Corticotrophin-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 protein (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 from 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 125I-labelled CRH tracer to five N-terminal and four C-terminal CRH antibodies. All anti-(1-20)CRH N-terminal antibody dilution curves displayed marked inhibition of binding in the presence of purified CRH-BP and human plasma in comparison with the curves with the control diluent or sheep plasma. Almost no inhibition of binding was obtained with any of the C-terminal antibodies (all directed against epitopes within the last six amino acids of CRH) and the four dilution curves were nearly superimposable. Liquid-phase CRH IRMAs were then developed with different combinations of two of each of the N- and C-terminal antibodies, using radiolabelled IgG prepared from purified C-terminal antisera as tracer and raw N-terminal antisera as the link antibodies to the separating system. The addition of dilutions of purified CRH-BP over the range 1.25-20 nmol/l to the IRMA standard curve in assay buffer resulted in a dose-dependent reduction in the signal; 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Linton
E A
EA
Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Oxford, UK.
Perkins
A V
AV
Hagan
P
P
Poole
S
S
Bristow
A F
AF
Tilders
F
F
Corder
R
R
Wolfe
C D
CD
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism*; D003346:Corticotropin-Releasing Hormone / Q000097:blood / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D015592:Immunoradiometric Assay
D000906:Antibodies; D002352:Carrier Proteins; C067133:corticotropin releasing factor-binding protein; D003346:Corticotropin-Releasing Hormone