Homogeneous and heterogeneous enzymeimmunoassays for the home detection of fertility : a thesis presented in partial fulfilment of the requirements of the degree of Doctor of Philosophy in Biochemistry at Massey University
The physiology of the menstrual cycle has been reviewed and the suitability of the ovarian steroid urinary metabolites estrone glucuronide and pregnanediol glucuronide as markers of fertility and as utilised by the home Ovarian Monitor fertility assay was discussed. The biomaterials for the homogeneous enzymeimmunoassay which forms the basis of the Ovarian Monitor home fertility assay were prepared. One of the major difficulties in preparing a signal generator for use in home tests is the separation of the unconjugated enzyme material from the desired signal generators. In this thesis a new procedure was developed for the purification and isolation of the complex range of signal generators formed during acylation of hen egg white lysozyme with estrone glucuronide. A cation exchange column in the presence of 7 M urea allowed the separation to be carried out in the absence of the hydrophobic effects which complicate other schemes. Even under these conditions complex behaviour was seen which could be rationalised in terms of the tertiary structures of the conjugates and their electrostatic fields. A second step involving hydrophobic interaction chromatography gave two pure mono conjugated estrone glucuronide lysozyme conjugates in good yield the activities of which were highly inhibited (>90%) by anti-estrone glucuronide antibodies. The availability of the pure conjugates allowed the effect of tertiary structure on the immunoassays to be evaluated. The results showed that both mono acylated hen egg white lysozymes could be used to give good standard curves for use in monitoring menstrual cycles for the naturally occurring periods of fertility and infertility. Since the specific activity of human lysozyme is three times that of hen egg white lysozyme, in an attempt to provide a more rapid test for fertility human lysozyme estrone glucuronide conjugates were synthesised with estrone glucuronide for the first time. However, despite the fact that these two enzymes had very similar tertiary structures they behaved completely differently in the protein chemistry and immunological experiments reported in this thesis. The human enzyme was more easily acylated to give pure mono acylated conjugates in high yield and the conjugates were more easily purified to give highly inhibitable conjugates (>95%). The differences in behaviour could be accounted for in terms of the sequence differences between the two lysozymes and the relative exposure of the lysine residues. A fast assay (1-2 minutes) was developed for urinary estrone glucuronide using the three new signal generators. However, the sensitivity of the assays was less than half that of the hen egg white conjugates making them unsuitable for use in home assays for fertility. The assays could be useful for women using fertility drugs such as clomiphene. The lack of sensitivity of the assays and other binding behaviour indicated a much tighter binding to the antibody than with the hen egg white conjugates. This important difference was accountable on the basis of the extra extension of the lysine residues in the human enzyme. A new method for producing estrone glucuronide conjugates of the active enzyme horse radish peroxidase was evaluated. The mono substituted hemin conjugates reconstituted with the apo protein to give active peroxidases with good specific activities (~50%) and good stability. The procedure is such that any small molecule can be attached to the enzyme using the same procedures and a large range of signal generators can be formed for immunoassays. However, both assay formats examined failed to produce an assay in this thesis. The reconstituted enzymes, although binding to the immobilised anti- estrone glucuronide antibodies as required did not produce the necessary colours. The reasons for this were discussed.