Enzymeimmunoassay of 17-alphahydroxyprogesterone
thesisposted on 11.03.2013 by David B. Henson
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21-hydroxylase deficiency is the commonest enzyme defect in congenital adrenal hyperplasia resulting in low circulating cortisol and, in severe cases, low aldosterone which may lead to cardiac arrest in the neonate. As the low levels of coitisol· lead to raised levels of adrenocorticotrophic hormone large concentrations of androgens are formed in utero which may lead to cliteromegaly and consequent confusion of the sexes at birth. High concentrations of the androgens give rise to rapid growth in early childhood and early fusion of the epiphyses results in short stature of the adult. 17-alphahydroxyprogesterone (170HP) is raised in the blood and saliva of children with 21-hydroxylase deficiency. 170HP is commonly measured by radioimmunoassay which limits the assay to being performed by laboratories equipped with beta or gamma counters. The aim of this project was to develop an enzymeimmunoassay which could be carried out using the minimum amount of equipment. Horseradish peroxidase was conjugated to 170HP-O-carboxymethyloxime using a mixed anhydride reaction. Separation of free and bound label was achieved using a second antibody linked to magnetisable particle solid phase. Measurement of the bound enzyme activity yielded colours which could be compared by eye, with known standards, to give a semi-quantitative assay. Alternatively the absorbance could be measured using a spectrophotometer for full quantitative results. The developed assays were evaluated, comparing the results with radioimmunoassay, and then used for measuring 170HP in blood and saliva in various clinical situations.