Development of a novel homogeneous immunoassay using mutant beta-glucuronidase

Conference Dates

September 24-28, 2017


Βeta-glucuronidase (GUS) catalyzes breakdown of complex carbohydrates, whose activity can be detected quantitatively and sensitively by using fluorogenic and chromogenic substrates. GUS is a tetramer composed of four identical subunits, and assembly of all these subunits is necessary to attain its activity. Based on a previous study, a set of interface mutations (M516K, Y517E) is known to effectively inhibit the assembly and makes it inactive [1]. Usually, the affinity between the two variable region domains (VH and VL) of an antibody recognizing a small molecule is relatively low. However, in the presence of antigen, this affinity becomes higher so that they bind each other more tightly [2]. This gives the idea that a fusion protein system comprising VH and VL of an antibody as the detector each tethered to a mutant GUS subunit (GUSm) as the reporter can be used as a biosensor for small molecules. In this study, we aimed at detecting 4-hydroxy-3-nitrophenylacetyl (NP) and bone Gla protein (BGP) as targets of this novel immunosensor (Fig. 1).

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