Nephelometric detection

The majoiity of the various analyte measurements made in automated clinical chemistry analyzers involve optical techniques such as absorbance, reflectance, luminescence, and turbidimetric and nephelometric detection means. Some of these ate illustrated in Figure 3. The measurement of electrolytes such as sodium and potassium have generally been accomphshed by flame photometry or ion-selective electrode sensors (qv). However, the development of chromogenic ionophores permits these measurements to be done by absorbance photometry also. 

The majority of the various analyte measurements made in automated clinical chemistry analyzers involve optical techniques such as absorbance, reflectance, luminescence, and turbidimctric and nephelometric detection means. 

The Schlieren effect has been investigated mainly in relation to UV—Visible spectrophotometry [3,28,92,93], but it is also important in fluorimetry. Analytical procedures involving chemi- or bioluminescence detection are less affected due to the geometry of the flow cell and the integrating nature of the light collection process. The influence of the Schlieren effect on turbidimetric and nephelometric detection is less pronounced because mixing tends to be more efficient due to the presence of solid particles in the flowing sample. 

Although very effective for compensating the influence of the Schlieren effect, Eq. 4.7 holds only when the intensity of the Schlieren effect is not wavelength dependent, otherwise, correction factors should be added. As the transient mirrors established between fluid elements of different refractive indices are not ideal and the incident light is also partially refracted, the refraction angle is strongly wavelength dependent (Eq. 4.15). Hence, the use of Eq. 4.7 for Schlieren compensation may be subject to restrictions. Moreover, it cannot be directly applied for Schlieren compensation in flow systems with turbidimetric or nephelometric detection. 

FIGURE 27.3 FBA system with nephelometric detection. Vj to Vj solenoid valves, Lys L-lysine, I/A isopropanol-acetone mixture, ISOP isopropanol, MC mixing chamber, SF spectrofluorometer. 

Fig. 3. Schematic of optical detection methods, (a) absorbance or colorimetric (b) turbidimetric and (c) nephelometric.

These measure turbidity, usually by sensing light scattered at 90° to the direction of the incident light beam. This type of turbidimeter is frequently termed a nephelo-meter or nephelometric turbidimeter. A typical arrangement is shown in Fig. 6.46. The nephelometer is commonly used for detecting solid particles present in water (e.g. waste water) or in air (e.g. smoke). The instrument requires periodic cleaning due to fouling of the optical system. 

Nephelometric methods in general are more sensitive than turbidimetric assays and have an average lower limit of detection of 0.1 to 10 mg/L for a serum protein. Lower detection limits are obtained in fluids, such as cerebrospinal fluid and urine, because of their lower lipid and protein concentrations, which results in a better signal-to-noise ratio. In addition, for low molecular weight proteins (e.g., myoglobin, MW 17,800), assay detection Emits can be lowered using a latex-enhanced procedure based on antibody-coated latex beads. ... 

Limit of Detection. Detection limits of approximately 10p,g/mL (lOmg/L) have been attained with routine nephelometric and turbidimetric methods this limit has been lowered by 10 to 100 fold using antibodies adsorbed onto latex particles. RID methods are able to detect down to a minimum of 10 to 20p.g/mL the detection limits have been lowered even further by using special low-level plates (Calbiochem-Behring Corp., La Jolla, Cahf.). Methods using labeled antibodies (e.g., RIA, EIA, chemiluminescence) are very sensitive and can measure levels as low as nanograms per milliliter level. 

The use of CRP for these purposes requires the use of hsCPR assays having detection limits less than 0.3 mg/L. " A number of automated immunoturbidimetric and inrnmno-nephelometric assays are commercially available and capable of sensitive and precise measurements at low concentrations of CRP. The analytical performance of nine of these assays has been evaluated. ... 

Fig. 5.21 Photometric detection unit of the CHEM-1 featuring (a) bubble, (b) spectrophotometric and (c) nephelometric optical detectors. (Courtesy of Technicon).

Detection By uv spectroscopy, by colorimetric determination of the hydrogen cyanide released under irradiation, biologically via lactic acid formation with Lac-tobacillus species or by nephelometric measurement of their multiplication. 

ABEI produces ECL when oxidized at 1.0 V versus Ag/AgCl in alkaline aqueous solution. In contrast to luminol, ABEI labels do not markedly lose their CL efficiency when conjugated with proteins. ECL immunoassays with a flow injection analysis (FIA) system using ABEl-isothiocyanate as a label were proposed, which have a better performance than either single-radial immunodiffusion or nephelometric immunoassays. ABEI can also be used as an oligonucleotide marker to label a DNA probe. The intensity of the ABEI ECL was linearly related to the concentration of the complementary sequence in the range 96-96 nM, and the detection limit was down to 30 pM. 

A 25 mm disc of 57% PAC media was challenged with a stream containing 250 NTU (nephelometric turbidity units) of A2 fine test dnst nntil the differential pressure reached 40 psi. The dirt holding capacity was 18 mg/cm as compared to 17 mg/cm for the nano aliunina media without PAC. It had been anticipated that the considerable amount of PAC in the structure would have an adverse affect on the retention of additional particles. The turbidity of both filtrates was less than detectable throughout the whole experimerrt. In both cases, the filtration efficiency exceeded 99.99%. In addition to demorrstrating... 

A nephelometric method has been used in this work for determination of dissociation constants of polyphosphonates at very low free-calcium ion concentrations (10 M). This method provides direct determination of the complex-calcium to ligand ratio. This direct determination of the calcium complex model is very important since anticipated models or extrapolation of the calcium complex model determined from other methods can lead to serious error. Model refers to the type of metal complex being detected as indicated by the mole ratio of metal to ligand. For example, the calcium ion activity electrode (Orion Research Inc., Cambridge, Mass.)... 

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