Background

Assay Standardization

 

An assay that is standardized by including standards and controls helps to provide the user with the guarantee that the assay is working correctly and that the results can be trusted. For this reason, all bioluminomics™ assays from HemoGenix® include standards and controls.

 

At HemoGenix®, we do NOT believe that standardization of an assay should involve costly and time-consuming training and non-regulated proficiency testing. Furthermore, high performance, quality controlled reagents should be expected from any commercial product. At HemoGenix®, we believe that an assay should be designed so that the investigator can be assured that results from one experiment can be compared with those of another experiment. In other words, the investigator does not have to worry about reagent or assay variability.

 

Measurement of a biochemical marker, such as intracellular ATP (iATP), that can provide information on metabolic viability, proliferation ability, cytotoxicity, cell number and even apoptotic state, allows an external ATP standard and ATP controls to be used for assay calibration and standardization. For this reason, an external ATP standard and high and low controls are included in every HemoGenix® bioluminomics™ assay kit.

 

Calibration, Standardization and Validation of Bioluminomics™ Assays

ProliferSTEM™, STEMpredict™, HALO®, LUMENESC™ and LumiSTEM™ Platforms require the measurement of bioluminescence using a plate luminometer. The output of a luminometer is in Relative Luminescence Units or RLU. This is a non-standardized unit because the number of RLUs measured varies from one manufacturer to another and even from one instrument to another. This means that it is very difficult, if not impossible, to compare multiple experimental results or even compare results between laboratories. Results can only be compared if the RLU output is converted to a standardized unit of measurement. By including an ATP standard and controls, all HemoGenix® bioluminomics™ assays allow:

• Calibration of the instrument
• Conversion of non-standardized RLU to standardized ATP concentrations
• Assurance that the reagents function as intended
• Controls pipetting error
• Assurance that the assay is working correctly
• Comparison of results over time
• Assay validation

 

The ATP standard curve shows results that should be obtained prior to measuring samples using any HemoGenix® bioluminomics™ assay platform.

 

Typical assay parameters and validation characteristics include:

• Assay linearity: >3 logs
• Assay cell sensitivity: 20-25 cells/well
• Sensitivity/Specificity (ROC curves): >0.75
• Precision (reliability & reproducibility): =< 15%
• Robustness: >95%