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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)



Table 4 shows the SOFTmax PRO printout of ID50s for inhibitors A-H.


CONCLUSION
Using SPECTRAmax 250 to determine ID50 has three major advantages:
1. High throughput. The protocol described enables 16 ID50s to be determined for 5 different enzymes in 3-4 hours (80 ID50s in total). SOFTmax PRO software rapid and accurate determination of ID50s without having to download data into other curve fitting software.

2. Flexibility of reporter substrate. β -lactamase assays performed on other plate readers are limited to using chromogenic substrates such as nitrocefin. However, as the SPECTRAmax 250 reads in the UV, other substrates can be utilized. This means that assays requiring UV monitoring were previously limited to standard spectrophotometers, but can now be converted to microtiter plate formats for high throughput. For example, nitrocefin is an extremely poor substrate for the CphA enzyme and an inappropriate reporter substrate. The UV capability of the SPECTRAmax 250 means that imipenem can be utilized as the substrate, enabling this enzyme to now be evaluated in the micro-titer plate format.

3. Use of small quantities of reagents. This system enables the β -lactamase ID50 assays to be performed on a microtiter plate system. If equivalent data was determined with a spectrophotometer using a method similar to that reported by Holt et al (1983), 20 times more reagent would be required.

This methodology could be utilized to measure the inhibition of a variety of other enzymes.

The introduction of the SPECTRAmax 250 spectrophotometric microplate reader together with its powerful data analysis software, SOFTmax PRO, has streamlined and greatly simplified the inhibitory screening of test compounds, allowing the rapid
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