Wilson, Karen; (1992) Design of a homogeneous fluorescent immunoassay suitable for antibody fermentation monitoring. Doctoral thesis (Ph.D.), University College London.

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Abstract

This thesis describes the development of a homogeneous fluorescent energy transfer immunoassay aimed at the measurement of large therapeutic proteins such as immunoglobulin G in fermentation broths. There is a requirement for increased control in fermentations to maximise product yields leading to a need for the development of a homogeneous immunoassay which may be readily automated and is capable of monitoring the chosen analyte against the complex background interference produced in fermentation broths. Current monitoring is usually carried out by off-line techniques. The energy transfer immunoassay described utilises an excimeric pyrenyl donor fluorophore with B-phycoerythrin as the acceptor. The use of an excimeric donor fluorophore was examined and the theory of excimeric energy transfer developed. It was demonstrated that the use of such a donor allowed the detection of IgGl and the dual monitoring of both donor quenching and acceptor enhancement which has not been possible using classical dyes. An extensive study of the kinetics of the labelling reaction of IgGl with succinimidyl-l-pyrenebutyrate was carried out. Excimer formation on the antibody surface produced by heavy labelling was modelled and optimised. An excimeric pyrenyl label was also synthesised to eliminate the need for heavy labelling of the capture antibody. The use of an excimeric donor was shown to have several major advantages over classical labels and its implementation extended the possible application of this immunoassay technique. The excimeric pyrenyl donor is long-lived affording the possibility of time-resolved spectroscopic detection to eliminate background and its large effective Stokes shift of 135 nm will allow the implementation of efficient optical filters to reduce interference. Homogeneous energy transfer immunoassays have previously been automated using flow injection analysis and the improved assay described here seems a promising prospect for the real-time monitoring of animal cell fermentations.

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