ELISA for Transthyretin Quantification

ELISA was carried out using commercially available anti-TTR monoclonal antibody (ProSci Antibody, USA). For comparison between patient and control group, 60 plasma samples from each group were taken. Individual plasma samples from the enrolled participants were diluted (1 μl/200 μl) in a coating buffer (0.01 M Na₂CO₃ and 0.035 M NaHCO₃, pH 9.6), coated on 96 well plates (Thermo Scientific, Nunc, USA) and incubated overnight at 4°C. Next day the plates were washed three times with 100 μl PBST and incubated with 100 μl blocking buffer (1% BSA in 1X PBS) for 1 h at RT. The blocking buffer was extracted, plates were washed and incubated with 100 μl of anti-TTR as a primary antibody (1∶2000 dilution) for 2 h at RT. The plates were then washed three times and incubated with 100 μl of anti-mouse HRP conjugate (1∶1000 dilution) for 1 h and developed with ortho phenylene diamine (1 mg/ml in 0.05 M citrate buffer and 5 μl/ml H₂O₂, Sigma-Aldrich, USA). The reactions were stopped by adding 2N H₂SO₄ in each well and binding efficiency was checked by reading absorbance at 492 nm in an ELISA reader (Spectramax Plus; Molecular Devices, USA) [20] (link). Plasma levels of TTR in RA patients was also compared from patients of different diseases.