Smarter staining: new biocatalytic systems for multiplexing histological staining

Immunohistochemistry (IHC) is an important technique with applications in disease diagnosis, biological research and drug development. Recent developments in personalised medicine, mean that there is a rising demand for visualising multiple antigens on a single tissue specimen. As yet, only a few systems have been developed which allow staining to be multiplexed in this way. Previously reported multiplexed IHC methods will be discussed in Chapter 1. Among the different approaches in IHC, enzyme-based chromogenic IHC detection holds advantages over other multiplexed IHC methods including higher stain stability, lower equipment costs and shorter image acquisition times. However, only a few applications of multiplex staining have been reported to date, due to the low numbers of enzymes which have been developed for use in IHC. In this project, a new combination of antibody-bound metallo-β-lactamase BcII and cephalosporin based pro-chromogenic/fluorescent substrates was developed, which is orthogonal to traditional IHC systems to enable multiplexed staining. In Chapter 2, the synthesis of a range of pro-chromogenic substrates with cephalosporin cores bearing different chromogenic/fluorescent moieties is described, together with an investigation of their solution phase turnover by BcII enzyme. In Chapter 3, the application of these first generation substrates to tissue staining with antibody-conjugated BcII enzyme is explored. The staining protocol was optimised to allow selective staining of the smooth muscle and skeletal muscle tumour marker, desmin, in human colon tissue samples. In Chapter 4, the development of a second generation of pro-chromogenic substrates is outlined, focusing on improving the staining sensitivity and aqueous solubility of the substrates. Finally, in Chapter 5, current synthetic routes to spiro-cephams are surveyed and the serendipitous exploitation of a side-reaction arising from the synthetic work described in Chapter 2 for the synthesis of novel spiro-cepham β-lactamase inhibitors is presented. In conclusion, a novel biocatalytic system using BcII enzyme has been developed for IHC and has been demonstrated on tissue specimens. Cephalosporin moiety was used as a core structure for the development of the BcII susbtrates. The aqueous solubility of the substrates has also been improved in order to make the substrates more suitable for IHC staining