Endocrine Abstracts

Corticosteroid-binding globulin (CBG) transports glucocorticoids in blood and is a serine protease inhibitor family member. Plasma CBG has a reactive center loop (RCL) structure that when cleaved by specific proteases, including neutrophil elastase, results in a loss of high affinity steroid-binding activity. Measurements of CBG levels are typically based on functional assays of its steroid-binding capacity or immunoassays, including enzyme-linked immunosorbant assays (ELISAs). Recently, CBG levels have been measured using different ELISAs that rely on monoclonal antibodies that discriminate between CBG molecules with an intact versus a cleaved RCL. In blood samples from healthy and diseased individuals, discrepancies in CBG levels measured using these ELISAs have been interpreted as evidence for CBG with a cleaved RCL and a low affinity for cortisol. We have questioned these assumptions by studying the steroid-binding activity and biochemical properties of CBG in blood samples in which there is a clear discrepancy in ELISA measurements. In addition, we sought to identify RCL-cleaved forms of human CBG in blood samples from patients suffering from acute inflammation in an intensive care unit (ICU) setting, in whom plasma CBG levels are very low. Our results found no evidence for cleavage of the RCL in CBG in blood samples from ICU patients, irrespective of whether their CBG ELISA measurements were concordant or discrepant. The absence of CBG with a cleaved RCL was also demonstrated using a heat-ramp polymerization assay in a serum sample that exhibits a discordancy in ELISA values. Moreover, when a monoclonal antibody designed to specifically recognize an intact RCL was used to immuno-absorb CBG from a discrepant sample, the residual CBG molecules had the same high affinity (Kd ~1.75 nM) for corticosterone as CBG in the sample prior to immuno-absorption (Kd ~1.72 nM) or in samples in which there is no discrepancy in ELISA values. It is therefore suggested that in some samples, CBG molecules react abnormally to ELISAs because of structural differences that influence the epitopes recognized by specific monoclonal antibodies.