AM assays

The calcein-AM assay [82-84] and cytotoxicity assays (e.g., performed with doxorubicin) [77, 78] are both basically competition assays. The accumulation of a primary substrate (e.g., calcein-AM or doxorubicin) in the cytosol of living cells is measured after addition of a second substrate (also called modifier or reverser) that reduces the efflux of the primary substrate. In the case of the calcein-AM assay, the primary substrate, calcein-AM, is hydrolyzed as soon as it reaches the cytosol, and the highly fluorescent hydrolysis product (calcein) can be determined using fluorescence spectroscopy. The more effective the reversal agent, the stronger is the increase in calcein fluorescence. Data can be quantified in terms of inhibitory constants, IQ, of the reversal agent. 

Accumulation/efflux studies can be performed on different cell systems or membrane vesicle preparations. In the accumulation assays, uptake of a probe over time, typically either fluorescent (e.g. calcein-AM (CAM) [25-27]) or radiolabeled, into the cell or membrane vesicles is measured in the presence or absence of a known P-gp inhibitor. As P-gp transports substrates out of the cells, the inhibition of the protein would result in an increase in the amount of the probe in the cell. Accumulation studies in cells that overexpress P-gp can be compared to those obtained in the parental cell line that does not have as high a level of P-gp expression. The probe in the absence of inhibitors shows lower accumulation in P-gp expressing cells than in P-gp deficient cells. Similarly, probe accumulation is increased under conditions where P-gp is inhibited such that the difference in accumulation in P-gp deficient and overexpressing cells, respectively, becomes smaller. Accumulation assays poorly distinguish substrates and inhibitors of P-gp and, as far as transport assays are concerned, are also influenced by a passive diffusion property of molecules [20]. In contrast to transport assays, both accumulation (i.e. calcein-AM assay) and ATPase assays tend to fail in the identification ofrelatively low permeable compounds as P-gp active compounds [20]. 

The most recent example of in silico efflux modeling has been based on Caco-2 permeability measured in the basal to apical direction [100]. This model can be very effective at ruling out compounds that most likely will show low in vivo intestinal absorption - however it carmot indicate which efflux pump(s) is/are responsible for that, making it more difficult for designers to circumvent the problem. Johnson [92] also included in his review an excellent summary of QSAR models and rules of thumb developed for P-gp substrates and inhibitors. These models are normally based on efflux ratios from MDCK/MDRl or Caco-2 cell lines - in the latter case it is important to notice that the data is combined with inhibition values from the calcein-AM assay, as the observed efflux might not be exclusively due to P-gp. 

B 10. What biomolecules would interfere with the measurement of nucleic acids using the spectrophotometric (Am]) assay ... 

The degree of inhibition of Pgp activity, calculated from the fluorescence increase of the Calcein-AM assay was used as activity data [3]. The experimental inhibition data are normalized to the value obtained for Cyclosporine A (which is a competitive inhibitor of Pgp [4])so that the value for untreated cells is 0% and 100% is the value for Cyclosporine A. The inhibition value of each substrate was then transformed in the logarithm in order to reduce the residuals for the larger values. The activity range spans from 2.32 to 0.37, covering 1.95 log units. 

AM methods, in contrast, depend on the modulation of the enzyme signal by competition of the test molecule for the same immunoreactant. Contrary to AA methods, the sensitivity of AM assays increases with lower immunoreactant concentrations since at low concentrations a variation in the amount of competing molecules has a larger impact on the interaction with the labeled species. According to the law of mass action, the low antigen and antibody concentrations reduce considerably the rate of complex formation. Moreover, at very low concentrations the accuracy tends to be poor. 

AM assays have an intrinsically higher specificity than AA assays. 

AA assays are faster than AM assays at their limiting detectabilities due to the excess of antibodies in AA assays and to the law of mass action. 

The detectability of AM assays is not better than that of saturation analysis RIA, in contrast to AA assays. 

Homogeneous, AM assays pose different requirements. Here, the enzyme should be easily conjugated near the active site without altering its activity. The reaction of hapten- or antigen-labeled enzyme with antibody should affect strongly the enzyme activity, e.g., through steric inhibition of the substrate at the catalytic site. The requirements for optimal ionic conditions and temperature for both enzyme activity and antigen-antibody interaction should be compatible. 

The enzymes used in the AA-assays can also be used for solid-phase AM-assays. 

The previously discussed enzymes may all be used for solid-phase AM assays. For homogeneous AM-type EIA, lysozyme, malate dehy-rogenase (MDase), glucose-6-phosphate dehydrogenase (GPDase), and ribonuclease A (RNase A), are used in addition to BGase. 

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