Enzyme assay detector concentrations

Figure 3.23 Typical amperometric (readout during automated flow injection assays of ethanol solutions of increasing concentrations in 2 x 1CL5M steps at a carbon paste enzyme electrode detector. Curves a-h 2 x 10 SM - 1.6 x lCUM ethanol.

Methods very similar to classical immunoassays in the sandwich format are easily implemented in flow systems (Fig. 2d). In this type of noncompetitive assays, again, antigen is captured and concentrated from an appropriate volume of sample on an immunosorbent (-Abi) column while nonantigenic components are eluted. Subsequent to the capture step, labeled second antibody (Abj-label) is introduced into the mobile phase and swept into the column, where it binds to the -Ab]-Ag complex to form -Ab -Ag-Ab2-label. Unbound Ab2-label is swept from the column, and when the label is an enzyme, antigen is quantitated indirectly by conducting an enzyme assay in the column. After substrate incubation, the reaction product is transported to a detector at the column terminus. Ag and Ab2-label can be introduced in the column sequentially or simultaneously. In some instances both modes led to similar sensitivity [55], and in other cases simultaneous injection produced a greater response than sequential injection [56]. The term sandwich has also been applied to the procedure carried out to quantitate Ab by capturing a complex Ab-Ag-label onto a protein G capillary column [57]. In this case detection is performed after elution. 

To apply this method to the problem at hand, the AMP kinase activity should be measured first. Clearly, it would be advantageous to carry out this assay under conditions similar to those that would be present when only ATP was added to the complex. Thus, one might set up a reaction mixture with about 1 mM ATP and with AMP in the nanomolar concentration range that is, at a concentration that would be expected if the AMP had been derived from reaction (2). In addition, to enable us to follow its fate, in reaction (3) the AMP should be added to the incubation mixture in a radiolabeled form. Reaction (3) is started by the addition of the enzyme complex, and samples should be removed from the incubation mixture at suitable intervals and injected onto the HPLC column for analysis. After separation, the eluent should be monitored by both radiochemical and UV (254 nm) detectors. 

The activity of both Novozym 435 and crosslinked Novozym 435 was measured using the standard PLU-assay. The PLU (propyl laurate units) assay is a method to determine the activity of immobilized lipases by ester synthesis. One PLU corresponds to 1 pmol of propyl laurate formed from condensation of lauric acid and 1-propanol per gram of enzyme per minute under specified conditions. The PLU value is determined by measuring the area under the peak of formed propyl laurate versus a standard sample of propyl laurate with known concentration using a gas chromatograph with an FID detector. Crosslinked Novozym 435 showed an activity of 4800 PLU g"1, whereas the activity of Novozym 435 before crosslinking was measured to be 12200 Pl.U g 1. 

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