A highly specific and sensitive enzyme-immunometric assay for calcitonin gene-related peptide based on enzyme amplification

Seth, Rashmi; Zaidi, Mone; Fuller, J Q; Self, Colin H.

doi:10.1016/0022-1759(88)90053-1

Cited by 13 publications

(2 citation statements)

References 12 publications

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“…The use of antibody bound to a solid phase eliminates separation procedures which contribute to the lower sensitivity of RIA. This also enables sequential addition of samples, antibody-enzyme conjugate and substrate allowing potentially interfering substances to be removed by washing (18). The ability of the assay to measure CRF-41 without the need for an extraction procedure is a significant improvement on RIA since differences in sample extraction have been implicated in the discrepancies between measurements?by different groups (8,9,19,20).…”

Section: Discussionmentioning

confidence: 99%

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

Milton

Hillhouse

Fuller³

et al. 1990

J Neuroendocrinology

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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...

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Section: Discussionmentioning

confidence: 99%

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

Milton

Hillhouse

Fuller³

et al. 1990

J Neuroendocrinology

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show abstract

“…CGRP levels are increased in patients with thyroid carcinoma (Mason et al, 1986;Carter et al, 1991;Schifter and Johnsen, 1994) or pheochromocytoma (Mouri et al, 1989) and, as noted, in neonatal blood from infants with neurodevelopmental disorders (Nelson et al, 2001). Conventionally, CGRP concentration has been measured by RIA (Schifter, 1991;Wimalawansa, 1993), EIA (Seth et al, 1988;Frobert et al, 1999) and RIC (Nelson et al, 2001). Due to low blood levels of CGRP and the small size of some of the samples such as blood spots that are available, a sensitive and reliable assay that allows the precise quantitation of this peptide in different biologic media is needed for the study of CGRP function and distribution in disease.…”

Section: Introductionmentioning

confidence: 98%

Measurement of CGRP in dried blood spots using a modified sandwich enzyme immunoassay

Song

VanDunk

Kuddo

et al. 2006

Journal of Neuroscience Methods

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Monoclonal antibodies distinguishing α and β forms of calcitonin gene-related peptide

Andrew

Bidgood

Bose

et al. 1990

Journal of Immunological Methods

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A highly specific and sensitive enzyme-immunometric assay for calcitonin gene-related peptide based on enzyme amplification

Cited by 13 publications

References 12 publications

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

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

Measurement of CGRP in dried blood spots using a modified sandwich enzyme immunoassay

Monoclonal antibodies distinguishing α and β forms of calcitonin gene-related peptide

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