Enzyme linked immunosorbent assay elisa

After recruitment and clinical interview, 5–8 ml of peripheral venous blood was obtained by trained phlebotomists. The blood was allowed to clot for 30 minutes at room temperature (RT) and serum was obtained by centrifuging the blood samples at 1300 g for 10 minutes at RT. Serum samples were aliquoted into 2 ml Eppendorf tubes and stored at −80 °C for subsequent analyses. One aliquot was sent to the NUH Department of Laboratory Medicine for serum C3 and C4, and anti-dsDNA assays by immunoturbidimetry and enzyme-linked immunosorbent assay (ELISA) (BioRad), respectively. Adipocyte fatty acid binding protein (aFABP) which was reported to be correlated with subclinical atherosclerosis in SLE^{14 (link)}, was determined by a commercially available ELISA kit (Aviscera Bioscience, Inc., Santa Clara, CA, USA) following the manufacturer’s instruction. The detection range was 1.56–100 ng/ml, with intra-assay and inter-assay precision of 4–6% and 8–10%, respectively. As per standard of care, serum total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-c) levels determined by the NUH Department of Laboratory Medicine were obtained for the SLE subjects.

For this experiment, 100 μl of heparinized whole blood was dispensed into 96 well plates (BIORAD, USA) in duplicate and was stimulated with or without 50 μM isopentenyl pyrophosphate (IPP) (Sigma Aldrich, USA) in RPMI 1640 medium supplemented with 10% Fetal Calf Serum, 1% L -glutamine, penicillin, and streptomycin. After 24 h incubation in 5% CO₂ incubator at 37 °C, supernatants were harvested from each well for detection of IPP-specific IFN-γ production using an Enzyme Linked Immunosorbent Assay (ELISA) (BIORAD, USA).
The data obtained in the presence of stimuli exhibited Optical Density (OD values above unstimulated controls, but were low and in the non-linear portion of the IFN-γ standard curve. Considering the extrapolation of OD values to IFN-γ concentrations therefore unreliable, and since all culture supernatants were evaluated by ELISA in the same experiment, we opted to simply express data as OD units in the presence of stimulus less that in the absence of stimulus.