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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)

es the UV capability of the SPECTRAmax 250 micro-plate spectrophotometer and can be readily modified to determine ID50 values of inhibitors of other enzymes requiring UV/Vis monitoring.


MATERIALS AND METHODS
Materials
1. SPECTRAmax 250 microplate spectrophotometer
2.SOFTmax PRO software
3.Quartz microplates
4. Deep well microplate
5. Multichannel pipette
6. DMSO
7. Imipenem and nitrocefin
8. β -lactamase preparation
9. Experimental compounds

Method for determining ID50 values following a 5 minute incubation of enzyme and inhibitor.

Step 1 Set the incubator temperature to 37C., then allow it to equilibrate for at least 30 minutes

Step 2 Set up SOFTmax PROs Instrument Settings dialog box for imipenem as shown in Figure 1 and for nitrocefin as shown in Figure 2.



Step 3 Dissolve the inhibitors in 25 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer, pH 7.0 with no added Zn2+ to produce a 3 mM solution. 5% DMSO can be used if needed to aid in dissolving the inhibitors.

Step 4 In a deep-well 96 well plate, pipette 600 l of 25 mM PIPES buffer, pH 7.0 into each well in columns 1-11.

Step 5 Pipette 1 mL 3 mM inhibitor solution into each well in column 12.

Step 6 Using a multichannel pipette, make a 1:3 serial dilution of the 3 mM solution down to column 3: take 300 l of 3 mM solution from column 12 and dispense into column 11. Mix, then take 300 l of column 11 and dispense into column 10, and so on across the columns to column 3 which will then be 0.15 M.

Step 7 Aliquot 50 l of the contents of the deep wells into the corresponding wel
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