Analysis of cytokines with Enzyme-Linked Immunosorbent Assay
Plasma was obtained from whole blood, drawn in EDTA tube, by
centrifuging at 400xg, for 10 min at room temperature. Plasma samples
from IgAN patients and HC were then analyzed with enzyme-linked
immunosorbent assay (ELISA) to determine the concentration of the
following factors; monocyte chemoattractant protein (MCP-1) (Human
CCL2/MCP-1 Quantikine ELISA Kit, R&D Systems, USA), soluble CD14 (Human
CD14 Quantikine ELISA Kit, R&D Systems), soluble CD40L (Human CD40
Ligand/TNFSF5 Quantikine ELISA Kit, R&D Systems), B cell activating
factor (BAFF) (Human BAFF Quantikine ELISA Kit, R&D Systems), IL-6
(Human IL-6 Quantikine HS ELISA Kit, R&D Systems), Fractalkine (Human
CX3CL1/Fractalkine Quantikine ELISA Kit, R&D Systems) and MIP-1 (Human
CCL3/MIP-1 alpha Quantikine ELISA Kit, R&D Systems). The samples were
assayed in duplicates and the average values were considered for further
analysis. The optical density (OD) in blank well was subtracted from the
OD values in samples before calculation of concentrations.