Characterization of an enzyme-linked immunosorbent assay for soluble CD163

Abstract

We have recently identified a soluble plasma form of CD163 sCD163, the macrophage receptor for clearance of haptoglobin-haemoglobin complexes, and we have observed highly elevated levels of sCD163 in subgroups of haematological patients. In the present study, we describe the optimization and characterization of a sandwich ELISA for the determination of the concentration of sCD163 in plasma and serum. The optimal concentrations of antibodies were determined systematically and the assay was calibrated by CD163 purified from human spleen membranes. The minimum detection limit was below 6.25 microg/L. Recovery of CD163 added to plasma samples was 106%. The concentration of sCD163 in paired serum and plasma samples correlated well (r2=0.99); however, serum levels were 1.1 times higher than the plasma levels. The addition of haptoglobin-haemoglobin complexes did not influence the assay. A very high stability of sCD163 was measured in whole blood and in plasma subjected to different temperatures and after repeated freezing and thawing.

Soluble CD163: a biomarker linking macrophages and insulin resistance

“…The limit of detection was 6.25 μg/l. Soluble CD163 is robust to thawing, and stability has been rigorously verified for at least 7 months at −20°C [16]. Postprandial levels of sCD163 (4 h after a meal) did not differ significantly from fasting levels in healthy individuals (data not shown).…”

“…We determined serum concentrations of sCD163 in duplicate samples that had been frozen for up to 7 years at −20°C by use of an in-house sandwich ELISA on a BEP-2000 ELISA analyser (Dade Behring, Marburg, Germany) [16]. In each run, we co-analysed control samples and serum calibrator with concentrations traceable to purified CD163.…”

Interleukin-37 Expression Is Increased in Chronic HIV-1-Infected Individuals and Is Associated with Inflammation and the Size of the Total Viral Reservoir

“…Plasma levels of sCD163 were determined using an in-house assay. This sandwich ELISA assay is using an polyclonal rabbit anti-CD163 IgG as capture antibody and a monoclonal anti-CD163 as detection, described in more details previously (42). Data from one patient in the sCD163 analysis were excluded as an outlier.…”

Molecular Characterization of the Haptoglobin·Hemoglobin Receptor CD163

“…Tris buffer (with CaCl 2 or EDTA) was used to equilibrate the column and elute the sample. Fractions of 0.5 ml were collected and analyzed for soluble CD163 in enzyme-linked immunosorbent assay as described (23). Hp phenotyping was performed by Western blotting of serum from healthy individuals.…”