Auto-analysers use ~ 0.315 mL reaction volumes and path-lengths between 4-8 mm which is similar to a standard 96-well microplate where a 0.315 mL reaction volume would give a path-length of ~ 6-7 mm. Therefore K-ACETAK, K-ACETAF or K-ACETGK can be used directly in a 96-well microplate format with minimal assay optimisation.
If preferred, K-ACET or K-ACETRM may also be easily converted for use in a 96-well microplate format. Basically, the assay volumes for the cuvette format must be reduced approximately 10-fold for use in a 96-well microplate. However, some assay optimisation may be required (e.g. increased enzyme concentration etc.) and unlike the cuvette which has a set path-length of 1 cm, the path-length in the microplate is dependent upon the volume of liquid in the well. Therefore to enable the calculation of the amount of analyte in the samples from tests performed in the microplate format one of the following must be done:
1. The easiest method is to use a microplate reader that has a path-length conversion capability (i.e. the microplate reader can detect the path-length of each well and convert the individual readings to a 1 cm path-length). This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm path-length) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values.