete MEM cell culture medium, at 37°C, 5% CO2. Cells were then stimulated with increasing concentrations of TGFbeta (0.2 to 20 ng/mL) under 600 µL of MEM + 1%. After 24h of stimulation, 16 µL of supernatant were transferred into a white detection plate to be analyzed using the Human CTGF assay. The HTRF signal was recorded after an overnight incubation.