Assay linearity range

The advantages of controlled-potential techniques include high sensitivity, selectivity towards electroactive species, a wide linear range, portable and low-cost instrumentation, speciation capability, and a wide range of electrodes that allow assays of unusual environments. Several properties of these techniques are summarized in Table 1-1. Extremely low (nanomolar) detection limits can be achieved with very small sample volumes (5-20 pi), thus allowing the determination of analyte amounts of 10 13 to 10 15 mol on a routine basis. Improved selectivity may be achieved via the coupling of controlled-potential schemes with chromatographic or optical procedures. 

Silverlight and Jackman developed an immunoassay for levamisole in meat and milk. The LOD in both milk and meat samples was 1 xg kg The assay was applied to milk directly, and muscle samples required only homogenization in the presence of 10-fold of buffer prior to analysis. The linear range of the assay was between 5 and 50 pg kg for meat and between 0.2 and 25 qg kg for milk. The linear range of the assay was below the MRL for milk (10 qg kg ) and meat (50 qg kg ). 

The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13-15]. When the tag is... 

HIV-1 RNA in plasma can also be quantitated by a branched-DNA (bDNA) signal amplification assay which has a quantitation limit of 1 x 104 HIV-1 eq/ml. A novel internally controlled PCR assay (ICPCR) has been used to quantitate HIV-1 Gag DNA and RNA in peripheral blood mononuclear cells and plasma. The linear range of amplification for the ICPCR assay is between 10° and 103 copies for HIV-1 DNA, while for HIV-1 RNA the amplification range is from 101 to 104 copies. The ICPCR assay correlates with the bDNA signal amplification assay for the quantitation of HIV-1 RNA, although subtle differences between the two assays were noted (G2). Nevertheless, the fall in HIV-1 RNA levels in plasma in response to antiretroviral therapy was comparable with both the bDNA and ICPCR assays. 

The purpose of sample acceptance criteria is to confirm that the run for a test article is suitable for quantification. The corrected peak area of the sample should be within linear range of the assay the baseline should be suitable for integration the corrected peak percentage should be in a reasonable range. Again, for a purity assay, migration time is usually not a critical parameter. 

It is an essential condition of biological assay methods that the tests on the standard preparation and on the sample whose potency is being determined should be carried out at the same time and, in all other respects, under strictly comparable conditions. The validation of microbiological assay method includes performance criteria (analytical parameters) such as linearity, range, accuracy, precision, specificity, etc. 

Depending on the use of the assay, different parameters will have to be measured during the assay validation. Validation of analytical assays is the process of establishing one or more of the following as appropriate to the type of assay accuracy precision (repeatability, intermediate precision), linearity, range, limit of detection, limit of quantification, specificity, and robustness [1]. For physicochemical methods there are accepted defined limits for these test parameters ... 

Glycerol kinase activity can be measured directly or indirectly however, these assays are not available as a clinical test and are done exclusively on a research basis. The direct method is as has been described previously [4, 6] and is used in the isotope dilution method indicated above. The amount of protein and the incubation time vary between cell types and it is important to be within the linear range of the assay for the given cell type. The indirect methods involve incorporation of 14C from glycerol into macromolecules and its subsequent oxidation to 14C02 [7,11]. 

Add 1 ml of DMB reagent to 100 pi of demineralized water (blank) or 100 pi of urine in a cuvette. In addition, 100 pi of each urine should be added to 1 ml of formiate buffer to assess the absorbance of the pure sample (sample blank). Each sample should be measured against pure formiate buffer (buffer blank, when measured separately). The color of the GAG-DMB complex is not stable over time, so that assay conditions have to be strictly standardized. Urine, standard, or water is added to all cuvettes first. The DMB reagent must then be added swiftly. All cuvettes are mixed with a spatula (10 x) and after 3 min samples are measured at 520 nm. When the absorbance exceeds the linear range, the urine has to be diluted. It is also recommended to measure all samples in duplicate using 50 and 100 pi of urine. This allows an evaluation of the plausibility of results. 

Fig. 18.6. Calibration curve obtained by the immuno-assay procedure in the detection of anti-E2 using the ITO-Poly (pyrrole-benzophenone) coated optical fibers. The linear range of the calibration curve was obtained for titer 1 64,000 and lower. The curve was fitted according to the equation y = A+B(x), where x is the human sera (anti-E2 antibodies) dilution value and y is the chemiluminescence response. The obtained correlation coefficient was R2 = 0.988.

GOD immobilisation technique Mediator Assay time (s) Lower linear range (pM) Upper linear range (mM) Applied potential (mV) Storage stability (weeks) Reference... 

Another common reason for having separate assay and impurity methods is the need to use more concentrated samples with the impurity assay to increase sensitivity for minor impurities. Modern HPLC systems have been shown to adequately detect low-level impurities (i.e., 0.05%) in chromatograms where the parent peak is still on scale (that is, within the linear range of the detector). This level of detection is usually adequate for screening methods therefore, the assay for loss of parent compound and the measurement of the increase in impurities can typically be done using a single HPLC method. 

Wahbi et al. [32] used a spectrophotometric method for the determination of omeprazole in pharmaceutical formulations. The compensation method and other chemometric methods (derivative, orthogonal function, and difference spectrophotometry) have been applied to the direct determination of omeprazole in its pharmaceutical preparations. The method has been validated the limits of detection was 3.3 x 10 2 /ig/ml. The repeatability of the method was found to be 0.3-0.5%. The linearity range is 0.5-3.5 /ig/ml. The method has been applied to the determination of omeprazole in its gastro-resistant formulation. The difference spectrophotometric (AA) method is unaffected by the presence of acid induced degradation products, and can be used as a stability-indicating assay method. 

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