| Assay Type |
Detection |
Parameters |
Advantages |
Limitations |
| Bi-directional Transport |
HPLC;
Radioactivity;
Fluorescence. |
Papp;
Efflux ratio;
Net transport. |
Representative of transport across cell barrier;
Directly measures efflux across cell barrier;
Indicates the bi-directional transport of compounds from apical to basolateral and basolateral to apical;
Evaluation of P-gp transport and inhibition;
Allow the localization of P-gp within apical or basolateral membrane;
Recommended by FDA. |
Intensive labour;
Requires culture of cells on filters;
Requires confluent monolayers with tight junctions and functional expression of transporters;
Underestimate substrate activity of high permeable compounds. |
| Accumulation/Efflux Assay |
Radioactivity;
Fluorescence. |
Inhibition of accumulation/ efflux of a fluorescent probe (Calcein-AM or Rho 123) |
Can use wide range of cells (adherent or in suspension) or membrane vesicles;
Primarily used for inhibition assay;
Assay is simple;
Readily adaptable for flow cytometry;
Easy and fast method for high throughput screening of drug efflux interactions (could be either substrate or inhibitor). |
Cannot distinguish substrate from inhibitor;
Tends to fail to identify substrate and/or inhibitor with low permeability;
Adsorption to cell and plastic can yield misleading results;
Underestimation of low permeable compounds;
High variability;
Localization of P-gp is difficult;
Limited sample analysis. |
| ATPase Assay |
Inhibition of photoaffinity labeling. |
ATPase stimulation |
High throughput and cost-effective for screening compound P-gp interaction;
Primarily used to complement other studies;
Commercially available;
Compound-independent colorimetric assay;
Uniform detection methods. |
High intra and inter-day variability;
Generate false positives and negatives;
Underestimation of low permeable compounds;
Localization of P-gp is difficult. |
