Kinetic turbidimetric assay

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. 

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. 

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. 

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). 

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 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. 

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... 

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