CORTICOTROPIN-RELEASING HORMONE (CRH)-BINDING PROTEIN INTERFERENCE WITH CRH ANTIBODY-BINDING - IMPLICATIONS FOR DIRECT CRH IMMUNOASSAY

Direct immunoassay of plasma corticotrophin-releasing hormone (CRH) is potentially subject to interference from high levels of CRH-binding p rotein (CRH-BP) that exist in the human circulation. In this study, we tested the effect of CRH-free, native CRH-BP (6.4 nmol/l) purified fr om human plasma, CRH-BP diluent alone, normal human plasma (containing 5.8 nmol endogenous CRH-BP/l) and normal sheep plasma (containing no CRH-BP) on the binding of I-125-labelled CRH tracer to five N-terminal and four C-terminal CRH antibodies. All anti-(1-20)CRH N-terminal ant ibody dilution curves displayed marked inhibition of binding in the pr esence of purified CRH-BP and human plasma in comparison with the curv es with the control diluent or sheep plasma. Almost no inhibition of b inding was obtained with any of the C-terminal antibodies (all directe d against epitopes within the last six amino acids of CRH) and the fou r dilution curves were nearly superimposable. Liquid-phase CRH IRMAs w ere then developed with different combinations of two of each of the N - and C-terminal antibodies, using radiolabelled IgG prepared from pur ified C-terminal antisera as tracer and raw N-terminal antisera as the link antibodies to the separating system. The addition of dilutions o f purified CRH-BP over the range 1.25-20 nmol/l to the IRMA standard c urve in assay buffer resulted in a dose-dependent reduction in the sig nal; with 5 nmol CRH-BP/l, a level commonly found in human plasma, the reduction in binding was 67% and 81% in two different IRMAs at a CRH concentration of 631 pmol/l. With endogenous CRH-BP in human plasma, a dose-dependent inhibition of binding similarly resulted, with the pla sma containing the most CRH-BP causing the greatest inhibition. Since plasma CRH-BP levels in humans vary widely, direct plasma IRMA using t hese type of antibodies will give inaccurate results and initial extra ction of the CRH is necessary. Methanol extraction of synthetic synthe tic or endogenous CRH is shown to be both highly efficient and unaffec ted by variable amounts of endogenous or exogenous CRH-BP; it is there fore suitable as the first step in plasma CRH measurement by IRMA.