Plate Assay

The protein concentration of the S-9 mix (S-9 fraction -i- cofactors) is adjusted to contain 400 /ig protein/0.1 ml S-9 mix. Portions of this preparation (0.1 ml) are incorporated into the 2 ml of soft agar overlay (HA-hT soft agar). The temperature of the soft agar should be 43-45 C. The indicator bacteria 

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As mentioned earlier, a-D-carba-galactopyranose (1) has been found in a fermentation broth of Streptomyces sp. MA-4145, as an antibiotic. The potency of the antibiotic was rather low. A concentration of—125 fig/mh is required in order to produce a standard inhibition zone of 25-mm diameter against Klebsiella pneumoniae MB-1264, using 13-mm assay discs in a disc-plate assay. A sample of the synthetic a-DL-carba-galactopyranose (17) was... 

Conventional plate assays require several hours ineubation and eonsequently the possibility of using rapid mierobiologieal assay methods has been studied. Two such methods are ... 

EIAs are more desirable for the measurement of agrochemicals than enzyme-linked immunosorbent assays (ELISAs) for several reasons. EIAs are easier to run, require minimal liquid transfers, and are completed in brief time frames, approximately 40 min for tube assays to 2.5 h for microtiter plate assays. In contrast, ELISAs are more complex, have many steps involving transfer of reagents, and require 6-8 h to complete. Most commercially available immunoassays utilize the EIA format. 

Four neutral lipid models were explored at pH 7.4 (1) 2% wt/vol DOPC in dode-cane, (2) olive oil, (3) octanol, and (4) dodecane. Table 7.5 lists the effective permeabilities Pe, standard deviations (SDs), and membrane retentions of the 32 probe molecules (Table 7.4). The units of Pe and SD are 10 6 cm/s. Retentions are expressed as mole percentages. Figure 7.22a is a plot of log Pe versus log Kd (octanol-water apparent partition coefficients, pH 7.4) for filters loaded with 2% wt/vol DOPC in dodecane (model 1.0, hlled-circle symbols) and with phospholipid-free dodecane (model 4.0, open-circle symbols). The dashed line in the plot was calculated assuming a UWL permeability (see Section 7.7.6) Pu, 16 x 10-6 cm/s (a typical value in an unstirred 96-well microtiter plate assay), and Pe of 0.8 x 10-6 cm/s... 

Kimball, S. R., Horetsky, R. L., and Jefferson, L. S. (2004). A microtiter plate assay for assessing the interaction of eukaryotic initiation factor eIF4E with eIF4G and eIF4E binding protein-1. Anal. Biochem. 325, 364—368. 

H. Harma, T. Soukka, S.Lonnberg, J. Paukkunen, P. Tarkkinen, and T. Lovgren, Zeptomole detection sensitivity of prostate-specific antigen in a rapid microtitre plate assay using time-resolved fluorescence. Luminescence 15, 351-355 (2000). 

Kavanagh and Dennen20 report microbiological turbidimetric and plate assays for erythromycin base in Analytical Microbiology,... 

Crespi, C.L., Miller, V.P., and Penman, B.W. 1997. Microtiter plate assays for inhibition of human, drug-metabolizing cytochromes P450. Anal. Biochem. 248 188. 

Naritomi, Y. et al. Utility of microtiter plate assays for human cytochrome P450 inhibition studies in drug discovery. Drug Metab. Pharmacokin. 19 55. 

Data Analysis. Data from the fluctuation test described above are analyzed by an appropriate statistical method as described in Robinson et al. (1989). Data from plate assays are analyzed as described in Arlett et al. (1989) for treat and plate tests. 

It was carried out according to Schwikkard et al. [9] and Actinomycin D was used as a positive control [ 16]. Cytotoxicity was determined against A2780 human ovarian cancer cells, using a microtiter plate assay. The plates were seeded with cells and... 

The most commonly used analgesic tests utilize a chemical, thermal or mechanical nociceptive stimulus, for example, rodent paw pressure, tail flick or hot plate assays. 

Edmiston, C.E., Jr., Goheen, M., Malaney, G.W., and Mills, W.E. Evaluation of carbamate toxicity acute toxicity in a culture of Paramecium multlmlcronucleatum upon exposure to aldicarb, carbaryl, and mexacarbate as measured by a Warburg respirometry and acute plate assay. Environ. Res., 36(2) 338-350, 1985. 

The sensitivity of the microfluidic system was determined by measuring calibration curves of four cathepsin B inhibitors. The inhibitors caused negative peaks in the product mass chromatograms by inhibiting cathepsin B and thus the substrate turnover. The measured order of afEnities of the four inhibitors is in agreement with the affinities determined in microtiter plate assays and the macro-scale system. 

P-glycoprotein-mediated efflux is a potential source of pecuHarities in drag pharmacokinetics, such as non-Hnearity. This includes dose-dependent absorption, drug-drag interactions, intestinal secretion and limited access to the brain. Assays are in development to quantify the interaction between transporters and drugs. One of the first is a 96-weU plate assay for P-gp binding [33, 34] and an MDRl ATPase test [35]. 

Higher throughput screening with human cytochrome P450 to study P450-medi-ated metabolism is now available [38-40]. Similarly, rapid microhtre plate assays to conduct for example, P450 enzyme inhibition studies have been developed, using individually expressed CYP enzymes (Supersomes) [41]. 

The Cup-Plate or Well Procedure. This procedure is similar to the cylinder-plate system except that wells are cut into the agar with cutters capable of cutting uniform, completely circular wells. As with the cylinder-plate assays, the wells are filled. Zones are measured after incubation and the concentration determined utilizing a comparison with a standard response line. 

In conjunction with the discussion of the receptor assay system, it is logical to discuss the variations of the plate assay systems and/or growth systems using colorimetric indicators of inhibition of metabolism or growth. 

Gowher, H., Zhang, X., Cheng, X. and Jeltsch, A. (2005) Avidin plate assay system for enzymatic characterization of a histone... 

Isolated compounds were collected after separation by HPLC and assayed for wr-induction in the strains described. It became apparent that the limited host range strain responded to all the substances that induced vir expression in the wide host range strain, but the LHR strain was less sensitive to the same substances and as a result was considered a less sensitive bioassay organism. Therefore work on isolation of grapevine-derived signal compounds continued using the more sensitive WHR strain and plate assay system described. 

An antibiotic inhibition zone often appears around Trichoderma spp. interacting with other fungi. The genus contains many species which produce secondary metabolites. Claydon et al. (23) have identified an antibiotic from T. harzianum as a volatile, 6-n-pentyl-2H-pyran-2-one this was recently shown to be an active antibiotic from T. koningii (24). The volatile appeared to be the factor responsible for the coconut smell of some biocontrol-effective strains of T. harzianum (25). However, in a Petri-plate assay, it can be difficult to be certain that antibiosis is involved. As well as competitive growth, lytic enzymes could also contribute to the action and Trichoderma has been shown to produce / -l,3-glucanase and chitinase (26-29). 

Bioassay may also be of low-precision design (multiple samples on large plates, i.e., >3 manual turbidimetric assays, small-plate assays). These types of assay are useful for trace analyses (cleaning validation), and are often used for the analysis of samples in body fluids as they are capable of dealing with the large numbers of samples that may be generated in these studies. 

Table 2 Inhibition of Human Cytochrome P450 3A4-, P450 3A5-, and P450 3A7-Mediated Metabolism of the Marker Substrate 7-Benzyloxyresorufin by Aqueous Extractions of SJW Teas in the Fluorescence Plate Assay and Testosterone HPLC Assay (in Brackets) (n — 3 25mg/mL Stock Solutions % Inhibition SD)...

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