Assay procedures

The procedure is carried out as described earlier, except that following distribution of the 1-ml portions into test tubes, some of the tubes are supplemented with 0.1 ml S-9 mix containing 400 /tig protein. The remainder of the procedure is carried out as described above. 

When performing the suspension assay coupled to the S-9 mix, it is important that the test agent be tested both in the presence and in the absence of S-9. Thus, in the data presented in Table 7 (below), 2-aminofluorene in the absence of S-9 is toxic to the cell as evidenced by a slight drop in the viable count however, this effect is not indicative of a preferential inhibition of the pol Ai , strain, since the survival index is 1.02. On the other hand, in the presence of the S-9 activation mixture, there is evidence of DNA modification as shown by a survival index below 1.0 (i.e., 0.73). 

A brief outline of the essential features of the standard plate incorporation assay (Maron and Ames, 1983) is given in Box 10.9. Protocols for pure chemicals and for complex mixtures of primary emissions and ambient aerosols are also described by Belser et al. (1981) and Alfheim et al. (1984b) intra- and interlaboratory comparisons are discussed later in this chapter. 

Specific activities of individual airborne PAHs and PACs determined by the standard plate incorporation assay cover a huge range e.g., the mono- and dinitro-PAHs in Table 10.16 have direct activities ( — S9) ranging from several hundred to 10A rev pg. This is also true for different isomers of the same compound. For example, the 1-, 3-, and 6-N02-BaP isomers formed in laboratory exposures of BaP particles to N02 (plus a trace of HN03) in air have direct activities (TA98, — S9) of 2500, 5300, and 1 rev /xg 1, respectively (Pitts et al., 1984b). 

The description of the assay, including a detailed stepwise procedure that was established during method development should be followed exactly as written during the validation and ultimately documented in a final analytical method or SOP. A description of the specific documentation requirements for the method can be found in Section 9.8.5. The method may also include references to other laboratory SOPs that describe routine procedures (e.g. equipment calibration). 

Preparation of Rhodamine Phalloidin-Labeled Actin Filaments 

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Both nicotinic acid and nicotinamide have been assayed by chemical and biological methods. Owing to the fact that niacin is found in many different forms in nature, it is important to indicate the specific analyte in question. For example, if biological assay procedures are used, it is necessary to indicate whether the analysis is to determine the quantity of nicotinic acid or if niacin activity is the desired result of the analysis. If nicotinic acid is desired, then a method specific for nicotinic acid should be used. If quantitation of niacin activity is the desired outcome, then all compounds (bound and unbound) which behave like niacin will assay biologically for this substance (1). 

Properties. Thienamycin is isolated as a colorless, hygroscopic, zwitterionic soHd, although the majority of carbapenems have been obtained as sodium salts and, in the case of the sulfated olivanic acids, as disodium salts (12). Concentrated aqueous solutions of the carbapenems are generally unstable, particularly at low pH. AH the substituted natural products have characteristic uv absorption properties that are often used in assay procedures. The ir frequency of the P-lactam carbonyl is in the range 1760 1790 cm . ... 

Board Rank Code Numbers are pardy assigned on the Gray-King assay and pardy on the volatile matter. The Gray-King assay procedure can also permit evaluation of yields of tar, gas, and Hquor. 

The guideline states that the objective of validation is to demonstrate that an analytical method is fit for its purpose and summarizes the characteristics required of tests for identification, control of impurities and assay procedures (Table 13-2). As such, it applies to chiral drug substances as to any other active ingredients. Requirements for other analytical procedures may be added in due course. 

Another extremely important application area for FNA and RFNA is to either directly nitrate or be used in mixed acids to nitrate raw materials to yield widely used expls and proplnt ingredients (Refs 29,31,33,38 39). Also see under Nitration in this Vol Analytical. Analysis and assay procedures for nitric acid may be found in Refs 1, 2,10,11,15, 17, 27, 29, 34, 35, and in this Vol under Nitrogen Determinations in Energetic Materials. 

In our laboratories the following organisms have been selected as representative and qualitative antimicrobial assays are carried out using the agar well diffusion assay procedure (5) ... 

It must be assumed that urine collections were accurately timed and that complete urine specimens were obtained at each collection time. It is also assumed that the assay procedure is accurate and reproducible. 

In an assay procedure the amine was heated with diluted nitric acid, and nearexplosive decomposition occurred. 

The residues from a cortisol assay procedure (5 cc dichloromethane, 2.5 cc of a fluorescent reagent in 15 85 ethanol-sulfuric acid) were added to a 500 cc bottle and screw capped. After a 90 s delay, the bottle burst violently and brown fumes were seen. It was surmised that a nitrate or nitrite contaminant in the bottle had... 

By use of model substrates and inhibitor studies, an esterase that is reactive in unbuffered sea water as well as in the disrupted algal tissue from C. taxifolia was identified which mediates cleavage of the acetyl residues of caulerpenyne (54) [121]. After complete deacetylation, oxytoxin 2 (64) appears as an unstable end-product. Due to the lack of an appropriate assay procedure for labile metabo-... 

Kobylinska et al. [62] described a high performance liquid chromatographic analytical method for the determination of miconazole in human plasma using solid-phase extraction. The method uses a solid-phase extraction as the sample preparation step. The assay procedure is sensitive enough to measure concentrations of miconazole for 8 h in a pharmacokinetic study of Mikonazol tablets and Daktarin tablets in human volunteers. The pharmacokinetics of the two formulations was equivalent. 

A rapid second-derivative spectrophotometric assay procedure was described for the simultaneous determination of niclosamide and thiabendazole in tablets [47]. The derivative absorbances were measured at 354 nm for niclosamide and at 242.5 nm for thiabendazole. 

It is essential to establish that the assay procedure is linear with time and input kinase over the range under study. This can be accomplished by performing pilot experiments using a range of amounts of kinase and taking samples for analysis at several times. 

Fluorescent labels, by contrast, can provide tremendous sensitivity due to their property of discrete emission of light upon excitation. Proteins, nucleic acids, and other molecules can be labeled with fluorescent probes to provide highly receptive reagents for numerous in vitro assay procedures. For instance, fluorescently tagged antibodies can be used to probe cells and tissues for the presence of particular antigens, and then detected through the use of fluorescence microscopy techniques. Since each probe has its own fluorescence emission character, more... 

Microbiological assay procedures for nalidixic acid have also been used for biological samples. Since nalidixic and hydroxynalidixic acids have the same order of antibacterial activity in-vitro, then cannot be determined separately. 

C Red No. 2) has been utilised by Hill2 and by Bufton and Saddlerl42 to assay neomycin in aqueous solution. Amaranth may also be used to determine neomycin in production samples from fermentation-recoveryl68. The dye, Orange II, has been similarly described(A max. = 484 nm). Complex formation between neomycin and the dye is very dependant on the ionic strength of the solution, thus necessitating careful control of reaction conditions to ensure complete precipitation of the complex during the assay procedure. Reaction of sodium 1,2-naphtho-quinone-4-sulphonate with... 

Although B12 can be assayed biologically in mice, chicks, and rats, especially with the use of radioactive cobalt, the microbiologic method of assay is preferred because it is economical and sensitive. One serious drawback of bacterial Bi2 assay procedures is the lack of specificity and sensitivity. The extreme sensitivity (1 X 10 12 g) and relative freedom of stimulation in blood, serum, and urine make protozoa the choice assay tools (B17, F3, H19). The most specific is O. malhamensis (B17, F3) Euglena is known to be stimulated by pseudo-B12 (F2). Serum and blood from normal subjects have a growth-promoting effect on Euglena (M12) above that seen with Ochromonas. The reason for such... 

The principle of the assay procedure for CMP-KDO synthetase had been developed previously, to assay for CMP-NeuAc synthetase in ex-... 

The complexity of the mixtures made it impossible to define the chemical composition so the commercial preparations were divided into four groups (Table 8.2) on the basis of a series of sophisticated chemical assay procedures. Caramel colorants must be compatible with the food products in which they are used, which usually means the absence of flocculation and precipitation in the food. These undesirable effects result from charged macromolecular components of caramel which react with the food. Hence the net ionic charge of the caramel macromolecules at the pH of the intended food product is the prime determinant of compatibility. Caramel colorants are used in a variety of foods (Table 8.2) but over 80% of the caramel produced in the US is used to color soft drinks particularly colas and root beers. 

A wide range of coupled assays involving ATP, NAD(P)H and FMN can be developed using these two enzymes and provide increased levels of sensitivity over other coupled assays (Procedure 8.7). 

Immersion Toxicity Assay Procedure, Criteria of Response and Data Analysis. The assay procedure was a modification of published methods (4, 5). Toxicity assays were conducted at 21 4 2°C in narrow-mouth, quart (0.95 L) glass jars containing conditioned Columbus tap water (pH 7.5 to 8.5, aged 24 hr). No food, substrate, or aeration were used during the test. Cannibalism did not occur with well-fed larvae and short term (24 hr) assays. 

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