Kinetic turbidimetric

To evaluate the influence of structural parameters governed by the cyclophosphazene core concerning the valency and the spatial orientation of epitopes, as well as the nature of linkers directly related to the ligation technique used for the mannoside incorporation, the authors performed preliminary kinetic turbidimetric assays with Con A. Insoluble cross-linked complexes formed rapidly for all compounds, without marked difference for the hexavalent analogues. On the other hand, the incorporation of additional mannosyl units led merely to statistical binding-affinity enhancements, notably for the less-dense decamer 194, which presents favorable extended intersugar distances. 

Sakai H, Hisamoto S, Fukutomi I, et al. Detection of lipopolysaccharide in hemoglobin-vesicles by Limulus amebocyte lysate test with kinetic-turbidimetric gel clotting analysis and pretreatment of surfactant. J Pharm Sci 2004 93 310. 

A short report demonstrated the absence of a reliable correlation between kiUing kinetics and normal laboratory tests for pristinamycin susceptibility testing of some pneumococci (42). Eight selected multiresistant clinical isolates and two reference pristinamycin-resistant Streptococcus pneumoniae strains were studied. Disk diffusion susceptibility and MICs were determined by the agar dilution method, and all clinical isolates appeared to be susceptible to pristinamycin, whereas the two reference strains were not. In contrast, time-kill experiments identified a limited bactericidal effect of pristinamycin in three clinical and both reference strains. These three strains had been classified as pristinamycin-resistant by the Vitek-II system, which uses a kinetic turbidimetric measurement of bacterial growth. Epidemiological information is hindered by the use of highly selected strains for the study. 

Reversed flow (Fig. 3.4e). This is a powerful tool for kinetic methods (see also 8.6.4). It requires a stop/start feature in addition to gearing for reverse drive, and has been exploited mainly in sequential and bead injection determinations. An example is the kinetic turbidimetric determination of phytic acid in food samples relying on the influence of the analyte concentration on the rate of calcium oxalate crystallisation [10]. The sample and reagents were aspirated and then the flow was reversed, directing the sample zone towards the flow cell, where its central portion was stopped for 30 s, monitored and discarded. 

J.G. March, B.M. Simonet, F. Grases, Kinetic-turbidimetric determination of phytic acid by sequential injection analysis, Anal. Chim. Acta 409 (2000) 9. 

Remillard J.F., Case Gould M., Roslansky PF. and Novitsky T.J. (1987) Quantitation of endotoxin in products using the LAL kinetic turbidimetric assay. In Detection of bacterial endotoxins with the Limuius amebocyte lysate test. Alan R. Liss, Inc, New York. 197-210. 

A two-point kinetic turbidimetric technique using commercially available monospecific antisera has been used for the determination of immunoglobulins G, A, and M in sera. The accuracy of the method when measuring idiotypic monoclonal proteins is greater than that of the radial diffusion method. 

In one case, a small peptide with enzyme-like capability has been claimed. On the basis of model building and conformation studies, the peptide Glu-Phe-Ala-Ala-Glu-Glu-Phe-Ala-Ser-Phe was synthesized in the hope that the carboxyl groups in the center of the model would act like the carboxyl groups in lysozyme 17). The kinetic data in this article come from assays of cell wall lysis of M. lysodeikticus, chitin hydrolysis, and dextran hydrolysis. All of these assays are turbidimetric. Although details of the assay procedures were not given, the final equilibrium positions are apparently different for the reaction catalyzed by lysozyme and the reaction catalyzed by the decapeptide. Similar peptide models for proteases were made on the basis of empirical rules for predicting polypeptide conformations. These materials had no amidase activity and esterase activity only slightly better than that of histidine 59, 60). 

In another paper (N5), Nielsen reports further kinetic studies of barium sulfate crystal growth, followed turbidimetrically and conducti-metrically. When the concentrations of Ba++ and S04 were larger... 

Although extended reaction time was also used by Selsted and Martinez (1980) in their turbidimetric method, they did not stress the importance of the kinetic order of cell lysis. McKenzie and White (1986) showed that, while the reaction is biphasic, simple kinetics are obeyed over a sufficiently long period that this property can be exploited in the determination. The kinetic order must be considered in the quantitative treatment of the results. The limit of detection was found to be 100 pg ml of reaction mixture, equivalent in the case of bovine milk lysozyme to 6 ng ml of skim milk, for a 50-/xl sample. 

The analysis of growth kinetics allows calculations of mutation frequencies and assessment of overall toxicity of tested compounds. Although this assay provides streamlined protocol and can be fully automated, the turbidimetric measurements of cell density are not highly sensitive or accurate. 

The samples of products are incubated with Limulus amoebocyte lysate at 37°C. If endotoxins are present a solid gel forms, indicating the presence of endotoxins. The British Pharmacopoeia (2002) describes six separate methodologies for the test for endotoxin. These are (A) gel-clot limit test (B) gel-clot semi quantitative (C) turbidimetric kinetic method (D) chromogenic kinetic method (E) chro-mogenic end-point method and (F) turbidimetric end-point method. There are checks for interfering factors. Any validated method may be used, but the gel-clot method is the referee test in the case of dispute. Coloured products cannot be tested by... 

The traditional way to measure aqueous solubility is by the shake-flask method, in which the compound is added in solvent and shaken until equilibrium is reached. This is, however, a time-consuming method. Another frequently used technique, the turbidimetric method, is based on aqueous titration of a DMSO solution of the compound under investigation. The solubility corresponds to the concentration at which precipitation occur. However, this method neglects the effect and particular kinetics of the solid state and... 

As with turbidimetric assays, many of the direct UV absorbance assays are set up to determine kinetic solubility. However, the UV absorbance method also lends itself well to thermodynamic solubility determination by extending the period of sample agitation prior to filtration to 24 h or more. This offers a number of advantages. The solubility data generated are less dependent on the physical form of the initial material precipitated from DMSO and are much closer to thermodynamic solubility values determined later in discovery and in early development. As such, it gives more consistent solubility data through the discovery phase and enables a better quality early assessment to be made of the likely difficulties or otherwise of progressing a lead series into development. 

Quantitative precipitation may be observed after a few seconds, and can be confirmed by potentiometry, but turbidity is sometimes observed only after several weeks [9]. Therefore, the timing of turbidimetric procedures is generally a very important aspect of system design [55]. In this context, the rate of turbidity formation can be selected as the measurement basis for kinetic procedures, as elegantly demonstrated in the flow injection determination of immunoglobulin G in serum [56]. 

The turbidimetric principle and automated microtiter plate reading apparatus are more commonly used in the kinetic rather than the end-point mode. Turbidity readings of each reaction mixture are taken at frequent intervals throughout an incubation period. The logarithm of the time (the onset time) taken to reach a specified level of turbidity is inversely proportional and linearly related to the logarithm of the concentration of endotoxin in the material under test. Standardization is necessary with each series of assays. This approach is only practical for routine application using microprocessor-controlled equipment. 

A nephelometric method was developed for the analysis of levallorphan tartrate in the presence of levorphanol tartrate. The method takes advantage of the kinetics of bromination of the allyl function on levallorphan which in acidic aqueous medium is faster than the bromination of the levorphanol. The bromination product is insoluble and hence lends itself to a turbidimetric determination (21). The procedure involves adding 1 mg of levallorphan tartrate (along with up to 10 mg of levorphanol tartrate) to 5 ml of 2.5N HCl and 15 ml of water in a glass stoppered 25-ml graduated cylinder. Bring to 25°C and add... 

Fig. 5.4 Analysis of the kinetics of the dispersion polymerization by turbidimetric measurements. The turbidimetry measured at different wavelengths given in the insets is plotted against time [10].

Turbidimetric and nephelometric assay Nephelo-metry and turbidimetry, because of their speed and ease of use, are most widely used. These techniques can be used either by measuring the amount of Ag-Ab complex formation (endpoint methods) or by measuring the rate of complex formation (kinetic methods). The kinetic methods are more rapid because measurements are accomplished within 20 s, and are more precise because sample blanks are not necessary. Kinetic assays are, however, somewhat less sensitive because low-affinity antibodies do not have time to react. The occurrence of Ag-Ab formation is related to the amount of light scattering and is used as the basis for antigen quantification. This approach has been accompanied by the development of... 

CrystaUisation of biliary cholesterol monohydrate is a multiphase process not yet fuUy understood [60]. Bile is normally supersaturated with respect to cholesterol [61] which is solubilised by bile salts (the soluble end product of cholesterol metabolism, such as sodium glycocholate and sodium taurocholate [9]) within micelles, whose solubilising capacity is considerably increased by the incorporation of phosphohpid molecules such as lecithin [62]. Biliary vesicles contain virtually no bile salts but may accumulate cholesterol up to a cholesterol/phosphohpid ratio of 2 1 (by phospholipid transfer to micelles) [63]. These thermodynamically unstable (but kinetically stabilised) vesicles then aggregate and nucleate cholesterol crystals [64,65]. The mechanism of this crucial miceUe-to-vesicle transition has been the subject of various physicochemical studies, including, e.g. calorimetric, turbidimetric, dynamic light and neutron scattering methods [66-69]. 

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