Studies on a Novel Steroid Radioimmunoassay and Its Application to the Neonatal Detection of Congenital Adrenal Hyperplasia

Thomson, Shirley (1991) Studies on a Novel Steroid Radioimmunoassay and Its Application to the Neonatal Detection of Congenital Adrenal Hyperplasia. MSc(R) thesis, University of Glasgow.

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Congenital adrenal hyperplasia (CAH) is the commonest adrenal disorder in childhood (worldwide incidence is 1:14,554) and because it is potentially fatal, early detection is necessary. Mass screening of newborn populations for other diseases, such as congenital hypothyroidism, phenylketonuria and galactosaemia, has been established based on blood samples collected onto filter paper (Guthrie cards). Elevated concentrations of the steroid, androstenedione (A4), occur in untreated, classical cases of CAH, and are modestly raised in late-onset cases. Some increase may also occur in polycystic ovarian disease. Several groups throughout the world have added screening (or pilot screening studies) for 21-hydroxylase deficiency, the most common form of congenital adrenal hyperplasia, to their existing neonatal programmes. From 1984, this was the case in Scotland but, due to the lack of central funding, screening was reluctantly stopped in 1986. The CAH screening programmes are based on the measurement of another steroid, 17-hydroxyprogesterone (17-OHP), also elevated in untreated cases. However, A4 determination offers greater potential as a diagnostic aid because it also can detect the second most common form of CAH, 11beta-hydroxylase deficiency. In addition, A4 measurement may be a better indicator of the efficacy of glucocorticoid replacement therapy in CAH patients than 17-OHP measurement. Unfortunately, because most A4immunoassays are based on a tritiated label, they are cumbersome, expensive and too insensitive to allow measurement in filter paper blood-spot samples. This makes them unsuitable for large-scale screening and home-monitoring of replacement therapy in patients with CAH. The aim of this investigation was to produce a sensitive, specific, precise and robust immunoassay to measure A4 in serum and blood eluted from filter paper samples. Antisera were raised in rabbits against A4 linked to bovine serum albumin (BSA) at positions 3, 6 or 11 on the steroid nucleus. Gamma-emitting labels were prepared by linking 125[I]-iodohistamine at positions 3, 6 or 11 on the steroid nucleus. Linkages were through either carboxymethyloxime (CMO) or hemisuccinate (HS) bridges. A major part of the work involved the selection of the best combination of A4 antibody and A4 label. Separation of antibody bound and free fraction was achieved by centrifugation of microencapsulated antibody or centrifugation after incubation with a second antibody. An antiserum raised against A4-CMO-BSA used with an A4-CMO-1255[I]-iodohistamine tracer was selected, on the basis of sensitivity and specificity, as the best combination of reagents. The performance of these reagents in an immunoassay for A4 extracted from serum or eluates of neonatal dried blood-spot samples was evaluated. A direct, non-extraction blood-spot immunnoassay was also evaluated to facilitate large-scale screening for CAH. Concentrations of A4 in serum under normal and pathological conditions, such as CAH and polycystic ovarian disease were investigated. Androstenedione was measured in blood spots from neonates born at term or prematurely, with respiratory distress syndrome, or with CAH. For the serum assay, the range of the standard curve was 1-21 nmol/1 of A4. The coefficients of variation of the within- and between-batches were 10% for low, medium and high serum pools. Mean analytical recovery of tritiated androstenedione or unlabelled androstenedione, which had been added to sera before solvent extraction, was >95% in both cases. Measurements of androstenedione in dilutions of sera from patients with increased A4 concentrations exhibited parallelism with the standard curve. Using this immunnoassay, concentrations of A4 in patients' sera showed good agreement with concentrations found with the routine method which employs a tritiated tracer. The correlation coefficient was 0. 94 with a slope of 1.02 and an intercept of 0.9 nmol/1 of A4. Results for samples from the U. K. National External Quality Assessment Scheme compared well with those from the other participating laboratories. The bias was +12.2% and the variability of bias was 6.9% over a six month period. Serum androstenedione concentrations were similar in men and women and both groups showed a reduction with age. (Abstract shortened by ProQuest.).

Item Type: Thesis (MSc(R))
Qualification Level: Masters
Keywords: Biochemistry
Date of Award: 1991
Depositing User: Enlighten Team
Unique ID: glathesis:1991-78261
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 28 Feb 2020 12:09
Last Modified: 28 Feb 2020 12:09

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