Duplication, assays

The rules for level I (screening) assays are shown in Table 13.1. An example of the type of samples where a level I assay could be used is the CARRS samples [85] that can be used for screening NCEs using a rat PK model [vide supra). The concept behind this assay is that it should use a small number of standards and a simple linear extrapolation. For level II assays (see Table 13.2) that might be used for discovery PK studies in preclinical species, a complete standard curve is required. In this case a complete standard curve is defined as 10-15 standards in duplicate assayed with at least five standards used in the final calibration curve. Neither level I nor level II assays require the use of quality control (QC) standards. When a compound is in the lead qualification stage, then a level III assay would be required. As shown in Table 13.3, the main distinction for level III assays is that they are required to include at least six QC standards. As described in Tables 13.1-13.3, these rules show the requirements for how an assay should be set up before the samples are assayed and then these rules describe the acceptance criteria for the assays after they have been performed. 

In routine use, the combined result of a series of independent assays spread over a number of days is a more reliable estimate of potency than that from a single assay. Minimum requirement for routine microbiological testing is duplicate assay. 

Perform the assay of six individual samples on a single day according to the method proposed with NMT 2% RSD. In most cases, a duplicate assay of the second lot should also be made to check the lot-to-lot variation. 

Fibroblasts are grown under standard conditions in Ham FI0 medium supplemented with 10% FCS. Cells are harvested by trypsinization, taken up in medium, counted, and reseeded in 6-well plates at a density of 200,000 cells per well (2.5 ml cell suspension, 80,000 cells per milliliter). A total of six wells on three different plates is needed per cell line (two wells per plate for duplicate assays). For each assay, one control and one NPC cell line with classical biochemical phenotype is included. Incubate for 3 days in Ham F-10 with 10% FCS, remove the culture medium and grow the cells in... 

The standard deviation (SD) of all the results from their group means was 0.07 mg/C. The SD from the results of duplicate assays on urine samples was 0.12 mg/f in the 1-10 mg/f range (30 samples with a mean pregnanediol concentration of 3.88 mg// ). This data implies that pregnanediol concentrations in urine of below 0.2 mg/C cannot be analyzed with acceptable precision by this method. 

Other advantages of microfabricated devices include faster response times, and the fabrication of multiple test sites for simultaneous replicate assays in one microfabricated device. This analytical redundancy provides a safeguard that is not easily attained in a conventional macroscale analyzer, where duplicate assays represent the usual extent of repetitive assay of a sample. Encapsulation technology used in the microelectronics industry may also be applicable to microscale devices and could be extended to operations over a wide range of environmental conditions of humidity, and temperature. 

The data files are ascii text files arranged in columns separated by space, tabs, or commas with header lines. Equilibrium files contain total ligand concentrations in the first column and free ligand concentrations in the second column. Initial velocity files contain substrate concentrations in the first column and initial velocities of duplicate assays in the subsequent columns. Progress curve files contain time in the first column and the measured experimental signal in the second column. 

Kafer, E., B.R. Scott, G.L. Dorn, and R. Stafford. Aspergillus nidulans Systems and results of tests for chemical induction of mitotic segregation and mutation. I. Diploid and duplication assay systems. 

Figure 9.56 Mouse brain spermidine synthetase activity as a function of time. Both the absorbance (O) and the radiometric ( ) determination of product formation represent the mean value of duplicate assays. The reproducibility was within 10%. (From Porta et al., 1981.)...

Figure 9.78 HPLC-based assay for sucdnyl-CoA synthetase from rabbit liver mitochondria. One hundred microliters of diluted supemate was added to 3.0 mL containing 50 mAf succinate, 33 mAf Na-Hepes, pH 7.4, 5 mAf MgCl2,1 mAf CoA, 1 mAf GTP, and 5 jug of oligomycin. The reaction was run at 30°C and aliquots of 0.30 mL were removed at 1-minute intervals and transferred to autosampler vials containing 0.2 mL of 0.2 Af formic acid. Nucleotides were separated by HPLC. The UV detector was set to 254 nm. The chromatographic profiles for the reaction after 0, 5, and 10 minutes are shown. Peaks (top profile) 1, GMP 2, GDP 3, GTP 4, CoA and 5, succinyl-CoA. (A) Linearity of succinyl-CoA (S-CoA) formation, expressed as the percentage conversion of CoA to sucdnyl-CoA, with time. (B) Linearity of S-CoA formation with micrograms of protein added for 5 minute assays carried out in a volume of 0.30 mL. The latter assays were carried out in duplicate. The values shown are averages of the duplicate assays. (From Lambeth and Muhonen, 1993.)...

Xi and X2 = duplicate assay results (pg/ml) standard deviation interval =... 

Figure 39-7 Polymerase chain reaction (PCR) analysis for clonal rearrangements of the antigen-receptor gene loci. PCR analysis of theTCR y-chain locus is shown. The upper and middle panels show duplicate assays with monoclonal capillary electrophoretic peaks of identical size ( 166 bp) in both replicates.The bottom panel shows a polyclonal pattern.

TIP Consistent sample preparation can eliminate questions about the validity of assay results and may eliminate the need for duplicate assays. This can save valuable time during scale-up runs. 

New impurity seen Confirm existence and level by duplicate assay. Confirm no change in quality of input batches. New impurities are especially important for API. 

We always analyze our zero-time aliquot in triplicate (i.e., six replicates for a duplicate assay to give three results) to overcome any random errors and take into account standard analytical errors, since stability data are often critical in dictating study logistics. 

Z.E. Perlman, T.J. Mitchison, T.U. Mayer, High-content screening and profiling of drug activity in an automated centrosome-duplication assay, Chembiochem 2005, 6,... 

An alternative to microarrays is the use of arrays of multiple parallel channels. Typically, such structures are used to carry out a number of duplicate assays on one or more samples, to perform a serial dilution of a single sample for quantitation of a given protein, or to detect multiple targets in a single sample. In the latter case, an array of channels may be used in conjunction with a printed array of spots or bands - the channel network is used for fluid delivery to the printed array (Fig. 2c). 

Figure 1. Effect of ASD-CoA (panel A) and 12-azidooleoyl-CoA (panel B) on DGAT activity in the particulate fraction. Membranes containing 68 fig protein were assayed for 10 min in the dark in reaction mixtures containing 2 ptM (O) or 14 /iM [l- CJoleoyl-CoA ( ) and 330 /iM exogenous 1,2-diolein as substrates. Data points are the means of duplicate assays.

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