The MDQuest MultiDrugQuant™ Kit is designed as a routine laboratory flow cytometry method for the measurement of the functional activity of the three clinically most relevant efflux transporters (MDR1, MRP1 and BCRP) on viable cells, offering several advantages:
Quantitative flow cytometry: results are expressed in harmonized MDR activity factor (MAF) values
Required specimen: cultured or collected cells from blood or other body fluid or bone marrow - Research Use Only
Potential for studying transporter malfunction diseases, even on in-house disease model samples
Compatible with cell surface markers for immunophenotyping and gating of cell populations during the FACS measurement
Different probe substrate for MDR1/MRP1 and BCRP
Highly selective inhibitors
Available as CE-IVD certified product as well, for clinical sample analysis (SOLVO MDQ KitTM)
Principle of the test
For the functional investigation of MDR1 and MRP1, the MDQuest MultiDrugQuant™ Kit applies the Calcein-assay technology patented by SOLVO Biotechnology. This assay has several advantages against other fluorescent dye accumulation tests: it is quick, quantitative and selective for MDR1 and MRP1 transporters, furthermore, it has validated internal standard, minimizing the batch to batch and interlab variations. Another big vantage of this assay is the direct activity measurement of the respective transporters versus mRNA and protein expression studies.
The Calcein assay utilizes the fluorogenic dye, calcein-acetoxymethyl ester (calcein-AM), a hydrophobic compound that readily penetrates the cell membrane. After entering the living cell, the non-fluorescent calcein AM is rapidly hydrolyzed by endogenous esterases to form a highly fluorescent acid derivative of the dye, which becomes trapped in the cytoplasm due to its hydrophilic character. Since calcein-AM is an excellent substrate of both MDR1 and MRP1, the activity of these efflux transporters results in a lower cellular accumulation of the fluorescent calcein. Addition of selective inhibitors of MDR1 and MRP1 in excess blocks the dye extrusion activity of the relevant transporter and increases calcein accumulation in the cells.
Activities of MDR1 and MRP1 transporters are reflected by the difference between the amount of calcein accumulated in the presence or absence of the selective inhibitors. This difference is normalized to the dye uptake measured in the presence of the inhibitor and the results of the test are expressed in MDR activity factor (MAF) values. Thus, the result of the test becomes independent from factors influencing the cellular accumulation of calcein other than the activity of the multidrug transporters. These variables include the differences in the cellular properties (membrane composition, intracellular esterase activity, cell size, cell surface, etc.); and the methodological differences (e.g. use of different equipment, amplification, and individual variables).
Since the influence of these factors is diminished by the simple normalization approach mentioned above, the intra and inter-laboratory comparison of MAF values is possible.
BCRP activity is measured using a similar principle: intracellular accumulation of the fluorescent BCRP-specific reporter substrate is measured in the presence and absence of the selective BCRP-inhibitor. However, the BCRP-specific reporter substrate is directly fluorescent and does not require cleavage by the intracellular esterases.