Assay sensitivity

One concern with equivalence and non-inferiority trials is that a positive conclusion of equivalence/non-inferiority could result from an insensitive trial by default. If, for example, equivalence is established then this could mean either that the two treatments are equally effective, or indeed equally ineffective. If chosen endpoints are insensitive, dosages of the drugs too low, patients recruited who are not really ill and the trial conducted in a sloppy fashion with lots of protocol deviators and dropouts, then the treatments will inevitably look very similar Clearly we must ensure that a conclusion of equivalence/non-inferiority from a trial is a reflection of the true properties of the treatments. The regulatory guidelines (ICH ElO) talk in terms of assay sensitivity as a requirement of a clinical trial that ensures this. 

ICH ElO (2001) Note for Guidance on Choice of Control Group in Clinical Trials  

Of course assay sensitivity applies in the same way to trials which evaluate superiority, but in those cases things take care of themselves. A conclusion of superiority by definition implies that the trial is a sensitive instrument, otherwise superiority would not have been detected. 

Further, the trial conduct should protect against any compromise of assay sensitivity and the following in particular should be avoided  

A further phrase that is used in this area is historical evidence of sensitivity to drug effects. This idea, introduced initially in ICH ElO, refers to the ability of effective treatments to consistently show an advantage over placebo in appropriately designed and conducted clinical trials. As mentioned in the previous paragraph, there are certain therapeutic settings where this is not the case. 

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Assay sensitivity is defined here as the concentration of analyte that inhibits the observed absorbance by 50% or the IC50. The lower limit of detection (LLD) is the lowest analyte concentration that elicits a detector response significantly different from the detector response in the absence of analyte. In some cases, the LLD is defined as three standard deviations from the mean of the zero analyte control. In other cases, the LLD is defined empirically by determining the lowest concentration of analyte that can be measured with a given degree of accuracy. Readers are referred to Grotjan and Keel for a simplified explanation and to Rodbard for the complete mathematics on the determination of LLD. 

Beasley et al. developed a panel of immunoassays to monitor DDT, its metabolites, and structurally related compounds, but they found that milk has a severe effect on the assay performance. They found that when directly utilizing whole milk, color development was completely inhibited. Even when using 1 100 dilutions of whole milk, the assay sensitivity was reduced by 90% (based on the IC50 shift, not simply the dilution factor). A number of procedures were evaluated to eliminate the interferences from the fat-soluble analytes. However, many of the procedures that removed interferences also removed the analytes. Extraction with a mixture of solvents and the use of similarly processed blank milk to prepare the standards ultimately yielded more accurate results. This article demonstrates the difficulties encountered in analyzing lipid-soluble analytes. 

During the last few years, miniaturization has become a dominant trend in the analysis of low-level contaminants in food and environmental samples. This has resulted in a significant reduction in the volume of hazardous and expensive solvents. Typical examples of miniaturization in sample preparation techniques are micro liquid/liquid extractions (in-vial) and solvent-free techniques such as solid-phase microextraction (SPME). Combined with state-of-the-art analytical instrumentation, this trend has resulted in faster analyses, higher sample throughputs and lower solvent consumption, whilst maintaining or even increasing assay sensitivity. 

Dean et al. [93] used a high performance liquid chromatographic method for the simultaneous determination of primaquine and carboxyprimaquine in plasma with electrochemical detection. After the addition of the internal standard, plasma was deproteinized by the addition of acetonitrile. Nitrogen-dried supernatants, resuspended in mobile phase were analyzed on a C8 reversed-phase column. Limits of detection for primaquine and carboxyprimaquine were 2 and 5 ng/mL with quantitation limits of 5 and 20 ng/mL, respectively. The assay sensitivity and specificity are sufficient to permit quantitation of the drug in plasma for pharmacokinetics following low dose (30 mg, base) oral administration of primaquine, typically used in the treatment of malaria and P. carinii pneumonia. 

Rhodes A, Jasani B, Anderson E, et al. Evaluation of HER-2/neu immunohisto-chemical assay sensitivity and scoring on formalin-fixed and paraffin-processed cell lines and breast tumors a comparative study involving results from laboratories in 21 countries. Am. J. Clin. Pathol. 2002 118 408-417. 

A common application for (strept)avidin-biotin chemistry is in immunoassays. The specificity of antibody molecules provides the targeting capability to recognize and bind particular antigen molecules. If there are biotin labels on the antibody, it creates multiple sites for the binding of (strept)avidin. If (strept)avidin is in turn labeled with an enzyme, fluorophore, etc., then a very sensitive antigen detection system is created. The potential for more than one labeled (strept)avidin to become attached to each antibody through its multiple biotinylation sites is the key to dramatic increases in assay sensitivity over that obtained through the use of antibodies directly labeled with a detectable tag. 

Wittekind E, Werner M, Reinicke A, Herbert A, Hansen P (1996) A microtiter-plate urease inhibition assay-sensitive rapid and cost-effective screening for heavy metals in water. Environ Technol 17 597-603... 

As described above, the dendrimer-coupled antibody conjugates show uniquely enhanced properties compared to the classical double antibody systems. Important characteristics such as complete solubility in aqueous buffers, flexibility in immunoassay format, ability to improve assay sensitivity, consistent, reproducible manufacturing and favorable stability has driven the utilization of these dendrimer-based reagents in Stratus CS, the latest member of the Stratus family of immunochemistry analyzers. In this new analyzer system, the primary... 

The assay sensitivity limit is found to be 100 pg of unlabelled-morhine per tube that caused 20% binding inhibition of labelled-dihydromorphine, (see Figure 32.3). 

For the determination of these compounds a binding inhibition immunoassay, consisting of the competitive immunoreaction of the unbound antibody present in an analyte-antibody mixture with the hapten derivative immobilized at the sensor surface, has been applied. With the aim of assuring the regeneration and reusability of the surface without denaturation of the immobilized molecule, the formation of an alkanethiol monolayer was carried out to provide covalent attachment of the ligand to the functionalized carbodiimide surface in a highly controlled way. For DDT, the assay sensitivity was evaluated in the 0.004 - 3545 pg/l range of pesticide concentration by the determination of the limit of detection 0.3 pg/1 and the I50 value 4.2 pg/1. 

Table 6.2 Assay Sensitivity for Antibody-Antigen Microarrays and Standard Sandwich ELISA... 

Concentrations decrease with increasing body weight by a mean of 3.3 pg/mL/kg. After capsule removal, mean concentrations drop to less than 100 pg/mL by 96 hours and to below assay sensitivity (50 pg/mL) by 5 to 14 days. 

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