The Production of Monoclonal Antibodies to Human TSH and Their Use in the Design of Immunometric Assays for Serum and Neonatal Blood Spot TSH

Medicine, Biochemistry, Endocrinology

Biggart, Elizabeth M

University:University of Glasgow

关键词: Medicine, Biochemistry, Endocrinology;   

Others  :  http://theses.gla.ac.uk/77844/1/10999377.pdf

来源: University of Glasgow

【 摘 要 】

The potential for the application of antibodies as analytical and preparative reagents was greatly expanded by the development of techniques for the production of monoclonal antibodies (Kohler and Milstein, 1975, 1976). The properties of monoclonal antibodies are particularly suited to application in immunometric assays and indeed overcome all the major problems associated with conventional antisera. Antibodies of only moderate affinity may be used in sensitive immunometric assays with detection limits up to two orders of magnitude greater than can be achieved in competitive immunoassays with the same antibody (Buchegger et al, 1981). The work of this thesis was concerned with the production of monoclonal antibodies to human TSH and their use in the design of immunometric assays for serum and neonatal blood spot TSH. Chapter 3 deals with the production of a variety of monoclonal antibodies to TSH. These antibodies were characterised in terms of affinity, specificity and isotype. The results indicated that 5H8 and 2G2 may be of extreme practical value in the development of a two-site immunoradiometric assay (IRMA) for TSH. A number of such assays for the measurement of TSH in serum and in neonatal blood spots were then developed using both monoclonal and polyclonal antisera. Chapter 4 seeks to illustrate the clinical role and suitability of the monoclonal-polyclonal serum TSH assay developed in this project. The assay successfully distinguished all cases of untreated primary hypothyroidism as having an elevated serum TSH and all cases of untreated hyperthyroidism as having an undetectable serum TSH (<0.2 mU/1). On the basis of this information, together with the performance data described in Chapter 3, the assay was introduced in 1984 as the 'routine" serum TSH assay for the laboratory at Glasgow Royal Infirmary, processing some 20,000 serum specimens per annum. Regarding the Scottish Screening Programme for congenital hypothyroidism, at the end of 1983 the monoclonal - polyclonal IRMA replaced the two-polyclonal IRMA of Sutherland et al (1981). Cumulative statistics and predictive values of the data obtained from The National Screening Programme with the two In-house IRMA TSH assays are recorded in Chapter 5 and they reveal that the monoclonal - polyclonal IRMA has continued to maintain the excellent clinical data obtained from the Scottish Screening Programme. Alternative high specific activity labels and the implications of these developments for the serum TSH assay service in Glasgow Royal Infirmary and the Scottish Screening Programme for congenital hypothyroidism are discussed in Chapter 6. It is almost certain that the clinical value of a serum TSH assay will be enhanced by improving the detection limit from 0.2 mU/1 to 0.02 mU/1. Such an improvement is not possible with the existing monoclonal and polyclonal antibodies and an 125I-label. The most straightforward way to achieve this improvement is to move to a non-isotopic label that is compatible with the available antibodies. The work presented in Chapter 4 of this thesis (Sections V and VI) has shown that many patients receiving thyroxine replacement therapy have undetectable serum TSH levels, both in the in-house IRMA and using the Delfia assay. The interpretation of these results is discussed in Chapter 6. However, the role of sensitive TSH assays in monitoring patients receiving thyroxine replacement has still to be fully evaluated.