Cooke, D.G. and Flight, S. and Smales, C.M. and Blackwell, L.F. (2003) Use of defined estrone glucuronide-hen egg white lysozyme conjugates as signal generators in homogeneous enzyme immunoassays for urinary estrone glucuronide. Journal of Immunoassay & Immunochemistry, 24 (2). pp. 147-172. ISSN 1532-1819. (The full text of this publication is not available from this repository)
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Three structurally characterized estrone glucuronide-lysozyme conjugates, El (a 60:40 mixture acylated at K3 and K97), E3 (acylated at K33), and E5 (acylated at both K33 and K97) were isolated and purified using a combination of cation-exchange chromatography on S-sepaharose in 7 M urea and hydrophobic interaction chromatography on butyl sepharose. Urea was essential to separate the conjugates into six chromatographically homogeneous fractions. In the absence of urea, complex mixtures of lysozyme and the six conjugate fractions were always encountered. The E1, E3, and E5 conjugates were highly inhibited by a sheep polyclonal anti-estrone glucuronide antibody only after the hydrophobic interaction chromatography step. The high level of inhibition enabled all three conjugates to be utilized as signal generators in homogenous enzyme immunoassays for urinary estrone glucuronide. Despite the apparently higher affinity of E3 for the antibody, both E1 and E3 gave standard curves that were indistinguishable provided that 1.7-fold more antiserum was used for E1. Both E1 and E3 yielded menstrual cycle urinary data that agreed with that provided by the Ovarian Monitor pre-coated assay tubes. Although, the menstrual cycle pattern was similar for the three signal generators, the E1G excretion rates yielded by E5 as the signal generator were only 60% of the reference values. Despite structural differences, there was no advantage gained in separating E1 and E3, but higher substituted conjugates such as E5 need removal for best assay performance.
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences|
|Depositing User:||Mark Smales|
|Date Deposited:||09 Sep 2008 05:02|
|Last Modified:||14 Jan 2010 14:23|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/6214 (The current URI for this page, for reference purposes)|