Corticotrophin‐Releasing Factor‐41 in the Human and Rat—Utility of a Highly Sensitive Enzyme Amplified Immunometric Assay

Abstract

RbstractW e have developed a highly sensitive and specific immunoassay for humanlrat corticotrophin-releasing factor-41 (CRF-41) to enable determination of immunoreactive CRF-41 levels in biological samples. To achieve high specificity, sensitivity and s p e e dwe have used two antisera in a sandwich enzyme immunoassay combined with enzyme amplification. The assay h a s a sensitivity of 0.08 fmollwell compared with radioimmunoassay sensitivities of 0.5 fmol/tube and is highly specific for the intact CRF-41 molecule. Measurement of samples is complete within 24 h compared with the 5 days required to obtain sensitive radioimmunoassay measurement. The assay has been used to measure both rat hypothalamic CRF-41 tissue content and release in vitro with good correlation when compared to radioimmunoassay measurement using antisera rC70 (0.983) or R1 (0.953). The assay only measures immunoreactive CRF-41 coeluting with humanlrat CRF-41 and its oxidized form Met [02'~38]CRF-41 in human and rat tissue extracts separated by high-performance liquid chromatography. The ability to measure immunoreactive CRF-41 in unextracted plasma allows rapid measurement and eliminates multiple extraction steps.( 'orticotrophin-releasing factor-41 (CRF-41) is a 41 amino-acid pcptide originally isolated from ovine hypothalamic tissue ( I ) . 7hc amino-acid sequences of human and rat CRF-41 are identical and differ from the ovine sequence at 7 residues (2, 3). CRF-41 cilso shares considerable sequence homology with sauvagine, a fi.ogskin peptide which has corticotrophin-releasing activity (4). 1,'arly assays for C R F relied on bioassay techniques ( 5 ) and were, therefore, susceptible to interfercnce from substances such as :idrenaline or arginine vasopressin which also have corticotrophinlcleasing activity (4). The development of RIAs for CRF-41 (6) allowed more specific and sensitive assay. However, the RIA tcchnique is time consuming and can give elevated measurements in the presence of CRF-41 peptide fragments or peptides with dcgrees of sequence homology with CRF-41 (4). Such RIAs also 1-cquire extraction of the material from biological fluids to reduce interference (7-9). The development of a 'two-site' immunoradiometric assay (IRMA) for CRF-41 (10) was a significant advance, reducing interference from fragments and allowing direct measurement of the peptide in biological fluids including plasma (1 1) In this study, we sought to construct an enzyme amplified ilnmunometric assay (EAIA) for human/rat CRF-41 using two polyclonal antisera raised against the whole CRF-41 molecule. The technique uses one antibody bound to a solid phase with sequential addition of samples, second antibody-enzyme conjugate and substrate. The assay was based on the technique of enzyme amplification (12) to improve sensitivity and reduce assay time (13). The performance of this assay has been compared t o two RIAs for CRF-41 in the measurement of rat hypothalamic CRF-41 content and release. The ability of the EAIA to measure human CRF-41 in both unextracted gest...

Effects of pyrogenic immunomodulators on the release of corticotrophin‐releasing factor‐41 and prostaglandin E2 from the intact rat hypothalamus in vitro

“…In our study, we used an ultrasensitive immunometric assay which is both more sensitive and specific for rat CRF-41 than available radioimmunoassays (Milton et al, 1990a). The basal release of irCRF-41 reported for the rat hypothalamic enn I fragment system corresponded to 8.6 ± 0.8 fmol/hypothalamus per 20 min (Tsagarakis et al, 1988) and is within the range found in the present study.…”

“…IrCRF-41 was measured with an ultra-sensitive, highly specific enzyme amplified immunometric assay (Milton et al, 1990a) modified with a monoclonal antibody (KCHMB003) to rat CRF-41 (Milton et al, 1990b …”

Corticotrophin-releasing hormone (CRH)-binding protein interference with CRH antibody binding: implications for direct CRH immunoassay

“…Of the three 'two-site' assays reported for CRH to date, two (Linton et al 1987, Milton et al 1990) were beheved to be suitable for direct plasma measurement despite the presence of the CRH-BP. Although there was concern that the binding protein would cause interference in one of these assays, it was assumed that the IRMA antibodies then in use were of sufficiently high affinity to dissociate CRH from the CRH:CRH-BP complex, rather than measure the complex intact (Linton et al 1988).…”

“…Many different CRH assays have now been described; most RIAs for plasma or serum CRH estimation require an initial extraction step to overcome plasma matrix effects and Sep-Pak C18 (Sasaki et al 1984), immunoaffinity (Suda et al 1985), vycor glass (Cunnah et al 1987) and methanol (Ellis et al 1988) have all been used for this purpose. Several two-site or sandwich immunoassays depending on the use of antibodies to distinct and non-overlapping regions of CRH have also been developed, two describing measurement in un¬ extracted samples (Linton et al 1987, Milton et al 1990), and a third which was markedly inhibited by human plasma (Hagan et al 1993). It is now known that a high-affinity binding protein (CRH-BP) for CRH exists in the human circulation and that most of the CRH in peripheral blood is present in its complexed form (Orth & Mount 1987, Linton et al 1988, Potter et al 1991.…”

The Effects of Rat Corticotrophin‐Releasing Factor‐41 Peptide Fragments on Bioassay and Immunoassay Determination of Corticotrophin‐Releasing Factor‐41 Levels

“…The bioassay chosen used freshly isolated fragments of anterior pituitary in a static incubation (14,15). The two RIAs used polyclonal antisera which showed different cross-reactivities with synthetic peptide fragments (16). The two-site EAIA used two polyclonal antisera and only measured the whole molecule (16).…”