Assay analytical sensitivity

The analytical sensitivities of the different quantitation methods have been compared using serial dilutions of patients specimens (Butterworth et al., 1996) and the Eurohep HBV DNA standards (Zaaijer et al., 1994). In both cases, bDNA was shown to be about 10-fold more sensitive than the liquid hybridization (Abbott) and the hybrid capture (Digene) assays. Using the Eurohep HB V standards, the detection limits were 2.5 X 106 genomes/ml for bDNA and 2.5 X 107 genomes/ml for both liquid hybridization (LH) and hybrid capture (HC) assays. 

A prototype bDNA assay was developed for quantification of HGV/GBV-C RNA in serum (Pessoa et al, 1997). The assay employed target probes based on the relatively conserved sequence in the 5 untranslated region of the HGV/GB V-C genome. Preamplifier molecules and incorporation of isoC and isoG into the sequences common to bDNA assays were used to enhance the analytical sensitivity. The provisional limit of detection was 32,500 genome equivalents/ml based on dilutions of a 700-nucleotide synthetic HGV/GBV-C RNA transcript. The run-to-run variance of the assay was <15%. 

A CL ISH assay for the detection of human papillomavirus (HPV) DNA was developed, in which the hybridization reaction was performed using either digoxigenin-, biotin-, or fluorescein-labeled probes [64], The hybrids were visualized using AP as the enzyme label and a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. This assay was applied to biopsy specimens from different pathologies associated with HPV, which had previously proved positive for HPV DNA by polymerase chain reaction (PCR). The analytical sensitivity was assessed using samples of HeLa and CaSki cell lines, whose content in HPV DNA is known (10-50 copies of HPV 18 DNA in HeLa cells and 400-600 copies... 

The enzyme attached to antibody 2 is critical for quantitative analysis. Figure 19-14 shows two ways in which the enzyme can be used. The enzyme can transform a colorless reactant into a colored product. Because one enzyme molecule catalyzes the same reaction many times, many molecules of colored product are created for each analyte molecule. The enzyme thereby amplifies the signal in the chemical analysis. The higher the concentration of analyte in the original unknown, the more enzyme is bound and the greater the extent of the enzyme-catalyzed reaction. Alternatively, the enzyme can convert a nonfluorescent reactant into a fluorescent product. Colorimetric and fluorometric enzyme-linked immunosorbent assays are sensitive to less than a nanogram of analyte. Pregnancy tests are based on the immunoassay of a placental protein in urine. 

Calibration Sensitivity, Analytical Sensitivity, and SEs for DNS, DNSsg, Nelson-Somogyi (Nelson lOmL), Modified Nelson-Somogyi (Nelson 5mL), PAHBAH, and BCA assays... 

In biochemical assays, additives such as detergents, DMSO, urea, BSA, and glycerol are commonly used to improve reaction performances and enzyme stability. However, these additives also act as crystallization disturbing agents preventing the formation of optimal crystals for the MALDI process. Analytical sensitivity and mass accuracy can be affected. The challenge is to develop bioassays that can perform optimally without crystallization disturbing additives. Often, it is necessary to use elaborate purification processes prior to analysis. 

The first ELISA designed for uPAR measurements with an analytical sensitivity of 19.5 pmol uPAR/liter was developed using two mAbs, R8 for catching and biotinylated R2 for detection [116], These mAbs recognize nonoverlapping epitopes on domain III of uPAR and therefore, this ELISA measured all known forms of uPAR except uPAR(I). A 20% decrease in the recovery was reported when uPA/uPAR complexes were measured. This assay was never used on patient material because of the... 

For high sensitivity immunoassay, the detection limit of the label must be in the sub-picomolar range. It is possible to have good sensitivity when two or three labels are used in assays but sensitivity is compromised when four labels are involved. Quadruple-label immunoassays of four analytes have been made [190] as shown in Fig. 12.35. 

The original assays used radioactive labels, but concerns about the safe handling and disposal of radioactive reagents and waste have led to the development of alternative non-isotopic labels (Table 9-1). In this section, the methodological principles on which these assays are based and the factors that affect their analytical sensitivity are discussed. In addition, specific examples of these assays and the types of labels that are used in them are evaluated. Commercial versions and applications of these assays are discussed in other chapters (see Chapters 3 and 11). 

Clmical performance of cardiac troponin assays has been shown to be strongly dependent on the analytical sensitivity and precision of measured concentrations around... 

As with the catecholamines, fluorescence methods have also been reported for urinary metanephrine analysis. Fluorescent derivatization of the metanephrines NM and MN by chemical oxidation was based on modification of the trihydroxyindole reaction used for catecholamines. The individual metanephrines were measured following chromatographic separation and fluorescent derivatization or through the formation of differential fluorescent compounds by oxidation at different pH levelsSince the stability of the fluorescent products was variable, with some products decomposing within 10 min,this method has limited application in current practice. Other early methods for analysis of NM and MN included electrophoresis and paper and thin-layer chromatography. These assays were technically complex and had poor analytical sensitivity. 

---