Dynamic range assays

When sufficient amounts of sample are available one tries to exploit the central part of the dynamical range because the signal-to-noise ratio is high and saturation effects need not be feared. (Cf. Figures 2.11 and 3.1.) Assays of a major component are mostly done in this manner. 

The use of Ct values also expands the dynamic range of quantitation because data are collected for every PCR cycle. A linear relationship between Ct and initial DNA amount has been demonstrated over flve orders of magnitude, compared with the one or two orders of magnitude typically observed with an endpoint assay. 

The version 2.0 assay uses a different set of probes designed to hybridize to genotypes 1 to 6 with equal efficacy (Fig. 4). The new probe set not only enhanced the efficiency of binding to genotypic variants but also lowered the LOQ from 3.5 X 105 to 2 X 105 HCV RNA equivalents/ml (Detmer et al., 1996). The version 2.0 assay displayed almost a 600-fold dynamic range up to 1.2 X 108 RNA equivalents/ml. The LOQ was set at 2 X 105 to ensure a specificity of 95%. The assay was reproducible, with a mean CV of 14% for replicates of low-, middle-, and high-titer sera. Serial dilutions of quality level 1 RNA transcripts (Collins et al,... 

Fig. 11.7 Equilibrium assay using immbolized mouse IgG antigen capturing anti mouse antibody from solution. The dynamic range is 300 1, showing 16% biological activity and an affinity of...

Fig. 11.10 Concentration recovery on the haptoglobin assay, (a) The recovered concentration against the applied concentration, (b) The precision of recovery, achieving 80% recovery over a dynamic range of 1,000 1...

The dynamic range of the standard curve for amitriptyline, nortriptyline, and imipramine was 25 to 250 /./g/mL it was 50 to 500 /ig/ml. for desipramine. Within-run and between-run coefficients of variation for the assay were below 10%. Up to 40 patient samples could be analyzed in 1 hr. 

MesoScale Discovery (MSD) succeeded in introducing product with a similar technology approach based upon ruthenium redox-mediated electrochemical detection (Figure 2.14). MSD is a joint venture of its parent company, MesoScale, and IGEN, a company that pioneered much of fhe work on electrochemical detechon based on the ruthenium redox system. MSD s Multi-Spot plates contain antibodies immobilized on multiple working electrode pads within each well, allowing each spot within the well to serve as an individual assay. Multiplexed cytokine immxmoassays can be performed in 96-well (4,7, or 10 spots per well) patterns with detection limits of 1 to 10 pg/mL and a linear dynamic range up to 3,000 pg/mL. Both 24-and 384-well electrode systems are available. 

Table 6.2 summarizes many of the studies discussed in this review. For comparison, the analyte LED values (pg/mL) have been recalculated in terms of analyte concentrations and reported in pM units in an attempt to easily identify assay formats leading to higher sensihvity. Where possible, we will also discuss dynamic range. 

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