Detection nephelometry

Light scattering (nephelometry) was used as a detection system for gly-cosaminoglycans from urine, eluted from a DEAE Sephadex (Pharmacia Biotechnology Uppsala, Sweden) A-25 column.68 This technique has been more recently applied to protein characterization.69 Interferometry was used for analysis of dextran eluted from a size exclusion column.70 One of the problems of electrochemical detection is that it is relatively insensitive to polymers. Because many of the materials discussed below (DNA, proteins, and polysaccharides) are polymeric, a brief mention of some alternative... 

In multiple myeloma, the neoplastic plasma cells secrete monoclonal proteins such as either IgG or IgA. Generally, either k or A. light chains are secreted. These M (monoclonal) proteins can be detected by serum and urine protein zone and immunofixation electrophoretic techniques, the latter providing better discrimination. The immunoglobulin levels exceed 25 g/L. Quantitation of immunoglobulins can be performed by rate nephelometry. 

Schultz-Ellison, G., Charland, C., Driscoll, J., and Thayer, W., A rapid method for immune complex detection PEG insolubilization combined with laser nephelometry. /. Immunol. Methods 31, 31-40 (1979). 

The quantitation of enzymes and substrates has long been of critical importance in clinical chemistry, since metabolic levels of a variety of species are known to be associated with certain disease states. Enzymatic methods may be used in complex matrices, such as serum or urine, due to the high selectivity of enzymes for their natural substrates. Because of this selectivity, enzymatic assays are also used in chemical and biochemical research. This chapter considers quantitative experimental methods, the biochemical species that is being measured, how the measurement is made, and how experimental data relate to concentration. This chapter assumes familiarity with the principles of spectroscopic (absorbance, fluorescence, chemi-and bioluminescence, nephelometry, and turbidimetry), electrochemical (poten-tiometry and amperometry), calorimetry, and radiochemical methods. For an excellent coverage of these topics, the student is referred to Daniel C. Harris, Quantitative Chemical Analysis (6th ed.). In addition, statistical terms and methods, such as detection limit, signal-to-noise ratio (S/N), sensitivity, relative standard deviation (RSD), and linear regression are assumed familiar Chapter 16 in this volume discusses statistical parameters. 

The alternative method to turbidimetric detection used for measuring solubility in early discovery is to quantify the aqueous supernatant directly via UV absorbance [13, 20, 21]. Typically, DMSO stock solution is added to aqueous buffer such that the final DMSO composition is kept to a minimum (5% or less) and the resulting precipitate is removed by filtration. A UV plate reader is then used to determine the aqueous solubility by comparing the filtrate absorbance against that of a calibration solution prepared in an identical solvent. It is important to match the sample and calibration solutions to prevent solvochromic effects. Care must also be taken in the selection of the filter plate since nonspecific binding of compound can occur with some filter materials leading to erroneously low solubility values [22], Like nephelometry, the plate-based UV detection approach is amenable to automation. 

Precipitate detection methods typically use light scattering techniques such as nephelometry, flow cytometry, and turbidity measurements to determine the amount of the precipitate formed during the incubation process. A major advantage with these types of techniques is the availability of particle size distribution and aggregation information. 

The applications of automatic continuous segmented analysers can aiso be classified according to the type of detection system involved. Thus, 70-75 of all the methodologies described on this topic used molecular UV absorption spectroscopy (spectrophotometry, photometry), followed by ISE potentiometry (10-15 ) and, much less often, nephelometry, fluorimetry, etc. The applications described below were mostly developed with the aid of Technicon technology and are classified according to this criterion —other applications to specific problems related to laboratory processes are described in the corresponding chapters. 

The method used for detection depends on the type of label used. Isotopic counting is employed for radioisotopes, colorimetry for enzyme assays, luminescence and fluorescence measurements can be achieved by means of photomultiplier tubes, while turbidimetry or nephelometry is used for particle enhanced assays. 

Antibodies have been used in analysis for over 60 years, and offer an unexpectedly wide range of techniques and applications. In some cases, the specific combination of an antibody with the corresponding antigen or hapten can be detected directly (e.g., by nephelometry), but more often such reactions are monitored by a characteristic label such as a radioisotope, fluorophore, etc. Since antibody reactions do not have a built-in amplification effect, these labels are frequently necessary to provide sufficient analytical sensitivity. Antibodies of different classes vary greatly in stability, but some are relatively robust proteins, and this contributes significantly to the range of methods available. 

Lysozyme (hen egg-white presumed) has been shown to complex with Pseudomonas aeruginosa lipopolysaccharide in two distinct stages. The initial stage did not produce turbidity detectable by nephelometry but did permit sedimentation of the complex. The association, which was considered to provide a model for investigation of in vivo protein-lipopolysaccharide interactions, was disrupted 100% by the action of Mg +. 

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