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A Microtiter-based Assay for the Determination of ID50s of b-lactamase Inhibitors Employing Reporter Substrates Detected at UV or Visible Wavelengths (MaxLine Application Note #20)

ls on the assay microtiter plate to create a range of inhibitor concentrations between column 3 and column 12. If the reporter substrate requires UV monitoring (e.g. imipenem), UV transparent microtiter plates (e.g., plastic or quartz SPECTRAplates, Molecular Devices Cat. no: R9012 or R8024, respectively) must be used.

Table I shows the final concentration of inhibitor in each well, taking into account the 50 l reporter substrate and 50 l enzyme that will be added to each well. 100 l and 50 l of 25 mM PIPES buffer, pH 7.0 are added to the Blank and Control wells, respectively.


Step 8
Addition of enzyme: both pure and crude β -lactamase samples (prepared as described in reference 5) are compatible with this screening format. Some of the enzymes used are shown in Table 2. The enzyme preparations for the assay are diluted in PIPES buffer, pH 7.0, as required. 50 l of the diluted enzyme is added to columns 212 of the assay microtiter plate. For MBLs, CfiA, BcII and L-1, 150 M ZnSO4 is added to the dilution buffer. For CphA, 1.5 M ZnSO4 is added. These levels of zinc are used to achieve optimal enzyme activity. No ZnSO4 is added to the buffer used to dilute the serine active site enzymes.


Step 9 Using SOFTmax PRO, open the SPECTRAmax 250 drawer, place the plate onto the carriage and close the drawer. Incubate the assay microplate in the SPECTRAmax 250 at 37 C. After 5 minutes, open the drawer of the SPECTRAmax 250 and remove the plate from the carriage.

Step 10 Add 50 l of reporter substrate to all 96 wells. 150 M ZnSO4 is added to the reporter substrate solutions used for assays with BcII, CfiA and L-1 metallo-β -lactamases. 1.5 M is added to CphA and no ZnSO4 is added to the reporter substrate solutions used to assay the serine β -lactamases. With the exception of the CphA β -lactamase, all ID
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