majority of the Megazyme test kits are developed to work in cuvettes using the
manual assay format, however the assay can be converted for use in a 96-well
microplate format. To do this the assay
volumes for the manual cuvette format are reduced by 10-fold. The calculation
of results for the manual assay format uses a 1 cm path-length, however the
path-length in the microplate is not 1 cm and therefore the MegaCalc
spreadsheet or the calculation provided in the kit booklet for the manual
format cannot be used for the micropalate format unless the microplate reader
being used can.
There a 3 main methods for calculation of results using the microplate format:
The easiest method is to use a microplate reader that has a path-length conversion capability (i.e. the microplater 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.
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).
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. Subsequent assays in the microplate format can then be converted from the calculated conversion factor.