Smarter staining: New biocatalytic systems for multiplexing histological staining

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Background: Histopathology is the workhorse of many medical laboratories, and can be used for diseasediagnosis, drug development and biological research. Histological staining is an essential tool for this process,providing valuable contrast and visually identifying microscopic features in samples. Since it is essential thatthis process is both robust and repeatable, innovation has been limited and only a very few technologies havebeen employed. Chief amongst these technologies are staining process which use horse radish peroxidase(HRP) to catalyze the oxidation of the chromogen diaminobenzidine (DAB) to give an insoluble brown stain.When coupled to an antibody recognition system this can provide a map of any individual target on thebiological tissue sample. With the advent of "personal medicine", multiplexing staining would provide animportant advance in histopathology in terms of the amount of information which could be generated fromany one patient sample, a particularly important feature where tissue is limited. At the moment multiplexingis limited to the use of mouse and rabbit antibodies linked to two different enzyme/chromogen systems: theHRP/DAB combination; and an orthogonal alkaline phosphatase (AP)/Fast Red combination.In this project: an innovative approach to multiplexing will be explored which relies upon the development ofnew polymer-bound biocatalyst /pro-chromogen substrate combinations, in which biocatalytic turnover ofthe pro-chromogen substrate can be used to multiplex staining.